Morphological constraints on calcium dependent glutamate receptor trafficking into individual dendritic spine

Cell Calcium 42 (2007) 41–57

Morphological constraints on calcium dependent glutamate receptor trafficking into individual dendritic spine Eduard Korkotian, Menahem Segal ∗ Department of Neurobiology, The Weizmann Institute, Rehovot 76100, Israel Received 8 October 2006; received in revised form 14 November 2006; accepted 16 November 2006 Available online 21 December 2006

Abstract Glutamate receptor trafficking into dendritic spines is a pivotal step in synaptic plasticity, yet the relevance of plasticity-producing rise of [Ca2+ ]i and of spine morphology to subsequent delivery of glutamate receptors into dendritic spine heads are still not well understood. Following chemical induction of LTP, an increase in eGFP-GluR1 fluorescence in short but not long dendritic spines of cultured hippocampal neurons was found. Repeated flash photolysis of caged calcium, which produced a transient rise of [Ca2+ ]i inside spine heads caused a selective, actin and protein synthesis dependent increase of eGFP-GluR1 in these spines. Strikingly, GluR1 increase was correlated with the ability of a calcium transient generated in the spine head to diffuse into the parent dendrite, and inversely correlated with the length of the spine: short spines were more likely to raise GluR1 than long ones. These observations link, for the first time, calcium transients in dendritic spines with spine morphology and its ability to undergo synaptic plasticity. © 2006 Elsevier Ltd. All rights reserved. Keywords: Dendritic spines; Flash photolysis; Hippocampus; Plasticity; GluR1

1. Introduction The pivotal role of glutamate receptor trafficking into and out of the synapse has been proposed to underlie the variations in postsynaptic strength associated with long term potentiation and depression of synaptic responses [1]. It is assumed that following intense activation of the NMDA receptor in the spine head, AMPA-type glutamate receptors are inserted into this spine head, converting the synapse from a ‘silent’ to an active one. Conversely, long term depression is assumed to involve removal of GluRs from the synapse. The likely track for insertion and removal of glutamate receptors in and out of a synapse which resides on a dendritic spine head is through the spine neck, which has been assumed to constitute a variable physical barrier in the link between the synapse and the parent dendrite. The transport of GluRs into the spine can take place either via intracellular routes or via diffusion along the membrane [2]. In either route, the spine neck is ∗

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a variable physical barrier. Considerable evidence indicates that the spine is a unique compartment, allowing calcium to increase locally without its spread into the parent dendrite [3,4]. The question of whether the spine morphology can regulate the transport of synaptic molecules into the spine head is still not entirely clear. While several molecular mechanisms have been proposed to regulate the trafficking of glutamate receptors [5,6], the relevance of morphological heterogeneity of dendritic spines to the mechanism of glutamate receptor insertion has not been clarified. It has been suggested that this heterogeneity may have functional relevance, in that large spines produce a larger response to photo-released glutamate but that smaller spines can undergo morphological plasticity more readily [7,8] indicating that morphological heterogeneity may have functional relevance. However, other studies in the same preparation of cultured slices [9] did not find such a correlation, and single spine responses to glutamate could undergo long term plasticity without a change in spine dimensions. We have now combined time lapse imaging of glutamate receptors linked to eGFP with local flash photolysis of caged calcium, to study the relations between spine

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morphology and accumulation of GluR1 in spine heads. Our results indicate that local, transient increases of [Ca2+ ]i as well as an intense synaptic activation of NMDA receptors which produces long term synaptic plasticity lead to accumulation of GluR1 clusters only in short and not in long spines.

2. Materials and methods 2.1. Culture preparation Cultures were prepared as detailed elsewhere [10]. Briefly, P1 rat pups were decapitated, their brains removed and placed in a chilled (4 ◦ C), oxygenated Leibovitz L15 medium (Biological Industries, Beit Haemek, Israel) enriched with 0.6% glucose and Gentamicin (Sigma, 20 ␮g/ml). Hippocampal tissue was dissociated and plated on 12 mm glass coverslips at 3–4 × 105 cells per well in a 24 well plate. The plating medium consisting of 5% heat inactivated horse serum (HS), 5% fetal calf serum (FCS), prepared in MEM-Earl salts (Biological Industries), enriched with 0.6% glucose, Gentamycin, and 2 mM glutamax. Cells were left to grow in the incubator at 37 ◦ C, 5% CO2 for 4 days, at which time the medium was changed to 10% HS in enriched MEM, plus a mixture of 5 -fluoro-2-deoxyuridine/uridine (Sigma, 20 and 50 ␮g/ml, respectively). The medium was changed 4 days later to 10% HS in enriched MEM. Cultures were used at 12–20 days in vitro, after they have been exposed to a chronic, 3–5 days blockade of NMDA receptors with APV (50 ␮M). 2.2. Transfection Transfection was routinely conducted with 8–10 DIV cells. A lipofectamine 2000TM (Invitrogen) mix was prepared at 1 ␮l/well with 50 ␮l/well optimemTM (Invitrogen), and incubated for 5 min at room temperature. This was mixed with 1.5 ␮g/well total DNA in 50 ␮l/well optimemTM , and incubated for 15 min at room temperature. The mix was then added to the transfected culture wells, and allowed to incubate for 4–6 h before a change of medium. In most cases, at least several primarily neurons were transfected. In all of the experiments reported below, GluR1-eGFP with CMV promoter was used. In several pilot experiments GluR2-eGFP plasmid was used. The distribution and pattern of the expression of the latter plasmid were similar to those of the GluR1 message, and further studies were conducted only with the GluR1-eGFP plasmid. Co-transfection efficiency for several plasmids using this method is nearly 100%. Experiments were conducted routinely at 3–7 days after transfection. 2.3. Imaging and electrophysiology Cultures were incubated for 1 h at room temperature with the standard recording medium containing TTX, and 2 ␮M Fluo-4AM and 6 ␮M caged EGTA-AM. Alternatively, the caged EGTA was introduced into the cell through the patch micropipette. For the GFP-GluR1 experiments Fluo-4 AM

was not used. Glasses were placed in the recording chamber, controlled by an automated X-Y stage (Luigs and Neumann, Ratingen, Germany). Cells were imaged thereafter on the stage of an upright Zeiss PASCAL confocal microscope. Standard recording medium contained (in mM); NaCl 129, KCl 4, MgCl2 1, CaCl2 2, glucose 10, HEPES 10, TTX 0.0005, and bicuculline, 0.02, pH was adjusted to 7.4 with NaOH, and osmolarity to 320 mOsm with sucrose. In some experiments neurons were recorded with a patch pipette containing (in mM) K-gluconate 140 (or K-gluconate 70 and CsCl 70), NaCl 2, HEPES 10, EGTA 0.2, Na-GTP 0.3, Mg-ATP 2, phosphocreatine 10, pH 7.4 having a resistance of 6–10 M. Signals were amplified with Axopatch 200A (Axon Instruments Inc., Foster City, CA), and were stored on IBM PC. Data were analyzed off-line using P-Clamp software. Images were taken at fast line scan mode for detecting rapid changes in [Ca2+ ]i. High resolution images were taken of the cell morphology using either DsRed transfected neurons, or cells that were loaded through the patch pipette with the calcium-insensitive fluorescent dye Alexa-545. GluR1 imaging was done in standard conditions of same pinhole diameter, PMT values and laser power. The optical system was set at low laser power so as to keep images far from saturation. 2.4. Flash photolysis Flash photolysis of caged molecules was described elsewhere [11]. Briefly, a UV laser (New Wave Inc. air cooled ND:YAG), emitting 355 nm, 4 ns light pulses, was focused through the objective lens (63×, 0.9NA Olympus, water immersion) into a spot of less than 1 ␮m2 . The size of the spot could be easily detected by flash photolysis of caged calcium or fluorescein in the spine head, where a distinct latency of response of the parent dendrite could be recorded, indicating that the photolysis did not spread beyond the spine neck, even for short spines. The UV spot was localized using a parallel red laser light, as detailed elsewhere [11]. UV pulses at 1 Hz could be applied repeatedly without noticeable tissue damage. Caged molecules were prepared in stock solution, aliquoted and stored frozen at −80 ◦ C. Caged glutamate (MNI-caged glutamate, Tocris, MO, USA) prepared from frozen stock solution, was used in the recording medium at a final concentration of 0.5 mM. 2.5. Drugs Latrunculin B (final concentration 5 ␮M, Sigma), Anisomycin (20 ␮M, Fluka), Bapta-AM (50 ␮M, Teflab), were all prepared from frozen stocks. Care was taken not to exceed a DMSO concentration of 0.5% solution. 2.6. Analysis Fluorescence measurements were made by producing a template from the DsRed image of a spine at about its largest

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cross-section area, and measuring the total fluorescence in the green image of the same template using Image-J and Photoshop software. Some measurements were made in a double blind procedure by an independent observer, to assure unbiased observations. In such cases, the independent observer (Y. Pilpel or S. Sapoznik) obtained unmarked images and analyzed them. Each experiment was repeated at least three times in different cultures. Line scan data were analyzed using home-made software written in Matlab. Electrophysiological data were analyzed with PClamp 8 software. Statistical comparisons were made using t-tests or ANOVA, as the case may be, in Matlab, KaleidaGraph and Origin software.

3. Results 3.1. Chronic blockade of NMDA receptors delays the maturation of dendritic spines and their glutamate receptors NMDA receptor activation is believed to play a major role in the maturation of dendritic spines, and in the formation of functional glutamate receptor clusters on them [12,13]. Accordingly, it is proposed that NMDA receptor activation, e.g. following tetanic stimulation which leads to NMDA gated influx of calcium, results in the migration of GluR’s into the synapse, converting it from a ‘silent’ to an active one. We compared spine properties, including size, length and density, as well as the presence of GluR1 clusters on spine heads in control cells and those treated chronically with the NMDA antagonist, APV. The two populations of neurons did not differ in gross or dendritic morphology. Dendritic spine density was slightly higher in control compared to APV-treated cells, but this difference was not significant statistically (n = 6 control cells, 30 fields, and 6 APV-treated cells, 35 fields, Fig. 1a–c). In sharp contrast, the proportion of GluR1-positive dendritic spines was significantly lower in the APV treated cells compared with controls. Whereas the majority (53.2%, n = 6 cells, 92 of 173 spines) of control dendritic spine heads contained a discernable GluR1 cluster, only 26.5% of the spines heads in 6 APV-treated cells, (39 of 147 spines) contained GluR1 clusters (p < 0.01). Interestingly, this difference was expressed primarily in the short (66.4 ± 4.3% of control versus 30.4 ± 4.6% of treated) GluR1-positive spines, while in both control and APV-treated cells only a minority (21.6 ± 6.1% and 17.8 ± 5.7%, respectively) of long spines (>1.5 ␮m) expressed GluR1 clusters even if their spines had clearly identified heads (Fig. 1c). This indicates that long spines are less favored for the insertion of GluR1 clusters in both control and APV treated cells. In addition, regardless of their lengths and exposure to APV, the maximal crosssection area of the heads was significantly larger in spines that contained GluR1 clusters (1.13 ± 0.05 ␮m2 for short GluR1positive spines and 1.61 ± 0.12 ␮m2 for long GluR1-positive spines compared to 1.05 ± 0.06 ␮m2 in short negative and 1.1 ± 1.04 ␮m2 long negative spines) (p < 0.01) (Fig. 1d).

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3.2. Glutamate receptors linked to GFP are functional and stable Before dwelling into the morphological and molecular attributes of GluR1 clusters on dendritic spines, two related questions are pertinent. Are the GFP-linked GluR1 functional, to the extent that their activation by glutamate can produce a distinct physiological response in the recorded cell, and second, is a glutamate receptor cluster, imaged through its link to eGFP, a true representative of native glutamate receptor, or is the GluR1-GFP an artificial entity, which does not reflect the native receptor distribution and function? To address these questions we patch clamped neurons that were co-transfected with eGFP-GluR1 and DsRed, identified dendritic spines that were/were not endowed with fluorescent GluR1 clusters, and recorded somatic responses to flash activation of caged glutamate, applied near the imaged spines or dendritic shafts (Fig. 2). In addition, comparisons were made between cultures that were exposed to APV for 3 days prior to the recording, and control cultures. In order to reduce subjective judgment on the presence of receptor clusters, the net amount of green eGFP fluorescence was measured in 1 ␮m circles around the center of the UV light spot, traveling along the dendrite. For the same neurons, there was a significant correlation between magnitude of eGFP fluorescence, be it spine or shaft, and the current response to glutamate flash photolysis recorded at the soma in patch clamped neurons (Fig. 2a–c). This indicates that both the shaft and spine GluR1 puncta represent viable membrane glutamate receptor clusters. In another series of experiments, there was a significant difference in the magnitude of somatic current responses to glutamate measured during the 2 ms period following its flash photolysis, between spines that expressed GluR1 clusters and those that did not, in the same sample of neurons (−46.2 ± 8.4 pA, n = 29, compared to −13.3 ± 1.9 pA, n = 51, respectively, p < 0.01, Fig. 2d). There was a lower proportion of eGFP clusters in APV treated (10 of 40 spines examined) than in control (19 of 48 spines examined) dendritic spines. When activated by uncaged glutamate, the APV-treated spines produced a significantly smaller somatic current response than the control spines (−15.8 ± 2.14 pA, n = 40, and −34 ± 6 pA, n = 48, respectively, p < 0.01). Short and long spines were then compared to find that the short ones produced larger responses than the long ones (Fig. 2e, n = 36 and 26 spines, respectively). Within the subpopulation of GluR1-punctated spines, the responses were even larger in the control compared to the APV treated cultures (−56.2 ± 12 pA, n = 19 versus −24.8 ± 5.1 pA, n = 10, Fig. 2f). To examine the stability of the GluR1 clusters in control cells, time-lapse images were taken at a rate of one frame per 10 s at high resolution to detect possible local motility of the green fluorescent puncta, as well as their stability over several hours of observation, in quiet conditions. While small ‘morphing’ of the spines could be detected, as reported

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Fig. 1. Chronic blockade of NMDA receptors reduces the spine expression of eGFP-GluR1 clusters. (a and b) Two sample illustrations of 14DIV cells growing in the presence or absence of 50 ␮M APV for 3 days prior to imaging. Left, low power, right three images, higher power images of the cells. Red is the DsRed expression, to outline the cell morphology. Green is the eGFP-GluR1 co-transfected in the same cells. Note the presence of large number of fluorescent GluR1 puncta in the controls, and their conspicuous absence in the APV-treated cell, despite the presence of normal-looking spines in the latter. (c) Summary diagrams of the distribution of short and long spines, and the presence of distinct GluR1 puncta in them, between control and APV treated cells. Note that while the total density of spines is only slightly larger in control than in APV treated cells, the proportion of GluR1 spines is significantly higher in control than in APV treated cells, especially in the short spines. The long ones do not contain much GluR1 puncta in either group. This is summarized in the normalized bar graphs on the right. (d) Head surface areas in control and APV treated cells. In both short and long spines, GluR1 puncta were associated with larger spine heads, although in control spines, the average size of spine heads, regardless of presence of GluR1 was slightly larger than in the APV treated cells. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

earlier [14], this was not accompanied by a significant change in either the spatial location of the GluR1 cluster, or its intensity (Fig. 3b). Also, no long term (up to 2 h) spontaneous changes in location of GluR clusters (Fig. 3a for younger cells and Fig. 3c for older cells; white arrows), nor their delivery into ‘naked’ spines were detected in 4 experiments with 29 analyzed spines (Fig. 3d). These results indicate that GFP-GluR1’s are stable functional receptors, such that when activated, produce large current responses in the recorded neurons. It is thus conceivable that the delivery of GluR1 into dendritic spines following conditioning stimulation will produce a genuine long lasting increase in efficacy of the postsynaptic responses to glutamate. We then commenced with the analysis of the mechanisms governing the delivery of GFP-GluR1 into dendritic spines.

3.3. Transient activation of NMDA receptors causes delivery of glutamate receptors into spine head A transient activation of the synaptic NMDA receptor in APV-treated cultures has been reported to cause a long lasting change in network activity as well as the formation of new spines, and the pruning of some existing ones [10,15,16]. We predicted that following this activation, there will be an increase in glutamate receptors in ‘silent’ synapses at the spine heads of the cultured neurons [12]. The possibility that synaptic activation of NMDA receptors leads to insertion of GluR1 clusters was therefore examined. Cultures were first imaged in the presence of 50 ␮M APV, and then perfused with a conditioning medium containing 3 mM CaCl, 100 ␮M glycine, 0 mM Mg, 0 ␮M APV, for 5 min, followed by a switch back to the standard recording medium, containing

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Fig. 2. Spines containing GluR1 puncta produce larger response to flash-photolysis of caged glutamate than ‘naked’ spines. (a) A dendrite trasfected with DsRed for visualization of morphology and with GFP-GluR1 was patch clamped at the soma, some 80 ␮m away from the image. Flash photolysis was made within the spots marked with O, along the dendrite, about the same distance from spines or the dendrite. The records marked with the arrowheads are illustrated in (b) which are averaged, overlapped current responses to flash activation of glutamate in these locations. (c) The correlation between GFP fluorescence and the slope of current response measured over 2 ms following the flash. (d–f) Group averaged responses to glutamate, comparing spine with and without GluR1 puncta (d), short and long spines (e, below and above 1.5 ␮m in length), in control and APV treated cells (f). All statistical comparisons were made using t tests, significance level = 0.01, n = number of spines, included within the bars.

50 ␮M APV and 1 mM Mg. There was a small increase in the total number of dendritic spines within 30–60 min after exposure to the conditioning medium, as reported before [10]. However, there was a highly significant increase in the number of dendritic spine heads that contained a new GluR1 cluster (from 0.67 to 3.48 spines/30 ␮m, p < 0.0001, 8 experiments, 42 fields, 267 spines analyzed before and 296 spines after the exposure to the conditioning medium) (Fig. 4). Strikingly, this increase in GluR1 fluorescence was restricted to short spines: Spines in this particular experiment were classified into those that did not change fluorescence (n = 26 spines), those where a moderate increase in fluorescence was detected (n = 36) and those where a marked increase in fluorescence was seen (n = 32). The mean length of the spine neck in the three groups was 0.72 ± 0.14, 0.46 ± 0.12 and 0.25 ± 0.08 ␮m, respectively (p < 0.003 for a t-test comparing group 1 and 3). Also, there was a slight but non-significant shrinkage of the spine neck length in the entire population of the monitored spines. When stubby spines that were initially short excluded from the analysis, shrinkage of the spine neck length became statistically significant (Fig. 4f2 and f3). In most cases examined, the rise in GFP-GluR fluorescence was gradual. However, there were some cases where fluorescent clusters were found to move along the spine neck

into the spine heads (Supplementary movie). Therefore, the possibility that the receptor is moving in a cluster cannot be ruled out. In any case, the analysis of fluorescence accumulation in the spine head was made by measuring all the change in the spine head, and this was fairly homogenous and gradual. Strikingly, the cross-section area of the spine heads having new GluR1 clusters increased significantly (by about 40%, p < 0.0001) but in spines that did not contain GluR1 fluorescence, there was no change in spine area (2% decrease, p = 0.8) (Fig. 4c and f). It should be noted that the crosssection area of the spine head was measured in the red channel, and there was no leak between the red and green channels, and thus it is unlikely that the added green fluorescence contributed to an over-estimation of the spine size. Importantly, the increase in spine head size was correlated with a reduction in neck length, bringing the spine head closer to the parent dendrite (Fig. 4g) in GluR1+ but not in GluR1-negative spines. This observation should have important implications with respect to the bidirectional synapse/dendrite communication (see below). Lastly, when stubby spines (having no spine neck) were excluded from the analysis, the net change in spine cross-section area was significantly larger for the GluR1-positive than for the GluR1-negative spines (Fig. 4h).

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Fig. 3. Stability of GluR1 clusters in dendritic spines. (a) Images of a double transfected DsRed/GluR1 were taken 1 h apart. Bottom, subtracted image, to show disparity between the two images. In a young cell (10DIV, 3 days after transfection), spines with GluR1 puncta are extremely stable, as are dendritic puncta. By comparison, spines that do not contain GluR1 are unstable. GluR1 puncta are highly stable. (b) Fast imaging of a section of the dendrite as in a, to show that even when the spine changes its shape, the GluR1 cluster is very stable. This observation was quantified below. (c) A more mature neuron, where most of its spines contain GluR1 clusters that are stable over 1 h of observation. For a and c: logical ‘difference’ is performed and contrast is enhanced. White arrows show highly stable puncta whereas the blue arrowheads mark puncta which slightly shifted during 1 h of detection. (d) 29 GluR1 puncta fluorescence averaged 1 h apart. f in d and d indicates arbitrary fluorescence units.

3.4. Variable calcium communication between spine head and parent dendrite The rapid delivery of GluR1 clusters into the spine head, and the preferential effect on short compared to long spines brings about two related issues: what is the signal for the delivery of GluR1 clusters into spine heads, and why is it different between short and long spines. Previous studies by others and us have indicated that short spines are more linked to the parent dendrite, in that a transient rise of intracellular calcium concentration in the spine head will spread to the parent dendrite only when the spine neck is short [17–19]. Otherwise the rise of [Ca2+ ]i will be restricted to the spine head. This is illustrated most dramatically when [Ca2+ ]i was raised by flash photolysis of caged calcium [17]: A single flash could cause an immediate, less than 1 ms delay, and rapidly decaying (t = 10–15 ms) rise of [Ca2+ ]i. This response was restricted to the green fluorescent line, and not to a red line that was used to identify and align the spine/dendrite pair (Supplementary Fig. 1). The response to flash activa-

tion of caged calcium was persistent over time, and was only changed in magnitude by speeding up the rate of photolysis (Supplementary Fig. 2). When the spine had a long neck, this change in [Ca2+ ]i was restricted to the spine head and not detected in the parent dendrite (Fig. 5b and c). However, following 30–60 flashes of calcium, the spine neck length could shrink (Fig. 5g2 (p < 0.001)), and consequently, the calcium transients were now detected in the parent dendrite (Fig. 5c and g1). For the same group of four spines, analysis of spine length and head size seemed to co-vary (Fig. 5e) such that only the spine that was flashed underwent significant length changes, whereas the other ones were not affected. Note that when the spine neck was long, the calcium transient decayed significantly slower than when it was short, indicating that the leak of calcium to the parent dendrite facilitated the decay of excess [Ca2+ ]i from the spine head (Fig. 5b2, c2 and d for a particular example and averaged data in g: 19 experiments, n = 19 spines). In general, there was a strong correlation between spine neck length and responses of the parent dendrite to flash activation of calcium in the spine

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Fig. 4. Transient activation of synaptic NMDA receptors causes delivery of eGFP-GluR1 clusters into spine heads. (a–d) Sample illustration of a APV-treated cell before (1) and 60 min (2) after a 5 min exposure to a conditioning medium containing 0 mM Mg, 3 mM Ca, 200 ␮M glycine and no APV. Two clusters of spines are shown at medium (b) and high (c and d) resolution. At the high resolution spines are shown in composite red/green combination (c1, c2, d1 and d2) and in green channel alone with red traces (c3, c4, d3 and d4). Note that two short spines (c) that did not contain fluorescent puncta before exposure to the conditioning medium, acquired them afterwards. A long spine unlike a short one (d) did not acquire the green fluorescent puncta after exposure to the medium, even becoming considerably shorter over time. (e) The proportion of spines containing GluR1 puncta increased significantly following exposure to the conditioning medium (e, right bars), although the total spine density was not changed significantly over 60 min of observation following a 5 min exposure to conditioning medium (e, left bars). (f) Changes in the morphological properties of spines following exposure to the conditioning medium. The population of spines is divided into GluR1 positive (n = 68) and GluR1-negative (n = 48) spines. Spine head volume, expressed as cross-section area, is increased following conditioning, only in the spines that now contained GluR1 puncta. The other spines did not change in size (f1). The effect of GluR1 on spine head area is clearly seen when the net change in spine area is calculated and stubby spines (not changing much) are excluded (h). Spine neck length went down in both groups, especially of the long ones (f2), becoming more obvious when stubby spines were not included (f3). Correlation between changes in morphological parameters of spines getting GluR1 is shown on g, left. Note lack of such a correlation in GluR1-negative spines. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

head (Fig. 5g1). Several control experiments were conducted to exclude the possibility that the flash induced photobleaching and photodamage (Supplementary Figs. 1 and 2). We found that UV flash-induced photobleaching may slightly affect both green and red channels but a careful “green by red” bleaching correction performed for a typical short and long spines did not affect the main results (Supplementary Fig. 1a and b). In order to verify that the changes in neck length and spine head area are not the result of damage due to excessive uncaging, we performed an additional experiment using the same spine/dendrite pair as shown in Fig. 5a–c (Supplementary Fig. 2a and b). The figure shows stability of

the repetitive responses to photolysis of caged calcium at a rate of a flash every 3 s (Supplementary Fig. 2a, upper panel and b, left). When the interval between pulses was reduced to 1 s, responses gradually decreased in the spine head and almost disappeared in the dendrite but a complete recovery could be seen in 30 s (Supplementary Fig. 2a, lower panel, b, right part, c and d) without affecting the half decay time (e). In addition, 3 control experiments without caged compound loaded into cells showed no calcium responses and no spine length or shape change following UV flashes (data not shown). In another set of 3 control experiments (n = 10 spines) cells were loaded with caged EGTA together with

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BAPTA-AM (20 ␮M) which completely prevented changes in spine morphology (see Fig. 7b and c). The contribution of passive diffusion to the difference between short and long spines was examined using caged fluorescein (6 experiments, 19 spines), as detailed before

[17]. While the initial response in both spine types was identical, and the decay of the fluorescence was much slower than that of photoreleased calcium, there was a marked contrast between the parent dendrites of the short (n = 10) and long spines (n = 9, each trace is an average of 14 sequen-

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tial trials): in both cases there was a significant delay in responses of the parent dendrites compared to the activated spines (Fig. 5f), indicating that it is not likely that the flash activated directly the parent dendrites, but the response of the parent dendrites of the long spines was far slower and smaller than that of the short ones. Finally, there were no interactions among adjacent spines, and each responded to the flashes independently (Fig. 5h). These results also illustrate the spatial resolution of the flash photolysis, which in our case is restricted to a sphere of less than 2 ␮m in diameter (see also [11]). 3.5. Local release of caged calcium facilitates Insertion of glutamate receptors into spine head If indeed the rise of intracellular calcium concentration in the spine head is a sufficient stimulus to trigger delivery of GluR1 clusters into the spine head, one can predict that a local release of calcium by flash photolysis should also produce this response. Indeed, repeated flash photolysis of calcium caused a gradual increase in GFP-GluR1 fluorescence in the spine heads of APV treated cultures, over a period of 10–45 min (Fig. 6). The increase in GluR1 fluorescence was gradual, and was sometimes preceded by a similar rise in fluorescence at the spine neck and the adjacent dendrite (Fig. 6). Once a fluorescent cluster accumulated in the spine head, it stayed there for as long as the spine was observed (up to 2 h after initial observation). Several control experiments were conducted to verify that the rise in GluR1 fluorescence is a specific response. In one such series of experiments, UV laser pulses were applied under identical conditions onto dendritic spines of cells that

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did not contain caged EGTA, and in these spines no new GFP fluorescence was detected (2 experiments, n = 6, data not shown). In another series of experiments, adjacent spines to the ones that were activated were imaged, and no GluR1 clusters developed in these spines spontaneously (Fig. 6a and b, white arrows) (4 experiments, n = 12). Additionally, when the cell was loaded with BAPTA intracellularly (20 ␮M, 3 cells, 10 spines, Fig. 7a2), there was no subsequent rise in GluR1 fluorescence. These experiments indicate that the increase in GluR1 fluorescence is a genuine response to the photolysis of calcium in the spine head and not a spontaneous event or a non-specific one, caused by flash illumination of the spine head. Interestingly, the accumulation of GluR1 clusters in dendritic spine heads was accompanied by their gradual expansion; spines that accumulated GluR1 clusters (26 experiments, n = 35 spines) gradually increased their volume (measured as maximal 2D cross-section space) (from 1.01 ± 0.07 ␮m2 to 1.24 ± 0.07 ␮m2 ) over a period of 30 min after a series of flashes. No such changes were observed in 34 spines without GluR1 accumulation (28 experiments, Fig. 8b and d). Net change of spine head volume was 0.22 ± 0.05 and −0.06 ± 0.06 ␮m2 , respectively (p = 0.0014). Finally, the accumulation of GluR1 in spine heads following flash photolysis of calcium in the spine head was accompanied by a rise of GluR1 fluorescence at the dendrite base of the affected spine, but not some 10–30 ␮m away from the affected spine (n = 30 randomly selected spines) (Fig. 7f). This indicates that the signal from the spine head affected the immediate vicinity of the spine, to cause an enhanced synthesis of GluR1 protein, before its delivery into the spine head (also, see below).

Fig. 5. Spine neck regulates calcium communication between spine head and parent dendrite. The cell, initially transfected with DsRed for imaging morphology, was loaded with Fluo-4 as calcium indicator, and NB-EGTA, both in cell permeant, AM form. a1 a low power image of a sample neuron with a rectangle zoomed in a2. Four spines color coded from blue to pink were imaged successively across 1 h of observation. A line was scanned between the spine head and the parent dendrite through the spine neck. The UV flash was aimed at the spine head, indicated by a bright spot on the red-marked spine in a2. b1 and c1 are two images of the same set of spines, taken 30 min apart. Note that the neck of the ‘red’ spine shrunk in length between b1 and c1. This was accompanied by an apparent increase in spine neck diameter. b2 and c2 are two sample line scans of the same spine at the corresponding time as in b1 and c1. Note that in b2, the rise in [Ca2+ ]i is restricted to the spine head, whereas in c2 the [Ca2+ ]i rise spread into the parent dendrite, to the left of the spine. b3 and c3 line scan analysis of the transient rises of [Ca2+ ]i, averaging 14 flashes each. In both cases, the blue trace is the summary of the [Ca2+ ]i changes in the spine head, and the red trace is of the parent dendrite. Note that in b3 there is no change in the parent dendrite [Ca2+ ]i level, whereas in c3 there is a small rise in [Ca2+ ]i in the parent dendrite following a larger, shorter latency and faster rise in the spine head. The two sets of experiments are normalized and plotted together in d for comparison. Note that b3 and c3 graphs are not normalized but show responses of about the same amplitude indicating similar concentrations of NP-EGTA in the same spine/dendrite pair (as in a1 and a2) at its longer (b) and shorter (c) states (averaged responses to 14 repetitive flash stimulations for every time point at 0 (b3) and 30 min (c3)). (e) Changes in morphological properties of the spines as a function of time, and successive flashes of UV light on one of the studied spines (the red one, shown also in b and c. Note that 5 (5p) and 25 (25p) flashes were not effective. Following first 60 flashes (60p) and then 66 flashes (66p) spine neck shrunk (e1) and spine head surface area increase (e2). There was a strong correlation between the two parameters (r = 0.98) unlike the other three spines sampled, which did not change in any systematic manner. (f) Caged fluorescein (Molecular Probes) was used to indicate difference in diffusion of a ‘neutral’ compound, different from the ‘active’ calcium into the dendrite of longer (doted lines) and shorter (solid lines) spines after uncaging. (g) Grouped data on calcium transients (g1) and changes in spine head and neck (g2) dimensions following flash photolysis of caged calcium in the spine heads. Using standard photolysis conditions, spine measurements were taken at 30 min after the onset of imaging. While the spine head area did not change significantly following 30 min of exposure to successive flashes of calcium, the spine neck shrunk significantly by about 1/3 of their original values. Despite the lack of a systematic change in spine head surface, there was a significant correlation between changes in spine head and neck (g3). For g1–g3, n = 19 for same spine/dendrite pairs measured before and after standard flashing protocol. (h) Line scans through the heads of three adjacent spines illustrate the localized responses to the flash. When the flash was aimed at the top spine, middle or lower spine, (red arrow), no responses were recorded in the adjacent ones (left). These experiments indicate that the spatial resolution of the flash photolysis is smaller than 2 ␮m, half the distance between adjacent spines. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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Fig. 6. Successive imaging of the same dendritic branch to show selective effects of flash photolysis of caged calcium on accumulation of GluR1 fluorescence in the affected spines. (a) cell was cotransfected with DsRed and eGFP-GluR1, and exposed to APV for 3 days and to NB-EGTA for 1.5 h. Images were taken over 100 min before and after application of flashes on the indicated spines (blue spots and blue arrows). Below are outlines of these 3 spines before and after the series of flashes. The spine on the left was flashed, it shrunk and a eGFP fluorescent cluster was added to it. In the middle, a spine remained small and stubby, and it received a fluorescent cluster (green arrows). The spine on the right has not been exposed to UV, and remained of the same dimensions over 45 min of observation. Several spines on the same dendrite were not activated by the flash, and remained without GluR1 puncta, and in fact, did not change their dimensions (for example, spine marked with white arrow). (b) Illustrates another dendritic segment, imaged over 60 min, with two spines, both receiving 2 × 25 flashes. The top one shrunk and was loaded with GluR1 fluorescence, and the bottom one did not change much and was not loading with the GluR1 fluorescence. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

3.6. Changes in native GluR1 following a rise of [Ca] in spine head One important control experiment involves the demonstration that the changes in GluR1 puncta observed in the current study, are not a unique result of the use of exogenous fluorescent GluR1. Will the native GluR1 also undergo upregulation following a rise of [Ca2+ ] in the spine head? To address this question we transfected neurons with DsRed without eGFPGluR, incubated them with caged calcium, imaged them, and applied the routine sequence of laser flashes on a random population of dendritic spines in these neurons. Following the experiment, cultures were fixed and immunostained for GluR1, and the transfected neurons/spines were identified in the fixed tissue and the fluorescence of their native immunostained GluR1 measured (Fig. 9). These experiments were conducted separately for control (Fig. 9c) and APV treated (Fig. 9a and b) cultures. As expected, the APV treated cells had lower native GluR1 fluorescence compared to the control, untreated cells (Fig. 9d, n = 234 APV treated spines, 6 experi-

ments, and 123 control spines in 4 experiments, p < 0.008). In the flash-activated spines, both for the controls and the APV treated, there was a significantly higher fluorescence than in the non-activated, quiet spines (n = 31 and 19 for APV treated and non-treated cells, respectively). p < 0.0001 for the APV treated flash activated, compared to their respective controls in the same cells, and p < 0.005 for the photoactivated, control spines compared to non-activated controls in the same cells. Altogether, there was a significantly higher fluorescence in the activated spines compared to their quiet controls in the same cells (Fig. 9d). This indicates that the eGFP-GluR1 accumulation in activated spine heads is by no means a unique artifact of the eGFP-GluR construct. 3.7. Molecular mechanisms underlying calcium induced GluR1 delivery into spine heads Since actin polymerization has been shown to affect spine shape, motility and function [20], we examined if it may also affect calcium mediated trafficking of GluR1 into dendritic

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Fig. 7. Flash photolysis of caged calcium caused an increase in GluR1 fluorescence, spine head expansion and neck shrinkage. Grouped data comprising 68 dendritic spines (54 experiments) are shown. (a1) Following exposure to 60 flashes, GluR1 fluorescence goes up gradually to reach plateau at about 30–40 min, in a group of 24 short spines. A much smaller response amplitude was seen in medium sized spines (n = 25), whereas long ones (n = 19) and a mixed group of short and medium sized control spines, loaded with BAPTA (20 ␮M, n = 10), only slightly changed across 30–80 min. The insert on top right indicates the case where flash photolysis spot was directed at the spine head. (a2) Control experiments demonstrating the lack of GluR1 accumulation in spine heads of cells pretreated with BAPTA or anisomycin (white and black dots, respectively, 20 ␮M for anisomycin, 3 experiments, n = 13 spines) before the NP-EGTA photolysis. (b) For the same groups, the change in spine head area across the same time scale is indicated. Only the heads of the short spines were changed over time. This change was rather rapid, and preceded the change in fluorescence. (c) changes in spine length. Interestingly, only the middle sized spine shrunk over time, whereas the other groups did not change much over time. (d) changes in spine parameters in two groups of shorter (n = 16) and longer (n = 17) spines (9 experiments), where flashes were applied on dendrites at the base of the spines. Fluorescence increase was obvious for both the short and the long spines. The two groups demonstrated the same time course of fluorescence change. The insert on top right indicates the case where flash photolysis spot was directed on the dendrite at the spine base. (e) UV flash photolysis was directed at a dendritic location (see inserts), where no adjacent spine was seen. Surprisingly there was a clear appearance of a new fluorescent cluster at the dendritic location, where the flash was applied (3 experiments, n = 15). (f) In a random subset of 30 spines, the GluR1 fluorescence intensity was measured in the parent dendrite, at the base of the spine (left) in the dendrite away from the spine base (middle) and in the spine heads (right) before and after a series of flashes aimed at the spine head. A clear and significant increase in GluR1 fluorescence was seen in the dendrite at the base of the spine, but not 10–30 ␮m away from the exposed spines. Obviously, a larger increase in GluR1 fluorescence was seen in the spine head, as seen before.

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Fig. 8. The morphological changes in spines were restricted to those that underwent an increase in GluR1 fluorescence, and not to those that remained unchanged. (a) Length remained unchanged for those spines that did not add green fluorescence to their heads. As indicated in Fig. 9, for the same data, the unresponsive spines were initially the long ones. (b) Averaged spine head size was significantly increased in GluR1 positive group (right dark bars). Heads in GluR1-negative group remained unchanged (left grey bars). (c and d) Correlations between spine length and fluorescence at 30 min after exposure, and between head size and length at the same time.

spine heads. Double transfected cells preloaded with caged calcium were incubated with latrunculin B (5 ␮M) before exposing individual spines to the flashes. Latrunculin blocked spontaneous motility of the spines (n > 100) (data not shown), and in its presence, the sequence of flashes did not cause the trafficking of GluR puncta into spine heads (8 experiments, n = 22) (Fig. 10a–c). Incidentally, changes in spine morphology that can be seen following a prolonged (hours) exposure to latrunculin, are not likely to contribute to the observed suppression of spine GluR1 clustering, which take place already during the first 30 min after drug application. (Fig. 10c1 and d3). Interestingly, in a subset of spines with existing GluR clusters, the presence of latrunculin caused a reduction in eGFP fluorescence in dendritic spines (2 experiments, n = 18) but not in the shaft (n = 14) (Fig. 10d1 and d2, and d3 dark and light blue), indicating that F-actin is instrumental in the on-going maintenance of GluR’s in dendritic spines. 3.7.1. Protein synthesis and GluR1 trafficking One important issue concerns the origin of the accumulated GluR1s in the spine head. Is the novel receptor cluster formed by its delivery from extra synaptic to synaptic sites or does it involve de-novo synthesis of proteins. Local dendritic protein synthesis has been known for some time [22].

In our experiments, the possibility that the accumulation of GluR1 in spine heads requires protein synthesis has not been examined before. We exposed cultures to the protein synthesis inhibitor anisomycin (20 ␮M) for 1 h before commencing the imaging experiments. In presence of anisomycin (3 experiments, n = 13 spines) there was no enhancement of spine GluR1 even after an extensive induction protocol (Fig. 7a2, thin line with black circles). In fact, there was not even a detectable reduction in GluR1 over 2 h of imaging in presence of anisomycin (3 experiments, data not shown). This experiment indicates that protein synthesis is required for the formation of new GluR1 clusters in the spine head. Anisomycin can affect the synthesis of other proteins which may be needed for the delivery/clustering of GluR1 at the synapse, not necessarily of GluR1 by itself. At any rate, since we have observed that the formation of a GluR1 clusters is not associated with a reduction in dendritic GluR fluorescence, it is likely that the new protein comes from its de-novo synthesis. 3.8. Spine neck length regulates insertion of glutamate receptors into the spine head The role of spine neck length in the communication between the spine head and its parent dendrite has been

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Fig. 9. The density of native GluR1 is higher following flash photolysis of calcium in spines than in control spines on the same dendrites. (a and b) Images of a live DsRed transfected neuron (a1 and b1) and following its fixation and immunostaining for GluR1 (a2 and b2). The images of the morphology (red) and the GluR1 immunoreactivity (green) are merged in a3 and b3. b is a zoomed section of a, marked with a rectangle. Arrows in b1 indicate the spines where flash photolysis of caged calcium was applied in the standard manner. These sites were identified in the fixed tissue, and the intensity of GluR1 immunofluorescence measured in them. c is another cell, live (c1) with 5 enlarged images of treated spines (blue arrows) and following fixation and immunostaining for GluR1 (c2) with same 5 spines shown in inserts. (d) Summed data for control, and APV pretreated cultures. The difference between the treated and quiet spines in both groups is highly significant. As seen before for the live GFP-GluR1 transfected neurons, the presence of APV reduced the intensity of native GluR1 immunostaining. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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studied extensively before [8,17–19]. An open question in these studies concerns the functional relevance of the linkage between spine head and the parent dendrite, i.e., is the ‘leak’ of excess calcium from the short spine into the parent

dendrite signaling anything that is not signaled in the long spine? We compared the accumulation of GluR1 in short and long spines, following flash activation of caged calcium. In 5 of 19 (26%) long spines (>2.5 ␮m) and in 12 of 25 (48%)

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of middle-size spines (<2.5 ␮m and >1.5 ␮m) there was a change in GluR1 fluorescence. By comparison, in 17 of 23 (75%) short spines (<1.5 ␮m) there was an increase in GluR1 fluorescence (Fig. 7a1). Strikingly, it is not the length of the spine neck in and of itself that prevents the accumulation of GluR1 clusters in the spine head. In 9 control experiments, the calcium photolysis was focused on the parent dendrite, at the base of a ‘naked’ spine. In 9 of 16 short and 11 of 17 long spines examined, irrespective of the initial length of the spine neck, the photolysis of calcium in the parent dendrite caused a gradual increase of GluR1 fluorescence in the spine heads (Fig. 7d, 2.6 ± 0.4 ␮m length for negative versus 2.5 ± 0.3 ␮m for positive groups). This indicates that the rise of calcium in the parent dendrite is likely to trigger the delivery of GluR1 into the spine heads regardless of the spine neck length. It is therefore concluded that the reason that long spines do not accumulate GluR1 clusters when calcium is released in them, is because in these cases the calcium signal does not reach the parent dendrite. As indicated before, spine length can vary spontaneously, or in response to a transient rise in [Ca2+ ]i [17], such that shrinkage of spine neck length can increase the coupling with the parent dendrite. Indeed, in 16 cases examined here, the long or middle-size spines did not accumulate GluR1 in response to a transient rise in [Ca2+ ]i. However, once they shrunk, GluR1 fluorescence began to accumulate in them (Figs. 6a and b, 7c, and 8a and c). The mean shrinkage in this group amounted to −0.46 ± 0.13 ␮m while the rest of long and middle-size spines length did not change (0.05 ± 0.13 ␮m). The difference in length change between the two groups was significant (p = 0.014). Overall, there was a negative correlation between spine length and the amount of GluR1 fluorescence (Fig. 8c) such that the shrinkage could be a good predictor for the inclusion of GluR1 in the spine head. Furthermore, there was a significant negative correlation between the change in spine head area and the change in spine length (Fig. 8d) (compare to Fig. 4g and Fig. 5e1–2 and g3).

4. Discussion The present results propose a link between the extensive knowledge of the synaptic activation of calcium transients in dendritic spines, that are instrumental in the establishment of long term potentiation of synaptic responses, and

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the regulation of glutamate receptor insertion into dendritic spines, which is proposed to underlie long term potentiation [1]. These results also address the long standing issue of the role of spine morphology in its ability to undergo plastic changes. Apparently, transient, local rises of intracellular calcium in dendritic spine heads were sufficient to cause delivery of eGFP-GluR1 into the spine heads. This delivery took place primarily in short spines, and was accompanied by swelling of the spine heads. Long spines could also load GluR1 clusters, but only if calcium was raised at the base of the spine, or after their shrinkage, following extensive flash photolysis of caged calcium in their heads, as seen before [17]. On the background of NMDA deprivation, which reduces the number of GluR1 in spine heads, a massive activation of the NMDA receptors caused a striking rise in the density of spines endowed with GluR1, to the extent that nearly 40–50% of the spines are now “unsilenced”. This indicates that the method of production of a ‘chemical’ LTP is highly effective. In fact, in a parallel study under the same conditions [16] we found a 60% increase in synaptic current evoked between random pairs of neurons, indicating that many of the synapses that had been silent, became active following this conditioning protocol. The time course of the morphological effect is rather slow, compared with the fast change in synaptic strength. This indicates that other mechanisms, e.g. phosphorylation of membrane receptors, underlie the immediate potentiation, which is then followed by an intermediate process involving the boosting of GluR1 in synapses, followed by an even slower process of formation of new spines. The role of spine morphology in its response to afferent input has been an enigma for some time; while spines vary in shape, size and length, the functional significance of these variations has not been analyzed until recently. Spine length has now been suggested to scale the communication between the synapse at the spine head and the parent dendrite [8,21,23,24,27]. Theoretical considerations suggest that this length may not be relevant to the electrical coupling between the synapse and the dendritic shaft, yet it has been shown to play an important role in calcium communication [4,19]. More recently, the size of the spine head was correlated with the magnitude of the cellular responses to caged glutamate [25]. Consequently, it has been suggested that small or thin spines are likely to undergo plastic changes, i.e., inflate, whereas large spines are likely to be mature and stable [25]. While our results concur with some of these sug-

Fig. 10. GluR1 trafficking into spines is dependent on the integrity of polymerized actin. a and b the same dendrite, imaged before (a) and after (b) exposure to latrunculin B. Initially, several spines were exposed to flash-photolysis of caged calcium, and 1 h later, they contained fluorescence GluR1. Subsequently, the culture was exposed to latrunculin B, and other spines were flash photolysed, and this time, no GluR1 cluster invaded the activated spines. c1 left: a sequence of raw images in control (top, black circle) and after latrunculin application (middle and low panels, blue circles) showing the same spines as in a and b at higher resolution, indicating little morphological changes in spines after latrunculin whereas GluR clustering is clearly blocked. Time points are indicated on the figure. c1 right: Summary of a single experiment shown in a, b and c1. c2: Summary of all experiments conducted with 16 spines, pretreated with latrunculin, showing lack of change in GluR1 fluorescence. Net fluorescence change (relative to −5 time-point) is shown in 2 bars of time 0 and 60 after exposure to the flash. (d) Latrunculin can reduce the intensity of existing GluR1 puncta without flash photolysis of caged calcium (d1 and d2). (d3) Intensity is reduced in 18 spines (d1–d3, light blue). The shaft GluR1 cluster did not change (d3) (d1–d3, dark blue). Note changes in spine morphology similar to those shown in b and c2 right and caused by latrunculin treatment. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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gestions; they are different in several respects: first, we do not observe rapid increases or decreases in volume of spine heads following exposure to the conditioning stimulation, be it the conditioning medium or the flash activation of caged calcium. Rather, the increase in spine volume grows slowly over a period of about 1/2 h. In this respect, our results are similar to those of Zhou et al. [26], and Otmakhov et al. [27] who find only small and slow changes in spine volume after induction of chemical LTP, which is a similar procedure to the one we used. Parenthetically, Bagal et al. [9] who used a pairing potentiation protocol, could not detect any change in spine volume in association with an immediate increase in reactivity to caged glutamate. Second, the spine head volume change was not restricted to the small spines, but to any spine that absorbed new fluorescent GluR1. In fact, the short and large spine heads are more likely to absorb new GluR1 puncta than the thin and long spines. Lastly, we find that the change in neck length is independent of the change in spine head volume; although in some cases an increase in volume was also reflected in shrinkage of the spine neck. The leak of calcium ions from the spine head, where they have been uncaged, into the dendrite is not large, and certainly not long lasting, but is likely to be responsible for the initiation of delivery of glutamate receptors into the spine head. First, there is a strong correlation, even in the same dendritic spines, between the calcium response in the parent dendrite and the entry of GluR1 into the spine head. While our results strongly suggest that a transient rise in [Ca2+ ]i is sufficient for the delivery of GluR1 into the synapse, it is highly likely that a number of other molecules that are dependent on changes in [Ca2+ ]i are involved in this process, and that the leak of calcium from the spine neck into the parent dendrite serves to boost such a process. On the other hand, it is unlikely that the changes in calcium are unrelated to the change in GluR1 and that other ions/molecules are responsible for this GluR1 delivery into the spines, since this entire process is blocked when calcium changes are chelated by BAPTA, Furthermore, our data indicate that the dendritic calcium signal is sufficient to induce the GluR1 delivery: flash photolysis of calcium in the parent dendrite at the base of the spine also caused delivery of GluRs into the spine head, regardless of the length of spine, indicating that a calcium transient in the parent dendrite triggers the delivery of GluR1 into the spine head. These results also indicate that it is the calcium change in the parent dendrite, and not some obstacles in the routing of GluR1 to the spine head, that determine the ability of GluR1 to accumulate in the spine head. This contrasts with a recent suggestion that spine neck is a barrier to the delivery of GluR1 into the spine head [24]. At any rate, the calcium dependent delivery is likely to use other adaptor and transduction proteins and there are probably other signaling molecules along the way that may interact with the released calcium, but it is obvious that the rise of calcium initiates this sequence of events. One related issue concerns the mode of insertion of the fluorescent GluR1 into the synapse. In most of our experi-

ments, time lapse imaging indicates that fluorescence level went up gradually in the affected spines, meaning that it is likely that newly synthesized GluR1 molecules accumulated in the spine head, and were not transported into it in a package. On the other hand, in some cases a distinct cluster was seen to travel along the spine neck and into the spine head, indicating that packages of new GluR1 can also travel together into affected dendritic spines. In no case did we notice a lateral movement of fluorescent particles along the membrane and into the spine head, as suggested elsewhere [28]. Thus it is unlikely that non-synaptic receptors contributed in our case to the insertion of synaptic receptors into the spine heads. The relations between spine size and responses recorded at the soma to glutamate application at the spine is complex indeed. On one hand, it has been suggested that spine neck cannot be a considerable barrier to the transfer of current to the dendritic shaft. On the other hand, we find that long spines are likely to produce a smaller synaptic current. In essence there are no contradictions between these two assertions, as the long spines are likely to contain fewer GluR1 puncta, hence their response to afferent input will be smaller than those short and stubby spines, which contain larger amounts of GluR’s. Hence, while the length of the spine, in and of itself may not be a barrier, it may indirectly affect the ability of the synapse to express mature properties. The maturation state of the cultured neuron may affect their reactivity to flash photolysis of caged calcium, as well as to the exposure to plasticity-producing chemical stimulation. The cultured neurons used in the current studies are about 12–20 DIV. Younger cells do not yet possess a significant proportion of mature-like spines, whereas older cultures are less likely to express plastic properties, as most of their spines are already large and stable. Thus, at the age that we examined the cultured neurons a whole variety of spine shapes and sizes can be found, making the correlation between structure and function more reliable. Finally, the slow delivery of GluR1 into the synaptic sites under our testing conditions indicate that this mechanism is not likely to underlie the fast rise in connectivity among neurons in culture, seen by us elsewhere [16], or the fast increase in EPSP’s seen in-situ [29,30]. What, then, is the mechanism responsible for this rapid change? While the role of other postsynaptic effectors cannot be ruled out (e.g. phosphorylation of receptor proteins), it is also possible that a presynaptic mechanism, (e.g. increase transmitter release) accounts for the initial phase of the potentiation, at least until delivery of receptor proteins and expansion of the postsynaptic spine takes place. These and similar mechanisms are still being investigated.

Acknowledgements The research was supported by grant #381/02 from the Israel Science Foundation and by a grant from the Ministry of Science within the cooperation program with the Ministry

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of Science and Technology, Korea. We thank V. Greenberger for the preparation of the cultures, Y. Pilpel for help with the transfection, S. Sapoznik for help with the double blind measurements, and A. Avital for help with the statistical analysis. The authors have no conflicting financial interests.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ceca. 2006.11.006.

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