L-AP3 blocks rises in intracellular calcium associated with hippocampal CA1 LTP

The involvement of type I metabotropic glutamate receptors in hippocampal CA1 long-term potentiation (LTP) depends on the applied tetanic stimulation protocol. Activation of these receptors may cause an elevation of intracellular calcium via the formation of the second messenger inositol triphosphate (IP3) and subsequent intracellular calcium release. It has been shown that the type I metabotropic receptors antagonist L-2-amino-3-phosphonopropionate (L-AP3) blocks CA1 LTP. Combining dendritic calcium and field potential measurements in CA1 hippocampal area, we found that L-AP3 did not affect single calcium transients but reduced the calcium changes evoked by a single tetanus, preventing the long-lasting calcium enhancements associated with CA1 LTP. These findings suggest that the formation of this type of LTP requires calcium release from IP3-sensitive stores.     

Properties of LTD and LTP of retinocollicular synaptic transmission in the developing rat superior colliculus

The developing retinocollicular pathway undergoes synaptic refinement in order to form the precise retinotopic pattern seen in adults. To study the mechanisms which underlie refinement, we investigated long-term changes in retinocollicular transmission in rats aged P0-P25. Field potentials (FPs) in the superior colliculus (SC) were evoked by stimulation of optic tract fibers in an in vitro isolated brainstem preparation. High intensity stimulation induced long-term depression (LTD) in the SC after both low (1000 stimuli at 1 Hz) and higher (1000 stimuli at 50 Hz) frequency stimulation. The induction of LTD was independent of activation of NMDA and GABA(A) receptors, because D-APV (100 microM) and bicuculline (10 microM) did not block LTD. Induction of LTD was dependent upon activation of L-type Ca(2+) channels as 10 microM nitrendipine, an L-type Ca(2+) channel blocker, significantly decreased the magnitude of LTD. LTD was down-regulated during development. LTD magnitude was greatest in rats aged P0-P9 and significantly less in rats aged P10-P25. Long-term potentiation (LTP) was induced by low intensity stimulation and only after high frequency tetanus (1000 stimuli at 50 Hz). LTP was NMDA receptor dependent because d-APV (100 microM) completely abolished it. LTP induction was also blocked by the L-type Ca2+ channel blocker nitrendipine. The magnitude of LTP first increased with age, being significantly greater at P7-P13 than at P0-3 and then decreased at P23-25. In summary, both LTD and LTP are present during retinocollicular pathway refinement, but have different transmitter and ionic mechanisms and time courses of expression.     

Acetylcholine-mediated axon-glia signaling in the developing insect olfactory system

In the olfactory system of the sphinx moth Manduca sexta, migration of neuropil glial cells is triggered by olfactory receptor axons and depends on intraglial Ca(2+) signaling. It is not known, however, how receptor axons and glial cells communicate and whether Ca(2+) signaling is a consequence of this communication. We studied Ca(2+) increases in glial cells in vivo and in situ, evoked by electrical stimulation of olfactory receptor axons in pupae and by odor stimulation of receptor neurons in adult moths. Axonal activity leads to Ca(2+) increases in neuropil glial cells that are blocked by nicotinic acetylcholine receptor antagonists and can be mimicked by acetylcholine and carbachol application. In addition, Ca(2+) transients were abolished by removal of external Ca(2+) and blockage of voltage-gated Ca(2+) channels. During development, acetylcholine-mediated Ca(2+) signaling could first be elicited at stage 6, the time when neuropil glial cells start to migrate. Glial migration was reduced after injection of nicotinic antagonists into pupae. The results show that Ca(2+) signaling can be induced by acetylcholine release from olfactory receptor axons, which activates nicotinic acetylcholine receptors and leads to voltage-gated Ca(2+) influx. The results further suggest that cholinergic signaling in the olfactory system is required for glial cell migration in Manduca.     

Calcium as a Trigger for Cerebellar Long-Term Synaptic Depression

Cerebellar long-term depression (LTD) is a form of long-term synaptic plasticity that is triggered by calcium(Ca2+) signals in the postsynaptic Purkinje cell. This Ca2+comes both from IP3-mediated release from intracellular Ca2+ stores, as well as from Ca2+ influx through voltage-gated Ca2+ channels. The Ca2+ signal that triggers LTD occurs locally within dendritic spines and is due to supralinear summation of signals coming from these two Ca2+ sources. The properties of this postsynaptic Ca2+signal can explain several features of LTD, such as its associativity, synapse specificity, and dependence on thetiming of synaptic activity, and can account for the slow kinetics of LTD expression. Thus, from a Ca2+ signaling perspective, LTD is one of the best understood forms of synaptic plasticity.     

Selective modulation of Ca(2+) influx pathways by 5-HT regulates synaptic long-term plasticity in the hippocampus

Both long-term potentiation (LTP) and long-term depression (LTD) can be induced in the Schaffer collateral-CA1 synapse of the hippocampus either by repetitive stimulation of afferent fibres with the frequency of the stimulation determining the polarity of the response or by associative pairing of pre- and postsynaptic activity. An increase in postsynaptic intracellular Ca(2+) concentration is an important signal for the induction of long-term synaptic plasticity. In patch-clamp experiments on hippocampal brain slices, we tested the modulation of different forms of synaptic plasticity by the neurotransmitter serotonin (5-HT) which is known to inhibit high-voltage activated Ca(2+) channels. 1 microM of 5-HT inhibited homosynaptic LTD induced by low frequency stimulation. This effect of 5-HT could be blocked by the selective 5-HT(1A) antagonist WAY 100635. Low frequency-induced LTD is both dependent on Ca(2+) influx through NMDA receptors and high-voltage activated Ca(2+) channels. It was blocked by the NMDA-receptor antagonist D-AP5 and by the N-type Ca(2+) channel antagonist omega-conotoxin GIVA. Tetanus induced LTP was not affected by low concentrations of 5-HT, whereas depotentiation of LTP by asynchronous pairing of EPSPs and postsynaptic action potentials was completely abolished with 5-HT in the bath solution. We conclude that those forms of plasticity which depend on Ca(2+) influx via high-voltage activated Ca(2+) channels are subject to modulation by 5-HT. This might be a relevant mechanism by which 5-HT modifies basic network properties in the brain.     

Long-term potentiation of transmitter exocytosis expressed by Ca2+-induced Ca2+ release from thapsigargin-sensitive Ca2+ stores in preganglionic nerve terminals

We have studied whether Ca(2+)-induced Ca(2+) release (CICR) is involved in the mechanism of long-term potentiation (LTP) at nicotinic synapses of bullfrog sympathetic ganglia. Fast excitatory postsynaptic potentials (fast EPSPs) were recorded in a low-Ca(2+), high-Mg(2+) solution and quantal analysis was applied. The conditioning stimulation of the B-type preganglionic nerve at 20 Hz for 4 min consistently enhanced the amplitude and quantal content of fast EPSP for > 2 h, but only sometimes enhanced the quantal size. The LTP of quantal content produced by the conditioning tetanus was blocked by thapsigargin, a blocker of Ca(2+) pumps at Ca(2+) stores, applied before or after the conditioning tetanus, and by Xestospongin C, a blocker of inositoltrisphosphate (IP(3)) receptors, applied before the tetanus. It was not, however, blocked by ryanodine, a blocker and/or activator of ryanodine receptors, or by propranolol, a blocker of beta-adrenergic receptors. Thus the long-lasting activity of the preganglionic nerve at a high frequency causes the LTP of impulse-evoked transmitter release by the activation of CICR from thapsigargin-sensitive Ca(2+) stores in the nerve terminals. It is likely that a large Ca(2+) entry into the nerve terminals during tetanic activity primes ryanodine-insensitive Ca(2+) release channels for activation.     

Stratum oriens stimulation-evoked modulation of hippocampal long-term potentiation involves the activation of muscarinic acetylcholine receptors and the inhibition of Kv7/M potassium ion channels

Acetylcholine is considered to be an endogenous modulator of hippocampal neurotransmission and synaptic plasticity. The activation of muscarinic acetylcholine receptors (mAChRs) reportedly enhances hippocampal synaptic plasticity, which plays an important role in memory function; however, the mechanism by which it enhances synaptic plasticity remains unclear. Here, we examined the involvement of the inhibition of Kv7/M K(+) channels, which are targets of mAChR modulation, during mAChR activation-induced enhancement of long-term potentiation (LTP) at rat hippocampal Schaffer collateral (SC)-CA1 synapses. When an electrical stimulus was applied to the stratum oriens before tetanic stimulation of the SCs, the magnitude of the induced SC-CA1 synapse LTP was enhanced as compared with that induced without stratum oriens stimulation. In the presence of the mAChR antagonist atropine, tetanic stimulation induced stable LTP, but the stratum oriens stimulation-evoked enhancement of LTP was abolished. The additional application of XE991, a selective blocker of Kv7/M K(+) channels, rescued the atropine-induced inhibition of LTP enhancement. The phospholipase C (PLC) inhibitor U-73122 inhibited the stratum oriens stimulation-evoked enhancement of LTP. Application of the T/R-type voltage-dependent Ca(2+) channel (VDCC) blocker Ni(2+) abolished the stratum oriens stimulation-evoked enhancement of LTP. In addition, tetanic stimulation with preceding stratum oriens stimulation was able to induce LTP during N-methyl-d-aspartate receptor blockade. We therefore propose that stratum oriens stimulation inhibits Kv7/M K(+) channels through mAChR activation-induced PLC activation, which leads to VDCC activation, and hence causes sufficient Ca(2+) influx to enhance LTP.     

Long-term depression is induced in Ca2+/calmodulin kinase-inhibited visual cortex neurons.

To elucidate a role of Ca2+/calmodulin-dependent protein kinase II (CaMKII) in induction of long-term potentiation (LTP), KN-62, a selective inhibitor for CaMKII, was injected into layer 2/3 neurons of sliced visual cortex obtained from young rats. Tetanic stimulation (5 Hz, 1 min) applied to the white matter after the KN-62 injection induced long-term depression (LTD) of excitatory postsynaptic potentials (EPSPs) evoked by test stimulation of the white matter in 9 of the 14 cells tested. However, EPSPs evoked by test stimulation of the non-tetanized site were not changed, indicating that the induction of LTD was input-specific. Simultaneously, recorded field potentials which were derived from neurons with intact CaMKII showed LTP. These results suggest that postsynaptic CaMKII plays a role in the induction of LTP/LTD in visual cortex.     

Calcium store-mediated signaling in sustentacular cells of the mouse olfactory epithelium

Sustentacular cells have structural features that allude to functions of secretion, absorption, phagocytosis, maintenance of extracellular ionic gradients, metabolism of noxious chemicals, and regulation of cell turnover. We present data detailing their dynamic activity. We show, using a mouse olfactory epithelium slice model, that sustentacular cells are capable of generating two types of calcium signals: intercellular calcium waves where elevations in intracellular calcium propagate between neighboring cells, and intracellular calcium oscillations consisting of repetitive elevations in intracellular calcium confined to single cells. Sustentacular cells exhibited rapid, robust increases in intracellular calcium in response to G-protein coupled muscarinic and purinergic receptor stimulation. In a subpopulation of sustentacular cells, oscillatory calcium transients were evoked. We pharmacologically characterized the properties of purinergic-evoked increases in intracellular calcium. Calcium transients were elicited by release from intracellular stores and were not dependent on extracellular calcium. BAPTA-AM, a cytosolic calcium chelator, and cyclopiazonic acid, an endoplasmic reticulum Ca(2+)-ATPase inhibitor irreversibly blocked the purinergic-induced calcium transient. Phospholipase C antagonist U73122 inhibited the purinergic-evoked calcium transient. 2-Aminoethoxydiphenyl borate, an inositol-1,4,5-trisphosphate (IP(3)) receptor antagonist, and the ryanodine receptor (RyR) antagonists tetracaine and ryanodine, inhibited the UTP-induced calcium transients. Collectively, these data suggest that activation of the phospholipase C pathway, IP(3)-mediated calcium release, and subsequent calcium-induced-calcium release is involved in ATP-elicited increases in intracellular calcium. Our findings indicate that sustentacular cells are not static support cells, and, like glia in the central nervous system, have complex calcium signaling.     

Axon-glia communication evokes calcium signaling in olfactory ensheathing cells of the developing olfactory bulb

Olfactory ensheathing cells (OECs) accompany receptor axons in the olfactory nerve and promote axonal growth into the central nervous system. The mechanisms underlying the communication between axons and OECs, however, have not been studied in detail yet. We investigated the effect of activity-dependent neuronal transmitter release on Ca(2+) signaling of OECs in acute mouse olfactory bulb slices using confocal Ca(2+) imaging. TTX-sensitive axonal activity upon electrical nerve stimulation triggers a rise in cytosolic Ca(2+) in OECs, which can be mimicked by application of DHPG, an agonist of metabotropic glutamate receptors (mGluRs). Both stimulation- and DHPG-induced Ca(2+) transients in OECs were abolished by depletion of intracellular Ca(2+) stores with cyclopiazonic acid (CPA). The mGluR(1)-specific antagonist CPCCOEt completely inhibited DHPG-evoked Ca(2+) transients, but reduced stimulation-induced Ca(2+) transients only partly, suggesting the involvement of another neurotransmitter. Application of ATP evoked CPA-sensitive Ca(2+) transients in OECs, which were inhibited by the P2Y(1)-specific antagonist MRS2179. Co-application of CPCCOEt and MRS2179 almost completely blocked the stimulation-induced Ca(2+) transients, indicating that they were mediated by mGluR(1) and P2Y(1) receptors. Our results show that OECs are able to respond to olfactory nerve activity with an increase in cytosolic Ca(2+) due to glutamate and ATP release.     

Thapsigargin blocks STP and LTP related calcium enhancements in hippocampal CA1 area

Multiple calcium signaling pathways, including intracellular calcium release that is mediated by inositol triphosphate (IP3) or ryanodine calcium store receptors, seem to be involved in CA1 hippocampal synaptic plasticity. We have addressed the role of dendritic calcium release in short- and long-term potentiation (STP and LTP) using thapsigargin, which depletes intracellular calcium stores. Measuring Fura-2 calcium signals and extracellular field potentials, we have found that thapsigargin did not affect single pre-tetanus calcium transients but reduced tetanically evoked calcium changes. The latter effect prevented the formation of short- and long-lasting calcium enhancements. These results are consistent with the idea that intracellular calcium release is not involved in baseline synaptic transmission but is essential for those forms of synaptic plasticity.     

Long-term depression but not potentiation is induced in Ca(2+)-chelated visual cortex neurons

An entry of Ca2+ into postsynaptic sites may play a role in the induction of long-term potentiation (LTP) of synaptic transmission in the visual cortex. To test this hypothesis, a Ca(2+)-chelator was injected into layer II/III neurons of sliced visual cortex obtained from young rats, and excitatory postsynaptic potentials (EPSPs) of these cells to test stimulation of the white matter were observed before and after tetanic stimulation of the same site. To confirm the effectiveness of the tetanus, field potentials reflecting the activities of many cells were recorded with another extracellular electrode. The chelator injection led to long-term depression (LTD) of EPSPs following tetanic stimuli which simultaneously induced LTP of field potentials derived from unchelated cells in most of the slices tested. This suggests that a low concentration of post-synaptic, free Ca2+, when associated with tetanic inputs, may lead to LTD while a rise of Ca2+ may lead to LTP.     

Localization of inositol 1,4,5-trisphosphate receptors in mouse retinal ganglion cells

Inositol 1,4,5-trisphosphate receptors (IP(3)R) are ligand-gated intracellular Ca(2+)channels that mediate release of Ca(2+) from intracellular stores into the cytosol on activation by second messenger IP(3.). Similarly, IP(3)R mediated changes in cytosolic Ca(2+) concentrations control neuronal functions ranging from synaptic transmission to differentiation and apoptosis. IP(3)R-generated cytosolic Ca(2+) transients also control intracellular Ca(2+) release and subsequent retinal ganglion cell (RGC) physiology and pathophysiology. The distribution of IP(3)R isotypes in primary adult mouse RGC cultures was determined to identify molecular substrates of IP(3)R mediated signaling in these neurons. Immunocytochemical labeling of IP(3)Rs in retinal sections and cultured RGCs was carried out using isoform specific antibodies and was detected with fluorescence microscopy. RGCs were identified by the use of morphologic criteria and RGC-specific immunocytochemical markers, neurofilament 68 kDa, Thy 1.1, and Thy 1.2. RGC morphology and immunoreactivity to neurofilament 68 kDa and Thy 1.1 or Thy 1.2 were identified in both RGC primary cultures and tissue cryosections. RGCs showed localization on intracellular membranes with a differential distribution of IP(3)R isoforms 1, 2, and 3. IP(3)R Types 1 and 3 were detected intracellularly throughout the cell whereas Type 2 was expressed predominantly in soma. Expression of all three IP(3)Rs by RGCs indicates that all IP(3)R types potentially play a role in Ca(2+) homeostasis and Ca(2+) signaling in these cells. Differential localization of IP(3) receptor subtypes in combination with biophysical properties of IP(3)R types may be an important molecular mechanism by which RGCs organize their cytosolic Ca(2+) signals.     

Effect of D-2 amino-5-phosphonopentanoate and nifedipine on postsynaptic calcium changes associated with long-term potentiation in hippocampal CA1 area

The induction of long-term potentiation (LTP) in CA1 hippocampal area requires a rise in intracellular postsynaptic calcium. Two major calcium mechanisms may mediate the transmembrane calcium influxes that contribute to this calcium accumulation: the N-methyl-D-aspartate (NMDA) receptor channels, which are voltage dependent and have large calcium permeability and voltage-dependent calcium channels (VDCCs). We have addressed the relative contribution of these routes of calcium entry before and during LTP expression, in synaptically evoked dendritic calcium transients from a population of CA1 pyramidal neurons. Combining the use of the fluorescent calcium indicator Fura-2 with field potential measurements, we observed that the calcium transients evoked by single stimuli, during the maintenance phase of LTP, were enhanced. These transients were not affected by D-2 amino-5-phosphonopentanoate (D-APV) (50 microM), an antagonist of NMDA receptors but were reduced by approximately one-quarter, in the presence of the L-type VDCCs blocker nifedipine (10 microM). During tetanic stimulation (100 Hz, 1 s) the components triggered by the activation of those two calcium mechanisms had comparable magnitudes representing the sum about half of the intracellular calcium accumulation. Thus, following both single and high frequency stimulation, a substantial fraction of calcium entry may occur through other types of VDCCs or be due to calcium release from intracellular stores.     

Skeletal muscle IP3R1 receptors amplify physiological and pathological synaptic calcium signals

Ca(2+) release from internal stores is critical for mediating both normal and pathological intracellular Ca(2+) signaling. Recent studies suggest that the inositol 1,4,5-triphosphate (IP(3)) receptor mediates Ca(2+) release from internal stores upon cholinergic activation of the neuromuscular junction (NMJ) in both physiological and pathological conditions. Here, we report that the type I IP(3) receptor (IP(3)R(1))-mediated Ca(2+) release plays a crucial role in synaptic gene expression, development, and neuromuscular transmission, as well as mediating degeneration during excessive cholinergic activation. We found that IP(3)R(1)-mediated Ca(2+) release plays a key role in early development of the NMJ, homeostatic regulation of neuromuscular transmission, and synaptic gene expression. Reducing IP(3)R(1)-mediated Ca(2+) release via siRNA knockdown or IP(3)R blockers in C2C12 cells decreased calpain activity and prevented agonist-induced acetylcholine receptor (AChR) cluster dispersal. In fully developed NMJ in adult muscle, IP(3)R(1) knockdown or blockade effectively increased synaptic strength at presynaptic and postsynaptic sites by increasing both quantal release and expression of AChR subunits and other NMJ-specific genes in a pattern resembling muscle denervation. Moreover, in two mouse models of cholinergic overactivity and NMJ Ca(2+) overload, anti-cholinesterase toxicity and the slow-channel myasthenic syndrome (SCS), IP(3)R(1) knockdown eliminated NMJ Ca(2+) overload, pathological activation of calpain and caspase proteases, and markers of DNA damage at subsynaptic nuclei, and improved both neuromuscular transmission and clinical measures of motor function. Thus, blockade or genetic silencing of muscle IP(3)R(1) may be an effective and well tolerated therapeutic strategy in SCS and other conditions of excitotoxicity or Ca(2+) overload.     

Blockade of M2‐like muscarinic receptors enhances long‐term potentiation at corticostriatal synapses

Acetylcholine (ACh) exerts a crucial role in learning and memory. The striatum contains the highest concentration of this transmitter in the brain. This structure expresses two different forms of synaptic plasticity, long-term depression (LTD) and long-term potentiation (LTP), which might contribute to the storage of motor skills and some cognitive processes. We have investigated the role of M2-like muscarinic receptors in striatal LTP by utilizing intracellular recordings in vitro from a rat corticostriatal slice preparation. Methoctramine (250 nm ), an antagonist of M2-like muscarinic receptors, enhanced striatal LTP induced in the absence of external magnesium (Mg²⁺) by high-frequency stimulation (HFS) of corticostriatal fibres. Methoctramine did not affect the amplitude of excitatory postsynaptic potentials (EPSPs) when bath applied either before or after the conditioning tetanus suggesting that a critical increase of ACh concentrations is produced only during HFS. Methoctramine per se failed to enhance the NMDA-mediated EPSPs recorded in the absence of external Mg²⁺ and in the presence of 10 μm CNQX. Methoctramine antagonized the presynaptic inhibitory action of neostigmine, an inhibitor of ACh-esterase, and oxotremorine, an agonist of M2-like muscarinic receptors. These data indicate that the activation of M2-like muscarinic receptors exerts a negative influence on striatal LTP, probably by reducing the release of glutamate from corticostriatal fibres and they suggest a complex modulatory effect of ACh in striatal synaptic plasticity.     

Input-specific induction of long-term depression in Ca(2+)-chelated visual cortex neurons

An input-dependent increase in postsynaptic Ca2+ may play a role in long-term potentiation (LTP) of synaptic transmission while no or subthreshold increase in Ca2+ is associated with long-term depression (LTD) in the developing visual cortex. To see whether LTD is induced only at tetanized synapses, a Ca(2+)-chelator was injected into layer 2/3 neurons in cortical slices from young rats, and excitatory postsynaptic potentials (EPSPs) of these cells, after test stimulation of the white matter and layer 1/2, were observed before and after tetanic stimulation of the former site. The chelator injection led to LTD of EPSPs at tetanized synapses, but no changes were seen at non-tetanized synapses. These results suggest that tetanic inputs induce LTD at tetanized synapses when they are associated with no or subtle increase in postsynaptic Ca2+.     

Rapid modulatory effect of estradiol on acetylcholine-induced Ca2+ signal is mediated through cyclic-GMP cascade in LHRH-releasing GT1-7 cells

Hypothalamic luteinizing hormone-releasing hormone neurons (LHRH) form the final pathway for the central control of reproduction through the release of LHRH into the pituitary-hypothalamic system. We previously found that LHRH-producing GT1-7 cells respond to acetylcholine (ACh) with an increase in intracellular calcium ([Ca2+]i) through activation of muscarinic receptors. This effect is acutely modulated by 17beta-estradiol in a manner compatible with specific membrane binding sites. Because increasing evidence suggests that second messengers are involved in the rapid action of estradiol, the aim of the present study was to identify the pathway underlying estrogen actions on ACh-induced Ca2+ signals. 8-Bromoguanosine 3',5'-cyclic monophosphate (10 microm) and C-type natriuretic peptide (10 microm) mimicked the effect of estradiol. On the contrary, neither dibutyryl cAMP (100 microm), forskolin (100 nm or 10 microm), or sodium nitroprusside (10 microm) induced any modification of [Ca2+]i in response to ACh. The effect of estradiol on calcium transients was totally blocked by two different cGMP-dependent protein kinase (PKG) inhibitors. In addition, phosphorylation of inositol 1,4,5-triphosphate (IP3) receptor was rapidly induced by estradiol but totally blocked when the cells were pretreated with a PKG inhibitor. We conclude that physiological concentrations of estradiol reduce ACh-induced Ca2+ transients via a mechanism involving a membrane-associated guanylate cyclase, which finally induces a PKG-dependent IP3 receptor phosphorylation that modifies calcium release from the endoplasmic reticulum.     

Endoplasmic reticulum dynamics in hippocampal dendritic spines induced by agonists of type I metabotropic glutamate but not by muscarinic acetylcholine receptors

Neurons in the hippocampus exhibit subpopulations of dendritic spines that contain endoplasmic reticulum (ER). ER in spines is important for synaptic activity and its associated Ca(2+) signaling. The dynamic distribution of ER to spines is regulated by diacylglycerol and partly mediated by protein kinase C, metalloproteinases and γ-secretase. In this study, we explored whether pharmacological activation of type I metabotropic glutamate receptors (mGluRs) and muscarinic acetylcholine receptors (mAChRs) known to activate phospholipase C would have any effect on spine ER content. We found that DHPG (100 μM) but not carbachol (10 μM) caused a reduction in the number of spines with ER. We further found that ER Ca(2+) depletion triggered by thapsigargin (200 nM) had no effect on ER localization in spines.