Calcium concentration changes during sensory transduction in spider mechanoreceptor neurons

Most mechanoreceptor neurons encode mechanical signals into action potential trains within the same cell. Evidence suggests that intracellular calcium ion concentration, [Ca2+], increases during mechanotransduction, either by direct entry through mechanically activated channels or indirectly through voltage-activated calcium channels. However, little is known about the amounts of calcium involved or its roles in mechanotransduction. We estimated [Ca2+] in mechanoreceptor neurons of the spider, Cupiennius salei, during mechanical stimulation using Oregon Green BAPTA-1, and a single-compartment model of [Ca2+] as a function of action potential firing rate. Resting [Ca2+] was approximately 400 nM and increased to up to 2 microM at 30 action potentials/s. Similar levels of resting and stimulated [Ca2+] were obtained in the cell soma, axon and two parts of the sensory dendrite, including the region immediately adjacent to the site of sensory transduction. The time constant of rise and fall of [Ca2+] was 1-5 s in the dendrite and axon, but up to 15 s in the soma. Calcium elevation was dependent on action potentials and could not be induced by the receptor potential alone. Blockade of voltage-activated calcium channels by nickel ions prevented calcium increase, but thapsigargin, which empties intracellular calcium stores, had no effect. Estimates of calcium entry per action potential from fluorescence changes agreed approximately with estimates based on action potential voltage-time profile and previous reports of calcium channel properties. This first report of calcium levels during transduction in spiking mechanoreceptors suggests that calcium signaling plays important roles in primary somatosensory neurons.     

Feedback modulation of transduction by calcium in a spider mechanoreceptor

Calcium ions play important roles in the adaptation of auditory hair cells, and there is evidence that they are involved in modifying the sensitivity and adaptation of a variety of vertebrate and invertebrate mechanoreceptors. However, there is little direct evidence concerning the concentration changes, signaling pathways or ultimate effects of these proposed modulatory mechanisms. We measured receptor potential, receptor current and action potentials intracellularly during mechanotransduction in a group of sensory neurons of the spider Cupiennius salei, which possesses low-voltage-activated calcium channels. Simultaneously, we elevated intracellular [Ca(2+) ] by UV light release from cage molecules, and observed increases in [Ca(2+) ] as changes in calcium-sensitive dye fluorescence. Increases of 10-15% in [Ca(2+) ] caused reductions of approximately 40% in receptor potential and approximately 20% in receptor current. Mechanically evoked action potential firing caused much larger increases in [Ca(2+) ], and the firing rate fell as [Ca(2+) ] rose during mechanical stimulation. Release of caged calcium just before mechanical stimulation significantly reduced peak firing. Dose-response measurements suggested that the binding of one or two intracellular calcium ions per channel reduces the probability of the mechanotransduction channel being open. Our data indicate that calcium regulates sensitivity in these mechanoreceptor neurons by negative feedback from action potentials onto transduction channels.     

Mechanisms regulating spill‐over of synaptic glutamate to extrasynaptic NMDA receptors in mouse substantia nigra dopaminergic neurons

N‐Methyl‐d ‐aspartate glutamate receptors (NMDARs) contribute to neural development, plasticity and survival, but they are also linked with neurodegeneration. NMDARs at synapses are activated by coincident glutamate release and depolarization. NMDARs distal to synapses can sometimes be recruited by ‘spill‐over’ of glutamate during high‐frequency synaptic stimulation or when glutamate uptake is compromised, and this influences the shape of NMDAR‐mediated postsynaptic responses. In substantia nigra dopamine neurons, activation of NMDARs beyond the synapse during different frequencies of presynaptic stimulation has not been explored, even though excitatory afferents from the subthalamic nucleus show a range of firing frequencies, and these frequencies change in human and experimental Parkinson's disease. This study reports that high‐frequency stimulation (80 Hz/200 ms) evoked NMDAR‐excitatory postsynaptic currents (EPSCs) that were larger and longer lasting than those evoked by single stimuli at low frequency (0.1 Hz). MK‐801, which irreversibly blocked NMDAR‐EPSCs activated during 0.1‐Hz stimulation, left a proportion of NMDAR‐EPSCs that could be activated by 80‐Hz stimulation and that may represent activity of NMDARs distal to synapses. TBOA, which blocks glutamate transporters, significantly increased NMDAR‐EPSCs in response to 80‐Hz stimulation, particularly when metabotropic glutamate receptors (mGluRs) were also blocked, indicating that recruitment of NMDARs distal to synapses is regulated by glutamate transporters and mGluRs. These regulatory mechanisms may be essential in the substantia nigra for restricting glutamate diffusion from synaptic sites and keeping NMDAR‐EPSCs in dopamine neurons relatively small and fast. Failure of glutamate transporters may contribute to the declining health of dopamine neurons during pathological conditions.     

mGluR7-like metabotropic glutamate receptors inhibit NMDA-mediated excitotoxicity in cultured mouse cerebellar granule neurons

Glutamate-induced glutamate release may be involved in the delayed neuronal death induced by N-methyl-D-aspartate (NMDA). In order to examine a possible modulatory effect of the presynaptic group III mGluRs on glutamate excitotoxicity, the effect of L-2-amino-4-phosphonobutyrate (L-AP4) was examined on NMDA-induced delayed death of mouse cerebellar granule neurons in culture. We found that L-AP4, at high concentration (in the millimolar range), inhibited in a non-competitive manner the NMDA-induced toxicity. This effect was mimicked by high concentration of L-serine-o-phosphate (L-SOP), and was inhibited by pertussis toxin (PTX) indicating the involvement of a Gi/o protein. This suggests the involvement of mGluR7 in the L-AP4 effect, and this was consistent with the detection of both mGluR7 protein and mRNA in these cultured neurons. To examine the mechanism of the L-AP4-induced protection from excitotoxic damage, the effect of L-AP4 on glutamate release was examined. L-AP4 (> or = 1 mM) noncompetitively inhibited by more than 60% the glutamate release induced by NMDA during the insult. We also observed that the 10-min NMDA receptor stimulation resulted in a dramatic increase in the extracellular glutamate concentration reaching 6000% of the control value 24 h after the insult. This large increase was also inhibited when NMDA was applied in the presence of > or = 1 mM L-AP4. Part of the L-AP4-induced protection from excitotoxic damage of granule neurons may therefore result from the inhibition of the vicious cycle: dying cells release glutamate, glutamate induced cell death. The present results add to the hypothesis that presynaptic mGluRs, probably mGluR7, may be the targets of drugs decreasing glutamate release and then neuronal death observed in some pathological situations.     

GABA and glutamate receptors have different effects on excitability and are differentially regulated by calcium in spider mechanosensory neurons

GABA and glutamate receptors belonging to the ligand‐gated chloride‐channel family are primary targets of insecticides and antiparasitics, so their molecular structure, pharmacology and biophysical properties have attracted significant attention. However, little is known about the physiological roles of these channels or how they regulate neuronal excitability and animal behavior. Mechanosensory neurons of VS‐3 slit sensilla in the patella of the tropical wandering spider, Cupiennius salei, react to the GABA_A‐receptor agonists, GABA and muscimol, with depolarization and an increase in intracellular [Ca²⁺] and, during random noise stimulation, with a mixed inhibitory–excitatory response. We established that the GABA_A‐receptors in all VS‐3 neurons are identical, but there are at least two types of glutamate receptors and some neurons do not respond to glutamate at all. Immunohistochemistry with antibodies against Drosophila inhibitory glutamate receptor (GluCls) α‐subunit suggests that in addition to VS‐3 neurons, these receptors may also be present in the efferent neurons surrounding the sensory neurons. Most VS‐3 neurons were inhibited but not depolarized by glutamate during random stimulation, but some depolarized and had a similar excitatory–inhibitory response to glutamate as to muscimol. The membrane‐permeable Ca²⁺‐chelator BAPTA‐AM abolished muscimol effects but potentiated glutamate effects, indicating that GABA and glutamate receptors are differentially modulated by Ca²⁺, leading to diverse regulation of neuronal excitability. We hypothesize that this could be achieved by different Ca²⁺‐triggered phosphorylation processes at each receptor type. These findings are important for understanding the significance of Ca²⁺‐mediated regulation of transmitter receptor molecules and its role in controlling excitability.     

Peripheral GABAergic inhibition of spider mechanosensory afferents

Spider mechanosensory neurons receive an extensive network of efferent synapses onto their sensory dendrites, somata and distal axonal regions. The function of these synapses is unknown. Peripheral synapses are also found on crustacean stretch-receptor neurons but not on mechanosensory afferents of other species, although inhibitory GABAergic synapses are a common feature of centrally located axon terminals. Here we investigated the effects of GABA receptor agonists and antagonists on one group of spider mechanosensory neurons, the slit sense organ VS-3, which are accessible to current- and voltage-clamp recordings. Bath application of GABA activated an inward current that depolarized the membrane and increased the membrane conductance leading to impulse inhibition. VS-3 neuron GABA receptors were activated by muscimol and inhibited by picrotoxin but not bicuculline, and their dose-response relationship had an EC(50) of 103.4 microm, features typical for insect ionotropic GABA receptors. Voltage- and current-clamp analysis confirmed that, while the Na(+) channel inhibition resulting from depolarization can lead to impulse inhibition, the increase in membrane conductance (i.e. 'shunting') completely inhibited impulse propagation. This result argues against previous findings from other preparations that GABA-mediated inhibition is caused by a depolarization that inactivates Na(+) conductance, and it supports those findings that assign this role to membrane shunting. Our results show that GABA can rapidly and selectively inhibit specific mechanoreceptors in the periphery. This type of peripheral inhibition may provide spiders with a mechanism for distinguishing between signals from potential prey, predators or mates, and responding with appropriate behaviour to each signal.     

Transmembrane topology of the glutamate receptors

The glutamate receptor subunits were first thought to cross the cell membrane four times in a manner analogous to the neuronal nicotinic acetylcholine, GABA_A, and glycine receptors. This model led the field for nearly five years, although it was frequently in conflict with the data. Recently, comparisons with bacterial proteins, epitope tagging experiments, and the construction of chimeras has produced a new model of glutamate receptor topology that is novel and quite unlike any of the other receptors.     

The role of glutamate and its receptors in central nervous system in stress-induced hyperalgesia

Purpose: To explore the potential mechanisms of glutamate and its receptors in stress-induced hyperalgesia.

Materials and methods: The stress-induced hyperalgesia, glutamate and its receptors are listed as key items in the pubmed database and the related articles are searched.

Results: Glutamate level is increased under stress and associated with stress-induced hyperalgesia. Moreover, the role of glutamate in stress-induced hyperalgesia depends on its subtypes of its receptors.

Conclusions: Increased glutamate during stress connect with ionotropic glutamate receptors can prompt hyperalgesia, but connect with metabotropic glutamate receptors can inhibit hyperalgesia.     

Gabab receptor-mediated modulation of glutamate signaling in cerebellar Purkinje cells

Since Purkinje cells are the sole output neurons of the cerebellar cortex, the postsynaptic integration of excitatory and inhibitory synaptic inputs in this cell type is a pivotal step for cerebellar motor information processing. In Purkinje cells, Gi/o protein-coupled B-type gamma-aminobutyric acid receptor (GABABR) is expressed at the annuli of the dendritic spines that are innervated by the glutamatergic terminals of parallel fibers. The subcellular localization of GABABR suggests the possibility of postsynaptic interplay between GABABR and glutamate signaling. It has recently been demonstrated that GABABR indeed modulates alpha amino-3-hydroxy-5-methyl-4-isoxalone propionate-type ionotropic glutamate receptor (AMPAR)-mediated and type-1 metabotropic glutamate receptor (mGluR1)-mediated signaling. Interestingly, GABABR exerts modulatory actions not only via the classical Gi/o protein-dependent signaling cascade but also via a Gi/o protein-independent interaction between GABABR and mGluR1. In this review, we compare the physiological nature, underlying mechanisms, and possible functional significance of these modulatory actions of GABABR.     

Differential blocking effects of a spider toxin on synaptic and glutamate responses in the afferent synapse of the acoustico-lateralis receptors of Plotosus

The hypothesis that glutamate is the afferent transmitter in the acoustico-lateralis receptors was examined in Plotosus electroreceptors. JSTX, a spider toxin known to specifically block glutamate receptors, irreversibly abolished afferent impulse discharges induced by iontophoretically applied glutamate, whereas those induced synaptically by focal stimulation of receptor cells were little affected. Such differential blocking effects by JSTX, complementary to other biochemical data, further provide pharmacological evidence against the glutamate hypothesis.     

Age‐dependent enhancement of inhibitory synaptic transmission in CA1 pyramidal neurons via GluR5 kainate receptors

Changes in hippocampal synaptic networks during aging may contribute to age‐dependent compromise of cognitive functions such as learning and memory. Previous studies have demonstrated that GABAergic synaptic transmission exhibits age‐dependent changes. To better understand such age‐dependent changes of GABAergic synaptic inhibition, we performed whole‐cell recordings from pyramidal cells in the CA1 area of acute hippocampal slices on aged (24–26 months old) and young (2–4 months old) Brown‐Norway rats. We found that the frequency and amplitude of spontaneous inhibitory postsynaptic current (IPSCs) were significantly increased in aged rats, but the frequency and amplitude of mIPSCs were decreased. Furthermore, the regulation of GABAergic synaptic transmission by GluR5 containing kainate receptors was enhanced in aged rats, which was revealed by using LY382884 (a GluR5 kainate receptor antagonist) and ATPA (a GluR5 kainate receptor agonist). Moreover, we demonstrated that vesicular glutamate transporters are involved in the kainate receptor dependent regulation of sIPSCs. Taken together, these results suggest that GABAergic synaptic transmission is potentiated in aged rats, and GluR5 containing kainate receptors regulate the inhibitory synaptic transmission through endogenous glutamate. These alterations of GABAergic input with aging could contribute to age‐dependent cognitive decline. © 2009 Wiley‐Liss, Inc.     

Activation by N-acetyl-L-aspartate of acutely dissociated hippocampal neurons in rats via metabotropic glutamate receptors

PURPOSE: We previously reported that an increase in the N-acetyl-L-aspartate (NAA) level due to the lack of aspartoacylase gene was found in the brain of the tremor rat (tm/tm), which is a mutant with a causative gene named tm that shows epileptic seizures. Therefore, NAA is suggested to be one of the factors involved in the induction of epileptic seizures. Patch-clamp studies were performed to determine whether NAA produces an excitatory effect on acutely dissociated rat hippocampal neurons. METHODS: Acutely dissociated hippocampal neurons were prepared from normal Wistar rats aged 3-4 weeks. NAA-induced currents were investigated by using the whole-cell voltage-clamp recording technique. RESULTS: Application of NAA at concentrations of 100 nM to 1 mM through a U-tube for 2 s produced an inward current in a concentration-dependent manner at a holding potential of -60 mV. When the current-voltage relation was examined, the reversal potential of the NAA-induced current was found to be approximately 0 mV. The NAA-induced current was inhibited by bath application of the metabotropic glutamate receptor (mGluR) antagonist (+/-)-alpha-methyl-4-carboxyphenylglycine (MCPG) and by intracellular application of guanosine 5'-O-(2-thiodiphosphate) (GDP-betaS), a nonhydrolyzable GDP analogue. However, the NAA-induced current remained unaffected by glutamic acid diethyl ester, a non-N-methyl-D-aspartate (NMDA)-subtype ionotropic glutamate receptor antagonist, or the voltage-dependent ion channel blockers tetrodotoxin, CdCl2, and tetraethylammonium-chloride. Conversely, the mGluR agonist, trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid (ACPD) also induced an inward current, with a reversal potential of 0 mV. The ACPD-induced current also was inhibited by MCPG. CONCLUSIONS: These results suggest that NAA acts on the G protein-coupled mGluRs to induce an inward current that results in excitation of the neurons, thereby contributing to the occurrence of epileptic seizures.     

Activation of Group II/III metabotropic glutamate receptors attenuates LPS-induced astroglial neurotoxicity via promoting glutamate uptake

Altered glial function that leads to oxidative stress and excitotoxicity may contribute to the initiation or progression of neuronal death in neurodegenerative diseases. We report the pivotal role of astroglial Group II and III metabotropic glutamate receptors (mGluR) against neurotoxicity. Activation of Group II or III mGluR on astrocytes with selective agonists DCG-IV or L-AP4 respectively inhibited astroglial lipopolysaccharide (LPS)-conditioned medium induced apoptosis of primary cultured mesencephalic neurons. Specific Group II or III mGluR antagonists APICA or MSOP completely abolished the neuroprotective effects of DCG-IV and L-AP4. Morphologic analysis showed that DCG-IV or L-AP4 could also attenuate the astroglial neurotoxicity to dopaminergic neurons. Measurement of extracellular glutamate concentration and [(3)H]-glutamate uptake showed that the restoration of glutamate uptake capability in LPS-treated astrocytes might be involved in the neuroprotective effects of activating astroglial Group II or III mGluR. Furthermore, we found that the repression of astroglial uptake function could be revived by GSH, and both Group II and III mGluR agonists could recover the endogenous reduced glutathione (GSH) level in LPS-treated astrocytes. These results suggested that the possible mechanisms of neuroprotection by either Type II or Type III mGluR activation may involve restoration of endogenous GSH, in turn affording recovery of astroglial capability to take up glutamate.     

Excitatory amino acids acting on metabotropic glutamate receptors broaden the action potential in hippocampal neurons

Activation of metabotropic glutamate receptors (mGluRs, QP or ACPD receptors) has recently been shown to cause depolarization, blockade of the slow after-hyperpolarization and depression of calcium currents in hippocampal pyramidal cells. Here, we report evidence for a new mGluR-mediated effect: slowing of the spike repolarization in CA1 cells in rat hippocampal slices. During blockade of the ionotropic glutamate receptors, the mGluR agonists trans-1-amino-cyclopentyl-1,3-dicarboxylate (t-ACPD), quisqualate or L-glutamate caused spike broadening. In contrast, the ionotropic receptor agonist alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) was ineffective. The spike broadening may act in concert with the other mGluR effects, e.g. by further increasing the influx of Ca2+ ions which, in turn, may contribute to synaptic modulation.     

Upregulation of Group I metabotropic glutamate receptors in neurons and astrocytes in the dorsal horn following spinal cord injury

Of the glutamate receptor types, the metabotropic glutamate receptors (mGluRs) are G proteins coupled and can initiate a number of intracellular pathways leading to hyperexcitability of spinal neurons. In this study, we tested the expression of mGluRs to determine which cell types might contribute to sustained neuronal hyperexcitability in the lumbar enlargement with postoperative day (POD) 7 (early), 14 (late), and 30 (chronic phase) following spinal cord injury (SCI) by unilateral hemisection at T13 in Sprague-Dawley rats. Expression was determined by confocal analyses of immunocytochemical reaction product of neurons (NeuN positive) and astrocytes (GFAP positive) in the dorsal horn on both sides of the L4 segment. Neurons were divided into two sizes: small (<20 microm) and large (>35 microm), for physiological reasons. We report a significant increase of mGluR(1) expression in large and small neurons of the dorsal horn on both sides of the cord in late and chronic phases when compared to control sham groups. Expression of mGluR(2/3) significantly increased in large neurons on the ipsilateral (hemisected) side in the late phase. Expression of mGluR(5) significantly increased in large neurons in early, late, and chronic phases. In addition, mGluR(1) and mGluR(5) expression after hemisection was significantly increased in astrocytes in early, late, and chronic phases; whereas mGluR(2/3) did not display any significant changes. In conclusion, our data demonstrate long-term changes in expression levels of Group I mGluRs (mGluR(1) and mGluR(5)) in both neurons and astrocytes in segments below a unilateral SCI. Thus, permanent alterations in dorsal horn receptor expression may play important roles in transmission of nociceptive responses in the spinal cord following SCI.     

Roles of metabotropic glutamate receptors in glial function and glial-neuronal communication

The amino acid glutamate plays a key role in brain function. One of the major roles of glutamate is to mediate fast excitatory neurotransmission via activation of ionotropic glutamate receptors (iGluRs). More recently, however, it has become clear that glutamate also serves a regulatory function through activation of receptors coupled to modulation of second messenger systems [metabotropic glutamate receptors (mGluRs)]. A body of evidence suggests that mGluRs regulate neuronal function through modulation of ion channels and enzymes to modulate cellular excitability and synaptic transmission. Interestingly, it has become clear that in addition to activation of neuronal receptors, glutamate can activate both iGluRs and mGluRs on glia. A growing body of evidence suggests that the mGluRs on glia play important roles in both glial function and mediation of intercellular signaling. © 1996 Wiley-Liss, Inc.     

The effect of lectins on desensitisation of locust muscle glutamate receptors

At the excitatory neuromuscular junction of the locust, Schistocerca gregaria, desensitisation to L-glutamate is blocked by the lectin concanavalin A (Con A). In this study a range of lectins has been used to assess the influence of simple sugar binding specificity on desensitisation block. Pea and lentil lectins have similar simple sugar specificities (mannose/glucose) to Con A and block desensitisation in a similar manner. Soybean and wheatgerm lectins have other simple sugar specificities and do not block desensitisation of the locust muscle glutamate receptor. Native Con A is a tetramer at pH 7 but at lower pH or following succinylation (S-Con A) it becomes a dimer, with reduced biological activity. S-Con A does not block desensitisation but it does bind to locust muscle and protects the glutamate receptor from the desensitisation block caused by Con A. When Con A is applied to a desensitised neuromuscular junction the ongoing desensitisation is not blocked. It appears that desensitisation block by Con A, pea and lentil lectins is dependent on lectin binding to mannose or glucose moieties on or in the region of the glutamate receptor, and that these moieties are masked from lectin when the receptor is in its desensitised state.