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Previous Article | Next Article
Volume 17, Number 19, Issue of October 1, 1997 pp. 7339-7350
Copyright ©1997 Society for NeuroscienceThe Development of Excitatory Synapses in Cultured Spinal Neurons
Richard J. O'Brien1, 2, 3, Andrew L. Mammen1, 2, Seth Blackshaw2, Michael D. Ehlers1, 2, Jeffrey D. Rothstein3, and Richard L. Huganir1, 2 1 Howard Hughes Medical Institute, and Departments of 2 Neuroscience and 3 Neurology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
ABSTRACT
Immunohistochemical studies of synapses in the CNS have demonstrated that glutamate receptors (GluRs) are concentrated at postsynaptic sites in vivo and in vitro (Baude et al., 1995
Key words: GluR1; synaptogenesis; spinal cord; glutamate transporter; glutamate receptors; rat; tissue culture). The mechanisms leading to receptor clustering at excitatory synapses are far less understood than those governing acetylcholine receptor accumulation at the neuromuscular junction (Hall and Sanes,1993
INTRODUCTION
The molecular cloning of the family of glutamate receptors (GluRs), which mediate excitatory synaptic transmission in the CNS, has allowed the development of subunit-specific antibodies that hold promise for elucidating the mechanisms of GluR regulation during central synaptogenesis (Hollmann and Heinemann, 1994
Work in vivo and in vitro using immunofluorescence and electron microscopy has shown that AMPA and NMDA receptors are concentrated at postsynaptic sites in dendritic spines (Petralia and Wenthold, 1992
MATERIALS AND METHODS
Rat spinal cord cultures. Embryonic day (E) 18 or 19 rat ventral spinal cords were digested for 45 min at 34°C in L-15 media supplemented with 1 mg/ml papain, 1 mM kynurenic acid, 2 mM cysteine, 10 mM HEPES, and 2.5 mM EDTA according to the method of Huettner et al. (1986)
Electrophysiology. Recordings from spinal cord neurons were performed using standard whole-cell patch-clamp recording at a holding potential of 60mV. Extracellular solution consisted of 130 mM NaCl, 4 mM KCl, 2 mM CaCl2, 1 mM NaH2PO4, 10 mM glucose, 12 mM HEPES, 10 µM glycine, 200 µM bicuculline, and 1 µM strychnine, pH 7.3. The osmolarity of the extracellular solution was adjusted to 280, and it was perfused over the neurons at a rate of 3 ml/min at 30°C. Intracellular solution consisted of 115 mM potassium gluconate, 10 mM KCl, 2 mM MgATP, 1 mM CaCl2, 11 mM EGTA, 10 mM HEPES, pH 7.3, 280 Osm. In some experiments neurobiotin (Vector Laboratories, Burlingame, CA) was included in the intracellular solution at 1 mg/ml. NMDA, 20 µM, was applied by rapid perfusion (Raymond et al., 1993
Immunohistochemistry. For standard immunohistochemistry, neuronal cultures were fixed in 4% paraformaldehyde, 4% sucrose in PBS for 30 min, permeablized in 0.5% Triton X-100, blocked in 10% goat serum at 30°C for 1 hr, and incubated with affinity-purified primary antibodies (see below) for 24 hr at 4°C. Rhodamine- or fluorescein-conjugated secondary antibodies (Jackson Immunochemicals, West Grove, PA) were then added at 10 µg/ml for 1 hr. Coverslips were mounted in Permafluor (Lipshaw) with 20 mg/ml Dabco (Aldrich, Milwaukee, WI). In cases in which two rabbit antisera were used, the first antiserum was incubated overnight, treated with FITC-conjugated anti-rabbit antibody for 1 hr, washed extensively, and then incubated with 5% rabbit serum for 30 min at room temperature to saturate reactive ends on the secondary antibody. The second rabbit antiserum [usually anti-GluR1 primarily coupled to Cy3 (CyDye Kit, Amersham, Arlington Heights, IL)] was then added overnight in 5% rabbit serum. Control experiments consisted of incubating a 20× concentrate of either the first or second rabbit antibody with appropriate peptide (0.2 mg/ml) for 1 hr. This solution was then diluted 1:20 to its final concentration and added to the coverslips as above. In all cases, incubation of the primary antibody with appropriate peptide eliminated all dendritic staining. The affinity-purified anti-peptide antibodies used in this paper have been well characterized previously. GluR1, GluR2/3, and GluR4 (Blackstone et al., 1992 
Fig. 2. Clustering of the AMPA receptor subunit GluR1. Fixed and permeablized spinal cord neurons were stained with a C-terminal antibody against GluR1 at 2 (A) and 11 (C) d in vitro. The diffuse pattern originally seen on day 2 is transformed into a highly clustered pattern by day 11, only at sites of synaptic contact between cells, defined by the presence of presynaptic synaptophysin stain (D). Selected synaptophysin clusters not associated with GluR1 clusters are designated with arrowheads. In E, an immunoblot of the C-terminal GluR1 antibody against 11 day cultured spinal cord neurons is shown, with a single protein at the appropriate size for GluR1. Cy3 and Nomarski images were superimposed at a 70:30 ratio in C and a 50:50 ratio in D using Adobe Photoshop.
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Fig. 5. GluR1 clusters in island cultures. Solitary neurons growing on islands of glia were analyzed for GluR1 staining and synaptophysin clusters. Neurons could be divided into those that did (A) or did not (B) form GluR1 clusters at each synapse. Synapses are designated with arrowheads in A and arrows in B. Neurites were not seen to extend from one island to another across the agarose barrier.
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Fig. 8. Colocalization of GluR1 and GluR4. As in Figure 7, neurons were double-labeled with antibodies against GluR1 and GluR4 after 8 d in culture. In a series of 85 neurons, 43 showed the pattern in A (R1+/R4
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Fig. 9. Localization of NR1 in spinal cord neurons. C-terminal antibodies to NR1 were used in fixed and permeablized spinal cord cultures (Fig. 10A) as described in Materials and Methods. N-terminal antibodies were added to live cultures for 1 hr, rinsed three times in media, and then processed as described in Materials and Methods (Fig. 10C). There was no evidence of significant NR1 clustering with either the C- or N-terminal antibody, and no correlation of NR1 staining with GluR1 clusters (compare Fig. 10A with B, and 10C with D). Note is also made that the background layer of glial cells, which is confluent under our conditions, is unstained with either the C- or N-terminal NR1 antibody, or with the GluR1 antibody. In E, immunoblots of spinal cord cultures with affinity-purified C-terminal (C) and N-terminal (N) NR1 antibodies are shown. Each shows a major protein at 116 kDa, the approximate size of NR1.
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Fig. 10. Distribution of glutamate transporters in cultured spinal cord cells. Spinal cord cultures were double-labeled with antibodies against GluR1 (B, F) and either EAAC1 (C) or GLT1 (E, G). The Nomarski image in A corresponds to images in B and C and is shown to illustrate the presence of presynaptic axons (arrows), which do not stain with EAAC1. In E, a low-power photomicrograph of GLT1 staining is included to demonstrate that only some glia stain for GLT1 (at these times in culture, the glial monolayer is confluent, including all the dark areas in E and >90% stain for GFAP). The boxed area in E is shown at higher magnification in F (GluR1) and G (GLT1) to demonstrate the lack of clustered glial transporter. Immunoblots of spinal cord cultures incubated with antibodies to EAAC1 and GLT1 are shown in D and H, respectively. Each demonstrates a single, appropriately sized protein. The scale bar shown in C applies to A-C; the scale bar in G applies to F, G.
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Fig. 4. Analysis of synaptic identity. After 10 d in culture, spinal cord neurons were fixed and stained with rabbit anti-synapsin (1:2500), mouse anti-GAD (2 µg/ml), and mouse anti-gephyrin (1:150). Secondary antibodies consisting of AMCA anti-rabbit and FITC anti-mouse were then added. Finally, Cy3-GluR1 was added as described in Materials and Methods. A series of eight GluR1-positive neurons were digitized using three different excitation wavelengths to distinguish synapsin (AMCA; Fig. 6A), GluR1 (Cy3; Fig. 6B), and GAD/gephyrin (FITC; Fig. 6C). Almost all synapses (synapsin clusters) can be accounted for either by inhibitory synapses (GAD/gephyrin) or GluR1 clusters. Two synapses with neither GluR1 nor GAD/gephyrin staining are identified in A (arrowheads).
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Fig. 7. Colocalization of GluR1 and GluR2/3. Neurons were fixed, permeablized, and double-labeled with antibodies against GluR1 and GluR2/3 as described in Materials and Methods. Although most early clusters showed clear colocalization of GluR1 and GluR2/3 (A, B), occasional GluR2/3 clusters were difficult to colocalize with GluR1 clusters because of high generalized dendritic stain (B, arrows). By Day 11, all GluR1 clusters clearly colocalized with GluR2/3 (C). In the 30% of neurons without GluR1 clusters, GluR2/3 clusters (D) had an appearance similar to those in neurons expressing GluR1. In E, a Western blot of GluR2/3 against spinal cord cultures demonstrates a single, appropriately sized band. The size calibration in A applies to A and B; the size calibration in D applies to C and D.
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Immunoblotting. Cell lysates from high-density cultures were solubilized in 1% Triton X-100, 0.2% SDS and subjected to SDS-Page (7.5% acrylamide). Proteins were transferred to Immobilon-P (Millipore, Bedford, MA), immunoblotted using the above-described antibodies at ~0.5 µg/ml, and visualized with enhanced chemiluminescence (Amersham).
RESULTS
The development of GluR1 clusters correlates with the onset of EPSCs
In cultures of rat ventral spinal cord, EPSCs can first be detected on day 3 in vitro and then gradually increase in frequency and distribution over the subsequent 7 d (Figs. 1, 3). By day 10, virtually all neurons display either continuous or bursting patterns of excitatory currents (Fig. 1A). These currents are completely abolished by low doses of the AMPA receptor antagonist CNQX (5 µM) (Fig. 1B), and they exist, albeit at a lower frequency, in the presence of TTX (1 µM) (Fig. 1C), identifying them as classic miniature EPSCs (mEPSCs). When neurons were simultaneously filled with neurobiotin (Fig. 1D), most synapses, identified with the synaptic vesicle protein synaptophysin (Fig. 1E), were seen to occur on neuritic shafts, although occasional synapses were seen on outpouchings that lacked the neck seen in true spines (Fig. 1D,E, arrowheads). Such morphology matches that seen in the spinal cord in vivo (Vaughn, 1989
Fig. 1. Synaptic currents in cultured spinal cord neurons. At 11 d in vitro, baseline recording at 30°C with a holding potential of
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Fig. 3. Correlation of GluR1 clustering and synaptic currents. A series of spinal cord neurons were stained with GluR1 at the designated times in culture. Simultaneously, additional neurons were assayed for the presence or absence of spontaneous excitatory synaptic activity. Each cell was scored for the number of GluR1 clusters within the first 60 µm of each major dendrite, as well as for the presence or absence of staining at the cell body. Each physiology time point represents 15 neurons from three different platings (3 × 5), and each immunohistochemical time point represents at least 50 neurons from three separate platings. Values for immunohistochemistry are expressed ±SEM. Continuous EPSCs (such as seen in Fig. 1A) are plotted as an indication of the increasing frequency of excitatory synaptic currents.
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Paralleling the onset of excitatory synaptic currents, the GluR subunit GluR1 changed its distribution from diffuse to highly clustered (Figs. 2, 3). After 1 week in vitro, multiple GluR1 clusters (Fig. 2C) were seen on 70% of the neurons, and they coincided in almost all cases with the presence of presynaptic terminals, identified with the synaptic vesicle protein synaptophysin (Fig. 2D). Clustered GluR1 immunostaining appeared to be exclusively postsynaptic, because it was seen only in those neurons whose cell body was also GluR1 immunopositive, in agreement with previous work (Petralia and Wenthold, 1992 Correlation of GluR1 clusters with excitatory nerve terminals
Although all GluR1 clusters on mature neurons were associated with presynaptic specializations in the form of synaptic vesicle accumulation, many synaptic terminals on GluR1-immunopositive neurons were not associated with GluR1 clusters (Fig. 2D, arrows). It was our initial assumption that these latter sites represented inhibitory synapses; however, given the recent interest in the possibility of dormant or "silent" excitatory synapses (Isaac et al., 1995GluR1 clustering in isolated neurons
To further study the specificity of AMPA receptor clustering, and to investigate the distribution of GluR1 in the absence of appropriate synaptic input, individual spinal cord neurons were grown for 8-10 d on glial islands, forcing them to form homogeneous autaptic connections. Under these circumstances neurons can be neatly divided into those that induce clusters of GluR1 at every synapse and those that do not. As shown in Figure 5, a total of 84 solitary neurons grown on islands of glia were studied, of which 64 were GluR1 immunopositive and were analyzed further. Twenty (31%) of the 64 showed a near-complete correlation between synaptophysin clusters (n = 154) and GluR1 clusters (n = 149) (Fig. 5A), 25 neurons (40%) had multiple synaptophysin clusters (n = 122) but no GluR1 clusters (Fig. 5B), and the remaining 19 neurons had neither GluR1 clusters (even though they were GluR1 immunopositive) nor synaptophysin clusters. Given that all GluR1-immunopositive neurons in mixed spinal cord cultures have GluR1 clusters (Fig. 3), these experiments demonstrate that in the absence of appropriate synaptic input, the AMPA receptor subunit GluR1 remains diffusely distributed.
A second series of experiments confirmed the specificity of cluster induction. Twenty-seven solitary R1-immunopositive neurons were stained for GAD immunoreactivity: nine were GAD positive and had no GluR1 clusters, 10 had GluR1 clusters and were GAD negative, and eight had neither GluR1 clusters nor GAD staining (data not shown). In sum, these experiments support the claim that ~30% of the ventral spinal cord neurons that grow under these conditions are capable of inducing clusters of postsynaptic GluR1 at each synaptic contact with a GluR1-expressing neuron. This contact results in a reciprocal accumulation of transmitter vesicles in the presynaptic neuron. The maturation of presynaptic terminals at sites of GluR1 clustering
Although mature excitatory synapses in cultured spinal cord neurons displayed a tight correlation between presynaptic vesicle accumulation and postsynaptic receptor clusters, such was not true for early contacts. As shown in Figure 6, the usual pattern of intense staining for the synaptic vesicle proteins synaptophysin (Fig. 6A,B) or synapsin 1 (Fig. 6C,D) was absent at nearly half the GluR1 clusters seen on days 2 and 3 in vitro (Table 1). These early receptor aggregates do not represent spontaneous receptor clustering such as seen in cultured myotubes (Anderson and Cohen, 1977
Fig. 6. Synaptic vesicle accumulation at newly formed synapses. In A and B, fluorescent images are superimposed on Nomarski images at a ratio of 70:30 (Cy3-GluR1) and 50:0 (FITC-synaptophysin) to demonstrate the induction of GluR clusters by cell-cell contact. In A and B, a day 3 neurite capable of clustering GluR1 is designated by open arrows as it crosses a GluR1-expressing dendrite, inducing two clusters of GluR1 (A, closed arrows) devoid of synaptophysin stain (B). Nearby synaptophysin stain serves as a positive control. In C-E, two day 3 axons stained with tau (E) contact a GluR1-expressing neuron inducing six clusters of GluR1 (C), two of which are devoid of associated synapsin 1 stain (C, arrows) and one of which is displaced from a nearby cluster of vesicles (C, asterisk). In the majority of cases, a complete lack of associated presynaptic vesicle accumulation was seen rather than a physical displacement.
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Table 1. Percentage of GluR1 clusters associated with synaptic vesicle protein accumulation
Day 2 Day 3 Day 4 Day 6 Synaptophysin + 56% (32/57) 70% (51/73) 91% (43/47) 97% (80/82) Synapsin 1+ 47% (19/40) 62% (49/78) 83% (43/52) 91% (47/52) Consecutive clusters of GluR1 were categorized in a series of two separate platings as either associated or unassociated with presynaptic staining for the synaptic vesicle proteins synaptophysin or synapsin 1, as described in Materials and Methods. A lack of synaptic vesicle accumulation could be attributable to either a complete absence of nearby synaptic vesicles (the majority of cases) or a clear misalignment of postsynaptic clusters and presynaptic vesicles (see Fig. 6 for examples). Cultures stained for synapsin 1 were also stained with an antibody to tau to identify the presence of axons at these early GluR1 clusters.
Consistent with the lack of significant synaptic vesicle accumulation at many early receptor clusters, we saw no evidence that AMPA or NMDA receptor activation played a role in receptor clustering. Craig et al. (1994) Coexpression of multiple AMPA receptor subunits
In addition to GluR1, cultures of rat ventral spinal cord express the AMPA receptor subunits GluR2/3 and GluR4, both by immunoblotting and immunocytochemistry. For the purposes of this study, GluR2 and GluR3 are referred to as a single entity, because our antibody recognizes the 20 amino acid C terminus common to both. Immunostaining for GluR2/3 is present in all neurons tested to date and is seen from the earliest time points. In 38 neurons that expressed both GluR1 and GluR2/3, GluR1 clusters were seen to clearly colocalize with GluR2/3 clusters in 171 of 174 cases (Fig. 7C). In addition to colocalization, the relative intensities of the signals from GluR1 and GluR2/3 appeared similar at different synapses within a given neuron. Even at the very earliest time points (Day 3), when clusters of GluR1 and excitatory synapses can first be detected, GluR2/3 staining was clearly seen to colocalize with GluR1 clusters (Fig. 7A), except for occasional instances (Fig. 7B, arrow) when high dendritic background staining made the detection of GluR2/3 clusters difficult. Coimmunoprecipitation experiments confirm the association between GluR2/3 and GluR1 as early as day 4, the first time at which GluR1 or GluR2/3 can be reliably immunoprecipitated (data not shown). In the 30% of neurons in which GluR1 was not expressed (Fig. 7D), clusters of GluR2/3 had an appearance, distribution, and number identical to those in neurons expressing GluR1, and they were always associated with presynaptic synaptophysin stain after 7 d in culture.
GluR4 was expressed in a pattern somewhat different from either GluR1 or GluR2/3. First, GluR4 was not detectable by immunohistochemistry or Western blot until 1 week in culture, by which time excitatory synapses were well established. In addition, specific staining for GluR4 was also present in glial cells, as has been described previously (Petralia and Wenthold, 1992
Because our previous experiments with tau immunostaining and neurobiotin injection had suggested that each cultured spinal cord neuron receives excitatory synaptic input from several other neurons, these results suggest that once spinal neurons express an AMPA receptor subunit, that subunit is expressed at all its excitatory synapses regardless of the source of synaptic input. In none of the neurons examined to date was there a suggestion that AMPA receptor subunit composition could vary within a postsynaptic neuron at sites of varying presynaptic input; however, because we are dealing with a rather homogeneous population of cells, it is possible that a more divergent source of synaptic input such as dorsal root ganglion cells could alter this interpretation. GluR2/3 is the only AMPA receptor subunit common to all excitatory synapses in all cultured spinal neurons. The NMDA receptor subunit NR1 is not clustered at excitatory synapses
As mentioned previously, mEPSCs in cultured rat spinal neurons are composed predominantly of AMPA-type receptor currents, despite the presence of electrophysiologically active NMDA channels. We investigated the immunohistochemical distribution of NR1, a subunit common to all NMDA receptors, using both C- and N-terminal antibodies, and compared it with the distribution of GluR1 in the same neurons. In all cells investigated, for periods up to 3 weeks in culture, NR1 staining was diffuse, without any relation to GluR1 or synaptophysin clusters. As shown in Figure 9, staining with both the C-terminal antibody (Fig. 9A), which in fixed and permeablized cells detects the entire pool of NR1, and the N-terminal antibody (Fig. 9C), which when applied to live cells detects the surface pool of NR1, failed to reveal any significant clustering of NR1 and showed no colocalization with GluR1 clusters (Fig. 9B,D). A similar diffuse staining pattern was seen with the well-characterized mouse mAb 54.1 (Huntley et al., 1994The distribution of neuronal and glial glutamate transporters in spinal cord cultures
Antibodies directed against the intracellular C terminus of both the neuronal and glial high-affinity glutamate transporters were used to stain cultures of rat spinal cord. Specific immunostaining for EAAC1 was distributed diffusely in the dendrites of all ventral spinal cord neurons for periods of up to 4 weeks in vitro (Fig. 10C). Although occasionally micropunctate, there was no correlation of transporter staining with postsynaptic GluR1 clusters (Fig. 10B). Although all GluR1-expressing dendrites stained for EAAC1, most presumptive axons (slender, unbranched, GluR1-negative processes ending in synapses) (Fig. 10A, arrows) and glia were unstained. In addition, because excitatory presynaptic terminals correlate with GluR1 clusters, these observations exclude a significant accumulation of glutamate transporter in presynaptic terminals. GLT1 was present in ~50% of the glial cells in our cultures and when present stained in a diffuse pattern. In Figure 10E, the central immunopositive glial cell is surrounded by glial cells that remain unstained. A neuron expressing multiple clusters of GluR1 lies on top of the GLT1-expressing glial cell (Fig. 10E, boxed area). That neuron, stained for GluR1, and the corresponding area of the glial cell, stained for GLT1, are shown at higher magnification in Fig. 10, F and G, respectively, to demonstrate the lack of clustered GLT1 staining at excitatory synapses in these cultures. We were not able to detect any specific immunostaining with antibodies to the less widely distributed glial transporter GLAST.
DISCUSSION
The development of AMPA receptor clusters during synaptogenesis
In cultures of rat spinal cord there exists a population of neurons, comprising 30% of those grown on glial islands, that are capable of inducing clusters of the AMPA receptor subunit GluR1 each time they form a synaptic connection with a GluR1-expressing dendrite. Clustering of postsynaptic receptors appears to be a nearly universal component of excitatory synaptogenesis in these cultures. In the absence of appropriate input, AMPA receptors are expressed but remain diffusely distributed. Although the onset of GluR1 clustering coincides with the first signs of spontaneous excitatory transmission, the activation of postsynaptic excitatory neurotransmitter receptors appears to play no role in the development of GluR1 clusters. The same cell-cell interaction that results in postsynaptic AMPA receptor clustering also results in a rapid, but not necessarily synchronous, accumulation of presynaptic vesicles. The induction of GluR clusters during synaptogenesis correlates well with previous studies of glutamate chemosensitivity in cultured neurons from the spinal cord and hippocampus (O'Brien and Fischbach, 1986bsubunit mRNA extends into the dendrite (Racca et al., 1997
Heteromeric synaptic receptors
In addition to GluR1, the AMPA receptor subunit GluR2/3 is present at the earliest sites of postsynaptic receptor clustering, likely as part of a heteromeric complex. GluR4 does not seem to be involved in early contacts, because it is expressed several days after the onset of synaptogenesis. By 11 d in vitro, all neurons that expressed GluR1 had multiple receptor clusters that also contained aggregated GluR2/3 and/or GluR4 at staining intensities that appeared similar from cluster to cluster within a given neuron. Because the multiple AMPA receptor clusters on any neuron reflect input from several different spinal neurons, their invariant subunit composition suggests that presynaptic terminals do not significantly influence the subunit expression pattern of postsynaptic receptor clusters. As mentioned previously, however, this concept requires further study.
Because the formation of receptor clusters appeared similar in all cultured spinal neurons despite differences in AMPA receptor subunit expression, it was natural to search for a subunit common to all heteromeric complexes that might serve as an anchor to postsynaptic cytoskeletal elements. By immunohistochemistry, GluR2 and/or GluR3 are present at nearly all receptor clusters in cultured spinal cord neurons. In addition, studies of AMPA receptor subunit expression in vivo have consistently shown that GluR2 and/or GluR3 are expressed in the vast majority of central neurons (Keinanen et al., 1990 The NMDA receptor subunit NR1 is not clustered at excitatory synapses
In cultured rat spinal neurons, the NMDA receptor subunit NR1 was not co-clustered with AMPA receptors at excitatory synapses. The antibodies used in our study span the entire length of NR1 and include both extracellular and intracellular pools. Because NR1 is a part of all NMDA receptor complexes (Nakanishi, 1992Glutamate transporters are not clustered at excitatory synapses
High-affinity transporters for glutamate have recently been cloned from both neurons and glia (Kanai and Hediger, 1992
FOOTNOTES
Received February 28, 1997; revised July 8, 1997; accepted July 14, 1997.
Correspondence should be addressed to Dr. Richard Huganir, Howard Hughes Medical Institute, PCTB 900, Johns Hopkins Medical School, 725 N. Wolfe Street, Baltimore, MD 21205.
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