Immunohistochemical and electrophysiological evidence for ω-conotoxin-sensitive calcium channels in unmyelinated C-fibres of biopsied human sural nerve

In vitro electrophysiological measurements of Ca²⁺ potentials in human sural nerve fascicles revealed that Ca²⁺ conductances might be present on unmyelinated C-fibres. Furthermore, these Ca²⁺ potentials were partially blocked by ω-conotoxin, a calcium antagonist for the N-type Ca²⁺ channels. Therefore, immunohistochemical staining with indirect immunofluorescent ω-conotoxin GVIA was used to localize N-type Ca²⁺ channels in intact and in enzymatically dissociated human sural nerve fascicles. Densities of toxin binding sites were highly heterogeneous throughout the different nerve fascicles investigated and putative N-type Ca²⁺ channels were localized in about 20% of the unmyelinated C-fibres. Myelinating Schwann cells as well as enzymatically demyelinated axons displayed no specific binding indicating the absence of N-type Ca²⁺ channels.     

Effects of ω-conotoxin MVIIC on veratridine-induced cytotoxicity and cytosolic Ca oscillations

External Ca²⁺ entry through various Ca t+-channel subtypes is responsible for the large oscillations of the cytosolic Ca²⁺ concentrations, [Ca²⁺]_i, and cell death induced by veratridine in primary cultures of bovine chromaffin cells. Blockade by ω-conotoxin GVIA (GVIA) of N-type Ca²⁺ channels, by ω-agatoxin GIVA (IVA) of P-type Ca²⁺ channels, or by furnidipine of L-type Ca²⁺ channels did not afford cytoprotection. However, ω-conotoxin MVIIC (MVIIC), a wide-spectrum blocker of N-, P- and Q-type Ca²⁺ channels greatly protected the cells against the cytotoxic effects of veratridine. Furnidipine further enhanced the cytoprotecting effects of MVIIC. MVIIC but not fumidipine, markedly reduced the oscillations of [Ca²⁺]_i induced by veratridine in single fura-2-loaded chromaffin cells. The results suggest that Ca²⁺ entry through any of the different Ca²⁺ channel subtypes present in bovine chromaffin cells might be cytotoxic. They also support two ideas: (i) that wide-spectrum neuronal Ca²⁺ channel blockers (i.e. MVIIC) might be better cytoprotecting agents than more specific neuronal Ca²⁺ channel blockers (i.e., GVIA, IVA, furnidipine); and (ii) that combined Ca²⁺ channel blockers may provide greater cytoprotection than single compounds.     

Signaling effects of α-thrombin and SFLLRN in rat glioma C6 cells

Effects of thrombin on brain cells, including change of neurite outgrowth and astrocyte shape, are described, but the molecular mechanisms are unclear. We investigated the effects of human α-thrombin and a six amino acid thrombin receptor activating peptide (TRAP-6, SFLLRN) on [Ca²⁺]₁, phosphoinositide hydrolysis, and protein kinase C in rat glioma C₆ cells. Stimulation of C₆ cells with both α-thrombin and TRAP-6 resulted in [Ca²⁺]₁ mobilization, [³H]Inositol phosphate response, and enhanced immunoreactivity of the protein kinase C (PKC) isoenzymes α, β, γ, δ, and ϵ. Results suggest that α-thrombin and TRAP-6 activate at least partially the same intracellular signaling pathways in rat glioma C₆ cells, which is evidence for involvement of “tethered ligand” receptor in thrombin induced signaling in glioma C₆ cells. © 1996 Wiley-Liss, Inc.     

Methamphetamine‐induced up‐regulation of α2/δ subunit of voltage‐gated calcium channels is regulated by DA receptors

This study was carried out to determine the roles of dopamine D1 and D2 receptors on the up‐regulation of α₂/δ subunit of voltage‐gated Ca²⁺ channels (VGCCs) induced by methamphetamine (METH). In the conditioned place preference paradigm, METH‐induced place preference suppressed with gabapentin, an antagonist for α₂/δ subunit. Under these conditions, the increase in α₂/δ subunit expression was found in the frontal cortex and limbic forebrain. In addition, the METH‐induced place preference was significantly attenuated by dopamine D1 and D2 receptor antagonists, SCH23390 and sulpiride, respectively. The expression of α₂/δ subunit protein and its mRNA was significantly enhanced in the METH‐treated cortical neurons. These increases in protein and mRNA of α₂/δ subunit were completely abolished by SCH23390 and sulpiride with simultaneous exposure to METH. These findings indicate that up‐regulation of α₂/δ subunit is regulated through the activation of dopamine D1 and D2 receptors during METH treatment. Synapse 64:822–828, 2010. © 2010 Wiley‐Liss, Inc.     

Inhibition of N-type Calcium Channels: The Only Mechanism by which Presynaptic α2-Autoreceptors Control Sympathetic Transmitter Release

α₂-Adrenoceptors are known to inhibit voltage-dependent Ca²⁺ channels located at neuronal cell bodies; the present study investigated whether this or alternative mechanisms, possibly downstream of Ca²⁺ entry, underlie the presynaptic α₂-adrenergic modulation of transmitter release from chick sympathetic neurons. Using chick sympathetic neurons, overflow of previously incorporated [³H]noradrenaline was elicited in the presence of extracellular Ca²⁺ by electrical pulses, 25 mM K⁺ or 10μM nicotine, or by adding Ca²⁺ to otherwise Ca²⁺-free medium when cells had been made permeable by the calcium ionophore A23187 or by α-latrotoxin. Pretreatment of neurons with the N-type Ca²⁺ channel blocker ω-conotoxin GVIA and application of the α₂-adrenergic agonist UK 14304 reduced the overflow elicited by electrical pulses, K⁺ or nicotine, but not the overflow caused by Ca²⁺ after permeabilization with α-latrotoxin or A23187. In contrast, the L-type Ca²⁺ channel blocker nitrendipine reduced the overflow due to K⁺ and nicotine, but not the overflow following electrical stimulation or α-latrotoxin- and A23187-permeabilization. The inhibition of electrically evoked overflow by UK 14304 persisted in the presence of nitrendipine and the L-type Ca²⁺ channel agonist BayK 8644, which per se enhanced overflow. In ω-conotoxin GVIA-treated cultures, electrically evoked overflow was also enhanced by BayK 8644 and almost reached the value obtained in untreated neurons. However, UK 14304 lost its effect under these conditions. Whole-cell recordings of voltage-activated Ca²⁺ currents corroborated these results: UK 14304 inhibited Ca²⁺ currents by 33%, nitrendipine caused a 7% reduction, and BayK 8644 increased the currents by 30%. Moreover, the dihydropyridines failed to abolish the inhibition by UK 14304, but pretreatment with ω-conotoxin GVIA, which reduced mean amplitude from 0.95 to 0.23 nA, entirely prevented α₂-adrenergic effects. Our results indicate that the α₂-autoreceptor-mediated modulation of noradrenaline release from chick sympathetic neurons relies exclusively on the inhibition of ω-conotoxin GVIA-sensitive N-type Ca²⁺ channels. Mechanisms downstream of these channels and voltage-sensitive Ca²⁺ channels other than N-type appear not to be important.     

Deletion of Cav2.1(α1A) subunit of Ca2+‐channels impairs synaptic GABA and glutamate release in the mouse cerebellar cortex in cultured slices

Deletion of both alleles of the P/Q‐type Ca²⁺‐channel Ca_v2.1(α_1A) subunit gene in mouse leads to severe ataxia and early death. Using cerebellar slices obtained from 10 to 15 postnatal days mice and cultured for at least 3 weeks in vitro, we have analysed the synaptic alterations produced by genetically ablating the P/Q‐type Ca²⁺‐channels, and compared them with the effect of pharmacological inhibition of the P/Q‐ or N‐type channels on wild‐type littermate mice. Analysis of spontaneous synaptic currents recorded in Purkinje cells (PCs) indicated that the P/Q‐type channels play a prominent role at the inhibitory synapses afferent onto the PCs, with the effect of deleting Ca_v2.1(α_1A) partially compensated. At the granule cell (GC) to PC synapses, both N‐ and P/Q‐type Ca²⁺‐channels were found playing a role in glutamate exocytosis, but with no significant phenotypic compensation of the Ca_v2.1(α_1A) deletion. We also found that the P/Q‐ but not N‐type Ca²⁺‐channel is indispensable at the autaptic contacts between PCs. Tuning of the GC activity implicates both synaptic and sustained extrasynaptic γ‐aminobutyric acid (GABA) release, only the former was greatly impaired in the absence of P/Q‐type Ca²⁺‐channels. Overall, our data demonstrate that both P/Q‐ and N‐type Ca²⁺‐channels play a role in glutamate release, while the P/Q‐type is essential in GABA exocytosis in the cerebellum. Contrary to the other regions of the CNS, the effect of deleting the Ca_v2.1(α_1A) subunit is partially or not compensated at the inhibitory synapses. This may explain why cerebellar ataxia is observed at the mice lacking functional P/Q‐type channels.     

Characterization of α-melanocyte-stimulating hormone (α-MSH)-like peptides in discrete regions of the rat brain. In vitro release of α-MSH from perifused hypothalamus and amygdala

The neuropeptide α-melanocyte-stimulating hormone (α-MSH) is synthesized by discrete populations of hypothalamic neurons which project in different brain regions including the cerebral cortex, hippocampus and amygdala nuclei. The purpose of the present study was to identify the α-MSH-immunoreactive species contained in these different structures and to compare the ionic mechanisms underlaying α-MSH release at the proximal and distal levels, i.e. within the hypothalamus and amygdala nuclei, respectively. The molecular forms of α-MSH-related peptides stored in discrete areas of the brain were characterized by combining high-performance liquid chromatography (HPLC) separation and radioimmunoassay detection. In mediobasal and dorsolateral hypothalamic extracts, HPLC analysis confirmed the existence of a major immunoreactive peak which co-eluted with the syntheticdes-Nα-acetyl α-MSH standard. In contrast, 3 distinct forms of immunoreactive α-MSH, which exhibited the same retention times as synthetic des-, mono- and di-acetyl α-MSH, were resolved in amygdala nuclei, hippocampus, cortex and medulla oblongata extracts. The proportions of acetylated α-MSH (authentic α-MSH plus diacetyl α-MSH) contained in these extrahypothalamic structures were, respectively, 78, 80, 60 and 92% of the total α-MSH immunoreactivity. In order to compare the ionic mechanisms underlaying α-MSH release from hypothalamic and extrahypothalamic tissues, we have investigated in vitro the secretion of α-MSH by perifused slices of hypothalamus and amygdala nuclei. High potassium concentrations induced a marked increase of α-MSH release from both tissue preparations. However, a higher concentration of KCl was required to obtain maximal stimulation of amygdala nuclei (90 mM) than hypothalamic tissue (50 mM). The effect of depolarizing concentrations of KCl was totally suppressed in the absence of Ca²⁺, indicating that high-K⁺ induced the opening of voltage-operated Ca²⁺ (VOC) channels. Veratridine (50 μM), a depolarizing agent which activates Na⁺ conductances, caused a robust stimulation of α-MSH release from hypothalamic slices but had virtually no effect on amygdala nuclei. ω-Conotoxin (1 μM), a peptide toxin which blocks L- and N-type VOC channels, caused a slight reduction of K⁺-evoked α-MSH release from hypothalamic slices but induced a dramatic decrease of α-MSH release from amygdala nuclei. These data suggest that acetylation of α-MSH to generate the biologically active forms of the peptide is a slow process which occurs gradually during axonal transport. Our results also indicate that release of α-MSH at the hypothalamic level mainly results from activation of T-type VOC channels whereas, in the amygdala nuclei, L- and (or) N-type VOC channels are involved in the regulation of α-MSH secretion.     

Blockade of N- and Q-type Ca channels inhibit K-evoked [H]acetylcholine release in rat hippocampal slices

In the present study, we examined the contribution of specific Ca²⁺ channels to K⁺-evoked hippocampal acetylcholine (ACh) release using [³H]choline loaded hippocampal slices. [³H]ACh release was Ca²⁺-dependent, blocked by the nonspecific Ca²⁺ channel blocker verapamil, but not by blockade of L-type Ca²⁺ channels. The N-type Ca²⁺ channel blocker, ω-conotoxin GVIA (ω-CgTx GVIA; 250 nM) inhibited [³H]ACh release by 44% and the P/Q-type Ca²⁺ channel blocker ω-agatoxin IVA (ω-Aga IVA; 400 nM) inhibited [³H]ACh release by 27%, with the combination resulting in a nearly additive 79% inhibition. Four hundred or one thousand nM ω-Aga IVa was necessary to inhibit [³H]ACh release, ω-Conotoxin MVIIC (ω-CTx-MVIIC) was used after first blocking N-type Ca²⁺ channels with ω-CgTx GVIA (1 μM). Under these conditions, 500 nM ω-CTx-MVIIC led to a nearly maximal inhibition of the ω-CgTx GVIA-insensitive [³H]ACh release. Based on earlier reports about the relative sensitivity of cloned and native Ca²⁺ channels to these toxins, this study indicates that N- and Q-type Ca²⁺ channels primarily mediate K⁺-evoked hippocampal [³H]ACh release.     

Distribution of various calcium channel α1 subunits in murine DRG neurons and antinociceptive effect of ω-conotoxin SVIB in mice

Immunohistological study revealed the differential localization of subtypes of voltage-dependent calcium channels in the dorsal root ganglion neurons. Intrathecal injection of ω-conotoxin SVIB, an analogue of ω-conotoxin GVIA, which acts on N-type voltage-dependent calcium channels, significantly shortened the licking time in the late phase of a formalin test.     

Lipopolysaccharides enhance the action of bradykinin in enteric neurons via secretion of interleukin‐1β from enteric glial cells

Functional changes of the enteric nervous system have been observed under inflammatory states of inflammatory bowel disease increasing the endotoxin level. The aim of the present study was to determine the effect of lipopolysaccharides (LPS) on myenteric neuron–glia interaction in vitro. We examined the increase of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) and the release of interleukin‐1β (IL‐1β) or prostaglandin E₂ (PGE₂) and COX‐2 expression in myenteric plexus cells from the rat intestine induced by LPS. LPS potentiated BK‐induced [Ca²⁺]_i increases in both myenteric neurons and enteric glial cells, which were suppressed by a B1R antagonist. Only in enteric glial cells, a B1R agonist increased [Ca²⁺]_i. The effects of LPS were blocked by pretreatment with an interleukin‐1 receptor antagonist or by reducing the density of enteric glial cells in culture. LPS prompted the release of IL‐1β from enteric glial cells. The augmenting effects of IL‐1β on the BK‐induced neural [Ca²⁺]_i increase and PGE₂ release from enteric glial cells were abolished by a phospholipase A₂ (PLA₂) inhibitor and a COX inhibitor, and partly suppressed by a COX‐2 inhibitor. IL‐1β up‐regulated the COX‐2 expression in enteric glial cells. LPS promotes IL‐1β secretion from enteric glial cells, resulting in augmentation of the neural response to BK through PGE₂ release via glial PLA₂ and COX‐2. The alteration of the regulatory effect of glial cells may be the cause of the changes in neural function in the enteric nervous system in inflammatory bowel disease. © 2009 Wiley‐Liss, Inc.     

β‐Amyloid activates presynaptic α7 nicotinic acetylcholine receptors reconstituted into a model nerve cell system: involvement of lipid rafts

Beta amyloid (Aβ) plays a central role in the pathogenesis of Alzheimer’s disease. Aβ is the major constituent of senile plaques, but there is a significant presence of Aβ in the brain in soluble forms. The results of functional studies indicate that soluble Aβ interacts with the α7 nicotinic acetylcholine receptor (nAChR) complex with apparent high affinity. However, conflicting data exist as to the nature of the Aβ–α7 nAChR interaction, and whether it is the result of specific binding. Moreover, both agonist‐like and antagonist‐like effects have been reported. In particular, agonist‐like effects have been observed for presynaptic nAChRs. Here, we demonstrate Aβ_1‐42‐evoked stimulatory changes in presynaptic Ca²⁺ level via exogenous α7 nAChRs expressed in the axonal varicosities of differentiated hybrid neuroblastoma NG108‐15 cells as a model, presynaptic system. The Aβ_1‐42‐evoked responses were concentration‐dependent and were sensitive to the highly selective α7 nAChR antagonist α‐bungarotoxin. Voltage‐gated Ca²⁺ channels and internal Ca²⁺ stores were both involved in Aβ_1‐42‐evoked increases in presynaptic Ca²⁺ following activation of α7 nAChRs. In addition, disruption of lipid rafts by cholesterol depletion led to substantially attenuated responses to Aβ_1‐42, whereas responses to nicotine were largely intact. These results directly implicate the nicotinic receptor complex as a target for the agonist‐like action of pico‐ to nanomolar concentrations of soluble Aβ_1‐42 on the presynaptic nerve terminal, including the possible involvement of receptor‐associated lipid rafts. This interaction probably plays an important neuromodulatory role in synaptic dynamics.     

Release of β-lipotropin and β-endorphin from rat hypothalami in vitro

Hypothalamic tissue extracts of rats were chromatographed and β-endorphin immunoreactivity (β-End_i) was measured. The two major peaks of β-End_i co-eluted with β-lipotropin (β-LPH) and β-End respectively. Hypophysectomy caused a local decrease of β-LPH and β-End concentrations in the mediobasal hypothalamus. During superfusion of hypothalamic tissue blocks in vitro, membrane depolarization by electric stimulation or 45 mM K⁺ induced a Ca²⁺-dependent release of both β-LPH and β-End.     

Sevoflurane neurotoxicity in neonatal rats is related to an increase in the GABAAR α1/GABAAR α2 ratio

Exposure of neonatal rat to sevoflurane leads to neurodegeneration and deficits of spatial learning and memory in adulthood. However, the underlying mechanisms remain unclear. The type A γ‐aminobutyric acid receptor (GABA_AR) is a target receptor for sevoflurane. The present study intends to investigate the changes in GABA_AR α1/α2 expression and its relationship with the neurotoxicity effect due to sevoflurane in neonatal rats. After a dose–response curve was constructed to determine minimum alveolar concentration (MAC) and safety was guaranteed in our 7‐day‐old neonatal rat pup mode, we conducted two studies among the following groups: (A) the control group; (B) the sham anesthesia group; and (C) the sevoflurane anesthesia group and all three groups were treated in the same way as the model. First, poly(ADP‐ribose) polymerase‐1 protein (PARP‐1) expression was determined in the different brain areas at 6 hr after anesthesia. Second, the expression of PARP‐1 and GABA_AR α1/GABA_AR α2 in the hippocampus area was tested by Western blotting at 6 hr, 24 hr, and 72 hr after anesthesia in all three groups. After 4 hr, with 0.8 MAC (2.1%) sevoflurane anesthesia, the PARP‐1 expression was significantly higher in the hippocampus than the other brain areas (p < .05). Compared with Groups A and B, the expression of PARP‐1 in the hippocampus of Group C significantly increased at 6 hr after sevoflurane exposure (216% ± 15%, p < .05), and the ratio of the α1/α2 subunit of GABA_AR surged at 6 hr (126% ± 6%), 24 hr (127% ± 8%), and 72 hr (183% ± 22%) after sevoflurane exposure in the hippocampus (p < .05). Our study showed that sevoflurane exposure of 0.8 MAC (2.1%)/4 hr was a suitable model for 7‐day‐old rats. And the exposure to sevoflurane could induce the apoptosis of neurons in the early stage, which may be related to the transmission from GABA_AR α2 to GABA_AR α1.     

Immunohistochemical localization of nicotinic acetylcholine receptors in the guinea pig bowel and pancreas

Although nicotinic acetylcholine receptors (nAChRs) are known to be present on neural elements in both the bowel and the pancreas, the precise location of these receptors has not previously been determined. Immunocytochemistry, by using a rat monoclonal antibody (mAb35), which recognizes α-bungarotoxin (α-Bgt)-insensitive nAChRs, and a polyclonal antibody raised against the α-Bgt-sensitive receptor subunit, α7, was used to locate receptor protein in guinea pig gut and pancreas. mAb35-receptor (mAb35-R) immunoreactivity was abundant in both enteric plexuses, enterochromaffin cells, and pancreatic ganglia. Immunostaining was associated with the cell membrane, and clusters of mAb35-R were observed on cell somas and dendrites. Receptor immunoreactivity was also observed on terminals and axons, suggesting that a subset of nAChRs is presynaptic. Internal sites of mAb35-R were observed in permeabilized ganglia. Cells expressing the receptors were closely associated with ChAT-immunoreactive nerve fibers. In addition, the majority of ChAT-positive neurons expressed both cell surface and internal stores of mAb35-R. In the bowel, clusters of mAb35-R were present on the soma and dendrites of Dogiel type I motorneurons and secretomotor neurons. Receptors were detected at the plasma membrane of calbindin-immunoreactive myenteric neurons. In contrast, calbindin-immunoreactive submucosal neurons did not express cell surface mAb35-R, supporting the idea that they are sensory neurons. A subset of enteric neurons expressed both mAb35-R and glutamate receptor (GluR1) immunoreactivity. In the pancreas, mAb35-R immunoreactivity was only observed in ganglia. α7-immunoreactivity was found on both enteric cell bodies and nerve fibers. Based on these results, it appears that visceral nAChRs are composed of at least four subunits and that both pre- and postsynaptic nAChRs are present in the gut and pancreas. J. Comp. Neurol. 390:497–514, 1998. © 1998 Wiley-Liss, Inc.     

Possible modulatory role of voltage-activated Ca currents determining the membrane properties of isolated pyramidal neurones of the rat dorsal cochlear nucleus

Voltage-activated Ca²⁺ currents have been studied in pyramidal cells isolated enzymatically from the dorsal cochlear nuclei of 6–11-day-old Wistar rats, using whole-cell voltage-clamp. From hyperpolarized membrane potentials, the neurones exhibited a T-type Ca²⁺ current on depolarizations positive to −90 mV (the maximum occurred at about −40 mV). The magnitude of the T-current varied considerably from cell to cell (−56 to −852 pA) while its steady-state inactivation was consistent (E₅₀=−88.2±1.7 mV, s=−6.0±0.4 mV). The maximum of high-voltage activated (HVA) Ca²⁺ currents was observed at about −15 mV. At a membrane potential of −10 mV the L-type Ca²⁺ channel blocker nifedipine (10 μM) inhibited approximately 60% of the HVA current, the N-type channel inhibitor ω-Conotoxin GVIA (2 μM) reduced the current by 25% while the P/Q-type channel blocker ω-Agatoxin IVA (200 nM) blocked a further 10%. The presence of the N- and P/Q-type Ca²⁺ channels was confirmed by immunochemical methods. The metabotropic glutamate receptor agonist (±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (200 μM) depressed the HVA current in every cell studied (a block of approximately 7% on an average). The GABA_B receptor agonist baclofen (100 μM) reversibly inhibited 25% of the HVA current. Simultaneous application of ω-Conotoxin GVIA and baclofen suggested that this inhibition could be attributed to the nearly complete blockade of the N-type channels. Possible physiological functions of the voltage-activated Ca²⁺ currents reported in this work are discussed.     

Val8‐GLP‐1 remodels synaptic activity and intracellular calcium homeostasis impaired by amyloid β peptide in rats

Type 2 diabetes mellitus (T2DM) is a risk factor for Alzheimer's disease (AD) in the elderly. Glucagon‐like peptide‐1 (GLP‐1), a modulator in T2DM therapy, has been shown to have neuroprotective properties. However, the native GLP‐1 can be rapidly degraded by the enzyme dipeptidyl peptidase IV (DPP IV); the neuroprotective mechanism of GLP‐1 in the central nervous system is still an open question, and whether GLP‐1 can prevent amyloid β (Aβ)‐induced synaptic dysfunction and calcium disorder is still unclear. The present study, by using patch clamp and calcium imaging techniques, investigated the effects of Val⁸‐GLP‐1(7–36), a GLP‐1 analogue with profound resistance to DPP IV, on the excitatory and inhibitory synaptic transmission and intracellular calcium concentration ([Ca²⁺]_i) in the absence or presence of Aβ1–40. The results showed that 1) Aβ1–40 significantly reduced the frequency of miniature excitatory postsynaptic currents (mEPSCs) and miniature inhibitory postsynaptic currents (mIPSCs) in CA1 pyramidal neurons of rat brain slices; 2) Val⁸‐GLP‐1(7–36) did not affect the activity of miniature postsynaptic currents but effectively protected against the Aβ1–40‐induced decrease in mEPSC and mIPSC frequency; 3) Aβ1–40 significantly increased [Ca²⁺]_i in primary neuronal cultures; and 4) Val⁸‐GLP‐1(7–36) alone did not change the intracellular calcium level but prevented Aβ1–40‐induced persistent elevation of [Ca²⁺]_i. These findings demonstrate for the first time that central application of Val⁸‐GLP‐1(7–36) could protect against Aβ‐induced synaptic dysfunction and intracellular calcium overloading, suggesting that the neuroprotection of GLP‐1 may be involved in the remodeling of synaptic activity and intracellular calcium homeostasis in the brain. © 2013 Wiley Periodicals, Inc.     

Mechanical activation of dorsal root ganglion cells in vitro: comparison with capsaicin and modulation by κ-opioids

The aim of this study was to characterize plasma membrane pathways involved in the intracellular calcium ([Ca²⁺]_i) response of small DRG neurons to mechanical stimulation and the modulation of these pathways by κ-opioids. [Ca²⁺]_i responses were measured by fluorescence video microscopy of Fura-2 labeled lumbosacral DRG neurons obtained from adult rats in short-term primary culture. Transient focal mechanical stimulation of the soma, or brief superfusion with 300 nM capsaicin, resulted to [Ca²⁺]_i increases which were abolished in Ca²⁺-free solution, but unaffected by lanthanum (25 μM) or tetrodotoxin (10⁻⁶ M). 156 out of 465 neurons tested (34%) showed mechanosensitivity while 55 out of 118 neurons (47%) were capsaicin-sensitive. Ninty percent of capsaicin-sensitive neurons were mechanosensitive. Gadolinium (Gd³⁺; 250 μM) and amiloride (100 μM) abolished the [Ca²⁺]_i transient in response to mechanical stimulation, but had no effect on capsaicin-induced [Ca²⁺]_i transients. The κ-opioid agonists U50,488 and fedotozine showed a dose-dependent inhibition of mechanically stimulated [Ca²⁺]_i transients but had little effect on capsaicin-induced [Ca²⁺]_i transients. The inhibitory effect of U50,488 was abolished by the κ-opioid antagonist nor-Binaltorphimine dihydrochloride (nor-BNI; 100 nM), and by high concentrations of naloxone (30–100 nM), but not by low concentrations of naloxone (3 nM). We conclude that mechanically induced [Ca²⁺]_i transients in small diameter DRG somas are mediated by influx of Ca²⁺ through a Gd³⁺- and amiloride-sensitive plasma membrane pathway that is co-expressed with capsaicin-sensitive channels. Mechanical-, but not capsaicin-mediated, Ca²⁺ transients are sensitive to κ-opioid agonists.     

Amyloid-β proteins activate Ca2+-permeable channels through calcium-sensing receptors

The amyloid-beta peptides (Aβ) are produced in excess in Alzheimer's disease (AD) and may contribute to neuronal dysfunction and degeneration. This study provides strong evidence for a novel cellular target for the actions of Aβ, the phospholipase C-coupled, extracellular Ca²⁺-sensing receptor (CaR). We demonstrate that Aβs produce a CaR-mediated activation of a Ca²⁺-permeable, nonselective cation channel (NCC), probably via elevation in cytosolic Ca²⁺ (Ca_i), in cultured hippocampal pyramidal neurons from normal rats and from wild type mice but not those from mice with targeted disruption of the CaR gene (CaR −/−). Aβs also activate NCC in CaR-transfected but not in nontransfected human embryonic kidney (HEK293) cells. Thus aggregates of Aβ deposited on hippocampal neurons in AD could inappropriately activate the CaR, stimulating Ca²⁺-permeable channels and causing sustained elevation of Ca_i with resultant neuronal dysfunction. © 1997 Wiley-Liss Inc.