Neurosteroid allopregnanolone inhibits glutamate release from rat cerebrocortical nerve terminals

Allopregnanolone, an active metabolite of progesterone, has been reported to exhibit neuroprotective activity in several preclinical models. Considering that the excitotoxicity caused by excessive glutamate is implicated in many brain disorders, the effect of allopregnanolone on glutamate release in rat cerebrocortical nerve terminals and possible underlying mechanism were investigated. We observed that allopregnanolone inhibited 4‐aminopyridine (4‐AP)‐evoked glutamate release, and this inhibition was prevented by chelating the extracellular Ca²⁺ ions and the vesicular transporter inhibitor. Allopregnanolone reduced the elevation of 4‐AP‐evoked intrasynaptosomal Ca²⁺ levels, but did not affect the synaptosomal membrane potential. In the presence of N‐, P/Q‐, and R‐type channel blockers, allopregnanolone‐mediated inhibition of 4‐AP‐evoked glutamate release was markedly reduced; however, the intracellular Ca²⁺‐release inhibitors did not affect the allopregnanolone effect. Furthermore, allopregnanolone‐mediated inhibition of 4‐AP‐evoked glutamate release was completely abolished in the synaptosomes pretreated with inhibitors of Ca²⁺/calmodulin, adenylate cyclase, and protein kinase A (PKA), namely calmidazolium, MDL12330A, and H89, respectively. Additionally, the allopregnanolone effect on evoked glutamate release was antagonized by the GABA_A receptor antagonist SR95531. Our data are the first to suggest that allopregnanolone reduce the Ca²⁺ influx through N‐, P/Q‐, and R‐type Ca²⁺ channels, through the activation of GABA_A receptors present on cerebrocortical nerve terminals, subsequently suppressing the Ca²⁺‐calmodulin/PKA cascade and decreasing 4‐AP‐evoked glutamate release.     

Endogenous calcium buffering at photoreceptor synaptic terminals in salamander retina

Calcium operates by several mechanisms to regulate glutamate release at rod and cone synaptic terminals. In addition to serving as the exocytotic trigger, Ca²⁺ accelerates replenishment of vesicles in cones and triggers Ca²⁺‐induced Ca²⁺ release (CICR) in rods. Ca²⁺ thereby amplifies sustained exocytosis, enabling photoreceptor synapses to encode constant and changing light. A complete picture of the role of Ca²⁺ in regulating synaptic transmission requires an understanding of the endogenous Ca²⁺ handling mechanisms at the synapse. We therefore used the “added buffer” approach to measure the endogenous Ca²⁺ binding ratio (κ_endo) and extrusion rate constant (γ) in synaptic terminals of photoreceptors in retinal slices from tiger salamander. We found that κ_endo was similar in both cell types—～25 and 50 in rods and cones, respectively. Using measurements of the decay time constants of Ca²⁺ transients, we found that γ was also similar, with values of ～100 s^?1 and 160 s^?1 in rods and cones, respectively. The measurements of κ_endo differ considerably from measurements in retinal bipolar cells, another ribbon‐bearing class of retinal neurons, but are comparable to similar measurements at other conventional synapses. The values of γ are slower than at other synapses, suggesting that Ca²⁺ ions linger longer in photoreceptor terminals, supporting sustained exocytosis, CICR, and Ca²⁺‐dependent ribbon replenishment. The mechanisms of endogenous Ca²⁺ handling in photoreceptors are thus well‐suited for supporting tonic neurotransmission. Similarities between rod and cone Ca²⁺ handling suggest that neither buffering nor extrusion underlie differences in synaptic transmission kinetics. Synapse 68:518–528, 2014 . © 2014 Wiley Periodicals, Inc.     

Local anesthetics inhibit glutamate release from rat cerebral cortex synaptosomes

Local anesthetics have been widely used for regional anesthesia and the treatment of cardiac arrhythmias. Recent studies have also demonstrated that low‐dose systemic local anesthetic infusion has neuroprotective properties. Considering the fact that excessive glutamate release can cause neuronal excitotoxicity, we investigated whether local anesthetics might influence glutamate release from rat cerebral cortex nerve terminals (synaptosomes). Results showed that two commonly used local anesthetics, lidocaine and bupivacaine, exhibited a dose‐dependent inhibition of 4‐AP‐evoked release of glutamate. The effects of lidocaine or bupivacaine on the evoked glutamate release were prevented by the chelation of extracellular Ca²⁺ ions and the vesicular transporter inhibitor bafilomycin A1. However, the glutamate transporter inhibitor dl ‐threo‐beta‐benzyl‐oxyaspartate did not have any effect on the action of lidocaine or bupivacaine. Both lidocaine and bupivacaine reduced the depolarization‐induced increase in [Ca²⁺]_C but did not alter 4‐AP‐mediated depolarization. Furthermore, the inhibitory effect of lidocaine or bupivacaine on evoked glutamate release was prevented by blocking the Ca_v2.2 (N‐type) and Ca_v2.1 (P/Q‐type) channels, but it was not affected by blocking of the ryanodine receptors or the mitochondrial Na⁺/Ca²⁺ exchange. Inhibition of protein kinase C (PKC) and protein kinase A (PKA) also prevented the action of lidocaine or bupivacaine. These results show that local anesthetics inhibit glutamate release from rat cortical nerve terminals. This effect is linked to a decrease in [Ca²⁺]_C caused by Ca²⁺ entry through presynaptic voltage‐dependent Ca²⁺ channels and the suppression of the PKA and PKC signaling cascades. Synapse 67:568–579, 2013 . © 2013 Wiley Periodicals, Inc.     

Two types of calcium currents of the mouse bipolar cells recorded in the retinal slice preparation

In the vertebrate retina, the bipolar cell makes reciprocal synapses with amacrine cells at the axon terminal. It has been postulated that amacrine cells may control the transmitter release from bipolar cells by modulating their calcium currents (I_Ca). To clarify this possibility calcium currents were studied in bipolar cells of the mouse retina using a slice preparation. I_Ca was identified by voltage clamp protocols, ionic substitution and pharmacological tools. Depolarization to –30 mV from a holding voltage of –80 mV induced an inward current consisting of an initial transient and a long-lasting sustained component. The transient component was inactivated by holding the membrane at more positive voltages. Addition of 100 μm nifedipine suppressed the sustained component, leaving the transient component almost intact. The sustained component was enhanced when external solution contained 0.1 μm Bay K 8644 or when the external Ca²⁺ was substituted by equimolar Ba²⁺. Omega-conotoxin (10 μm ω-ctxn GVIA) did not alter either component. We concluded that the transient component is a low-voltage activated T-type I_Ca, while the sustained component is a high-voltage activated L-type I_Ca. T-type I_Ca was recorded in all cells tested, while L-type I_Ca was found only in cells that retained axon terminals ramifying in the inner plexiform layer. Thus, it is highly likely that L-type I_Ca is generated at the axon terminal and contributes to the transmitter release from the bipolar cell. The present results confirm that in addition to the T-type I_Ca that had been previously described, bipolar cells of the mammalian retina also contain L-type I_Ca similar to the one that has been reported in bipolar cells of the goldfish. The use of retinal slice preparation allowed us to record this current that was not seen previously in the dissociated mouse bipolar cells.     

Facilitation of glutamate release from rat cerebrocortical glutamatergic nerve terminals (synaptosomes) by phosphatidylserine and phosphatidylcholine

Phosphatidylserine (PS) and phosphatidylcholine (PC) have been shown to enhance cognitive function. Considering that brain glutamatergic system is thought to participate in cognitive processing, our objective was to determine the effect of PS and PC on glutamate release from the nerve terminal (synaptosome) freshly isolated from rat cerebral cortex. Data showed that both PS and PC potently facilitate 4‐aminopyridine (4‐AP)‐evoked Ca²⁺‐dependent and Ca²⁺‐independent glutamate release. Facilitation of glutamate release by PS or PC was associated with an increase of 4‐AP‐evoked depolarization and downstream elevation of cytoplasmic free calcium concentration ([Ca²⁺]_c). In addition, glutamate release elicited by direct Ca²⁺‐entry with Ca²⁺‐ionophore (ionomycin) was also facilitated by PS or PC. Furthermore, PS‐ or PC‐mediated facilitation of 4‐AP‐evoked glutamate release was superseded or suppressed by protein kinase C (PKC) activator and inhibitor, respectively. Together, these results suggest that PS or PC effects a facilitation of glutamate exocytosis by increasing nerve terminal excitability and Ca²⁺ influx into cerebrocortical nerve terminals through a signaling cascade involving PKC. Synapse 63:215–223, 2009. © 2008 Wiley‐Liss, Inc.     

Lack of protein-tyrosine sulfation disrupts photoreceptor outer segment morphogenesis, retinal function and retinal anatomy

To investigate the role(s) of protein‐tyrosine sulfation in the retina, we examined retinal function and structure in mice lacking tyrosylprotein sulfotransferases (TPST) 1 and 2. Tpst double knockout (DKO; Tpst1^?/?/Tpst2 ^?/?) retinas had drastically reduced electroretinographic responses, although their photoreceptors exhibited normal responses in single cell recordings. These retinas appeared normal histologically; however, the rod photoreceptors had ultrastructurally abnormal outer segments, with membrane evulsions into the extracellular space, irregular disc membrane spacing and expanded intradiscal space. Photoreceptor synaptic terminals were disorganized in Tpst DKO retinas, but established ultrastructurally normal synapses, as did bipolar and amacrine cells; however, the morphology and organization of neuronal processes in the inner retina were abnormal. These results indicate that protein‐tyrosine sulfation is essential for proper outer segment morphogenesis and synaptic function, but is not critical for overall retinal structure or synapse formation, and may serve broader functions in neuronal development and maintenance.     

Glycine Release Is Potentiated by cAMP via EPAC2 and Ca2+ Stores in a Retinal Interneuron

Neuromodulation via the intracellular second messenger cAMP is ubiquitous at presynaptic nerve terminals. This modulation of synaptic transmission allows exocytosis to adapt to stimulus levels and reliably encode information. The AII amacrine cell (AII-AC) is a central hub for signal processing in the mammalian retina. The main apical dendrite of the AII-AC is connected to several lobular appendages that release glycine onto OFF cone bipolar cells and ganglion cells. However, the influence of cAMP on glycine release is not well understood. Using membrane capacitance measurements from mouse AII-ACs to directly measure exocytosis, we observe that intracellular dialysis of 1 mm cAMP enhances exocytosis without affecting the L-type Ca²⁺ current. Responses to depolarizing pulses of various durations show that the size of the readily releasable pool of vesicles nearly doubles with cAMP, while paired-pulse depression experiments suggest that release probability does not change. Specific agonists and antagonists for exchange protein activated by cAMP 2 (EPAC2) revealed that the cAMP-induced enhancement of exocytosis requires EPAC2 activation. Furthermore, intact Ca²⁺ stores were also necessary for the cAMP potentiation of exocytosis. Postsynaptic recordings from OFF cone bipolar cells showed that increasing cAMP with forskolin potentiated the frequency of glycinergic spontaneous IPSCs. We propose that cAMP elevations in the AII-AC lead to a robust enhancement of glycine release through an EPAC2 and Ca²⁺ store signaling pathway. Our results thus contribute to a better understanding of how AII-AC crossover inhibitory circuits adapt to changes in ambient luminance.SIGNIFICANCE STATEMENT The mammalian retina operates over a wide dynamic range of light intensities and contrast levels. To optimize the signal-to-noise ratio of processed visual information, both excitatory and inhibitory synapses within the retina must modulate their gain in synaptic transmission to adapt to different levels of ambient light. Here we show that increases of cAMP concentration within AII amacrine cells produce enhanced exocytosis from these glycinergic interneurons. Therefore, we propose that light-sensitive neuromodulators may change the output of glycine release from AII amacrine cells. This novel mechanism may fine-tune the amount of tonic and phasic synaptic inhibition received by bipolar cell terminals and, consequently, the spiking patterns that ganglion cells send to the upstream visual areas of the brain.     

Glutamate-like Immunoreactivity in Retinal Terminals of the Mouse Suprachiasmatic Nucleus

With a view to identifying the neurotransmitter content of retinal terminals within the mouse suprachiasmatic nucleus, a highly specific antiserum to glutaraldehyde-coupled glutamate was used in a postembedding immunogold procedure at the ultrastructural level. Retinal terminals were identified by cholera toxin–horseradish peroxidase transported anterogradely from the retina and reacted with tetramethyl benzidine/tungstate/H₂O₂, or by their characteristically pale and distended mitochondria with irregular cristae. Controls included model ultrathin sections containing high concentrations of various amino acids. Alternate serial sections were labelled with anti-glutamate and anti-γ-aminobutyric acid (GABA). Data were analysed by computer-assisted image analysis. Density of glutamate labelling (gold particles per μm²) on whole retinal terminals was > 3 times higher than that on postsynaptic dendrites, and > 5 times higher than that on miscellaneous non-retinal non-glutamatergic terminals in the suprachiasmatic nucleus. The overall density of gold particles over retinal terminals was ～ 3 times higher than that over GABAergic terminals, in which glutamate-like immunoreactivity was mainly mitochondrial. Labelling of vesicles in retinal terminals was almost 5 times greater than the apparent labelling of vesicles in GABAergic terminals, underscoring the location of transmitter glutamate within synaptic vesicles in retinal terminals. In the retino-recipient region of the suprachiasmatic nucleus there was also a small population of non-retinal glutamatergic terminals. Their overall immunoreactivity was similar to or exceeded that of retinal terminals, but morphological features clearly distinguished between these two types of glutamate-containing terminals. The present results indicate that the vast majority of retinal terminals may use glutamate as a transmitter, in keeping with electrophysiological and neuropharmacological data from other sources. The possibility of cotransmitters within retinal terminals, suggested by the presence of dense-core vesicles among the glutamate-containing synaptic vesicles, has still to be addressed.     

Curcumin inhibits glutamate release in nerve terminals from rat prefrontal cortex: possible relevance to its antidepressant mechanism

There is abundant evidence suggesting the relevance of glutamate to depression and antidepressant mechanisms. Curcumin, a major active compound of Curcuma longa, has been reported to have the biological function of antidepressant. The aim of the present study was to investigate the effect of curcumin on endogenous glutamate release in nerve terminals of rat prefrontal cortex and the underlying mechanisms. The results showed that curcumin inhibited the release of glutamate that was evoked by exposing synaptosomes to the K⁺ channel blocker 4-aminopyridine (4-AP). This phenomenon was blocked by the chelating the extracellular Ca²⁺ ions, and by the vesicular transporter inhibitor bafilomycin A1, but was insensitive to the glutamate transporter inhibitor DL-threo-β-benzyl-oxyaspartate (DL-TBOA). Further experiments demonstrated that curcumin decreased depolarization-induced increase in [Ca²⁺]_C, whereas it did not alter the resting membrane potential or 4-AP-mediated depolarization. Furthermore, the inhibitory effect of curcumin on evoked glutamate release was prevented by blocking the Ca_v2.2 (N-type) and Ca_v2.1 (P/Q-type) channels, but not by blocking intracellular Ca²⁺ release or Na⁺/Ca²⁺ exchange. These results suggest that curcumin inhibits evoked glutamate release from rat prefrontocortical synaptosomes by the suppression of presynaptic Ca_v2.2 and Ca_v2.1 channels. Additionally, we also found that the inhibitory effect of curcumin on 4-AP-evoked glutamate release was completely abolished by the clinically effective antidepressant fluoxetine. This suggests that curcumin and fluoxetine use a common intracellular mechanism to inhibit glutamate release from rat prefrontal cortex nerve terminals.     

Inhibition of glutamate release by bupropion in rat cerebral cortex nerve terminals

Central glutamate neurotransmission has been postulated to play a role in pathophysiology of depression and in the mechanism of antidepressants. The present study was undertaken to elucidate the effect and the possible mechanism of bupropion, an atypical antidepressant, on endogenous glutamate release in nerve terminals of rat cerebral cortex (synaptosomes). Result showed that bupropion exhibited a dose-dependent inhibition of 4-aminopyridine (4-AP)-evoked release of glutamate. The effect of bupropion on the evoked glutamate release was prevented by the chelating the intrasynaptosomal Ca²⁺ ions, and by the vesicular transporter inhibitor, but was insensitive to the glutamate transporter inhibitor. Bupropion decreased depolarization-induced increase in [Ca²⁺]_C, whereas it did not alter the resting synaptosomal membrane potential or 4-AP-mediated depolarization. The effect of bupropion on evoked glutamate release was abolished by the N-, P- and Q-type Ca²⁺ channel blocker, but not by the ryanodine receptor blocker, or the mitochondrial Na⁺/Ca²⁺ exchanger blocker. In addition, the inhibitory effect of bupropion on evoked glutamate release was prevented by the mitogen-activated/extracellular signal-regulated kinase kinase (MEK) inhibitors. Western blot analyses showed that bupropion significantly decreased the 4-AP-induced phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2), and this effect also was blocked by MEK inhibitor. These results are the first to suggest that, in rat cerebrocortical nerve terminals, bupropion suppresses voltage-dependent Ca²⁺ channel and MEK/ERK activity and in so doing inhibits evoked glutamate release. This finding may provide important information regarding the beneficial effects of bupropion in the brain.     

Synaptic Integration of Subquantal Neurotransmission by Colocalized G Protein-Coupled Receptors in Presynaptic Terminals

In presynaptic terminals, membrane-delimited G_i/o-mediated presynaptic inhibition is ubiquitous and acts via Gβγ to inhibit Ca²⁺ entry, or directly at SNARE complexes to inhibit Ca²⁺-dependent synaptotagmin-SNARE complex interactions. At CA1-subicular presynaptic terminals, 5-HT_1B and GABA_B receptors colocalize. GABA_B receptors inhibit Ca²⁺ entry, whereas 5-HT_1B receptors target SNARE complexes. We demonstrate in male and female rats that GABA_B receptors alter P_r, whereas 5-HT_1B receptors reduce evoked cleft glutamate concentrations, allowing differential inhibition of AMPAR and NMDAR EPSCs. This reduction in cleft glutamate concentration was confirmed by imaging glutamate release using a genetic sensor (iGluSnFR). Simulations of glutamate release and postsynaptic glutamate receptor currents were made. We tested effects of changes in vesicle numbers undergoing fusion at single synapses, relative placement of fusing vesicles and postsynaptic receptors, and the rate of release of glutamate from a fusion pore. Experimental effects of P_r changes, consistent with GABA_B receptor effects, were straightforwardly represented by changes in numbers of synapses. The effects of 5-HT_1B receptor-mediated inhibition are well fit by simulated modulation of the release rate of glutamate into the cleft. Colocalization of different actions of GPCRs provides synaptic integration within presynaptic terminals. Train-dependent presynaptic Ca²⁺ accumulation forces frequency-dependent recovery of neurotransmission during 5-HT_1B receptor activation. This is consistent with competition between Ca²⁺-synaptotagmin and Gβγ at SNARE complexes. Thus, stimulus trains in 5-HT_1B receptor agonist unveil dynamic synaptic modulation and a sophisticated hippocampal output filter that itself is modulated by colocalized GABA_B receptors, which alter presynaptic Ca²⁺. In combination, these pathways allow complex presynaptic integration.SIGNIFICANCE STATEMENT Two G protein-coupled receptors colocalize at presynaptic sites, to mediate presynaptic modulation by Gβγ, but one (a GABA_B receptor) inhibits Ca²⁺ entry whereas another (a 5-HT_1B receptor) competes with Ca²⁺-synaptotagmin binding to the synaptic vesicle machinery. We have investigated downstream effects of signaling and integrative properties of these receptors. Their effects are profoundly different. GABA_B receptors alter P_r leaving synaptic properties unchanged, whereas 5-HT_1B receptors fundamentally change properties of synaptic transmission, modifying AMPAR but sparing NMDAR responses. Coactivation of these receptors allows synaptic integration because of convergence of GABA_B receptor alteration on Ca²⁺ and the effect of this altered Ca²⁺ signal on 5-HT_1B receptor signaling. This presynaptic convergence provides a novel form of synaptic integration.     

Presynaptic cannabinoid CB2 receptors modulate [3H]‐Glutamate release at subthalamo‐nigral terminals of the rat

Recent studies suggested the expression of CB2 receptors in neurons of the CNS, however, most of these studies have only explored one aspect of the receptors, i.e., expression of protein, messenger RNA, or functional response, and more complete studies appear to be needed to establish adequately their role in the neuronal function. Electron microscopy studies showed the presence of CB2r in asymmetric terminals of the substantia nigra pars reticulata (SNr), and its mRNA appeared is expressed in the subthalamic nucleus. Here, we explore the expression, source, and functional effects of such receptors by different experimental approaches. Through PCR and immunochemistry, we showed mRNA and protein for CB2rs in slices and primary neuronal cultures from subthalamus. GW833972A, GW405833, and JHW 133, three CB2r agonists dose‐dependent inhibited K⁺‐induced [³H]‐Glutamate release in slices of SNr, and the two antagonist/inverse agonists, JTE‐907 and AM630, but not AM281, a CB1r antagonist, prevented GW833972A effect. Subthalamus lesions with kainic acid prevented GW833972A inhibition on release and decreased CB2r protein in nigral synaptosomes, thus nigral CB2rs originate in subthalamus. Inhibition of [³H]‐Glutamate release was PTX‐ and gallein‐sensitive, suggesting a Gi_βγ‐mediated effect. P/Q Ca²⁺‐type channel blocker, ω‐Agatoxin‐TK, also inhibited the [³H]‐Glutamate release, this effect was occluded with GW833972A inhibition, indicating that the βγ subunit effect is exerted on Ca²⁺channel activity. Finally, microinjections of GW833972A in SNr induced contralateral turning. Our data showed that presynaptic CB2rs inhibit [³H]‐Glutamate release in subthalamo‐nigral terminals by P/Q‐channels modulation through the Gi_βγ subunit and suggested their participation in motor behavior.     

Osthole or imperatorin‐mediated facilitation of glutamate release is associated with a synaptic vesicle mobilization in rat hippocampal glutamatergic nerve endings

Osthole and imperatorin, two active compounds of Cnidium monnieri (L.) Cusson, have previously been shown to facilitate depolarization‐evoked glutamate release from rat hippocampal nerve terminals by increasing voltage‐dependent Ca²⁺ entry. In this study, we further investigated whether osthole and imperatorin possess an action at the exocytotic machinery itself, downstream of a Ca²⁺ influx. Our data showed that ionomycin‐induced glutamate release and KCl‐evoked FM1‐43 release were facilitated by osthole and imperatorin, suggesting that some steps after Ca²⁺ entry are regulated by these two compounds. Consistent with this, osthole or imperatorin‐mediated facilitation of ionomycin‐induced glutamate release was occluded by cytochalasin D that inhibits actin polymerization, implying that the disassembly of cytoskeleton is involved. In addition, the facilitatory action of osthole or imperatorin on ionomycin‐induced glutamate release was attenuated by the Ca²⁺/calmodulin‐dependent kinase II (CaMKII) inhibitor KN62. Furthermore, Western blotting analysis further showed that osthole or imperatorin significantly increased ionomycin‐induced phosphorylation of CaMKII and synapsin I, the main presynaptic target of CaMKII. These results suggest, therefore, that osthole or imperatorin‐mediated facilitation of glutamate release involves modulation of downstream events controlling synaptic vesicle recruitment and exocytosis, possibly through an increase of CaMKII activation and synapsin I phosphorylation, thereby increasing synaptic vesicle availability for exocytosis. Synapse 64:390–396, 2010. © 2009 Wiley‐Liss, Inc.     

Somatic and neuritic spines on tyrosine hydroxylase–immunopositive cells of rat retina

Dopamine‐ and tyrosine hydroxylase–immunopositive cells (TH cells) modulate visually driven signals as they flow through retinal photoreceptor, bipolar, and ganglion cells. Previous studies suggested that TH cells release dopamine from varicose axons arborizing in the inner and outer plexiform layers after glutamatergic synapses depolarize TH cell dendrites in the inner plexiform layer and these depolarizations propagate to the varicosities. Although it has been proposed that these excitatory synapses are formed onto appendages resembling dendritic spines, spines have not been found on TH cells of most species examined to date or on TH cell somata that release dopamine when exposed to glutamate receptor agonists. By use of protocols that preserve proximal retinal neuron morphology, we have examined the shape, distribution, and synapse‐related immunoreactivity of adult rat TH cells. We report here that TH cell somata, tapering and varicose inner plexiform layer neurites, and varicose outer plexiform layer neurites all bear spines, that some of these spines are immunopositive for glutamate receptor and postsynaptic density proteins (viz., GluR1, GluR4, NR1, PSD‐95, and PSD‐93), that TH cell somata and tapering neurites are also immunopositive for a γ‐aminobutyric acid (GABA) receptor subunit (GABA_AR_α1), and that a synaptic ribbon‐specific protein (RIBEYE) is found adjacent to some colocalizations of GluR1 and TH in the inner plexiform layer. These results identify previously undescribed sites at which glutamatergic and GABAergic inputs may stimulate and inhibit dopamine release, especially at somata and along varicose neurites that emerge from these somata and arborize in various levels of the retina. J. Comp. Neurol. 525:1707–1730, 2017. © 2016 Wiley Periodicals, Inc.     

Glutamate‐induced calcium increase mediates magnesium release from mitochondria in rat hippocampal neurons

Excess administration of glutamate is known to induce Ca²⁺ overload in neurons, which is the first step in excitotoxicity. Although some reports have suggested a role for Mg²⁺ in the excitotoxicity, little is known about its actual contribution. To investigate the role of Mg²⁺ in the excitotoxicity, we simultaneously measured intracellular Ca²⁺ and Mg²⁺, using fluorescent dyes, Fura red, a fluorescent Ca²⁺ probe, and KMG‐104, a highly selective fluorescent Mg²⁺ probe developed by our group, respectively. Administration of 100 μM glutamate supplemented with 10 μM glycine to rat hippocampal neurons induced an increase in intracellular Mg²⁺ concentration ([Mg²⁺]_i). Extracellular Mg²⁺ was not required for this glutamate‐induced increase in [Mg²⁺]_i, and no increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) or [Mg²⁺]_i was observed in neurons in nominally Ca²⁺‐free medium. Application of 5 μM carbonyl cyanide p‐(trifluoromethoxy) phenylhydrazone (FCCP), an uncoupler of mitochondrial inner membrane potential, also elicited increases in [Ca²⁺]_i and [Mg²⁺]_i. Subsequent administration of glutamate and glycine following FCCP treatment did not induce a further increase in [Mg²⁺]_i but did induce an additive increase in [Ca²⁺]_i. Moreover, the glutamate‐induced increase in [Mg²⁺]_i was observed only in mitochondria localized areas. These results support the idea that glutamate is able to induced Mg²⁺ efflux from mitochondria to the cytosol. Furthermore, pretreatment with Ru360, an inhibitor of the mitochondrial Ca²⁺ uniporter, prevented this [Mg²⁺]_i increase. These results indicate that glutamate‐induced increases in [Mg²⁺]_i result from the Mg²⁺ release from mitochondria and that Ca²⁺ accumulation in the mitochondria is required for this Mg²⁺ release. © 2010 Wiley‐Liss, Inc.     

Adenosine modulates the excitability of layer II stellate neurons in entorhinal cortex through A1 receptors

Stellate neurons in layer II entorhinal cortex (EC) provide the main output from the EC to the hippocampus. It is believed that adenosine plays a crucial role in neuronal excitability and synaptic transmission in the CNS, however, the function of adenosine in the EC is still elusive. Here, the data reported showed that adenosine hyperpolarized stellate neurons in a concentration‐dependent manner, accompanied by a decrease in firing frequency. This effect corresponded to the inhibition of the hyperpolarization‐activated, cation nonselective (HCN) channels. Surprisingly, the adenosine‐induced inhibition was blocked by 3 μM 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX), a selective A₁ receptor antagonists, but not by 10 μM 3,7‐dimethyl‐1‐propargylxanthine (DMPX), a selective A₂ receptor antagonists, indicating that activation of adenosine A₁ receptors were responsible for the direct inhibition. In addition, adenosine reduced the frequency but not the amplitude of miniature EPSCs and IPSCs, suggesting that the global depression of glutamatergic and GABAergic transmission is mediated by a decrease in glutamate and GABA release, respectively. Again the presynaptic site of action was mediated by adenosine A₁ receptors. Furthermore, inhibition of spontaneous glutamate and GABA release by adenosine A₁ receptor activation was mediated by voltage‐dependent Ca²⁺ channels and extracellular Ca²⁺. Therefore, these findings revealed direct and indirect mechanisms by which activation of adenosine A₁ receptors on the cell bodies of stellate neurons and on the presynaptic terminals could regulate the excitability of these neurons. © 2010 Wiley‐Liss, Inc.     

Localization of the glutamate transporter protein GLAST in rat retina

Glutamate is a neurotransmitter in retina. Glutamate transporter proteins keep the resting extracellular glutamate concentration low. This is required for normal neurotransmission and prevents the extracellular concentration of glutamate from reaching toxic levels. Here we describe the light and electron microscopic localization of the glutamate transporter protein GLAST in rat retina using an antibody raised and affinity purified against a peptide corresponding to amino acid residues 522–541. The strongest immunocytochemical labelling was observed in the outer plexiform layer, ganglion cell layer, and optic disc. GLAST was found in Müller cell processes in all retinal layers, notably ensheathing the photoreceptor terminals in the outer plexiform layer, and in astrocytes close to vessels in the inner retina and optic disc. No labelling was observed in neurons. The electrophoretic mobility of GLAST in retina was similar to that in cerebellum. In conclusion, the findings are in agreement with those reported by Derouiche and Rauen [7], except that we did not detect any GLAST in the retinal pigment epithelium.     

Distinctive patterns of alterations in proton efflux from goldfish retinal horizontal cells monitored with self‐referencing H+‐selective electrodes

The H⁺ hypothesis of lateral feedback inhibition in the outer retina predicts that depolarizing agents should increase H⁺ release from horizontal cells. To test this hypothesis, self‐referencing H⁺‐selective microelectrodes were used to measure extracellular H⁺ fluxes from isolated goldfish horizontal cells. We found a more complex pattern of cellular responses than previously observed from horizontal cells of other species examined using this technique. One class of cells had an initial standing signal indicative of high extracellular H⁺ adjacent to the cell membrane; challenge with glutamate, kainate or high extracellular potassium induced an extracellular alkalinization. This alkalinization was reduced by the calcium channel blockers nifedipine and cobalt. A second class of cells displayed spontaneous oscillations in extracellular H⁺ that were abolished by cobalt, nifedipine and low extracellular calcium. A strong correlation between changes in intracellular calcium and extracellular proton flux was detected in experiments simultaneously monitoring intracellular calcium and extracellular H⁺. A third set of cells was characterized by a standing extracellular alkalinization which was turned into an acidic signal by cobalt. In this last set of cells, addition of glutamate or high extracellular potassium did not significantly alter the proton signal. Taken together, the response characteristics of all three sets of neurons are most parsimoniously explained by activation of a plasma membrane Ca²⁺ ATPase pump, with an extracellular alkalinization resulting from exchange of intracellular calcium for extracellular H⁺. These findings argue strongly against the hypothesis that H⁺ release from horizontal cells mediates lateral inhibition in the outer retina.     

Voltage‐induced Ca2+ release by ryanodine receptors causes neuropeptide secretion from nerve terminals

Depolarisation‐secretion coupling is assumed to be dependent only on extracellular calcium ([Ca²⁺]_o). Ryanodine receptor (RyR)‐sensitive stores in hypothalamic neurohypophysial system (HNS) terminals produce sparks of intracellular calcium ([Ca²⁺]_i) that are voltage‐dependent. We hypothesised that voltage‐elicited increases in intraterminal calcium are crucial for neuropeptide secretion from presynaptic terminals, whether from influx through voltage‐gated calcium channels and/or from such voltage‐sensitive ryanodine‐mediated calcium stores. Increases in [Ca²⁺]_i upon depolarisation in the presence of voltage‐gated calcium channel blockers, or in the absence of [Ca²⁺]_o, still give rise to neuropeptide secretion from HNS terminals. Even in 0 [Ca²⁺]_o, there was nonetheless an increase in capacitance suggesting exocytosis upon depolarisation. This was blocked by antagonist concentrations of ryanodine, as was peptide secretion elicited by high K⁺ in 0 [Ca²⁺]_o. Furthermore, such depolarisations lead to increases in [Ca²⁺]_i. Pre‐incubation with BAPTA‐AM resulted in > 50% inhibition of peptide secretion elicited by high K⁺ in 0 [Ca²⁺]_o. Nifedipine but not nicardipine inhibited both the high K⁺ response for neuropeptide secretion and intraterminal calcium, suggesting the involvement of CaV1.1 type channels as sensors in voltage‐induced calcium release. Importantly, RyR antagonists also modulate neuropeptide release under normal physiological conditions. In conclusion, our results indicate that depolarisation‐induced neuropeptide secretion is present in the absence of external calcium, and calcium release from ryanodine‐sensitive internal stores is a significant physiological contributor to neuropeptide secretion from HNS terminals.