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Distribution of synapses of CA1 neurons

Distribution of synapses of CA1 neurons


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In the Wikipedia article on dendritic spikes I read:

In the hippocampus, the CA1 neurons contain two distinctive regions that receive excitatory synaptic inputs: the perforant path (PP) through the apical dendritic tuft (500-750 μm from soma) and the Schaffer-collateral (SC) through the basal and apical dendrites (250-500 μm from soma).

I wonder how the distinctiveness of these two regions does appear when plotting the number of synapses as a function of the distance to the soma:

More like the gray, or more like the red, or more like the green curve? Or which other?


One source for this estimate is Megias et al. 2001, an electron microscopy study in CA1 of the rat hippocampus.

I plot their data from Table 3 in the following graph.

The X-axis is not in micrometers. Rather it represents dendritic subclasses. $Ori$ stands for Stratum Oriens, $Rad$ for S. Radiatum, $L-M$ for Lacunosum-Moleculare, $T$ for thick dendrites, $t$ for thin dendrites, $prox/med/dist$ for proximal, medial, and distal, respectively. Stratum Oriens represents basal dendrites that are close to the cell body, stratum Radiatum apical trunks, and stratum Lacunosum-Moleculare the apical tufts. The approximate locations of the layers in reference to the cell body are (in micrometers): $Ori = (-100, 0)$, $Rad = (100, 350)$, $L-M = (350,550)$. The Y-axis represents the total number of synapses, both excitatory and inhibitory.


Total number and distribution of inhibitory and excitatory synapses on hippocampal CA1 pyramidal cells

The integrative properties of neurons depend strongly on the number, proportions and distribution of excitatory and inhibitory synaptic inputs they receive. In this study the three-dimensional geometry of dendritic trees and the density of symmetrical and asymmetrical synapses on different cellular compartments of rat hippocampal CA1 area pyramidal cells was measured to calculate the total number and distribution of excitatory and inhibitory inputs on a single cell.A single pyramidal cell has approximately 12,000 microm dendrites and receives around 30,000 excitatory and 1700 inhibitory inputs, of which 40 % are concentrated in the perisomatic region and 20 % on dendrites in the stratum lacunosum-moleculare. The pre- and post-synaptic features suggest that CA1 pyramidal cell dendrites are heterogeneous. Strata radiatum and oriens dendrites are similar and differ from stratum lacunosum-moleculare dendrites. Proximal apical and basal strata radiatum and oriens dendrites are spine-free or sparsely spiny. Distal strata radiatum and oriens dendrites (forming 68.5 % of the pyramidal cells' dendritic tree) are densely spiny their excitatory inputs terminate exclusively on dendritic spines, while inhibitory inputs target only dendritic shafts. The proportion of inhibitory inputs on distal spiny strata radiatum and oriens dendrites is low ( approximately 3 %). In contrast, proximal dendritic segments receive mostly (70-100 %) inhibitory inputs. Only inhibitory inputs innervate the somata (77-103 per cell) and axon initial segments. Dendrites in the stratum lacunosum-moleculare possess moderate to small amounts of spines. Excitatory synapses on stratum lacunosum-moleculare dendrites are larger than the synapses in other layers, are frequently perforated ( approximately 40 %) and can be located on dendritic shafts. Inhibitory inputs, whose percentage is relatively high ( approximately 14-17 %), also terminate on dendritic spines. Our results indicate that: (i) the highly convergent excitation arriving onto the distal dendrites of pyramidal cells is primarily controlled by proximally located inhibition (ii) the organization of excitatory and inhibitory inputs in layers receiving Schaffer collateral input (radiatum/oriens) versus perforant path input (lacunosum-moleculare) is significantly different.


Sonic hedgehog distribution within mature hippocampal neurons

Sonic hedgehog (Shh) regulates neural progenitor cells in the adult brain but its role in postmitotic mature neurons is not well understood. Using immunoelectron microscopy, we have recently demonstrated the postsynaptic distribution of Patched (Ptch) and Smoothened (Smo), the receptors for Shh, in hippocampal neurons of the adult rat brain. In this study, we describe the distribution of Shh protein in these adult hippocampal neurons. We find that Shh is present in both presynaptic and postsynaptic terminals. In presynaptic terminals, Shh is located either at the center or on the side of the synaptic junction. In postsynaptic terminals, Shh is mostly located on the side of the synaptic junction. We also find Shh in dendrites. Synaptic and dendritic Shh often reside in or are associated with vesicular structures that include dense-cored vesicles, synaptic vesicles, and endosomes. Thus, our subcellular map of Shh and its receptors provides a foundation for elucidating the functional significance of Shh signaling in mature neurons.

Sonic hedgehog (Shh) plays at least two important roles in the nervous system. One is to stimulate the production of stem/progenitor cells, which include granule cell precursors in the young cerebellum 1 - 3 and neural stem cells in the specific brain regions of the adult brain. 4 - 8 The other is to promote axon growth of young neurons, which include spinal cord commissural neurons, 9 , 10 retinal ganglion cells, 11 olfactory sensory neurons, 12 and midbrain dopaminergic neurons. 13 Evidence has indicated that Shh and its signaling components also exist in mature neurons, neurons that do not have progenitor properties. 14 - 16 However, where Shh signaling takes place in these mature neurons and how Shh signaling affects them remain largely unknown.

We have recently described the subcellular distribution of Patched (Ptch) and Smoothened (Smo), the receptor and transducer for Shh respectively, in hippocampal neurons of adult rats. 17 Multiple types of hippocampal neurons express Ptch and Smo, which are particularly concentrated in their dendrites, spines and postsynaptic terminals. 17 Here, we studied the distribution of Shh protein within adult hippocampal neurons.

We used the same hippocampal tissue samples from adult rats that were used in our previous ultrastructural analysis of Ptch and Smo. 17 We performed postembedding immunogold labeling (two animals) using monoclonal anti-Shh antibody (5E1 Developmental Studies Hybridoma Bank). The 5E1 antibody was generated against the N-terminus of Shh (aa1� of rat Shh) 18 and its specificity has been characterized. 10 , 15 , 18 , 19

Because of the preferential distribution of Ptch and Smo in the postsynaptic terminals of hippocampal neurons, 17 we wondered whether Shh also was located near or at the synapse. We examined several hippocampal regions that include the CA1 stratum pyramidal and stratum radiatum, the molecular layer of the dentate gyrus, and the CA3 stratum lucidum. Synapses from these regions exhibited different morphological characteristics. Nevertheless, we found Shh labeling present in all of these synapses ( Fig.ਁ ). Figureꀚ is an example of an inhibitory synapse based on its symmetric appearance. Shh labeling was seen in the presynaptic compartment as well as in the postsynaptic compartment. Within both compartments, the Shh labeling was similarly distributed toward the side rather than the center of the synaptic junction ( Fig.ꀚ ). Upon closer examination, the postsynaptic Shh labeling appeared to be associated with pit-like structures, which were situated opposite or across from the presynaptic Shh labeling ( Fig.ꀚ ).

Distribution of Shh at the synapses in CA1, CA3, and dentate gyrus of the adult rat hippocampus. Immunoelectron micrographs showing Shh immunolabeling at synaptic sites in: the CA1 stratum pyramidal (A,C), the CA1 stratum radiatum (B,D-G), the molecular layer of the dentate gyrus (H), and the CA3 stratum lucidum (I-M). Shh labeling (10 or 15 nm gold particles) is indicated by arrowheads. (A) shows an inhibitory synapse in which Shh labeling is in the presynaptic terminal (p) as well as the postsynaptic soma (so). In the case of both the presynaptic and postsynaptic compartment, Shh labeling is not in the active zone of the synaptic junction but is extrasynaptic. Also notice that the postsynaptic labeling is associated with pit-like structures, which are located opposite and across from the presynaptic labeling. (B)-(D) are examples of excitatory synapses in all of which Shh labeling is in the presynaptic terminal – near the center of the synaptic junction and directly contacting the presynaptic membrane. s, postsynaptic spine. (E)-(G) are also examples of excitatory synapses but in these cases, Shh labeling is found postsynaptically and it concentrates near the side of the postsynaptic membrane. (H) is an excitatory synapse in which Shh labeling is located both in the center and extrasynaptic side of the presynaptic membrane. (I)-(M) show Shh labeling in the mossy fiber terminal (m) and the postsynaptic thorny excrescence (te). Presynaptic Shh labeling is often associated with various vesicular structures (arrowheads in I,J,M) and dense-cored vesicles (arrows in J-M). Postsynaptic Shh labeling is seen in tubulovesicular organelles (I) or associated with the membrane (L). sa in K, spine apparatus * in M marks a spinule. Scale bars are 100 nm.

Figureꀛ-H show typical excitatory synapses based on their prominent postsynaptic density. In these synapses, presynaptic Shh labeling displayed a slightly different pattern from postsynaptic Shh labeling. Presynaptic Shh labeling could be found either directly at the membrane of the synaptic junction ( Fig.ꀛ-D ) or on the side of the terminal ( Fig.ਁH ). Postsynaptic Shh labeling, on the other hand, was found mostly on the side, away from the center of the synaptic junction ( Fig.ꀞ-G ).

FigureਁI-M shows examples of mossy fiber synapses. As for other types of synapses, Shh was found in both the presynaptic mossy terminals and the postsynaptic thorny excrescences. Within the presynaptic mossy terminals, most Shh labeling was clearly seen associated with either dense-cored vesicles (arrows in FigureਁJ-M ), or other smaller vesicular structures (arrowheads in FigureਁI,J,M ). Within the postsynaptic thorny excrescences, Shh was associated with tubulovesicular organelles in some cases (arrowheads in FigureਁI ), or positioned quite close to the postsynaptic membrane in other cases (arrowhead in FigureਁL ).

In addition to synaptic localizations, Shh labeling was found in various tubulovesicular organelles in the soma and dendrites of hippocampal neurons ( Fig.ਂ ). Interestingly, some of these Shh-labeled organelles made direct contact with the cell membrane surface, typically near contacts with adjacent processes ( Fig.ꀪ-C ).

Shh (arrowheads 10 or 15 nm gold particles) is found in tubulovesicular organelles that are located in soma (so) or dendrites (de). These Shh-containing organelles often make direct contact with the membrane surface of the neuron, near adjacent cell processes (pr in A-C). (A), the CA1 stratum pyramidal (B), the CA1 stratum radiatum (C), the molecular layer of the dentate gyrus (D), the CA3 stratum lucidum. Scale bars are 100 nm.

Our previous immunoelectron microscopic work has described the postsynaptic localization of Ptch and Smo in adult hippocampal neurons. 17 The present findings showing the presence of Shh in the presynaptic terminal, in particular localized in close vicinity to or even directly at the synaptic contact, raises the possibility that Shh signaling may occur across the synapse in these neurons. It is then tempting to ask what form of Shh protein is being released from the presynaptic terminal. In the photoreceptor neurons of the developing Drosophila retina, while the Hedgehog (Hh) C-terminus harbors the axonal targeting signal, the N-terminal domain, and a small amount of the full-length Hh, also travel along the axon. 20 Our results obtained using the 5E1 antibody - specific to the Shh N-terminus, could reflect the full-length as well as the N-terminal domain of Shh. To definitively identify the Shh forms that are present and possibly released from the presynaptic terminal will require further studies, including the use of the Shh C-terminus specific antibody.

We also observed an interesting subcellular distribution of Shh in postsynaptic spines and dendrites of hippocampal neurons. Several studies have shown that the dendrite and the postsynaptic terminal of neurons can release transmitters 21 or growth factors. 22 Moreover, studies of other Hh-producing cells have shown that the release of Hh occurs on the apical and the basal side of the Drosophila photoreceptor neurons. 20 Likewise, in Drosophila wing disk epithelium, Hh is found in the apical and basolateral plasma membranes. 23 It will be interesting to investigate whether Shh in mammalian hippocampal neurons is also released from both pre- and post-synaptic sites. The mapping of Shh protein and its receptors at the subcellular level will advance the understanding of where and how Shh signaling occurs, and what roles Shh plays in the function and plasticity of mature neurons.


Results

Generation of 2D electrophoresis maps of synaptosomes from CA3-CA1 hippocampal cultures

We have previously employed a classical fractionation approach for the purification of synaptic membranes and cytosol from CA3-CA1 hippocampal synapses [19]. The starting material was obtained from dissociated cultures, which were prepared from neonatal rat brain (P4-P5) after microdissection of the CA3-CA1 region. In the present study the same source of synaptic material was used to obtain pure synaptosomes and generate 2DE maps of this subpopulation of hippocampal synapses. To evaluate the extent of enrichment in synaptic proteins and the degree of contamination with glia and myelin proteins, subcellular fractions were analyzed by standard electron microscopy (EM) and by western blotting (see Additional file 1). These two methods showed that the degree of sample pureness was indeed much greater in protein extracts from cultures than in extracts from CA3-CA1 hippocampal tissue ([19] and unpublished data).

In figure ​ figure1 1 is presented the 2D reference map of synaptosomes purified from CA3-CA1 cultured neurons. Around 1000 spots were detected in the size range 15� kDa with isoelectric point (pI) values comprised between 4 and 9. The general appearance of these 2D maps, the molecular weight (MW) and pI distributions, the number and the relative intensity of individual protein spots were all reproducible in different experiments (See below n = 12, number of gels used for this study to. This number includes n = 9 analytical and preparative gels, and n = 3 gels used for Western blot analysis the number of spots detected in the best analytical silver stained gels which were used for differential expression analysis is n = 1154 ± 231, mean ± sem n = 3 gels.)


Discussion

The findings presented here show that optogenetic activation of CA1 pyramidal neurons in the hippocampus resulted in a distal relocation of the AIS, but no change in the position of axo-axonic synapses, creating a mismatch between the AIS and its GABAergic inputs. More importantly, our model predicts that this configuration has multiple and far-reaching effects on AP initiation and properties, including modulation of the timing and amplitude of back-propagating APs, both of which are likely to affect long-term plasticity of synaptic inputs. The current threshold to short stimuli is also increased, making it more efficient at filtering out depolarizing synaptic inputs, thus resulting in a homeostatic decrease in neuronal excitability. Interestingly, our model predicted that a relocation of the synapses together with the AIS would have had the opposite effect, an increase in neuronal excitability compared with controls. It appears that the dissociation between the AIS and its axo-axonic synapses leads to an optimal configuration for a homeostatic response to long-term stimulation. Our findings reveal a novel form of plasticity where the position of synapses and their target can be modified with exquisite precision to fine-tune neuronal output.

Distribution of Axo-Axonic GABAergic Synapses in the Hippocampus.

An activity-dependent change in AIS position has previously been reported in dissociated hippocampal neurons (17). Here we show, to our knowledge for the first time, that a similar form of plasticity also occurs in CA1 neurons in organotypic slices, a preparation where the architecture of the network and its connections is preserved. In addition, slices can be taken from postnatal tissue, assuring that the late-born chandelier cells have migrated to the hippocampus before slice preparation (9). As a result, we were able to visualize the GABAergic axo-axonic synapses made by chandelier neurons onto the AIS of pyramidal cells and follow their behavior during AIS plasticity. Axo-axonic synapses formed all along the AIS and were not biased toward the distal end, as observed in cortical neurons. Strikingly, we also observed that synapses were not spatially constrained to the AIS but extended beyond it (19, 23). Although it is unclear what role these synapses play, it is likely that they modulate AP propagation down the axon. In the context of AIS plasticity, these synapses ensure that the AIS is still innervated even after it relocates distally, so that axo-axonic synapses can continue to modulate neuronal output.

Along the axon of CA3 neurons, axo-axonic synapses often fitted in the gaps or holes in the AnkG stain of the AIS. This punctured distribution of AnkG has been previously described in a number of different neurons, including in CA1 and CA3 cells, using superresolution structured illumination microscopy (24). The gaps in AnkG were filled mainly by clusters of the voltage-gated potassium channel Kv2.1, the cisternal organelle marker synaptopodin, and GABAergic synapses. Further high-resolution imaging will need to be done following activity-dependent AIS relocation, especially on the thin axons of CA1 pyramidal cells, to establish what happens to the ultrastructure of the AIS, its synapses, and other proteins associated with axo-axonic synapses. The Kv2.1 channel is particularly interesting because it does not have an AnkG binding domain (unlike other ion channels at the AIS) and undergoes an activity-dependent dispersion (loss of clustering) and change in gating properties thought to be homeostatic in nature (30, 31). These events are calcineurin-dependent (30), much like the AIS relocation in dissociated neurons (16), implying that similar signaling cascades may be acting on both compartments and could thus be responsible for the mismatch between axo-axonic synapses and the AIS observed here.

Our data strongly suggest that there are two spatial domains at the AIS with distinct molecular identities, one based on the scaffolding protein AnkG and the other on the GABAergic scaffolding protein gephyrin. However, recent data in hippocampal neurons has shown that AnkG is generally found adjacent to GABAergic synapses in the somatodendritic compartment and actually interacts with it through its binding of GABARAP (32). The interaction with AnkG is thought to stabilize GABAergic synapses by opposing endocytosis of GABARs (32). Although we found that AnkG levels in the space between the AIS and the soma were either strongly reduced or absent following AIS relocation, in many of our images we do still see weakly labeled clusters of AnkG staining, which may be sufficient to stabilize the synapses left behind after the relocation of AnkG. Alternatively, these particular synapses may not need AnkG to remain stable. Purkinje cells, for example, show no AnkG label in the soma (32), yet they still receive inhibitory inputs, suggesting that the presence of AnkG is not always a prerequisite for a stable GABAergic synapse. In any case, our data did not reveal a change in the number of axo-axonic synapses, although whether the strength of these synapses changed remains to be established (33 ⇓ –35). Previous work has shown that seizures in the brain can lead to a loss of both axo-axonic and axo-somatic GABAergic inputs that may then lead to intractable forms of epilepsy (36, 37). Most of this can be explained by a loss of interneurons through excitotoxic damage, likely caused by the high levels of activity in the network. Our experimental procedure made sure that activity was only increased in single pyramidal cells, by using optogenetic stimulation of pharmacologically isolated neurons. As a result, only cell-autonomous events would be induced that would bypass network hyperactivity and therefore avoid interneuron excitotoxicity.

AIS Plasticity and Neuronal Output.

We observed that CA1 neurons lowered their excitability levels after long-term stimulation. This result is likely because of the change in AIS position, as well as the decrease in Rin. We also found that the shape of the AP changed. The second derivative of the AP in control neurons showed two clear peaks that correspond to the axonal and somatic AP, in that order, and these two peaks were found further apart in stimulated neurons with a more distal AIS (Fig. S5 C and D). The simplest explanation for this delay in the second peak is the larger distance that the AP needs to travel as it back-propagates to the soma. In fact, taking the 12-μm distal relocation of the AIS measured structurally together with the 60-μs increase in delay between the peaks results in an AP propagation speed of 0.2 m/s. This finding is in line with previous measures of AP propagation along unmyelinated axons in the hippocampus (38 ⇓ –40) and was also confirmed in our model, where relocating the AIS caused a similar delay in the second peak (Fig. S6A).

AP shape in stimulated and control CA1 pyramidal neurons. (A) Average AP trace (Left), first derivative (Center), and phase plot (Right) of the first AP elicited in response to 10-ms current injections at threshold. (B) Average AP trace (Left), first derivative (Center), and phase plot (Right) of the first AP elicited in response to 500-ms current injections. (C) Average second derivative of the first AP elicited in response to 10-ms (Upper) or 500-ms current injections (Lower). (D) Delay between the first and second peak in the second derivative of the AP waveform (means ± SEM **P < 0.01 n = 14 ChR2 and ChR2-stimulated, n = 16 Neighbor).

Characterization of AP output from the computational model. (A) AP waveform, its first derivative, phase plot, and second derivative in two different conditions: AIS proximal (AIS distance to soma 3.5 μm) and AIS distal (AIS distance 15 μm). (B) Input-output curve for the two different conditions as described for A, in response to an 800-ms current injection at the soma.

Importantly, our computational model also allowed us to investigate the consequence of GABAergic synapse position on neuronal output and explore different scenarios. It revealed that moving the AIS distally could have opposite effects on AP generation, depending on the position of axo-axonic synapses. When the AIS is moved distally while leaving axo-axonic synapses close to the soma, AP amplitude decreased and latency to the first AP as well as current threshold increased, all indicating a decrease in excitability compared with controls. However, if the synapses were to relocate together with the AIS, moving away from the soma, we found an opposite effect on these parameters, suggesting an increase in excitability compared with controls. One possible explanation for this surprising result is that proximal synapses create a shunt that will partially prevent the charging of the membrane in response to depolarizing inputs and will affect both axonal and nearby somatic membranes. As a result, sodium channels in both compartments will be affected. However, if synapses are moved distally, the shunting effect is also displaced away from the soma, resulting in modulation occurring mainly at the AIS and less so at the soma. Although the AP in our model is initiated at the distal end of the AIS, where membrane conditions are ideal for spike initiation, somatic sodium channels also contribute to AP generation and AP shape in the soma. Proximal synapses will thus prevent activation of both somatic and AIS sodium channels, whereas distal synapses will preferentially act on AIS channels only, resulting in the opposing effects on AP properties shown in this study.

In conclusion, the distal relocation of the AIS together with the proximal distribution of axo-axonic synapses results in the ideal configuration for decreasing neuronal excitability of CA1 pyramidal neurons, and thus for a homeostatic response to long-term stimulation.


Total number and distribution of inhibitory and excitatory synapses on hippocampal CA1 pyramidal cells

The integrative properties of neurons depend strongly on the number, proportions and distribution of excitatory and inhibitory synaptic inputs they receive. In this study the three-dimensional geometry of dendritic trees and the density of symmetrical and asymmetrical synapses on different cellular compartments of rat hippocampal CA1 area pyramidal cells was measured to calculate the total number and distribution of excitatory and inhibitory inputs on a single cell.

A single pyramidal cell has ∼12,000 μm dendrites and receives around 30,000 excitatory and 1700 inhibitory inputs, of which 40% are concentrated in the perisomatic region and 20% on dendrites in the stratum lacunosum-moleculare. The pre- and post-synaptic features suggest that CA1 pyramidal cell dendrites are heterogeneous. Strata radiatum and oriens dendrites are similar and differ from stratum lacunosum-moleculare dendrites. Proximal apical and basal strata radiatum and oriens dendrites are spine-free or sparsely spiny. Distal strata radiatum and oriens dendrites (forming 68.5% of the pyramidal cells’ dendritic tree) are densely spiny their excitatory inputs terminate exclusively on dendritic spines, while inhibitory inputs target only dendritic shafts. The proportion of inhibitory inputs on distal spiny strata radiatum and oriens dendrites is low (∼3%). In contrast, proximal dendritic segments receive mostly (70–100%) inhibitory inputs. Only inhibitory inputs innervate the somata (77–103 per cell) and axon initial segments. Dendrites in the stratum lacunosum-moleculare possess moderate to small amounts of spines. Excitatory synapses on stratum lacunosum-moleculare dendrites are larger than the synapses in other layers, are frequently perforated (∼40%) and can be located on dendritic shafts. Inhibitory inputs, whose percentage is relatively high (∼14–17%), also terminate on dendritic spines.

Our results indicate that: (i) the highly convergent excitation arriving onto the distal dendrites of pyramidal cells is primarily controlled by proximally located inhibition (ii) the organization of excitatory and inhibitory inputs in layers receiving Schaffer collateral input (radiatum/oriens) versus perforant path input (lacunosum-moleculare) is significantly different.


Distinct mechanisms regulate GABAA receptor and gephyrin clustering at perisomatic and axo-axonic synapses on CA1 pyramidal cells

Pyramidal cells express various GABA(A) receptor (GABA(A)R) subtypes, possibly to match inputs from functionally distinct interneurons targeting specific subcellular domains. Postsynaptic anchoring of GABA(A)Rs is ensured by a complex interplay between the scaffolding protein gephyrin, neuroligin-2 and collybistin. Direct interactions between these proteins and GABA(A)R subunits might contribute to synapse-specific distribution of GABA(A)R subtypes. In addition, the dystrophin-glycoprotein complex, mainly localized at perisomatic synapses, regulates GABA(A)R postsynaptic clustering at these sites. Here, we investigated how the functional and molecular organization of GABAergic synapses in CA1 pyramidal neurons is altered in mice lacking the GABA(A)R α2 subunit (α2-KO). We report a marked, layer-specific loss of postsynaptic gephyrin and neuroligin-2 clusters, without changes in GABAergic presynaptic terminals. Whole-cell voltage-clamp recordings in slices from α2-KO mice show a 40% decrease in GABAergic mIPSC frequency, with unchanged amplitude and kinetics. Applying low/high concentrations of zolpidem to discriminate between α1- and α2/α3-GABA(A)Rs demonstrates that residual mIPSCs in α2-KO mice are mediated by α1-GABA(A)Rs. Immunofluorescence analysis reveals maintenance of α1-GABA(A)R and neuroligin-2 clusters, but not gephyrin clusters, in perisomatic synapses of mutant mice, along with a complete loss of these three markers on the axon initial segment. This striking subcellular difference correlates with the preservation of dystrophin clusters, colocalized with neuroligin-2 and α1-GABA(A)Rs on pyramidal cell bodies of mutant mice. Dystrophin was not detected on the axon initial segment in either genotype. Collectively, these findings reveal synapse-specific anchoring of GABA(A)Rs at postsynaptic sites and suggest that the dystrophin-glycoprotein complex contributes to stabilize α1-GABA(A)R and neuroligin-2, but not gephyrin, in perisomatic postsynaptic densities.

Figures

Figure 1. Characterization of α2-KO mice

Figure 1. Characterization of α2-KO mice

Figure 2. Morphological characterization of GABAergic components…

Figure 2. Morphological characterization of GABAergic components in α2-KO mice

Figure 3. Effect of Gabra2 deletion on…

Figure 3. Effect of Gabra2 deletion on GABAergic mIPSCs and their sensitivity to zolpidem in…

Figure 4. Differential alterations in postsynaptic marker…

Figure 4. Differential alterations in postsynaptic marker distribution in CA1 neurons of α2-KO mice, as…

Figure 5. Intact dystrophin, α1 subunit and…

Figure 5. Intact dystrophin, α1 subunit and NL2 clustering in perisomatic synapses of CA1 pyramidal…

Figure 6. Loss of GABAergic postsynaptic markers…

Figure 6. Loss of GABAergic postsynaptic markers in the AIS of mutant CA1 pyramidal cells…

Figure 7. Preservation of presynaptic terminals in…

Figure 7. Preservation of presynaptic terminals in perisomatic and axo-axonic synapses in CA1 neurons of…


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Synaptic Transmission at the CA3-CA1 Glutamatergic Synapse

We reconstructed a 6x6x5 μm3 piece of the rat CA1 hippocampal neuropil containing 465 synapses and parts of 450 axons, 150 dendrites and a single astrocyte (Fig. 1), and used MCell to study the dynamics of transmitter release at presynaptic terminals (Modchang, Nadkarni et al. 2010 Nadkarni, Bartol et al. 2010), the diffusion and spillover of glutamate in extracellular space (ECS) (Kinney Submittted) and the dynamics of calcium in postsynaptic spines (Keller, Franks et al. 2008). An HD video of the reconstruction can be viewed online at: http://www.youtube.com/watch?v=FZT6c0V8fW4.


Fig 1. Frame from an MCell movie shortly after release of glutamate (yellow) from an axon (gray) opposed to a dendritic spine (blue) in reconstructed CA1 neuropil in which only a few of the axons and dendrites are shown (Scientific American, March 2011).

We used MCell to model the entry and diffusion of calcium in the presynaptic terminal and calcium binding to calcium sensors on synaptogamin. We identified three time scales for synchronous and asynchronous vesicle release that were insensitive to the spatial details of the synaptic ultrastructure and accounted for a wide range of experimental results. The model predicted that approximately 64 High-Voltage Calcium Channels were needed in the terminal at an average distance of 300 μm from the calcium sensors to account the range of release probabilities and paired-pulse facilitation (Modchang, Nadkarni et al. 2010 Nadkarni, Bartol et al. 2010).

The 3D geometry of the extracellular space is visualized in Fig. 2 as a solid. In contrast to the accepted value of 20 nm for the extracellular spacing, the reconstruction revealed an interconnected network of large diameter (60 nm) tunnels, formed at the junction of three or more cellular processes, spanned by sheets between pairs of cell surfaces with nearly uniform width (Kinney Submittted).

We used MCell to simulate the entry of calcium through NMDA receptors in the postsynaptic spine and showed that at normal calcium pump densities, the calcium dynamics in spines is almost independent of the spine neck length. This implies that spines can change the length of their neck to make synaptic connections with nearby axons without affecting the critical timing of the calcium signals that control synaptic plasticity (Keller, Franks et al. 2008).


Fig 2. The geometry of extracellular space (ECS) has thin sheet-like surfaces between networks of wide tunnels. (B) ECS vertices were classified as sheet-like or tunnel-like according to the number of neighboring objects. (C) Cross-section through the raw reconstruction showing approximate location of vertices identified as being tunnel-like (blue) and sheet-like (red). Scale bar: 500 nm. (D) One cubic micron showing the ECS as a solid with the intracellular space invisible. ECS width is color coded. (Kinney Submittted)

Affiliations

Pasteur Institute Rome-Department of Physiology and Pharmacology, Sapienza University of Rome, Italy

Giampaolo Milior, Maria Amalia Di Castro, Livio Pepe’ Sciarria, Stefano Garofalo, Davide Ragozzino, Cristina Limatola & Laura Maggi

Inserm U1127, CNRS UMR7225, Sorbonne Universités, UPMC UMR S1127, Institut du Cerveau et de la Moelle épinière, Paris, 75013, France

Department of Cell Biology and Neurosciences, Section of Behavioural Neurosciences, Istituto Superiore di Sanità, Rome, Italy


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