1. Effects Of Amyloid β-protein Fragment 31-35 On BK Channels And [Ca～(2+)]_i Of Hippocampal CA1 Neurons: Observation By Whole Cell Clamping And Ion Fluorescence Imaging Techniques 2. Humanin, A Novel Rescue Factor, Antagonize Aβ31-35-triggered Neurot

Partâ… Effects of amyloidβ-protein fragment 31-35 on BK channels and [Ca²⁺]_i of hippocampal CA1 neurons: observation by whole cell clamping andion fluorescence imaging techniquesThe excessive extracellular deposition of amyloid-βpeptide (Aβ) in brain areas forming senile plaques and the degeneration of neurons in cortical and hippocampal regions are the invariant characteristic features of Alzheimer's disease (AD). Aβis a polypeptide of 39-43 amino acids, and is generated by enzymatic cleavages from amyloid precursor protein. Up to now, it is generally accepted that Aβ1-42 and Aβ25-35 possess similar neurotoxicity. Recently, our lab and others have showed that Aβ31-35, a shortest fragment of Aβ, also possesses similar neurotoxicity and thus this peptide has been used as a tool in analysis the effects of Aβon hippocampal and cortical neurons.However, the mechanism of Aβ-induced neurotoxicity has not been well understood by far. Some studies have proved the neurotoxicity of Aβare associated with the overloading of intracellular Ca²⁺, direct neurotoxicity, free radical production, formation of new ion channels and so on. Others begin to emphasize Aβ-induced suppression of potassium currents more and more, which brings on the longer time course of membrane repolarization and action potential, and the latter leads to the elevation of [Ca²⁺]_i, the change of neural excitability and the increase of discharge frequency. A large-conductance Ca²⁺-activated K⁺ channel (BK channel), an important K⁺ channel, is extensively distributed in excitable tissue, including nervous system. BK channels can be activated following membrane depolarization and the elevation of [Ca²⁺]_i, and plays a key role in the course of membrane repolarization. Such as the promotion of repolarization course, the suppression of Ca²⁺ influx and the less release of neurotransmitter owing to the decrease of [Ca²⁺]_i.Our previous researches have indicated that Aβ31-35 may affect neuronal electrophysiological properties of BK channel by using inside-out patch clamp technique in isolated hippocampal neurons of rats. In present experiments, we observed that Aβ31-35-mediated affection to BK channel by using whole-cell model of patch clamp technique, and that to [Ca²⁺]_i by using Ca²⁺ fluorescence imaging, to try to explore neurotoxicity induced by Aβfurther.The properties of BK channels of whole cell in acutely isolated hippocampal CA1 neurons of ratsWhole cell outward currents were evoked by depolarizing steps from -60 to +45 mV (increment 15 mV) in bathing solution with Ca²⁺, which contained a mixture of channel blockers: 4-AP, 5 mM, for transient voltage-gated K⁺ current and SK current; glybenclamide, 5μM, for ATP-sensitive K⁺ current. 4-AP and glybenclamide were included routinely in bathing solution unless otherwise indicated. On the conditions, the very strong outward currents appeared, and the first peak amplitude of the outward currents neared 3000 pA (membrane potential at +45 mV) and was nearly suppressed by TEA (20 mM), which suggested that the outward currents belonged to K⁺ currents. According to Sun XL et al., the outward currents are composed of rapidly activating, transient outward currents mediated by extracellular Ca²⁺ mostly, and slowly activating, sustained outward currents. And BK channels mediate a major part of the transient outward. The sustained outward currents are composed of multiple components. Therefore, a variation of the peak amplitude in transient outward currents can be mostly considered to the change of BK currents. In the present experiment below, Aβ31-35 was applied in transient currents above, thus we might observe the affection of BK currents mediated by Aβ31-35.Aβ31-35 suppressed BK currents of whole cell in acutely isolated hippocampal CA1 neurons of ratsIn one experimental group, the application of Aβ31-35 (5μM) in bathing solution including Ca²⁺, whole cell outward currents recorded by voltage clamp showed that the first peak amplitude of transient outward currents reduced from 2722±201 pA to 2203±195 pA, showing a reduction rate of 19.07%±3.96% (n= 8,P<0.01).Aβ31-35 and Aβ25-35 elevated the [Ca²⁺]_i in acutely isolated hippocampal CA1 neurons of ratsIn the present study, the change of [Ca²⁺]_i induced by two Aβfragments, Aβ31-35 at different dose (5, 12.5 or 25μM) or Aβ25-35 (25μM), was investigated in acutely isolated hippocampal CA1 neurons by fluorescence imaging technique using the Ca²⁺-sensitive dye Furo-2/AM. The results showed that Aβ31-35 elevated [Ca²⁺]_i at 12.5μM-dose group, and the value of R (F340/380) increased from 880.42±11.84 to 1002.20±47.70, showing a growth rate of 12.0%±3.5%(n=5, P<0.05). Aβ31-35 induced a greater elevation of [Ca²⁺]_i at 25μM-dose group, and the value of R increased from 873.58±7.78 to 1311.40±112.04, showing a growth rate of 32.7%±5.4% (n=5, P<0.01).When the neurons were exposed to 5μM-Aβ31-35, the elevation of[Ca²⁺]_i was not significant difference.In the experiment of application of Aβ25-35, the dose of 25μM was carried out alone. 25μM-Aβ25-35 also elevated [Ca²⁺]_i, and the value of R increased from 862.21±13.1 to 1323.2±115.13, showing a growth rate of 34.1%±5.4% (n=5,P<0.01). There was significant difference between before and after application of Aβ25-35, but no markedly difference between Aβ31-35 and Aβ25-35 at 25μM-dose group.We can draw such conclusions according to the above: (1) BK currents in hippocampal CA1 neurons can be recorded by using whole-cell model of patch clamp technique. Aβ31-35 can be surely deemed the necessary shorter active sequence in Aβ-mediated neurotoxicity, just as Aβ1-40-and Aβ25-35-mediated neurotoxicity; (2) Aβ31-35 can lessen BK currrents by the suppression of BK channels; (3) Aβ31-35 can elevate [Ca²⁺]_i and trigger Ca²⁺ overloading in hippocampal CA1 neurons; (4) The influences of Aβ31-35 to [Ca²⁺]_i and BK channels might be carried out through different pathways. Partâ…¡Humanin, a novel rescue factor, antagonize Aβ31-35-triggered neurotoxicity by the suppression of Ca²⁺ influxAlzheimer's disease (AD) is characterized by overproduction ofβamyloid peptides (Aβ) in the brain with progressive loss of neuronal cells. Some studies have showed that Aβ31-35, a shorter active fragment of Aβ, possesses the similar neurotoxicity as the full length of Aβor Aβ25-35. Humanin (HN), a recently discovered rescue factor, is reported to protect neuronal cells from various AD-relevant. Our previous studies have showed that Aβ31-35 can suppress the BK currents and prevent the repolarization of membrane and generation of the fast after-hyperpolarization (fAHP). Meanwhile, it is generally accepted that the overloading of intracellular Ca²⁺ is an initial factor and one of the key mechanisms for multiple insults. So, in the present of HN, BK currents and voltage-dependent Ca²⁺ currents will be surveyed by using whole cell patch clamp. And HN-induced [Ca²⁺]_i will also be detected by using calcium imaging.The results showed that the BK currents, in the same neuron, were suppressed from 3303.67±332.70 pA in bathing solution (Control) to 2715.12±82.99 pA in bathing solution with Aβ31-35 (P<0.005) and to 2225.54±254.29 pA in the presence of Aβ31-35 and HN (P< 0.001). BK currents, after solitary application of HN, reduced from 1405.97±350.87 pA to 999.74±247.72 pA, showing a reduction rate of 28.4%±10.4% (P<0.05) at 0mV membrane potential. Ca²⁺ currents, in the present of HN, reduced from -622.30±118.13 pA to -467.40±112.28pA, showing a reduction rate of 25.2%±6.9% (P<0.05, n=7) at 0mV membrane potential. At 0 mV membrane potential, the reduction rate of the peak amplitude of Ca²⁺ currents (25.2%±6.9%) match that of BK currents (28.4%±10.4%). There is no significant difference between them (P>0.05). In addition, [Ca²⁺]_i were measured by using laser-scanning confocal imaging system. Cutured cortical neurons were respectively observed before and after application of Aβ31-35 (25μM), Aβ31-35 (25μM) and HN (20μM), and Aβ31-35 (25μM) and nicardipine (20μM). All neurons were first detected the fluorescence intensity (F) of basal [Ca²⁺]_i for 5 min, then that of [Ca²⁺]_i after agent treatments for 20 min. The results showed that F/F_b of [Ca²⁺]_i, after application of Aβ31-35, Aβ31-35 and HN, and Aβ31-35 and nicardipine, increased respectively from 1 to 1.75±0.06 (n= 5, P<0.005), 1.25±0.04 (n= 7,P<0.01) and 1.32±0.02 (n= 6,P<0.01), but there were no significant difference between HN and nicardipine.Therefore we presume that (1) Aβcan increase [Ca²⁺]_i, through L-type Ca²⁺ channels; (2) HN can antagonize Ca²⁺ influx triggered by Aβ31-35; (3) L-type Ca²⁺ channel may be the common target factor of HN and Aβ.According to the above, we can draw such conclusions: 1) Aβcan elevates [Ca²⁺]_i, and suppresses BK currents; 2) Aβ-triggered Ca²⁺ is the reason of its neurotoxicity; 3) HN can reduce Aβ-induced Ca²⁺ overloading by the supression of L-type Ca²⁺ channels; 4) L-type Ca²⁺ channel may be the common target factor of HN and Aβ; 5) Because HN can suppressed either Ca²⁺ or BK currents solitarily and comparably, it is credible that, owing to the suppression of Ca²⁺ channels by HN, the reduction of Ca²⁺ influx results in the decrease of BK currents, but HN has no direct effect to BK channels. Partâ…¢Group I metabotropic glutamate receptors facilitate synaptic transmission of nociceptive inputs at the spinal level in ratsThe excitatory amino glutamate plays a major role in nociceptive processing at the spinal level. Glutamate receptors are divided into two types of receptors named ionotropic receptors (iGluRs) and metabotropic receptors (mGluRs), and most of the previous investigations are focused on the contribution of iGluRs on the spinal processing of the nociceptive inputs. In the present study, we carried out observations to evaluating if two subtypes of mGluRs, mGluR₁ and mGluR₅, were involved in the spinal processing and transmission of nociceptive inputs at the spinal level in rats by intrathecal pretreatment with selective antagonists of mGluR₁ and mGluR₅, namely CPCCOEt and MPEP, respectively, and then examined the changes in both rats' nociceptive behavior and their Fos-like immunoreactive (FLI) neurons in the spinal dorsal horn induced by formalin injection into the plantar aspect of one hind paw.The results showed that the tonic phase of behavioral nociceptive licking and biting induced by formalin injection was significantly suppressed in MPEP and CPCCOEt groups, showing decreases from 131.3±26.5 s in vehicle control group to 64.0±28.4 s (P<0.01) and 58.7±38.7 s (P<0.05) in two experimental groups, respectively; while the number of FLI neurons also decreased from 154.9±21.4 in control group to 98.3±17.0 (P<0.01) and 101.5±21.8 (P <0.05) in experimental groups, respectively. The acute phase of behavioral nociceptive responses did not change markedly by i.t. injection of CPCCOEt and MPEP either.The present experiments indicate that mGluR₁ and mGluR₅ are involved in the facilitation upon the spinal perception and transmission of the peripherally-evoked nociceptive inputs, though the effectiveness of them is less as estimated by immunocytochemical examination than that by behavioral examination. It is proposed that these receptors may be located postsynaptically, as generally thought before, but also located presynaptically on the primary afferent terminals in the dorsal horn, and the activation of these autoreceptors might increase the release of excitatory transmitters from these terminals, and thus facilitates the synaptic transmission.