GABAergic synapses are formed without the involvement of dendritic ...

© 2008 Nature Publishing Group http://www.nature.com/natureneuroscience

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GABAergic synapses are formed without the involvement of dendritic protrusions Corette J Wierenga, Nadine Becker & Tobias Bonhoeffer Synaptogenesis and the role of dendritic protrusions in this process are well studied in glutamatergic synapses. Much less is known about the formation of GABAergic synapses, which are located predominantly on the dendritic shaft. We used genetically labeled interneurons in mature hippocampal slice cultures and two-photon laser-scanning microscopy to examine contact formation between GABAergic axons and the dendrites of CA1 pyramidal cells. Dendritic protrusions distinguished and selected between glutamatergic and GABAergic boutons. In contrast with contacts with glutamatergic boutons, which can be long lasting, the contacts of dendritic protrusions with GABAergic boutons were always short lived. Similarly, the contacts made by GABAergic axonal protrusions were always transient. New putative GABAergic synapses were formed exclusively by new boutons appearing at pre-existing axon-dendrite crossings without the involvement of any dendritic or axonal protrusions. These findings imply that fundamentally different mechanisms underlie the generation of GABAergic and glutamatergic synapses.

Chemical synapses are the key connective elements in neuronal networks. They are not only crucial for information processing, but their plasticity also endows the brain with its outstanding capacity for adaptation to the environment. Understanding synapses and how they are formed is therefore a fundamentally important task in neuroscience. So far, the majority of studies have focused on the formation of glutamatergic synapses, whereas the formation of the other major type of synapse in the brain, which uses the inhibitory transmitter GABA, remains less examined, in spite of the fact that GABAergic synapses form 10–20% of all synapses in the brain and are indispensable for the proper and stable functioning of the brain1. Glutamatergic synapses on excitatory neurons in the hippocampus occur almost exclusively on dendritic spines2,3. It is thought that these spines and their precursors, dendritic filopodia, are actively involved in the formation of new glutamatergic synapses. Dendrites grow small protrusions that make contact with presynaptic axons and boutons4–8. Only a subset of these initial contacts is selected through filopodial signaling and forms mature, functional synapses9–11. During further maturation of a new synaptic contact, the dendritic protrusion shrinks as its head expands, turning into a mature spine4,12,13. In this scenario, dendritic filopodia precede fully mature spines and it has been shown that long, thin dendritic protrusions that are reminiscent of filopodia can have (sometimes even multiple) synaptic contacts5,6,14. Although the above is probably not the only way to form new synapses on spines15, the studies cited above provide ample evidence of a role for outgrowing protrusions in the formation of glutamatergic synapses. In contrast to glutamatergic synapses, GABAergic synapses are usually not located on spines but are found directly on the dendritic shaft1,3,16. It has been suggested that dendritic protrusions can also

serve to guide presynaptic axons to the dendritic shaft, thereby mediating the formation of shaft synapses5,6. However, it is unresolved to date how often this occurs and whether alternative scenarios, possibly involving a more active role of the GABAergic axon, are also important in the formation of GABAergic contacts. We used high-resolution two-photon imaging of genetically labeled GABAergic neurons and their postsynaptic counterparts to examine how new GABAergic shaft synapses are formed in the CA1 area of organotypic hippocampal cultures. We found that new GABAergic boutons, probably indicating new GABAergic synapses, were formed exclusively at pre-existing axon-dendrite crossings, without the involvement of either dendritic or axonal protrusions. This indicates that there are fundamentally different mechanisms for the formation of glutamatergic and GABAergic synapses. RESULTS To visualize GABAergic axons, we used hippocampal slice cultures of GAD65-GFP mice. In these mice, 30–50% of GABAergic interneurons in the hippocampus express GFP at embryonic ages and well into adulthood17. Dendrites of 2–5 CA1 pyramidal neurons were filled with Alexa Fluor 594 through a patch pipette before imaging. This resulted in a sparse overlap between GFP-labeled GABAergic axons and Alexa 594–labeled dendrites, allowing the contacts between GABAergic axons and boutons with labeled dendritic structures to be resolved in detail with two-photon laser-scanning microscopy (Fig. 1a,b). We took high-resolution image stacks every 30 min for a total period of 3–6 h and examined the formation of contacts between GABAergic axons and apical dendrites of CA1 pyramidal neurons. In total, we analyzed the time-lapse series

Max Planck Institute of Neurobiology, Cellular and Systems Neurobiology, Am Klopferspitz 18, 82152 Martinsreid, Germany. Correspondence should be addressed to C.J.W. ([email protected]). Received 19 March; accepted 30 June; published online 24 August 2008; doi:10.1038/nn.2180

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of 21 image stacks, in which we observed 4400 pre-existing, stable contacts (Table 1). From the high-resolution images, we could clearly identify the locations where a GABAergic bouton was in contact with a labeled dendrite (see Methods for our criteria). Although synapses require the presence of an axonal bouton, axonal varicosities or swellings do not necessarily imply the presence of synapses18,19. To examine whether the physical contacts between GABAergic boutons and dendrites corresponded to synapses, we carried out post hoc immunostaining on contacts between boutons and dendrites that were previously identified in two-photon image stacks. We found that 80% of the contacts (56 out of 70, 5 slices) that were classified as making physical contact on the basis of the two-photon images did indeed show postsynaptic gephyrin staining, indicating that the majority of physical contacts represent GABAergic synapses (Fig. 1c,d). For 10 of the 14 gephyrin-negative contacts, the bouton showed gephyrin staining that did not overlap with the labeled dendrite, suggesting that the GABAergic bouton made a synapse to a neighboring unlabeled structure. Using the mirror image of the gephyrin channel reduced the number of gephyrin-positive contacts from 80% to 47%, indicating that gephyrin staining was specific (P o 0.001, w2 test). We also examined locations where a bouton was close to a labeled dendrite, but the overlap between the red and green channel in the two-photon images was too small to be classified as a contact by our criteria. However, 19% of these ‘noncontacts’ showed gephyrin staining (10 out of 54, 5 slices). This indicates that our criteria for identifying GABAergic synapses from our two-photon images might lead to an error in assessing synaptic contacts of no more than B20% in both directions, which should be unproblematic for the conclusions presented below. From the immunostaining experiments, we therefore inferred that at least 80% of boutons contacting a dendrite, identified on the basis of the two-photon images, represent actual synaptic contacts. The locations where we observed a GABAergic axon crossing or touching a dendrite, but which lacked a bouton, are probably nonsynaptic contact points18,19 and will be referred to as axondendrite crossings.

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Figure 1 Imaging GABAergic synapses in GAD65-GFP slice cultures. (a) Maximal intensity projection image of the CA1 region, showing GFP-labeled GABAergic interneurons (green) and Alexa 594–labeled pyramidal neurons (red). (b) A single section from an image stack (raw data) from the boxed area indicated in a showing clear labeling of GABAergic axons and boutons and their overlap with labeled dendrites. (c,d) Two examples of synaptic contacts (white arrows) between GABAergic boutons (green) and labeled dendrites (red). Single sections of two-photon image stacks are shown to the left with the corresponding confocal sections after immunofluorescence staining for gephyrin (blue) to the right. Areas with all three labels appear white. In these images (but nowhere else), twophoton images were filtered to allow for better comparison with the confocal images.

Transient contacts are formed by protrusions In principle, new GABAergic synapses could Post hoc immunostaining be formed with or without the involvement of dendritic or axonal protrusions. During the 3–6-h imaging period, we observed many dendritic protrusions on labeled dendrites forming, retracting or changing size and/or shape. In 19 cases, a dendritic protrusion could be seen making contact with a GFP-labeled GABAergic bouton (two examples are shown in Fig. 2a,b). In the first example, a dendritic protrusion (Fig. 2a) grew out from the shaft and touched the GABAergic bouton of a neighboring axon (Fig. 2a). At the next time point, 30 min later, the dendritic protrusion had almost completely retracted to the shaft. In the second example (Fig. 2b), the dendritic protrusion grew out from a spine that was present during the entire imaging period. As in the first example (Fig. 2a), the contact between the dendritic protrusion and the GABAergic bouton lasted for only a single time point (complete time series for these two examples are shown in Supplementary Fig. 1 online). The majority of contacts between a new dendritic protrusion and a GABAergic bouton (12 out of 19) were present at only one time point (0.5 h) and none lasted longer than 1.5 h. There was no difference in the lifetime of the contact between protrusions that grew directly from the shaft (13 out of 19) and protrusions that grew from stable spines (6 out of 19). These observations indicate that dendritic protrusions do make contact with GABAergic boutons in a similar fashion to glutamatergic boutons. However, these contacts were always transient, and therefore, Table 1 Number of observations Pre-existing stable boutons Transient contacts

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New contact by movement of bouton or dendrite Lost contact by movement of bouton or dendrite Data from 21 experiments with imaging durations of 3–6 h.

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Figure 2 Transient contacts by dendritic and axonal protrusions. Threedimensional representations (left) and single sections (right) of two-photon image stacks at the indicated time points. GABAergic axons are shown in green (GFP) and dendrites in red (Alexa 594). Overlap (yellow) of the red and green channels indicates contact between the axonal and dendritic structures. Some nonrelevant structures that are visible in the single sections were removed from the three-dimensional reconstructions for clarity. Threedimensional reconstructions were made for visualization purposes only; analysis was always done on individual image sections. (a) A dendritic protrusion (DP) grew out from the dendritic shaft and made transient contact with a GABAergic bouton. (b) A DP emerged from a stable spine and made transient contact with a nearby GABAergic bouton. (c) An axonal protrusion (AP) grew out from a GABAergic bouton that was contacting the labeled dendrite. The protrusion made contact with the dendrite twice (see Supplementary Fig. 1), but the contact was not present at the end of the imaging period. (d) An AP grew out and formed a transient contact with the labeled dendrite. Both boutons did not form contacts with the labeled dendrite. Scale bars represent 1 mm.

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protrusion (and its contact) was not present the next day and the axon and dendrite appeared to be unchanged. This shows that, even in our longest-lasting contact between axonal protrusion and dendritic shaft, the contact did not seem to be transformed into a stable synapse. These observations suggest that axonal protrusions grow out and make transient contacts with the dendritic shaft, but they are not transformed into long-lasting, stable contacts.

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in contrast with glutamatergic synapses, the formation of stable GABAergic synapses is probably not mediated by dendritic protrusions. We also looked at the outgrowth of axonal protrusions from labeled GABAergic axons during the 3–6-h imaging period. Consistent with previous reports20–22, these were usually smaller and shorter lived than dendritic protrusions and occurred exclusively at boutons. In 25 cases, we observed an axonal protrusion touching a labeled dendrite (two examples are shown in Fig. 2c,d; complete time series for these two examples are shown in Supplementary Fig. 1). In the first example (Fig. 2c), a GABAergic bouton contacted the labeled dendrite during the entire imaging period. After 1.5 h, an axonal protrusion (Fig. 2c,d) grew out from the bouton along the dendritic shaft of the labeled neuron but retracted before the end of the imaging period. The axonal protrusion in the second example (Fig. 2d) grew from a bouton that was not in contact with the labeled dendrite. It appeared to form a new bouton on contact with the dendrite (t ¼ 4.5 h). As with the first example, however, the contact did not last. In all cases where an axonal protrusion made contact with a labeled dendrite, the contact was only transient (o2.5 h for all but one). There was no difference in lifetime of the contact between small protrusions (such as in Fig. 2c; 17 out of 26) or longer filopodia-like protrusions (Fig. 2d; 9 out of 26). Only once did we observe a new contact by an axonal protrusion that lasted until the end of the imaging period (4.5 h). In this case, we could image the same contact the following day (see below). We found that the axonal

New stable boutons are formed on dendritic shafts In addition to transient contacts by protrusions, we also observed longlasting changes in GABAergic contacts during the 3–6-h imaging period (Fig. 3). At locations where a labeled GABAergic axon crossed a labeled dendritic shaft, a new bouton occasionally appeared that then stayed present for the rest of the imaging period. We observed this in 12 cases (Figs. 3a,b and 4a). The appearance of a new bouton did not seem to be influenced by the presence (4/12; Fig. 3a) or absence (8/12; Fig. 3b) of a neighboring bouton that was already contacting the dendritic shaft (complete time series of the two examples in Fig. 3 are shown in Supplementary Fig. 2 online). Not all new GABAergic boutons were stable from their first appearance. Some boutons were present for only a short period (Figs. 3c and Fig. 4b), whereas others disappeared and reappeared during the imaging period (Figs. 3d and Fig. 4b). We observed 30 of these transient boutons. In some cases, the new bouton appeared to make small movements to and from the dendrite before and after making contact (Fig. 3c and Supplementary Fig. 2). In other cases, the new bouton appeared or disappeared at the location of the contact without noticeable movement (Fig. 3d). The detailed time courses of the appearance of individual boutons on axon-dendrite crossings clearly shows the difference between boutons that were stable from their first appearance (Fig. 4a) and transient boutons, which disappeared and sometimes reappeared during the imaging period (Fig. 4b). These observations indicate that stable GABAergic boutons can be formed over a period of several hours on a similar time scale as has been reported for the formation of glutamatergic synapses23–25. However, in contrast with glutamatergic contacts, dendritic or axonal protrusions do not seem to be involved in the establishment of long-lasting contacts. Instead, new stable GABAergic boutons appear at pre-existing axon-dendrite crossings. Although the majority of inhibitory synapses in the brain are located directly on the dendritic shaft, some of them occur on dendritic spines3,26,27. In hippocampal slice cultures of GAD65-GFP mice, in which only a subset of GABAergic interneurons are labeled, 21% of the labeled GABAergic contacts that were present before and during the

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Figure 3 New GABAergic boutons appear at axon-dendrite crossings. Threedimensional representations (left) and single sections (right) of two-photon microscopy image stacks at the indicated time points (see Fig. 2 for details). (a) A stable bouton (SB) appeared at an axon-dendrite crossing and stayed in contact with the dendrite for the rest of the imaging period. The lower bouton formed a stable contact with the dendrite for the entire imaging period. (b) An SB appeared at an axon-dendrite crossing at t ¼ 2.5 h and stayed in contact with the dendrite for the rest of the imaging period. (c) A transient appearance of a bouton (TB) at an axon-dendrite crossing. (d) A TB at an axon-dendrite crossing appeared, disappeared and reappeared several times. Scale bars represent 1 mm.


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entire imaging period were located on spines (90 out of 423 pre-existing contacts). We asked whether the appearance of a new bouton preferentially occurred at axons crossing the dendritic shaft or whether they also occurred at spines. Two of the 12 new stable boutons and 5 of the 30 transient boutons appeared at dendritic spines. The fraction of new boutons appearing on spines versus shafts was not significantly different from the fraction of pre-existing GABAergic contacts made on spines (P 4 0.5, w2 test). This suggests that the formation of new GABAergic synapses occurs through the appearance of a new bouton at pre-existing axon-dendrite crossings, irrespective of whether the new bouton is made on a spine or on the dendritic shaft of the postsynaptic neuron. Lifetime of transient contacts and new boutons As described above, we observed three types of transient events. Transient contacts were initiated by the dendrite through the outgrowth of a dendritic protrusion that touched a GABAergic bouton but could also be initiated by the outgrowth of an axonal protrusion contacting the dendrite. Furthermore, boutons transiently appeared at an existing axon-dendrite crossing. For each of these events, we


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quantified the duration of each episode of contact or bouton appearance, the number of repeated contacts or bouton appearances during the imaging period, and the total percentage of the imaging period in which the contact or bouton was present. The durations for all types of transient events were fairly similar and transient events were much shorter compared to the time a new stable bouton was present (Fig. 4c). Notably, repeated appearance and disappearance of a transient bouton at an axon-dendrite crossing was fairly common, whereas contacts made by protrusions, either dendritic or axonal, were usually made only once during the imaging period (Fig. 4d). When a transient bouton appeared at an axon-dendrite crossing, it was present, on average, for almost 40% of the total imaging period, whereas a transient contact made by a dendritic protrusion was present for o20% of the time (Fig. 4e). These data support the notion that new GABAergic synapses are formed by the occurrence of a new GABAergic bouton at an existing axon-dendrite crossing, whereas contacts made by protrusions, either axonal or dendritic, have a very low probability of being transformed into stable synapses. Long-lasting contacts We next examined whether GABAergic boutons that were formed during the imaging period persisted into the next day. After a night in the incubator, the Alexa 594 dye was often cleared by the CA1 pyramidal neurons and had gathered mostly in the soma, leaving only weakly staining in the dendrites. In five experiments, the dendrites were bright enough to image the same region with sufficient detail the next day (examples are shown in Supplementary Fig. 3 online). The GABAergic axons and boutons were as bright as the day before and had largely unchanged morphology. Of the 86 contacts between GABAergic boutons and labeled dendrites that were present before and during the entire imaging period on the first day, 81 were also observed the next day (94%), corroborating their classification as pre-existing stable contacts. Two stable boutons had formed during the experiment at an axon-dendrite crossing that had stayed present for the rest of the imaging period on the first day. Both boutons were still present and in contact with the red dendrite the following day, indicating that the newly formed boutons were indeed long lasting. Of the 15 boutons that qualified as transient on the first day, seven were present the next day at the location where the bouton had first occurred. In the other eight cases, the axon-dendrite crossing was still present, but a bouton could not be discerned. This suggests that at least a subset of transient boutons is transformed over time into stable boutons, possibly forming synapses. Of the transient contacts made by 11 axonal protrusions and 3 dendritic protrusions that were observed on the first day, none were present the next day and the axons and dendrites appeared to be unchanged (see Table 2). Although it is possible that small dendritic protrusions were missed as a result of the weak dendrite labeling on the second day, these observations are consistent with the idea that transient contacts formed by axonal or dendritic protrusions are generally not transformed into GABAergic synapses.

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Lifetime of protrusions We noticed that dendritic protrusions often retracted soon after contacting a GABAergic bouton, whereas axonal protrusions appeared to live longer. We examined whether this was the result of a general difference in lifetime between dendritic and axonal protrusions or whether the transient contact with the GABAergic bouton or dendritic shaft, respectively, affected the lifetimes of the protrusions. We therefore measured the lifetime of an arbitrarily chosen population of newly emerging (that is, not present at the first time point) axonal and dendritic protrusions that were not contacting any labeled structure. Axonal protrusions that had contacted a labeled dendrite had a range of lifetimes that was indistinguishable from the range of lifetimes of the population of arbitrarily chosen GABAergic axonal protrusions (Fig. 5a). For dendritic protrusions, the picture was very different. Although the arbitrarily chosen dendritic protrusions included both short-lived and long-lived protrusions, all dendritic protrusions that contacted a GABAergic bouton had short lifetimes (Fig. 5b). This suggests that only short-lived dendritic protrusions contact GABAergic boutons or, somewhat more likely, that contacting a GABAergic bouton shortens the lifetime of a dendritic protrusion. Alternatively, some of the arbitrarily chosen protrusions contacted something that prolonged their lifetimes. The arbitrarily chosen population of dendritic protrusions probably contacts a rather inhomogeneous selection of structures, including GABAergic and glutamatergic axons and boutons, glial processes, and other dendrites28. We therefore tested more specifically whether the lifetime of dendritic protrusions depends on whether they contact a glutamatergic or GABAergic bouton. To this end, we labeled glutamatergic axons from CA3 neurons with a bolus-loading technique (N.B., C.J.W., Fonseca, R., T.B. and Na¨gerl, U.V., unpublished data). In these experiments, we observed 20 dendritic protrusions growing from labeled CA1 pyramidal neurons that contacted labeled glutamatergic boutons. In contrast with the protrusions contacting GABAergic boutons, a substantial fraction of these protrusions had lifetimes that were greater than 2 h (Fig. 5b). In fact, in 5 of the 20 cases, the protrusion was still present and contacting the glutamatergic bouton at the last imaging time point (Fig. 5c). In many cases, the dendritic protrusions stayed in contact for much longer than was observed for dendritic protrusions contacting a GABAergic bouton. Nevertheless, not all contacts between dendritic protrusions and glutamatergic boutons were long lasting. Retraction of dendritic protrusions shortly

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Figure 4 Lifetime of transient contacts and boutons. (a,b) Time course of new stable (a, n ¼ 12) and transient boutons (b, n ¼ 30). Individual boutons are represented in different colors, and the average time course is given by the thick black curve. The number of overlapping pixels between bouton and shaft (normalized to maximum overlap) is plotted for all time points, aligned to the time when the bouton (first) appeared. New stable boutons always stayed in contact with the shaft, whereas transient boutons disappeared and sometimes reappeared at later time points. In some experiments, we examined the boutons on the day following the imaging period (1 d, see also Table 2). (c) Average duration of single episodes of contact by dendritic and axonal protrusions and transient and stable bouton occurrences. (d) Number of repeated contacts or bouton appearances. (e) Percentage of the imaging period that the transient contact or bouton was present. The numbers at the base of the bars reflect the number of observations. * P o 0.05, *** P o 0.001, ANOVA with post hoc Tukey HSD test. Error bars reflect s.e.

after contacting glutamatergic boutons also occurred, similar to protrusions contacting GABAergic boutons. Taken together, these data suggest that some, but not all, of the contacts with glutamatergic boutons were transformed into stable glutamatergic spine contacts, consistent with the proposed role of dendritic protrusions in the formation of glutamatergic spine synapses4–6,9. Our finding that dendritic protrusions distinguish between GABAergic and glutamatergic boutons is further supported by the individual time courses of contact formation by dendritic protrusions (Fig. 5d,e). These data strongly suggest that, although dendritic protrusions are important in the formation of glutamatergic spine contacts, contacts between dendritic protrusions and GABAergic boutons are short lived and do not normally result in new GABAergic synapses. New boutons can form synapses in several hours The results described above suggest that new GABAergic boutons appearing at pre-existing axon-dendrite crossings may be important for establishing new GABAergic synapses. However, our two-photon data show anatomical apposition as well as a morphological change into a bouton, but they do not conclusively demonstrate that these new boutons actually form functional synapses, nor do they indicate at what time scale these new synapses form. For glutamatergic synapses, it has been shown that the presynaptic active zone can be functional in 30–60 min after the initial contact between axon and dendrite and that postsynaptic scaffold proteins and receptors are recruited soon thereafter23,25,29,30 (but also see refs. 7,8). At present, detailed information on the time course of the recruitment of synaptic molecules to new GABAergic synapses is almost completely lacking31–33. To start addressing this issue, we carried out time-lapse imaging of GFP-labeled GABAergic axons, followed by post hoc immunostaining for GABAergic pre- and postsynaptic markers, vesicular GABA transporter (VGAT) and gephyrin (Fig. 6). For comparison, we immunostained slice cultures without previous time-lapse imaging and found that the vast majority of GABAergic boutons contained VGAT and were associated with gephyrin (Fig. 6c,d). In fact, only 2.4% of GFP-positive boutons Table 2 Contacts that were present the following day Pre-existing stable boutons

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(total 1,317 boutons, 12 slices) lacked a synaptic marker. This indicates that, in general, GABAergic boutons reliably reflect GABAergic synapses. During 3 h of time-lapse imaging, most GABAergic boutons were present for the entire imaging period and only 5–10% of boutons were plastic, that is, appeared or disappeared. Post hoc immunostaining a bouton that appeared at t ¼ 1.5 h during the imaging period (Fig. 6a,b) showed that, at the time of fixation (2 h after first appearance), the new bouton (yellow circle) had accumulated VGAT, while the postsynaptic scaffolding protein gephyrin was not (yet) present (Fig. 6b). Stable boutons on the same and nearby axons (blue circles) were immunopositive for VGAT and gephyrin (Fig. 6b), presumably reflecting stable GABAergic synapses. When we examined multiple new boutons that appeared at various intervals before fixation, we found that the majority of new boutons acquired VGAT in the first hour after appearance (all time points are different from background, P o 0.01, w2 test), whereas gephyrin accumulated at a slower rate (Fig. 6c). We found that B70% of new boutons were immunopositive for both synaptic markers 3 h after their first appearance, presumably reflecting new GABAergic synapses (Fig. 6d). At locations where boutons had disappeared during the two-photon imaging period (at various intervals before fixation), the presynaptic protein VGAT was not present above background levels, whereas postsynaptic gephyrin could occasionally be found up to 1 h after the bouton disappeared (data not shown). This might indicate that presynaptic changes precede postsynaptic alterations during GABAergic synapse formation and disassembly, but more detailed studies will be needed to fully resolve these processes. Notably, our findings indicate that a large fraction of newly appearing GABAergic boutons forms new GABAergic synapses with a time constant on the order of a few hours.


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Figure 5 Lifetime of dendritic and axonal protrusions. (a) The mean (upper) and cumulative distributions (lower) of the lifetimes of axonal protrusions that contacted a labeled dendrite were not different from those of arbitrarily selected axonal protrusions. (b) The mean lifetime (upper) of dendritic protrusions that contacted a GABAergic bouton was significantly shorter than that of the arbitrarily chosen dendritic protrusions (P ¼ 0.03, Mann-Whitney U test). The cumulative distributions also clearly show the shorter lifetimes of GABA-contacting protrusions (lower, gray solid lines; P o 0.01, w2 test). Dendritic protrusions that contacted a glutamatergic bouton showed a much broader range of lifetimes, with some short- and some long-lived protrusions (lower, gray dotted line; significantly different from gray solid line, P o 0.001, w2 test). (c) Example of a long-lasting contact made by a dendritic protrusion and a glutamatergic bouton. The dendritic protrusion and the contact were still present at the end of the imaging period. Scale bar represents 1 mm. (d,e) Time course of contact formation for dendritic protrusions with GABAergic (d, n ¼ 19) and glutamatergic (e, n ¼ 20) boutons. Individual protrusions are represented in different colors and the average time course is given by the thick black curve. The numbers of overlapping pixels between protrusion and bouton (normalized to maximum overlap) are plotted for all time points, aligned to the time of first contact. Contacts with GABAergic boutons were always short lived, whereas longer-lived and repeated contacts occurred with glutamatergic boutons.

Axon-dendrite crossings Our data show that new GABAergic synapses are formed at pre-existing axon-dendrite crossings. Taking this result into consideration, a flexible, modifiable GABAergic system would require a considerable fraction of axon-dendrite crossings that do not (yet) have synaptic contacts so that new synapses can still be formed when needed. We therefore estimated the percentage of GABAergic axon-dendrite crossings with and without boutons. We found that only 42 ± 8% (mean ± s.d.; 21 slice cultures) of axon-dendrite crossings contained one or more boutons. This indicates that there are still a substantial number of available axon-dendrite crossings where new GABAergic contacts can be formed. Notably, the percentage of axon-dendrite crossings with boutons was similar in all slices (range of 30–56%, with one exception of 21%; 21 slices) and did not depend on the total number of crossings. This suggests that the fraction of axon-dendrite crossings that have synapses may be tightly controlled by the GABAergic system so that the generation of new synaptic contacts is always permitted. DISCUSSION The overwhelming majority of studies on synapse formation in the CNS have been carried out on glutamatergic synapses. However, besides the greater part of synapses, which are glutamatergic and often located on spines, a substantial fraction of synapses in the brain are GABAergic. These synapses are located mostly (although not exclusively) on dendritic shafts. Our experiments were conducted in vitro on a specific subset of GABAergic neurons. Therefore, care needs to be taken before generalizing these results to all GABAergic axons and/ or the in vivo situation. Nevertheless, we demonstrate that, at least in the CA1 region of the hippocampus and under our experimental circumstances, GABAergic synapses are formed via a fundamentally different

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Figure 6 New GABAergic boutons form synapses. (a) Time lapse two-photon images (projection of three z sections) showing the appearance of a new bouton (yellow circle) on a GFP-expressing GABAergic axon. (b) Post hoc immunostaining (projection of three z sections) of the same region as in a. The dashed box is enlarged on the right side (merge) and the separate channels are shown. The new bouton (yellow circle) was immunopositive for VGAT (blue), but not for gephyrin (red). Stable boutons (blue circles) were immunopositive for both markers (appearing white in merged image). Scale bars represent 1 mm. (c) Left, percentage of new boutons that were immunopositive for VGAT (black bars) and gephyrin (gray bars), grouped according to their lifetime before fixation. Right, percentage of boutons that were immunopositive for VGAT and gephyrin in slices in which no previous time lapse imaging was performed. Background staining levels of boutons were determined after horizontal or vertical reflection of the VGAT and gephyrin channels (‘randomized’). (d) Same data as in c, but the bars indicate the number of synaptic markers that were present at each bouton. * P o 0.05, *** P o 0.001, w2 test, difference from background. The numbers at the base of the bars reflect the number of boutons examined.

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process than glutamatergic spine synapses. Although, in the case of glutamatergic synapses, dendrites grow small protrusions, such as spines and filopodia, to form new synaptic contacts with nearby axons, such protrusions do not seem to mediate the formation of GABAergic synapses. In contrast, new GABAergic synapses appear to be formed exclusively at locations where GABAergic axons and postsynaptic dendrite are already in close proximity. Although the above caveat about preparation and molecular/genetic cellular identity holds, our data make a strong case that GABAergic and glutamatergic synapse formation occur according to very different schemes. Our immunofluorescence labeling (Figs. 1 and 6) showed that the great majority of stable GABAergic boutons probably represent actual synapses. However, from the two-photon imagery, it is impossible to definitively tell whether a GABAergic bouton synapses onto the labeled dendrite or onto another nonlabeled nearby dendrite. Indeed, B15–20% of the light microscopy–identified contacts were close to gephyrin-labeled structures that were not on the labeled dendrite but were instead elsewhere adjacent to the bouton. This drawback, inherent to conventional light-microscopy imaging, does not conflict with or devaluate our main observation that new GABAergic boutons are formed at pre-existing axon-dendrite crossings without mediating protrusions. Our post hoc immunostaining of newly formed GABAergic boutons showed that boutons can acquire pre- and postsynaptic markers in a period of several hours after their first appearance, suggesting that new functional GABAergic synapses can be formed on a similar time scale as has been previously shown for glutamatergic synapses23,25,29,30. However, for both types of synapses, the formation of morphologically fully mature synapses (with membrane specializations as observed at the electron-microscopy level) may take longer, perhaps even days7,8,23,34.

Dendritic protrusions that contacted GABAergic boutons had substantially shorter lifetimes than protrusions that contacted glutamatergic boutons. This suggests that only short-lived protrusions make contact with GABAergic boutons, that contact with GABAergic boutons promotes the retraction of dendritic protrusions or, perhaps most likely, that contact with glutamatergic boutons can prolong the lifetime of dendritic protrusions. It has been shown that dendritic protrusions can detect and grow toward glutamate35,36 and that dendritic protrusions often make contacts with pre-existing glutamatergic boutons that are already part of a synapse7,8. Stabilization of dendritic protrusions has been shown to be correlated with (appearance or) growth of the postsynaptic density, and this process depends on the activation of glutamatergic receptors9. This suggests that contact with glutamatergic boutons indeed stabilizes dendritic protrusions. The observation that some spines have GABAergic contacts seems to argue against GABA as a retraction signal, although previous studies have shown that spines with GABAergic synapses usually also have a glutamatergic synapse3,26,37,38, potentially stabilizing the spine and ‘protecting’ it against negative GABAergic effects. In any case, our findings clearly indicate that outgrowing dendritic protrusions can distinguish between potential presynaptic partners, even before synapses are formed10,11. It should be kept in mind that the mechanism for the formation of new GABAergic synapses could change with changing circumstances. For instance, during early development, when GABA is depolarizing and can induce calcium influx, GABA might stabilize dendritic protrusions, possibly leading to the formation of GABAergic synapses on dendritic protrusions. Our main finding is that new GABAergic synapses appear to be exclusively formed by the appearance of new boutons at pre-existing axon-dendrite crossings. Although this is in marked contrast with the formation of glutamatergic synapses, in which the outgrowth of dendritic protrusions often has a major role, it is not unique, as glutamatergic axons can also form new boutons at pre-existing spines39.

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ARTICLES A recent study showed that axons appear to have preferred locations for forming new boutons, even in the absence of postsynaptic structure40. At present, it is not clear how the locations for new boutons are determined. One possibility is that these locations are determined by the presence of cell adhesion or other signaling molecules. Indeed, recent studies have shown that the formation of GABAergic and glutamatergic synapses is regulated by different synaptic adhesion molecules41–43. Differential expression of such molecules on dendritic shafts and protrusions could provide an explanation for the different mechanisms underlying the formation of glutamatergic and GABAergic synapses. Our findings imply that GABAergic axons in a mature network can make new synapses only with postsynaptic partners that are in their immediate neighborhood, which is in marked contrast to the way glutamatergic synapses are made. Our findings do not address whether GABAergic and glutamatergic synapses differ in their capacity to undergo plasticity, but they suggest that plasticity in GABAergic connections is more restricted than that in glutamatergic connections. The degree of this restriction depends on the number of available axondendrite crossings. An adaptable GABAergic system requires that GABAergic axons cross and touch the dendrites of a sufficiently large number of potential postsynaptic partners. We found that, in our slice cultures, B60% of axon-dendrite crossings do not have a GABAergic bouton and therefore leave ample opportunity for generating new GABAergic connections. Obviously, plasticity can also be achieved by changing the strength of pre-existing synapses, but so far as synaptic strength is controlled by synaptogenesis, the different rules for plasticity in the mature network may require different developmental strategies for GABAergic and glutamatergic axons44,45. In this context, it should be noted that anatomical studies have shown that many GABAergic axons are highly complex with often tortuous paths, whereas glutamatergic axons tend to follow more linear trajectories45–47. A theoretical analysis of the trajectories of axons revealed that GABAergic axons have substantially larger overlap with the dendritic trees of their potential target neurons than expected from chance, whereas this is not the case for glutamatergic axons44. These findings suggest that glutamatergic axons grow relatively straight, as nearby postsynaptic partners can later grow spines to form synaptic contacts. Conversely, GABAergic axons may cross and touch dendrites of many postsynaptic neurons to enhance their future potential for synapse formation. Taken together, our findings imply that fundamentally different mechanisms underlie the generation of GABAergic and glutamatergic synapses, at least as far as the involvement of dendritic and axonal protrusions are concerned. Glutamatergic synapses use short connecting processes such as filopodia and spines to enable new synaptic connections, whereas GABAergic synapse rely on pre-existing axon-dendrite crossing. This puts substantial structural constraints on the generation and plasticity of GABAergic and glutamatergic synapses, which will be important to keep in mind when trying to understand development and plasticity of the intricate neural networks of the brain. METHODS Cultures. Hippocampal slices (300 mm thick) were prepared from postnatal day 2–5 GAD65-GFP mice17 and maintained using the roller tube technique48. In these mice, 30–50% of hippocampal GABAergic interneurons express GFP, with a steady expression level from early embryonic age to adulthood17. Slices were kept in culture for at least 1 week before the experiments (range, 7–20 d in vitro; mean ± s.d., 12.2 ± 3.8 d in vitro). At this developmental stage, the hippocampal network is mostly mature, but synapse formation still takes place. For the experiments, cultures were transferred into a recording chamber, where they were continuously perfused with carbogenated (95% O2, 5% CO2) artificial cerebrospinal fluid containing 126 mM NaCl, 3 mM KCl, 2.5 mM CaCl2, 1.3 mM MgCl2, 1.25 mM NaH2PO4, 26 mM NaHCO3, 20 mM glucose,


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1 mM pyruvate and 1 mM Trolox. The temperature was maintained at 35 1C. CA1 pyramidal neurons were filled through a patch pipette and 250 mM Alexa Fluor 594 was added to the pipette solution. For experiments with labeled glutamatergic axons, slice cultures of wild-type C57 BL/6 mice were used. Glutamatergic axons were labeled via extracellular bolus loading from a pipette in the CA3 pyramidal layer, as will be described elsewhere (N.B., C.J.W., Fonseca, R., T.B. and Na¨gerl, U.V., unpublished data)29. Imaging. Time-lapse images were acquired using a custom-made two-photon laser-scanning microscope based on an Olympus IX70 microscope with a 40, 1.2-NA water-immersion objective (Olympus). GFP and Alexa Fluor 594 were simultaneously excited using a laser beam tuned to 855 nm (5W Mira-Verdi laser system, Coherent). Laser power at the objective was 4–5 mW. Fluorescence was detected by external photomultipliers (Hamamatsu). The imaged regions varied between 100 and 140 mm (1,024 1,024 pixels) and stacks consisted of 60–99 z layers (step size Dz ¼ 0.5 mm), depending on the overlap between GFP axons and labeled dendrites. Images were taken every 30 min, for a total of 3–6 h. Small misalignments of images between time points as a result of drift were compensated for during the analysis. Data analysis. Image stacks were visually inspected in ImageJ (US National Institutes of Health) to determine all of the locations at which a GFP-labeled axon or bouton was in close proximity to labeled dendritic structures. These locations were subsequently examined in detail at all time points and z sections to detect possible contacts using software written in Matlab (Mathworks). To reduce noise, images were filtered with a 2 2-pixel Wiener filter. Analysis was always carried out on individual image sections. Volume renderings were made in Imaris 5.0.1 (Bitplane) and were used for illustrational purposes only. The overlap between the red and the green channel was determined after thresholding the channels independently. Thresholds were empirically defined by a semi-automatic two-step procedure. In the first step, the background was distinguished from neuronal structures such as dendrites and axons (threshold ¼ mean + 3 s.d. of intensity values of a local 200 200-pixel area). In the second step, boutons were distinguished from axonal shafts (threshold ¼ mean + 1.5 s.d. of axon pixel intensities). In this way, thresholds were objective and automatically corrected for bleaching (B5% in the green and 15–20% in the red channel over the entire imaging period). Nevertheless, we also verified that changing these thresholds only marginally changed the numbers but did not affect our general conclusions. For simplicity, we refer to all axonal swellings or varicosities as boutons, although we are aware that some of them might not contain presynaptic specializations. Conversely, some small presynaptic structures might go unnoticed by this procedure. Furthermore, to avoid ambiguities, all dendritic structures that formed during our observation are simply denoted as protrusions, irrespective of whether they were filopodia like or spine like. We use the term ‘spine’ for stable dendritic structures that were present before and during the entire imaging period. Contacts between a GABAergic bouton and a labeled dendrite were considered to be physical contacts only if the red and the green channel had overlapping pixels in at least two z sections and if the overlap covered at least 10% of the bouton pixels (mean ± s.d. bouton volume ¼ 126 ± 86 pixels, n ¼ 221 boutons). For contact with spines, the value of 10% of the smaller structure (that is, bouton or spine) was used. Because of their small size and low fluorescence levels, outgrowing dendritic or axonal protrusions were considered to be making contact when they showed any overlap (Z1 pixel) with a labeled bouton or dendrite, respectively. The minimal volume for new or transient boutons was set at 15 pixels. The lifetime of transient contacts and protrusions were determined by counting each time point at which the contact or protrusion had been present at 30 min. In principle, for every time point, the actual lifetime could have been between 1 and 59 min. One should also realize that lifetimes beyond the imaging period (3–6 h) are artificially shortened by the end of the imaging period and therefore the distributions (Fig. 5a,b) are inevitably biased toward shorter lifetimes. Immunofluorescence staining. For post hoc immunofluorescence analysis, the GFP signal in GABAergic axons was amplified by immunofluorescence staining (chicken antibody to GFP, 1:1,000, Chemicon). GABAergic synapses were

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ARTICLES visualized by fluorescent labeling of VGAT (rabbit antibody to VGAT, 1:200, Synaptic Systems) and the postsynaptic scaffold protein gephyrin (mouse antibody to gephyrin, 1:400, Synaptic Systems). Slice cultures were fixed in 4% paraformaldehyde (wt/vol, pre-warmed to 35 1C) for 4 h at 4 1C, washed extensively in 0.1 M phosphate buffer and removed from their coverslips to be processed as free-floating slices. Permeabilization and blocking was achieved by incubation of the sections for 24 h at 4 1C in 0.1 M phosphate buffer, 0.4% Triton X-100 (vol/vol) and 10% horse serum (vol/vol). Primary antibodies were applied overnight at 4 1C in 0.1 M phosphate buffer with 0.4% Triton and 5% horse serum. Following extensive washing, appropriate secondary antibodies (Molecular Probes, Invitrogen) were applied overnight at 4 1C at a concentration of 1:200. For the immunostaining of contacts after two-photon imaging, patch pipettes contained an additional 0.1% biocytin, which was visualized after fixation by Avidin Texas Red (Vector Labs). Immunofluorescence labeling was analyzed from high-resolution confocal image stacks (Dz ¼ 0.35–0.4 mm). The association of GFP-labeled boutons with pre- and postsynaptic markers was assessed in individual z sections after filtering and thresholding each channel independently.

1. Freund, T.F. & Buzsaki, G. Interneurons of the hippocampus. Hippocampus 6, 347–470 (1996). 2. Harris, K.M. & Kater, S.B. Dendritic spines: cellular specializations imparting both stability and flexibility to synaptic function. Annu. Rev. Neurosci. 17, 341–371 (1994). 3. Megı´as, M., Emri, Z., Freund, T.F. & Gulya´s, A.I. Total number and distribution of inhibitory and excitatory synapses on hippocampal CA1 pyramidal cells. Neuroscience 102, 527–540 (2001). 4. Ziv, N.E. & Smith, S.J. Evidence for a role of dendritic filopodia in synaptogenesis and spine formation. Neuron 17, 91–102 (1996). 5. Fiala, J.C., Feinberg, M., Popov, V. & Harris, K.M. Synaptogenesis via dendritic filopodia in developing hippocampal area CA1. J. Neurosci. 18, 8900–8911 (1998). 6. Petrak, L.J., Harris, K.M. & Kirov, S.A. Synaptogenesis on mature hippocampal dendrites occurs via filopodia and immature spines during blocked synaptic transmission. J. Comp. Neurol. 484, 183–190 (2005). 7. Knott, G.W., Holtmaat, A., Wilbrecht, L., Welker, E. & Svoboda, K. Spine growth precedes synapse formation in the adult neocortex in vivo. Nat. Neurosci. 9, 1117–1124 (2006). 8. Na¨gerl, U.V., Kostinger, G., Anderson, J.C., Martin, K.A. & Bonhoeffer, T. Protracted synaptogenesis after activity-dependent spinogenesis in hippocampal neurons. J. Neurosci. 27, 8149–8156 (2007). 9. De Roo, M., Klauser, P., Mendez, P., Poglia, L. & Muller, D. Activity-dependent PSD formation and stabilization of newly formed spines in hippocampal slice cultures. Cereb. Cortex 18, 151–161 (2008). 10. Lohmann, C., Finski, A. & Bonhoeffer, T. Local calcium transients regulate the spontaneous motility of dendritic filopodia. Nat. Neurosci. 8, 305–312 (2005). 11. Lohmann, C. & Bonhoeffer, T. A role for local calcium signaling in rapid synaptic partner selection by dendritic filopodia. Neuron 59, 253–260 (2008). 12. Dailey, M.E. & Smith, S.J. The dynamics of dendritic structure in developing hippocampal slices. J. Neurosci. 16, 2983–2994 (1996). 13. Marrs, G.S., Green, S.H. & Dailey, M.E. Rapid formation and remodeling of postsynaptic densities in developing dendrites. Nat. Neurosci. 4, 1006–1013 (2001).

14. Saito, Y., Song, W.J. & Murakami, F. Preferential termination of corticorubral axons on spine-like dendritic protrusions in developing cat. J. Neurosci. 17, 8792–8803 (1997). 15. Yuste, R. & Bonhoeffer, T. Genesis of dendritic spines: insights from ultrastructural and imaging studies. Nat. Rev. Neurosci. 5, 24–34 (2004). 16. Somogyi, P., Tama´s, G., Lujan, R. & Buhl, E.H. Salient features of synaptic organization in the cerebral cortex. Brain Res. Brain Res. Rev. 26, 113–135 (1998). 17. Lo´pez-Bendito, G. et al. Preferential origin and layer destination of GAD65-GFP cortical interneurons. Cereb. Cortex 14, 1122–1133 (2004). 18. Kalisman, N., Silberberg, G. & Markram, H. The neocortical microcircuit as a tabula rasa. Proc. Natl. Acad. Sci. USA 102, 880–885 (2005). 19. Shepherd, G.M. & Harris, K.M. Three-dimensional structure and composition of CA3CA1 axons in rat hippocampal slices: implications for presynaptic connectivity and compartmentalization. J. Neurosci. 18, 8300–8310 (1998). 20. Meyer, M.P. & Smith, S.J. Evidence from in vivo imaging that synaptogenesis guides the growth and branching of axonal arbors by two distinct mechanisms. J. Neurosci. 26, 3604–3614 (2006). 21. De Paola, V. et al. Cell type–specific structural plasticity of axonal branches and boutons in the adult neocortex. Neuron 49, 861–875 (2006). 22. Ruthazer, E.S., Li, J. & Cline, H.T. Stabilization of axon branch dynamics by synaptic maturation. J. Neurosci. 26, 3594–3603 (2006). 23. Ahmari, S.E., Buchanan, J. & Smith, S.J. Assembly of presynaptic active zones from cytoplasmic transport packets. Nat. Neurosci. 3, 445–451 (2000). 24. Friedman, H.V., Bresler, T., Garner, C.C. & Ziv, N.E. Assembly of new individual excitatory synapses: time course and temporal order of synaptic molecule recruitment. Neuron 27, 57–69 (2000). 25. Bresler, T. et al. Postsynaptic density assembly is fundamentally different from presynaptic active zone assembly. J. Neurosci. 24, 1507–1520 (2004). 26. Kubota, Y., Hatada, S., Kondo, S., Karube, F. & Kawaguchi, Y. Neocortical inhibitory terminals innervate dendritic spines targeted by thalamocortical afferents. J. Neurosci. 27, 1139–1150 (2007). 27. Knott, G.W., Quairiaux, C., Genoud, C. & Welker, E. Formation of dendritic spines with GABAergic synapses induced by whisker stimulation in adult mice. Neuron 34, 265–273 (2002). 28. Risher, W.C., Ostroff, L.E. & Harris, K.M. What dendritic filopodia induced by LTP encounter along their path through the neuropil of PN15 rat hippocampus. Abstr. Soc. Neurosci. 135.4 (2006). 29. Ziv, N.E. & Garner, C.C. Cellular and molecular mechanisms of presynaptic assembly. Nat. Rev. Neurosci. 5, 385–399 (2004). 30. Gerrow, K. et al. A preformed complex of postsynaptic proteins is involved in excitatory synapse development. Neuron 49, 547–562 (2006). 31. Maas, C. et al. Neuronal cotransport of glycine receptor and the scaffold protein gephyrin. J. Cell Biol. 172, 441–451 (2006). 32. Chattopadhyaya, B. et al. GAD67-mediated GABA synthesis and signaling regulate inhibitory synaptic innervation in the visual cortex. Neuron 54, 889–903 (2007). 33. Di Cristo, G. et al. Activity-dependent PSA expression regulates inhibitory maturation and onset of critical period plasticity. Nat. Neurosci. 10, 1569–1577 (2007). 34. Ahmari, S.E. & Smith, S.J. Knowing a nascent synapse when you see it. Neuron 34, 333–336 (2002). 35. Portera-Cailliau, C., Pan, D.T. & Yuste, R. Activity-regulated dynamic behavior of early dendritic protrusions: evidence for different types of dendritic filopodia. J. Neurosci. 23, 7129–7142 (2003). 36. Richards, D.A. et al. Glutamate induces the rapid formation of spine head protrusions in hippocampal slice cultures. Proc. Natl. Acad. Sci. USA 102, 6166–6171 (2005). 37. de Zeeuw, C.I., Ruigrok, T.J., Holstege, J.C., Jansen, H.G. & Voogd, J. Intracellular labeling of neurons in the medial accessory olive of the cat. II. Ultrastructure of dendritic spines and their GABAergic innervation. J. Comp. Neurol. 300, 478–494 (1990). 38. Cope, D.W. et al. Cholecystokinin-immunopositive basket and Schaffer collateralassociated interneurones target different domains of pyramidal cells in the CA1 area of the rat hippocampus. Neuroscience 109, 63–80 (2002). 39. Pratt, K.G., Taft, C.E., Burbea, M. & Turrigiano, G.G. Dynamics underlying synaptic gain between pairs of cortical pyramidal neurons. Dev. Neurobiol. 68, 143–151 (2008). 40. Sabo, S.L., Gomes, R.A. & McAllister, A.K. Formation of presynaptic terminals at predefined sites along axons. J. Neurosci. 26, 10813–10825 (2006). 41. Graf, E.R., Zhang, X., Jin, S.X., Linhoff, M.W. & Craig, A.M. Neurexins induce differentiation of GABA and glutamate postsynaptic specializations via neuroligins. Cell 119, 1013–1026 (2004). 42. Chih, B., Engelman, H. & Scheiffele, P. Control of excitatory and inhibitory synapse formation by neuroligins. Science 307, 1324–1328 (2005). 43. Chubykin, A.A. et al. Activity-dependent validation of excitatory versus inhibitory synapses by neuroligin-1 versus neuroligin-2. Neuron 54, 919–931 (2007). 44. Stepanyants, A., Tama´s, G. & Chklovskii, D.B. Class-specific features of neuronal wiring. Neuron 43, 251–259 (2004). 45. Thomson, A.M. & Morris, O.T. Selectivity in the inter-laminar connections made by neocortical neurones. J. Neurocytol. 31, 239–246 (2002). 46. Karube, F., Kubota, Y. & Kawaguchi, Y. Axon branching and synaptic bouton phenotypes in GABAergic nonpyramidal cell subtypes. J. Neurosci. 24, 2853–2865 (2004). 47. Shepherd, G.M., Raastad, M. & Andersen, P. General and variable features of varicosity spacing along unmyelinated axons in the hippocampus and cerebellum. Proc. Natl. Acad. Sci. USA 99, 6340–6345 (2002). 48. Ga¨hwiler, B.H. Organotypic monolayer cultures of nervous tissue. J. Neurosci. Methods 4, 329–342 (1981).

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Statistics. Unless otherwise stated, means are always given ± standard error. Means were compared using the Mann-Whitney U test. Distributions were compared using the w2 test. Multiple comparisons were made by an ANOVA test, followed by a Tukey Honestly Significant Difference (HSD) test. Note: Supplementary information is available on the Nature Neuroscience website.

ACKNOWLEDGMENTS We would like to thank G. Sza´bo for kindly providing the GAD65-GFP mice, U.V. Na¨gerl for help with the experimental setup and comments on the manuscript, N. Sto¨hr and C. Huber for technical assistance, and T. Mrsic-Flo¨gel, C. Lohmann and V. Stein for critical reading of the manuscript. This work was supported by the Max Planck Gesellschaft, the Alexander von Humboldt Stiftung, a Marie Curie Intra-European fellowship (C.J.W.) and the Boehringer Ingelheim Fonds (N.B.). AUTHOR CONTRIBUTIONS C.J.W. designed and conducted the experiments and analyzed the data. N.B. carried out the experiments on glutamatergic boutons. C.J.W. and T.B. conceived the project and wrote the manuscript. Published online at http://www.nature.com/natureneuroscience/ Reprints and permissions information is available online at http://npg.nature.com/ reprintsandpermissions/

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