Nicotinic Acetylcholine Receptor Mechanism Of Amyloid β-Peptide Neurotoxicity: Electrophysiological And Intracellular Calcium Imaging Study

Alzheimer's disease (AD) is the most common form of dementia among the elderly, which causing severe impairment of learning and memory. One prominent feature of AD is the presence of high density of senile plaques in the brain. The main constituent of senile plaque is amyloid P-peptides (Aβ), which consisting of 39-43 amino acids and coming from the proteolysis of amyloid precursor protein (APP). As reported widely, the full-length fragment of Aβmolecules, no matter in vivo or in vitro experiments, is neurotoxic.A shorter, hydrophobic fragment of Aβ, Aβ_25-35, though not present in biological systems, is widely used together with, or instead of, the endogenous fragment by a number of investigators and is found to be at least as toxic as the full-length fragment. In addition, our previous study indicates that Aβ_31-35, a shorter fragment than Aβ_25-35, can also induce the apoptosis of rat cortical neurons. Another characteristic of AD is degeneration of basal forebrain cholinergic neurons and ensuing deficient cholinergic functions in cortex and hippocampus. Cholinergic receptors include muscarinic and nicotinic receptors. Relative to muscarinic and other receptors, a large decrease in brain nicotinic receptor levels occurs in AD. Nicotinic acetylcholine receptors (nAChRs) are a multigene family of ligand-gated ion channels that participate in various cognitive brain functions. It is reported that Aβ_1-42 binds toα7 andα4β2 subtypes of nAChRs with high affinity in the picomolar and nanomolar range, respectively. Thereinto, neurons possessingα7 andα4β2 nAChRs are particularly vulnerable.Hippocampus in the central nervous system has been widely considered to be a crucial center for learning and memory. Hippocampal long-term potentiation (LTP) is an activity-dependent increase in the synaptic response and has been used as a cellular model of learning and memory. It is reported that different Aβfragments can affect hippocampal LTP in vivo and in vitro. However, the shortest active sequence and the mechanism of Aβin depressing LTP are still not well known. Especially, whether and how nAChRs are involved in the Ap-induced LTP suppression is still an open question. Although previous reports showed that nAChRs agonist can induce LTP in hippocampus and cigarette smoking is negatively associated with AD, the recent studies indicated that theα4β2 nAChRs specific agonist can directly depress LTP induction in rat dentate gyrus in vivo, and current smokers have an increased risk of AD compared with never smokers. Therefore, the effects of nAChRs in LTP induction and AD progress are still controversial up to now. On the other hand, high amounts of nAChRs have been found in the hippocampus of the mammalian animals, which can be divided into two categories: high-affinity receptors such asα4β2 nAChRs and low-affinity receptors such as homomericα7 nAChRs. Different subtypes of nicotinic currents have been recorded on hippocampal slice and cultured rat hippocampal neurons. However, it is not well known whether and how nAChR-mediated currents are modulated by neurotoxic Ap. And also, it is still arguable whetherα7 orα4β2 nAChRs expresses on the acutely isolated hippocampal CA1 neurons. At the same time, it is widely accepted that the disturbances of intracellular calcium homeostasis play a proximal pathological role in the neurodegeneration associated with AD, and Aβinduced [Ca²⁺]_i increase may be a final common path in the neurotoxicity of Aβ. However, the mechanisms by which Ap increased [Ca²⁺]_i are still not well known. It is reported that Aβ_1-42 binds toα7 andα4β2 subtypes of nAChRs with high affinity, and the neuronal nAChRs have a high relative permeability to calcium. Probably, Aβelevates [Ca²⁺]_i through binding and impairing nAChRs.Therefore, the purposes of the present study are as follows: (1) clarifying the effects of three Aβfragments (namely Aβ_25-35, Aβ_31-35 and Aβ_35-31) on the induction of hippocampal LTP in vivo and the possible nAChRs mechanism; (2) clarifying whether nAChRs expresses on the acutely isolated hippocampal CA1 neurons and the modulation effects of Aβon nAChRs; (3) investigating the mechanism by which Aβincreased [Ca²⁺]_i.especially whether and how nAChRs are involved in the Ap-induced [Ca²⁺]_i elevation; and (4) additionally, identifying whether Aβ_31-35 is the shorter active center than Aβ_25-35 in full-length of Aβmolecule by comparing the effects of these three Aβfragments on LTP, whole-cell currents and [Ca²⁺]_i elevation.Partâ… : Study on the nAChRs Mechanisms of Amyloidβ-Peptides in Inhibiting Hippocampal Long-Term Potentiation In VivoThe present study observed the effects of Aβfragments on hippocampal LTP in area CA1 of urethane anesthetized rats, and investigated the probable nAChRs mechanism by which Aβinsults LTP. The results showed that: (1) In control group (i. c. v. injection of saline), by applying high-frequency stimulus (HFS), the amplitudes of the field excitatory postsynaptic potentials (fEPSPs) increased to 205.1±10.0% immediately after HFS, as compared to that measured before HFS and set arbitrarily as 100%, and remains at 160.8±7.3 % 60 min after HFS (n=7). (2) I.C.V. injection of 25 nmol Aβ_25-35, Aβ_31-35 and Aβ_35-31 had no effect on baseline synaptic transmission; but Aβ_25-35 and Aβ_31-35 similarly and significantly depressed the induction of LTP, being 118.5±3.0 % (n=7) and 114.3±4.1% (n=8) at 60 min after HFS, respectively; while the reversal sequence of Aβ_31-35, fragment 35-31, had no effect on the induction of LTP, being 155±3.9% (n=6) at the same time point, similar to the effect in control group. (3)α4β2 subtype nAChRs specific agonist epibatidine impaired LTP in a dose-dependent manner. After i.c.v. injection of 0.01 ug, 0.05 ug and 0.5 ug epibatidine, the LTP at 60 min post HFS decreased from 160.8±7.3% in control to 129.7±4.4% (n=6), 122.3±6.5% (n=7) and 107.6±4.1% (n=6) in three epibatidine groups, respectively. (4) Co-injection of 0.05 ug epibatidine and 25 nmol Aβ_31-35 did not further enhance the Aβ_31-35-induced suppression of LTP, and the LTP value was 118.3±1.8% (n=6) 60 min after HFS, similar to the effect when using 25 nmol Aβ_31-35 alone (114.3±4.1%, n=8). (5) Application ofα4β2 subtype nAChRs specific antagonist dihydro-beta-erythroidine (D-beta-E) alone had no effect on baseline synaptic transmission and hippocampal LTP, but co-injection of D-beta-E and Aβ_31-35 partly reversed the depression of LTP by Aβalone, returning to 132.2±7.5% (n=6) at 60 min after HFS from 114.3±4.1% of Aβ+_31-35 group. (6) Application of vehicle, Aβfragments, epibatidine, D-beta-E and co-application of epibatidine and Aβ_31-35, D-beta-E and Aβ_31-35 showed no significant effect on paired pulse-evoked facilitation.These results demonstrate that: (1) Aβ_31-35, similar to Aβ_25-35, can impair the induction of hippocampal LTP in vivo, suggesting Aβ_31-35 may be a shorter active fragment of Aβ. (2)α4β2 subtype nAChRs are required for the Aβpeptides-induced suppression of LTP in hippocampal CA1 area in vivo. (3) LTP suppression induced by Ap/epibatidine and the reverse effect of D-beta-E are not associated with presynaptic neurotransmitter release in the CA1 region in vivo.Partâ…¡: Effects of Amyloid p-Peptide on nAChRs-Mediated Currents in Acutely Isolated Rat Hippocampal CA1 NeuronsTo investigate whether the nAChRs such asα7 andα4β2 subtypes express on the acutely isolated hippocampal CA1 neurons and whether nAChR-mediated currents are modulated by neurotoxic Aβ, the present study recorded nicotine (non-specific nAChRs agonist)-, choline (specificα7 nAChRs agonist)-, and epibatidine (specificα4β2 nAChRs agonist)-evoked currents on acutely isolated hippocampal neurons in rats with patch clamp technique and examined the effect of Aβfragments on these nAChRs-mediated whole cell currents. The results showed that: (1) Application of AP25.35, Aβ_31-35 or Aβ_35-31 alone did not directly induce any membrane current even at a higher concentration (10μM). (2) Nicotine effectively evoked inward currents on hippocampal CA1 neurons in a dose-dependent manner, the average current density being 13.4±0.83 pA/pF (n=16), 18.9±0.96 pA/pF (n=13) and 27.0±1.74 pA/pF (n=15) when applying 10μM, 100μM and 1mM of nicotine, respectively. (3) The specificα7 nAChRs agonist choline andα4β2 nAChRs agonist epibatidine all evoked whole cell currents on hippocampal CA1 neurons, the average current density being 21.4±1.52 pA/pF (n=12) and 11.2±0.87 pA/pF (n=10), respectively. (4) The desensitization of choline-induced current is quicker, the fast time constants (Ï„_f) and slow time constants (Ï„_s) being 224.52±39.73 ms and 2850.49±491.23 ms (n=12), respectively; the desensitization of epibatidine-induced current is slower, theÏ„_f andÏ„_s being 604.69±89.08 ms and 8809.43±1033.03 ms (n=10), respectively. (5) Pretreatment with AP25.35 reversibly suppressed the nicotine (1 mM)-induced current in a concentration-dependent manner, from 100% (set arbitrarily) in control to 68.5±7.2% (n=8), 54.5±7.9% (n=8) and 40.3±4.0% (n=8) 3 min after perfusion with 0.1, 1 and 10μM Aβ_25-35, respectively. (6) Aβ_31-35(1μM), similar to Aβ_25-35,also suppressed the currents evoked by nicotine, decreased to 54.1±6.7% (n=8) from 100% in control. (7) Aβ_35-31, a reverse sequence of Aβ_31-35, had no effect on the nicotine-induced current, still being 96.9±1.3% (n=8). (8) The whole cell currents evoked by choline and epibatidine were also suppressed by pretreatment with Aβ_31-35, being 57.6±5.3% (n=8) and 72.1±6.7% (n=8), respectively.These results demonstrate that: (1) Nicotine, choline and epibatidine all can effectively evoke inward current in acutely isolated hippocampal CA1 neurons, suggesting that bothα7 andα4β2 nAChRs exist on the membrane of hippocampal neurons and they have different desensitization characteristic. (2) The reversible suppression of nicotine-, choline- and epibatidine-induced currents by the pretreatment of Aβfragments suggests thatα7 andα4β2 nAChRs may be the biological targets of Aβmolecules in AD process. (3) The similar effects of Aβ_25-35 and Aβ_31-35 in suppressing nicotinic currents suggest that the fragment 31-35 may be a shorter active fragment in full length of Aβpeptide responsible for the suppression of nicotinic currents.Partâ…¢: Effects of Amyloidβ-Peptide on Intracellular Ca²⁺ Concentration and Possible nAChRs MechanismThe present study, utilizing calcium imaging via laser-scanning confocal imaging system, observed the effects of Aβfragments on [Ca²⁺]_i in primary cultured cortical neurons, and investigated the probable cholinergic mechanism by which Aβinduced [Ca²⁺]_i elevation. The results showed that: (1) Both Aβ_25-35 and Aβ_31-35 all can induce [Ca²⁺]_i elevation in a concentration dependent manner. After application of 6.25μM, 12.5μM and 25μM Aβ_25-35 to primary cultured cortical neurons.the relative fluorescent intensity of neurons obviously increased from 100% in control to 119.5±3.9% (n=36), 136.8±7.7% (n=21), and 160±10.9% (n=20), respectively. With the same concentration of Aβ_31-35, similar elevation in [Ca²⁺]_i is also found, from 100% in control to 122.4±4.18% (n=28), 134.4±7.9% (n=37), and 151.2±6.4% (n=26), respectively. However, at the same concentration, the effects of Aβ_25-35 and Aβ_31-35 on [Ca²⁺]_i elevation does not show any statistical significance. As a control, Aβ_35-31, the reverse peptide of Aβ_31-35, had no effect on [Ca²⁺]_i, from 100% to 101.2±6.0% (n=18). (2) Non-specific nAChRs antagonist mecamylamine (MCA) can partly block the [Ca²⁺]_i elevation induced by Aβ_25-35 or Aβ_31-35 in a concentration dependent manner. 1μM, 10μM and 100μM MCA were used to pretreat the cultured neurons for 30 min, then the effect of 25μM Aβ_25-35 or Aβ_31-35 on [Ca²⁺]_i was observed. We found that, with the increase of MCA concentration, the [Ca²⁺]_i elevation induced by AP25.35 or Aβ_31-35 decreased. After co-application of MCA and Aβ_25-35, the relative fluorescent intensity, at three different concentration of MCA, was 138.5±6.5% (n=27), 126.2±6.7% (n=17), and 117.9±9.9% (n=20), respectively. After co-application of MCA and Aβ_31-35, the relative fluorescent intensity was 132.1±8.9% (n=17), 123.4±8.9% (n=22) and 114.8±8.3% (n=20), respectively. Compared with application of AP25.35 or Aβ_31-35 alone, the [Ca²⁺]_i elevation in co-application of MCA and Aβ_25-35 or Aβ_31-35 significantly decreased (P<0.01). (3) Pretreatment with 10μM dihydro-beta-erythroidine (D-beta-E), a specificα4β2 subtype nAChRs antagonist, also significantly decreased the [Ca²⁺]_i elevation induced by Aβ_25-35 and Aβ_31-35.Compared with application of Aβ_25-35 or Aβ_31-35 alone, the relative fluorescent intensity in co-application of D-beta-E and Aβ_25-35 or Aβ_31-35 reduced to 150.3±8.5% (n=28, P<0.05) and 141.0±7.3% (n=22, P<0.05) from 160±10.9% (n=20) and 151.2±6.4% (n=26), respectively.These results demonstrate that: (1) both Aβ_25-35 and Aβ_31-35 can elevate [Ca²⁺]_i in primary cultured cortical neurons, suggesting Aβ_31-35 may be a shorter active fragment in full length of Aβresponsible for Aβ-induced intracellular calcium overloading; (2) nAChRs, includingα4β2 subtype of nAChRs, at least partly participate in the Aβ_25-35 and Aβ_31-35 induced [Ca²⁺]_i elevation; (3) the impairment of neuronal nAChRs by Aβfragments is probably implicated in the intracellular Ca²⁺ overloading and the deficit in cognitive function.In conclusion, the present study, by using field potential recording, whole-cell patch clamp technique and calcium imaging via laser-scanning confocal imaging system, observed the effects of Aβ_25-35, Aβ_31-35 and the reverse peptide Aβ_35-31 on hippocampal LTP in vivo, postsynaptic nAChRs current of acute dissociated CA1 neurons and intracellular Ca²⁺ concentration of cultured cortical neurons. The roles of nAChRs in Aβ-induced LTP suppression and [Ca²⁺]_i elevation were investigated. The results demonstrate that Aβ_31-35, just as Aβ_25-35, can strongly suppress the induction of hippocampal LTP in vivo andα4β2 subtype nAChR is required for the Aβ-induced suppression of LTP; Aβ_25-35 and Aβ_31-35 can directly block the activation of postsynaptic nAChRs, includingα7 andα4β2 subtypes, in acutely isolated hippocampal neurons; both Aβ_25-35 and Aβ_31-35 can elevate [Ca²⁺]_i in primary cultured cortical neurons by altering nAChRs includingα4β2 subtype. These results might be useful in understanding the cognitive impairment in AD and provide some clues about protecting neurons against Aβneurotoxicity. Additionally, the results support our previous hypothesis that Aβ_31-35 may be a shorter active center than Aβ_25-35 in full-length of Aβmolecule.