Regulation of DARPP-32 dephosphorylation at PKA- and Cdk5-sites by NMDA and AMPA receptors: distinct roles of calcineurin and protein phosphatase-2A

Glutamatergic inputs from corticostriatal and thalamostriatal pathways have been shown to modulate dopaminergic signaling in neostriatal neurons. DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of M (r) 32 kDa) is a signal transduction molecule that regulates the efficacy of dopamine signaling in neostriatal neurons. Dopamine signaling is mediated in part through phosphorylation of DARPP-32 at Thr34 by cAMP-dependent protein kinase, and antagonized by phosphorylation of DARPP-32 at Thr75 by cyclin-dependent protein kinase 5. We have now investigated the effects of the ionotropic glutamate NMDA and AMPA receptors on DARPP-32 phosphorylation in neostriatal slices. Activation of NMDA and AMPA receptors decreased the state of phosphorylation of DARPP-32 at Thr34 and Thr75. The decrease in Thr34 phosphorylation was mediated through Ca(2+) -dependent activation of the Ca(2+) -/calmodulin-dependent phosphatase, calcineurin. In contrast, the decrease in Thr75 phosphorylation was mediated through Ca(2+) -dependent activation of dephosphorylation by protein phosphatase-2A. The results provide support for a complex effect of glutamate on dopaminergic signaling through the regulation of dephosphorylation of different sites of DARPP-32 by different protein phosphatases.     

CB1 receptors diminish both Ca(2+) influx and glutamate release through two different mechanisms active in distinct populations of cerebrocortical nerve terminals

We have investigated the mechanisms by which activation of cannabinoid receptors reduces glutamate release from cerebrocortical nerve terminals. Glutamate release evoked by depolarization of nerve terminals with high KCl (30 mmol/L) involves N and P/Q type Ca(2+)channel activation. However, this release of glutamate is independent of Na(+) or K(+) channel activation as it was unaffected by blockers of these channels (tetrodotoxin -TTX- or tetraethylammonium TEA). Under these conditions in which only Ca(2+) channels contribute to pre-synaptic activity, the activation of cannabinoid receptors with WIN55,212-2 moderately reduced glutamate release (26.4 +/- 1.2%) by a mechanism that in this in vitro model is resistant to TTX and consistent with the inhibition of Ca(2+) channels. However, when nerve terminals are stimulated with low KCl concentrations (5-10 mmol/L) glutamate release is affected by both Ca(2+) antagonists and also by TTX and TEA, indicating the participation of Na(+) and K(+) channel firing in addition to Ca(2+) channel activation. Interestingly, stimulation of nerve terminals with low KCl concentrations uncovered a mechanism that further inhibited glutamate release (81.78 +/- 4.9%) and that was fully reversed by TEA. This additional mechanism is TTX-sensitive and consistent with the activation of K(+) channels. Furthermore, Ca(2+) imaging of single boutons demonstrated that the two pre-synaptic mechanisms by which cannabinoid receptors reduce glutamate release operate in distinct populations of nerve terminals.     

A D2 class dopamine receptor transactivates a receptor tyrosine kinase to inhibit NMDA receptor transmission

Receptor tyrosine kinases (RTKs) are membrane spanning proteins with intrinsic kinase activity. Although these receptors are known to be involved in proliferation and differentiation of cells, their roles in regulating central synaptic transmission are largely unknown. In CA1 pyramidal neurons, activation of D2 class dopamine receptors depressed excitatory transmission mediated by the NMDA subtype of glutamate receptor. This depression resulted from the quinpirole-induced release of intracellular Ca(2+) and enhanced Ca(2+)-dependent inactivation of NMDA receptors. The dopamine receptor-mediated depression was dependent on the "transactivation" of PDGFRbeta. Therefore, RTK transactivation provides a novel mechanism of communication between dopaminergic and glutamatergic systems and might help to explain how reciprocal changes in these systems could be linked to the deficits in cognition, memory, and attention observed in schizophrenia and attention deficit hyperactivity disorder.     

Activation of P2X(7) receptors decreases glutamate uptake and glutamine synthetase activity in RBA-2 astrocytes via distinct mechanisms

Glutamate clearance by astrocytes is critical for controlling excitatory neurotransmission and ATP is an important mediator for neuron-astrocyte interaction. However, the effect of ATP on glutamate clearance has never been examined. Here we report that treatment of RBA-2 cells, a type-2-like astrocyte cell line, with ATP and the P2X(7) receptor selective agonist 3'-O-(4-benzoylbenzoyl) adenosine 5'-triphosphate (BzATP) decreased the Na+-dependent [3H]glutamate uptake within minutes. Mechanistic studies revealed that the decreases were augmented by removal of extracellular Mg2+ or Ca2+, and was restored by P2X7 selective antagonist , periodate-oxidized 2',3'-dialdehyde ATP (oATP), indicating that the decreases were mediated through P2X(7) receptors. Furthermore, stimulation of P2X7 receptors for 2 h inhibited both activity and protein expression of glutamine synthetase (GS), and oATP abolished the inhibition. In addition, removal of extracellular Ca(2+) and inhibition of protein kinase C (PKC) restored the ATP-decreased GS expression but failed to restore the P2X(7)-decreased [3H]glutamate uptake. Therefore, P2X7-mediated intracellular signals play a role in the down-regulation of GS activity/expression. Activation of P2X7 receptors stimulated increases in intracellular Na+ concentration ([Na+](i)) suggesting that the P2X(7)-induced increases in [Na+](i) may affect the local Na+ gradient and decrease the Na+-dependent [3H]glutamate uptake. These findings demonstrate that the P2X7-mediated decreases in glutamate uptake and glutamine synthesis were mediated through distinct mechanisms in these cells.     

Metabotropic glutamate receptor 5 and calcium signaling in retinal amacrine cells

To begin to understand the modulatory role of glutamate in the inner retina, we examined the mechanisms underlying metabotropic glutamate receptor 5 (mGluR5)-dependent Ca(2+) elevations in cultured GABAergic amacrine cells. A partial sequence of chicken retinal mGluR5 encompassing intracellular loops 2 and 3 suggests that it can couple to both G(q) and G(s). Selective activation of mGluR5 stimulated Ca(2+) elevations that varied in waveform from cell to cell. Experiments using high external K(+) revealed that the mGluR5-dependent Ca(2+) elevations are distinctive in amplitude and time course from those engendered by depolarization. Experiments with a Ca(2+) -free external solution demonstrated that the variability in the time course of mGluR5-dependent Ca(2+) elevations is largely due to the influx of extracellular Ca(2+). The sensitivity of the initial phase of the Ca(2+) elevation to thapsigargin indicates that this phase of the response is due to the release of Ca(2+) from the endoplasmic reticulum. Pharmacological evidence indicates that mGluR5-mediated Ca(2+) elevations are dependent upon the activation of phospholipase C. We rule out a role for L-type Ca(2+) channels and cAMP-gated channels as pathways for Ca(2+) entry, but provide evidence of transient receptor potential (TRP) channel-like immunoreactivity, suggesting that Ca(2+) influx may occur through TRP channels. These results indicate that GABAergic amacrine cells express an avian version of mGluR5 that is linked to phospholipase C-dependent Ca(2+) release and Ca(2+) influx, possibly through TRP channels.     

An inwardly rectifying whole cell current induced by Gq-coupled receptors

Ca(2+) influx across the plasma membrane after stimulation of G protein-coupled receptors is important for many physiological functions. Here we studied the regulation of an inwardly rectifying whole cell current and its putative role in Ca(2+) entry in Xenopus oocytes. Expression of P2Y(1) or M1 receptors in Xenopus oocytes elicited a characteristic inwardly rectifying current without receptor stimulation. This current displayed distinct activation and inactivation kinetics and was highly Ca(2+)-dependent. After stimulation of endogenous G(q)-coupled receptors in water-injected cells similar currents were observed. We therefore speculated that the current could be activated via Ca(2+) store depletion induced by constitutive stimulation of the IP(3) cascade in cells overexpressing G(q)-coupled receptors. Receptor-independent Ca(2+) store depletion also induced the current. In conclusion, this current is activated after store depletion suggesting a role in Ca(2+) entry after stimulation of G(q)-coupled receptors. Finally, our data do not support the proposed ionotropic properties of the P2Y(1) receptor.     

Bidirectional regulation of thermotaxis by glutamate transmissions in Caenorhabditis elegans

In complex neural circuits of the brain, massive information is processed with neuronal communication through synaptic transmissions. It is thus fundamental to delineate information flows encoded by various kinds of transmissions. Here, we show that glutamate signals from two distinct sensory neurons bidirectionally affect the same postsynaptic interneuron, thereby producing the opposite behaviours. EAT-4/VGLUT (vesicular glutamate transporter)-dependent glutamate signals from AFD thermosensory neurons inhibit the postsynaptic AIY interneurons through activation of GLC-3/GluCl inhibitory glutamate receptor and behaviourally drive migration towards colder temperature. By contrast, EAT-4-dependent glutamate signals from AWC thermosensory neurons stimulate the AIY neurons to induce migration towards warmer temperature. Alteration of the strength of AFD and AWC signals led to significant changes of AIY activity, resulting in drastic modulation of behaviour. We thus provide an important insight on information processing, in which two glutamate transmissions encoding opposite information flows regulate neural activities to produce a large spectrum of behavioural outputs.     

Presynaptic Glutamate Receptors Modulate Dopamine Release from Striatal Synaptosomes

The wide-ranging neuronal actions of glutamate are thought to be mediated by postsynaptic N-methyl-D-aspartate (NMDA) and non-NMDA receptors. The present report demonstrates the existence of presynaptic glutamate receptors in isolated striatal dopaminergic nerve terminals (synaptosomes). Activation of these receptors, by NMDA in the absence of Mg2+ and presence of glycine and by non-NMDA agonists in the presence of Mg2+, results in Ca(2+)-dependent release of dopamine from striatal synaptosomes. The release stimulated by NMDA is blocked by Mg2+ and by selective NMDA antagonists, whereas the release stimulated by selective non-NMDA agonists is blocked by a non-NMDA antagonist but not by Mg2+ or NMDA antagonists. Thus, these presynaptic glutamate receptors, localized on dopaminergic terminals in the striatum, appear to be pharmacologically similar to both the NMDA and the non-NMDA postsynaptic receptors. By modulating the release of dopamine, these presynaptic receptors may play an important role in transmitter interactions in the striatum.     

Purinergic receptors mediate two distinct glutamate release pathways in hippocampal astrocytes

The purinergic P2X(7) receptor (P2X(7)R) can mediate glutamate release from cultured astrocytes. Using patch clamp recordings, we investigated whether P2X(7)Rs have the same action in hippocampal astrocytes in situ. We found that 2- and 3-O-(4-benzoylbenzoyl)ATP (BzATP), a potent, although unselective P2X(7)R agonist, triggers two different glutamate-mediated responses in CA1 pyramidal neurons; they are transient inward currents, which have the kinetic and pharmacological properties of previously described slow inward currents (SICs) due to Ca(2+)-dependent glutamate release from astrocytes, and a sustained tonic current. Although SICs were unaffected by P2X(7)Rs antagonists, the tonic current was inhibited, was amplified in low extracellular Ca(2+), and was insensitive to glutamate transporter and hemichannel inhibitors. BzATP triggered in astrocytes a large depolarization that was inhibited by P2X(7)R antagonists and amplified in low Ca(2+). In low Ca(2+) BzATP also induced lucifer yellow uptake into a subpopulation of astrocytes and CA3 neurons. Our results demonstrate that purinergic receptors other than the P2X(7)R mediate glutamate release that evokes SICs, whereas activation of a receptor that has features similar to the P2X(7)R, mediates a sustained glutamate efflux that generates a tonic current in CA1 neurons. This sustained glutamate efflux, which is potentiated under non-physiological conditions, may have important pathological actions in the brain.     

A crucial role for cAMP and protein kinase A in D1 dopamine receptor regulated intracellular calcium transients

D1-like dopamine receptors stimulate Ca(2+) transients in neurons but the effector coupling and signaling mechanisms underlying these responses have not been elucidated. Here we investigated potential mechanisms using both HEK 293 cells that stably express D1 receptors (D1HEK293) and hippocampal neurons in culture. In D1HEK293 cells, the full D1 receptor agonist SKF 81297 evoked a robust dose-dependent increase in Ca(2+)(i) following 'priming' of endogenous G(q/11)-coupled muscarinic or purinergic receptors. The effect of SKF81297 could be mimicked by forskolin or 8-Br-cAMP. Further, cholera toxin and the cAMP-dependent protein kinase (PKA) inhibitors, KT5720 and H89, as well as thapsigargin abrogated the D1 receptor evoked Ca(2+) transients. Removal of the priming agonist and treatment with the phospholipase C inhibitor U73122 also blocked the SKF81297-evoked responses. D1R agonist did not stimulate IP(3) production, but pretreatment of cells with the D1R agonist potentiated G(q)-linked receptor agonist mobilization of intracellular Ca(2+) stores. In neurons, SKF81297 and SKF83959, a partial D1 receptor agonist, promoted Ca(2+) oscillations in response to G(q/11)-coupled metabotropic glutamate receptor (mGluR) stimulation. The effects of both D1R agonists on the mGluR-evoked Ca(2+) responses were PKA dependent. Altogether the data suggest that dopamine D1R activation and ensuing cAMP production dynamically regulates the efficiency and timing of IP(3)-mediated intracellular Ca(2+) store mobilization.     

Ionotropic glutamate receptors trigger microvesicle-mediated exocytosis of L-glutamate in rat pinealocytes

Rat pinealocytes receive noradrenergic innervation that stimulates melatonin synthesis. Besides melatonin, we showed previously that pinealocytes accumulate L-glutamate in microvesicles and secrete it through an exocytic mechanism. The secreted glutamate binds to the class II metabotropic glutamate receptor and inhibits norepinephrine-stimulated melatonin synthesis in neighboring pinealocytes through an inhibitory cyclic AMP cascade. In this study, it was found that, in addition to metabotropic receptors, pinealocytes express functional ionotropic receptors. RT-PCR and northern analyses indicated the expression of mRNA for GluR1, KA2, and NR2C in pineal gland. The presence of GluR1 protein was confirmed by immunological techniques, but neither KA2 nor NR2C was detected. Consistent with this observation, the presence of (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid or kainate, non-N-methyl-D-aspartate receptor agonists, transiently stimulated increased the intracellular Ca(2+) concentration of cultured pinealocytes, whereas N-methyl-D-aspartate did not. These responses were prevented by 6-cyano-7-nitroquinoxaline-2,3-dione, a selective antagonist for non-N-methyl-D-aspartate receptors, by L-type Ca(2+) channel blockers such as nifedipine, or by omitting Ca(2+) or Na(+) in the medium. In the presence of Ca(2+) and Na(+), (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid or kainate evoked glutamate secretion from the cultured cells, which was prevented by 6-cyano-7-nitroquinoxaline-2,3-dione, L-type Ca(2+) channel blockers, type E or B botulinum neurotoxin, or incubation at <20 degrees C. These results strongly suggest that GluR1 is functionally expressed in pinealocytes and triggers microvesicle-mediated exocytosis of L-glutamate via activation of L-type Ca(2+) channels. It is possible that GluR1 participates in a signaling cascade that enhances and expands the L-glutamate signal throughout the pineal gland.     

The astrocyte excitability brief: from receptors to gliotransmission

Astrocytes do not merely serve as the supporting cast and scenery against which starring roles would be played by neurons. Rather, these glial cells are intimately involved in many of the brain's functions by responding to neuronal activity and modulating it. Such interplay between two principle neural cells, neurons and astrocytes, is evidenced in bi-directional glutamatergic astrocyte-neuron signaling. A key feature in this signaling pathway is astrocytic excitability based on variations of cytosolic Ca(2+). It enables astrocytes, through the activation of their glutamatergic receptors, to respond to the same signal used by nearby neurons in synaptic transmission. Furthermore, increases in cytosolic Ca(2+) in astrocytes can subsequently lead to Ca(2+)-dependent exocytotic secretion of gliotransmitter glutamate that in turn can signal to adjacent neurons. Astrocytic secretory machinery includes an assortment of exocytotic proteins which governs a merger of secretory vesicles to the plasma membrane. A cumulative knowledge on astrocytic excitability will aid better understanding of operating procedures in the brain in health and disease.     

Molecular mechanisms of calcium-dependent neurodegeneration in excitotoxicity

Excitotoxicity contributes to neuronal degeneration in many acute CNS diseases, including ischemia, trauma, and epilepsy, and may also play a role in chronic diseases, such as amyotrophic lateral sclerosis (ALS). Key mediators of excitotoxic damage are Ca ions (Ca(2+)), which under physiological conditions govern a multitude of cellular processes, including cell growth, differentiation, and synaptic activity. Consequently, homeostatic mechanisms exist to maintain a low intracellular Ca(2+) ion concentration so that Ca(2+) signals remain spatially and temporally localized. This permits multiple independent Ca-mediated signaling pathways to occur in the same cell. In excitotoxicity, excessive synaptic release of glutamate can lead to the disregulation of Ca(2+) homeostasis. Glutamate activates postsynaptic receptors, including the ionotropic N-methyl-D-aspartate (NMDA), 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl) proprionate (AMPA), and kainate receptors. Upon their activation, these open their associated ion channel to allow the influx of Ca(2+) and Na(+) ions. Although physiological elevations in intracellular Ca(2+) are salient to normal cell functioning, the excessive influx of Ca(2+) together with any Ca(2+) release from intracellular compartments can overwhelm Ca(2+)-regulatory mechanisms and lead to cell death. Although Ca(2+) disregulation is paramount to neurodegeneration, the exact mechanism by which Ca(2+) ions actually mediate excitotoxicity is less clear. One hypothesis outlined in this review suggests that Ca(2+)-dependent neurotoxicity occurs following the activation of distinct signaling cascades downstream from key points of Ca(2+) entry at synapses, and that triggers of these cascades are physically co-localized with specific glutamate receptors. Thus, we summarize the importance of Ca(2+) regulation in mammalian neurons and the excitotoxicity hypothesis, and focus on the molecular determinants of glutamate receptor-mediated excitotoxic mechanisms.     

Modulation of Ca2+ signals by phosphatidylinositol-linked novel D1 dopamine receptor in hippocampal neurons

Recent evidence indicates the existence of a putative novel phosphatidylinositol-linked D1 dopamine receptor in brain that mediates phosphatidylinositol hydrolysis via activation of phospholipase Cbeta. The present work was designed to characterize the Ca(2+) signals regulated by this phosphatidylinositol-linked D(1) dopamine receptor in primary cultures of hippocampal neurons. The results indicated that stimulation of phosphatidylinositol-linked D1 dopamine receptor by its newly identified selective agonist SKF83959 induced a long-lasting increase in basal [Ca(2+)](i) in a time- and dose-dependent manner. Stimulation was observable at 0.1 microm and reached the maximal effect at 30 microm. The [Ca(2+)](i) increase induced by 1 microm SKF83959 reached a plateau in 5 +/- 2.13 min, an average 96 +/- 5.6% increase over control. The sustained elevation of [Ca(2+)](i) was due to both intracellular calcium release and calcium influx. The initial component of Ca(2+) increase through release from intracellular stores was necessary for triggering the late component of Ca(2+) rise through influx. We further demonstrated that activation of phospholipase Cbeta/inositol triphosphate was responsible for SKF83959-induced Ca(2+) release from intracellular stores. Moreover, inhibition of voltage-operated calcium channel or NMDA receptor-gated calcium channel strongly attenuated SKF83959-induced Ca(2+) influx, indicating that both voltage-operated calcium channel and NMDA receptor contribute to phosphatidylinositol-linked D(1) receptor regulation of [Ca(2+)](i).     

Regulation of neurotransmitter release by metabotropic glutamate receptors

The G protein-coupled metabotropic glutamate (mGlu) receptors are differentially localized at various synapses throughout the brain. Depending on the receptor subtype, they appear to be localized at presynaptic and/or postsynaptic sites, including glial as well as neuronal elements. The heterogeneous distribution of these receptors on glutamate and nonglutamate neurons/cells thus allows modulation of synaptic transmission by a number of different mechanisms. Electrophysiological studies have demonstrated that the activation of mGlu receptors can modulate the activity of Ca(2+) or K(+) channels, or interfere with release processes downstream of Ca(2+) entry, and consequently regulate neuronal synaptic activity. Such changes evoked by mGlu receptors can ultimately regulate transmitter release at both glutamatergic and nonglutamatergic synapses. Increasing neurochemical evidence has emerged, obtained from in vitro and in vivo studies, showing modulation of the release of a variety of transmitters by mGlu receptors. This review addresses the neurochemical evidence for mGlu receptor-mediated regulation of neurotransmitters, such as excitatory and inhibitory amino acids, monoamines, and neuropeptides.     

Glutamatergic regulation of the p70S6 kinase in primary mouse neurons

Brief glutamatergic stimulation of neurons from fetal mice, cultured in vitro for 6 days, activates the mTOR-S6 kinase, ERK1/2 and Akt pathways, to an extent approaching that elicited by brain-derived neurotrophic factor. In contrast, sustained glutamatergic stimulation inhibits ERK, Akt, and S6K. Glutamatergic activation of S6K is calcium/calmodulin-dependent and is prevented by inhibitors of calcium/calmodulin-dependent protein kinase 2, phosphatidylinositol 3-OH-kinase and by rapamycin. 2-Amino-5-phosphonovaleric acid, an inhibitor of N'-methyl-D-aspartate receptors, abolishes glutamatergic activation of ERK1/2 but not the activation of mTOR-S6K; the latter is completely abolished by inhibitors of voltage-dependent calcium channels. Added singly, dopamine gives slight, and norepinephrine a more significant, activation of ERK and S6K; both catecholeamines, however, enhance glutamatergic activation of S6K but not ERK. After 12 days in culture, the response to direct glutamatergic activation is attenuated but can be uncovered by suppression of gamma-aminobutyric acid interneurons with bicuculline in the presence of the weak K(+) channel blocker 4-aminopyridine (4-AP). This selective synaptic activation of mTOR-S6K is also resistant to APV and inhibited by Ca(2+) channel blockers and higher concentrations of glutamate. Elongation factor 2 (EF2) is phosphorylated and inhibited by the eEF2 kinase (CaM kinase III); the latter is inhibited by the S6K or Rsk. Bicuculline/4-AP or KCl-induced depolarization reduces, whereas higher concentrations of glutamate increases, EF2 phosphorylation. Thus the mTOR-S6K pathway in neurons, a critical component of the late phase of LTP, is activated by glutamatergic stimulation in a calcium/calmodulin-dependent fashion through a calcium pool controlled by postsynaptic voltage-dependent calcium channels, whereas sustained stimulation of extrasynaptic glutamate receptors is inhibitory.     

Presynaptic kainate receptor-mediated facilitation of glutamate release involves Ca(2+) -calmodulin at mossy fiber-CA3 synapses

J. Neurochem. (2012) 122, 891-899. ABSTRACT: Presynaptic kainate receptors (KARs) modulate the release of glutamate at synapses established between mossy fibers (MF) and CA3 pyramidal cells in the hippocampus. The activation of KAR by low, nanomolar, kainate concentrations facilitates glutamate release. KAR-mediated facilitation of glutamate release involves the activation of an adenylate cyclase/cyclic adenosine monophosphate/protein kinase A cascade at MF-CA3 synapses. Here, we studied the mechanisms by which KAR activation produces this facilitation of glutamate release in slices and synaptosomes. We find that the facilitation of glutamate release mediated by KAR activation requires an increase in Ca(2+) levels in the cytosol and the formation of a Ca(2+) -calmodulin complex to activate adenylate cyclase. The increase in cytosolic Ca(2+) underpinning this modulation is achieved, both, by Ca(2+) entering via Ca(2+) -permeable KARs and, by the mobilization of intraterminal Ca(2+) stores. Finally, we find that, congruent with the Ca(2+) -calmodulin support of KAR-mediated facilitation of glutamate release, induction of long-term potentiation at MF-CA3 synapses has an obligate requirement for Ca(2+) -calmodulin activity.     

Group I metabotropic glutamate receptors, mGlu1a and mGlu5a, couple to cyclic AMP response element binding protein (CREB) through a common Ca2+ - and protein kinase C-dependent pathway

Coupling of the group I metabotropic glutamate receptors, mGlu1a and mGlu5a, to the cAMP response element binding protein (CREB) has been studied in Chinese hamster ovary cell lines where receptor expression is under the control of an inducible promoter. Both receptors stimulate CREB phosphorylation with similar time courses, and agonist potency was also comparable between the two receptors. Stimulation of cells in Ca(2+)-free medium containing EGTA (100 microm), with or without the additional depletion of intracellular stores, caused marked decreases in agonist-mediated responses in both cell lines. Down-regulation of protein kinase C (PKC) activity by phorbol ester treatment, or treatment with the broad spectrum PKC inhibitor Ro 31-8220, partially attenuated both mGlu1a and mGlu5a receptor-mediated responses. Furthermore, stimulation of cells in the absence of extracellular Ca(2+) following prior PKC down-regulation resulted in additive inhibitory effects. The involvement of extracellular signal-regulated kinases (ERK1/2), Ca(2+)/calmodulin or Ca(2+)/calmodulin-dependent protein kinases was assessed using pharmacological inhibitors. Results indicated that coupling of the group I mGlu receptors to CREB phosphorylation occurs independently of these pathways. Thus, although the [Ca(2+)](i) signatures activated by these mGlu receptors differ, they couple to CREB with comparable potency and recruit similar downstream components to execute CREB phosphorylation.     

M1 muscarinic receptors boost synaptic potentials and calcium influx in dendritic spines by inhibiting postsynaptic SK channels

Acetylcholine release and activation of muscarinic cholinergic receptors (mAChRs) enhance synaptic plasticity in?vitro and cognition and memory in?vivo. Within the hippocampus, mAChRs promote NMDA-type glutamate receptor-dependent forms of long-term potentiation. Here, we use calcium (Ca) imaging combined with two-photon laser glutamate uncaging at apical spines of CA1 pyramidal neurons to examine postsynaptic mechanisms of muscarinic modulation of glutamatergic transmission. Uncaging-evoked excitatory postsynaptic potentials and Ca transients are increased by muscarinic stimulation; however, this is not due to direct modulation of glutamate receptors. Instead, mAChRs modulate a negative feedback loop in spines that normally suppresses synaptic signals. mAChR activation reduces the Ca sensitivity of small conductance Ca-activated potassium (SK) channels that are found in the spine, resulting in increased synaptic potentials and Ca transients. These effects are mediated by M1-type muscarinic receptors and occur in a casein kinase-2-dependent manner. Thus, muscarinic modulation regulates synaptic transmission by tuning the activity of nonglutamatergic postsynaptic ion channels.