CA1 pyramidal neurons are a major output of the hippocampus and encode features of experience that constitute episodic memories. Feature-selective firing of these neurons results from the dendritic integration of inputs from multiple brain regions. While it is known that synchronous activation of spatially clustered inputs can contribute to firing through the generation of dendritic spikes, there is no established mechanism for spatiotemporal synaptic clustering. Here we show that single presynaptic axons form multiple, spatially clustered inputs onto the distal, but not proximal, dendrites of CA1 pyramidal neurons. These compound connections exhibit ultrastructural features indicative of strong synapses and occur much more commonly in entorhinal than in thalamic afferents. Computational simulations revealed that compound connections depolarize dendrites in a biophysically efficient manner, owing to their inherent spatiotemporal clustering. Our results suggest that distinct afferent projections use different connectivity motifs that differentially contribute to dendritic integration.
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We thank S. Viswanathan and L. Looger (Janelia Research Campus) for sharing the AAV2/1-FLEX-V5 virus, A. Karpova and G. Tervo (Janelia Research Campus) for sharing the AAV2retro-Cre virus, B. Mensh and D. Hunt for critical discussions, and D. Otstot for genotyping and breeding. This work was made possible by funding from the Howard Hughes Medical Institute.
Integrated supplementary information
(a) Example excitatory and inhibitory synapse morphology in SLM (from n = 1 EM volume). Left, serial TEM sections through an asymmetric, excitatory synapse (Gray’s type 1) onto a dendritic spine head. Note the consistently round appearance of the synaptic vesicles and the prominent postsynaptic density (arrowheads). Right, serial TEM sections through a symmetric, inhibitory synapse (Gray’s type 2) onto a dendritic shaft. Note the flattened appearance of many synaptic vesicles and the absence of the postsynaptic density. The scale bar applies to both sets of images. (b) Reconstructed segment lengths from SR (n = 8 independent reconstructed branch segments) and SLM (n = 12 independent reconstructed branch segments) (two-sided Mann-Whitney test, p = 0.91). (c) Excitatory inputs to SR (n = 8 independent reconstructed branch segments) and SLM (n = 12 independent reconstructed branch segments) pyramidal cell dendrites. Left, dendritic spine density as a function of segment length (***two-sided Mann-Whitney test, p < 0.0001). Center, axodendritic excitatory synapse density as a function of segment length (**two-sided Mann-Whitney test, p = 0.002). Right, the distance between neighboring dendritic spines from SR (n = 445 pairs) and SLM (n = 236 pairs) (black lines, separate Gaussian fits). (d) Dendritic spine morphology. Left, cumulative frequency plots of spine volume from individual SR (from n = 616 reconstructed spines) or SLM branches (from n = 322 reconstructed spines). Individual branches shown in thin lines, and pooled data from each domain is shown in thick lines; two-sided KS test, p < 0.0001). Right, histogram showing the percentage of spine volumes from each branch type (p < 0.0001, χ2 test); inset, mean spine volume (***two-sided Mann-Whitney test, p < 0.0001). Data in (d, inset) represent mean ± SEM.
(a) All reconstructed pyramidal cell dendritic segments from SR (top, n = 8) and SLM (bottom, n = 12). The dendritic cable is in gray, and dendritic spines from single connections are shaded orange while those from compound connections are blue and indicated by arrowheads. Note that axodendritic compound connections are not shown, and the dendritic branch may obscure some spines. (b) Density of compound synapses on reconstructed dendritic segments (from n = 8 SR segments and n = 12 SLM segments; ***two-sided Mann-Whitney test, p < 0.0001). (c) Distribution of axon path lengths (left) and angular trajectories (right) in SR (from n = 259 axons) and SLM (from n = 152 axons). Left inset, mean axon path length: **two-sided Mann-Whitney test, p = 0.003. Right inset, mean axon angular trajectory: **two-sided Mann-Whitney test, p = 0.002. (d) SLM path lengths (left) and angular trajectories (right) do not differ between axons making single (from n = 127 SLM axon segments) or compound connections (from n = 25 SLM axon segments). Left, mean path length: two-sided Mann-Whitney test, p = 0.67. Right, mean axon angular trajectory: two-sided Mann-Whitney test, p = 0.29. All box and whisker plots in (c) and (d) have the box depicting the 25th percentile, median, and 75th percentile, and the whiskers depicting 5-95 percentile. Data points outside these ranges are shown as individual circles. (e) Ultrastructural features of sets of coupled synaptic connections on SLM dendrites (from n = 72 compound connections, including the data from the axon-based dataset).
(a) Left, mean spine volume from single synapses (orange, from n = 195 reconstructed spines) and compound synapses (blue, from n = 56 reconstructed spines) (***p < 0.0001, Mann-Whitney test). Right, mean PSD area for the same spines (**two-sided Mann-Whitney test, p = 0.007). Data represent mean ± SEM. (b) Left, relationship between spine volume and spine neck diameter for single (orange, from n = 195 reconstructed spines) and compound synapses (blue, from n = 56 reconstructed spines) (single: Spearman’s correlation, r = 0.31, p < 0.0001; compound: Spearman’s correlation, r = 0.12, p = 0.37; difference between best-fit slopes, p = 0.25). Center, mean spine neck diameter (two-sided Mann-Whitney test, p = 0.32, data represent mean ± SEM.). Right, histogram showing the percentage of spine neck diameters from single (orange) and compound (blue) connections (Χ2 test, p = 0.55). Inset, cumulative frequency plot (two-sided KS test, p = 0.46). (c) Left, a spine (yellow; from n = 251 reconstructed spine synapses) containing both a perforated PSD (denoted by arrowheads) and a spine apparatus (denoted by arrows). Center, cumulative frequency plot of volumes from spines with or without a perforated PSD (two-sided KS test, p < 0.0001). Right, cumulative frequency plot of volumes from spines with or without a spine apparatus (two-sided KS test p < 0.0001). (d) Percentage of single or compound dendritic spine synapses that contain a perforated PSD, a spine apparatus, both, or neither (from n = 195 reconstructed single connection spines and n = 56 compound connection spines). (e) A table of spine morphological parameters (from n = 195 single connection spines and n = 56 compound connection spines).
Supplementary Figure 4 Synaptic vesicles at single and compound SLM spine synapses; related to Fig. 4.
(a) Synaptic vesicle numbers (≤ 50 nm from the presynaptic active zone membrane) at single and compound synapses (from n = 195 reconstructed single connection synapses and n = 56 compound connection synapses). Left, mean number of vesicles per synapse (***two-sided Mann-Whitney test, p < 0.0001). Right, synaptic vesicle density from the same synapses (defined as vesicle number per µm2 of active zone; ***two-sided Mann-Whitney test, p < 0.0001). Bar graphs represent mean ± SEM. (b) The relationship between the number of synaptic vesicles (located ≤ 50 nm from the presynaptic active zone membrane) and the size of the active zone in single and compound synapses (Spearman correlation: single, n = 195, r = 0.84, p < 0.0001; compound, n = 56, r = 0.65, p < 0.0001; comparison between best-fit slopes, p = 0.053). (c) Synapses containing presynaptic mitochondria are associated with larger active zones (AZ) (from n = 251 reconstructed synapses: ***two-sided Mann-Whitney test, p < 0.0001), an increased number of docked vesicles per synapse (vesicles with centers ≤25 nm of the AZ; from n = 251 reconstructed synapses: ***two-sided Mann-Whitney test, p < 0.0001), and a higher density of docked vesicles (vesicles with centers ≤25 nm of the AZ normalized per µm2 of AZ; (from n = 251 reconstructed synapses: ***two-sided Mann-Whitney test, p < 0.0001). Bar graphs represent mean ± SEM.
(a) A subset of reconstructed axon segments from SR (n = 10), with postsynaptic spines colored orange (single connections only on these axon segments). (b) Top, SLM axon segments forming at least one compound connection (n = 25; postsynaptic single connection synapses are orange and compound synapses are blue); bottom, axons forming single connections only (n = 18; postsynaptic single connection synapses are orange). (c) Lengths of SLM reconstructed segments (n = 18 segments making single connections only and n = 25 segments making a compound connection; two-sided Mann-Whitney test, p = 0.06). (d) Single (n = 234 reconstructed spines) and compound (n = 74 reconstructed spines) spines reconstructed from the axonal dataset. Left, volumes of single and coupled spines (χ2 test, p = 0.049); inset, cumulative frequency plot (two-sided KS test, p < 0.0001). Right, the relationships between spine volumes from compound connections (Spearman correlation: n = 42 pairs; r = 0.31, p = 0.04). (e) The number (left) and density (right) of axospinous and axodenritic synapses on reconstructed axon segments from SLM (black horizontal bars represent group means; ***two-sided Mann-Whitney test, p < 0.0001 for all axospinous vs. axodendritic comparisons). (f) The relationship between the segment length and the number of synapses on each segment (Spearman correlations: single: n = 18 axon segments; r = 0.57, p = 0.014; compound: n = 25 axon segments; r = 0.82, p < 0.0001; difference in best-fit slopes, p < 0.0001).
Supplementary Figure 6 Array tomography of retrogradely labeled EC→CA1 synaptic connections; related to Fig. 6.
(a) A retrograde strategy to label EC→CA1 afferent projections. AAV2retro-Cre virus was injected into the SLM area of Thy1 YFP-H mice to drive Cre expression in neurons projecting to the ipsilateral hippocampus. Cre-dependent virus encoding the V5 construct was injected locally into the MEC or LEC to label hippocampal-projecting EC neurons. Note that the axons of EC neurons projecting to the dentate gyrus were also infected in this preparation. (b) Left, maximum intensity projection of the MEC→CA1 array (YFP pyramidal cells in green, MEC axons in magenta). Right, maximum intensity projection of the LEC→CA1 array (YFP pyramidal cells in green, LEC axons in magenta) (from n = 1 large volume array for each projection, though these data replicate the data using the anterograde strategy shown in Fig. 6). Note the reciprocal proximal-distal gradient of axon density in CA1 SLM and the reciprocal labeling in the molecular layers of the dentate gyrus. (c) Similar to the anterograde strategy, the occurrence of compound synapses from retrograde labeling of MEC and LEC afferents differed (n = 149 identified MEC synapses and n = 66 identified LEC synapses: *two-sided Fisher’s exact test, p = 0.018).
(a) A retrograde strategy to selectively label CA1-targeting EC projections. Top, rAAV2retro-Cre virus was injected into the contralateral CA1 SLM, and Cre-dependent viruses encoding Cre-dependent GFP or tdTomato were injected separately into the ipsilateral LEC or MEC, respectively. Bottom, images of GFP-expressing LEC neurons (left), and tdTomato-expressing MEC neurons (from n = 3 independent animal replicates). (b) CA1-targeting afferents in the ipsilateral and contralateral CA1 spanned a large portion of the anterior-posterior axis of dorsal CA1. In CA1 from both hemispheres, a strong proximal-distal gradient is apparent (from n = 3 independent animal replicates). (c) Top, a horizontal slice showing strong labeling of both LEC and MEC afferents in SLM of the ipsilateral hemisphere. Bottom, higher magnification image from the same slice of the contralateral CA1. Note that the imaging settings were changed in order to visualize the fewer axons present (from n = 3 independent animal replicates).
(a) Comparison between a compound synapse pair and the 2x synapse configuration across the entire dendritic morphology of the pyramidal cell. Note that the advantage of compound connectivity is present in the oblique dendrites to the same extent as the tuft dendrites (compare to results from Fig. 8). (b) Left, comparison between the compound synapse configuration and the scaled single input condition (note: the compound synapse pair is the same data from Fig. 8c). Right, in order to produce the same peak dendritic voltage change as the scaled single input in the dendritic shaft, the synaptic conductance in the “2x synapse” condition needs to be increased three-fold. (c) Electrical advantages of the different compound synapse configurations observed by ssTEM. Left, the recording configuration of the ball-and-stick model. Right, peak voltage changes in spine 1, spine 2, and in the dendritic shaft for the 2x configuration and for various different compound synapse configurations. The dashed line is the maximal dendritic depolarization in the 2x synapse configuration.
Serial section TEM of area CA1
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BMC Biology (2018)