Abstract
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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References
Eichenbaum, H. A cortical-hippocampal system for declarative memory. Nat. Rev. Neurosci. 1, 41–50 (2000).
Eichenbaum, H. On the integration of space, time, and memory. Neuron 95, 1007–1018 (2017).
Brun, V. H. et al. Place cells and place recognition maintained by direct entorhinal-hippocampal circuitry. Science 296, 2243–2246 (2002).
Ito, H. T., Zhang, S. J., Witter, M. P., Moser, E. I. & Moser, M. B. A prefrontal-thalamo-hippocampal circuit for goal-directed spatial navigation. Nature 522, 50–55 (2015).
Hargreaves, E. L., Rao, G., Lee, I. & Knierim, J. J. Major dissociation between medial and lateral entorhinal input to dorsal hippocampus. Science 308, 1792–1794 (2005).
Jankowski, M. M. et al. Nucleus reuniens of the thalamus contains head direction cells. Elife 3, e03075 (2014).
Nakashiba, T., Young, J. Z., McHugh, T. J., Buhl, D. L. & Tonegawa, S. Transgenic inhibition of synaptic transmission reveals role of CA3 output in hippocampal learning. Science 319, 1260–1264 (2008).
McHugh, T. J. et al. Dentate gyrus NMDA receptors mediate rapid pattern separation in the hippocampal network. Science 317, 94–99 (2007).
Brun, V. H. et al. Impaired spatial representation in CA1 after lesion of direct input from entorhinal cortex. Neuron 57, 290–302 (2008).
Golding, N. L., Mickus, T. J., Katz, Y., Kath, W. L. & Spruston, N. Factors mediating powerful voltage attenuation along CA1 pyramidal neuron dendrites. J. Physiol. (Lond.) 568, 69–82 (2005).
Stuart, G. & Spruston, N. Determinants of voltage attenuation in neocortical pyramidal neuron dendrites. J. Neurosci. 18, 3501–3510 (1998).
Nicholson, D. A. et al. Distance-dependent differences in synapse number and AMPA receptor expression in hippocampal CA1 pyramidal neurons. Neuron 50, 431–442 (2006).
Jarsky, T., Roxin, A., Kath, W. L. & Spruston, N. Conditional dendritic spike propagation following distal synaptic activation of hippocampal CA1 pyramidal neurons. Nat. Neurosci. 8, 1667–1676 (2005).
Takahashi, H. & Magee, J. C. Pathway interactions and synaptic plasticity in the dendritic tuft regions of CA1 pyramidal neurons. Neuron 62, 102–111 (2009).
Stuart, G. J. & Spruston, N. Dendritic integration: 60 years of progress. Nat. Neurosci. 18, 1713–1721 (2015).
Losonczy, A. & Magee, J. C. Integrative properties of radial oblique dendrites in hippocampal CA1 pyramidal neurons. Neuron 50, 291–307 (2006).
Gasparini, S. & Magee, J. C. State-dependent dendritic computation in hippocampal CA1 pyramidal neurons. J. Neurosci. 26, 2088–2100 (2006).
Ariav, G., Polsky, A. & Schiller, J. Submillisecond precision of the input-output transformation function mediated by fast sodium dendritic spikes in basal dendrites of CA1 pyramidal neurons. J. Neurosci. 23, 7750–7758 (2003).
Legenstein, R. & Maass, W. Branch-specific plasticity enables self-organization of nonlinear computation in single neurons. J. Neurosci. 31, 10787–10802 (2011).
Govindarajan, A., Kelleher, R. J. & Tonegawa, S. A clustered plasticity model of long-term memory engrams. Nat. Rev. Neurosci. 7, 575–583 (2006).
Takahashi, N. et al. Locally synchronized synaptic inputs. Science 335, 353–356 (2012).
Kleindienst, T., Winnubst, J., Roth-Alpermann, C., Bonhoeffer, T. & Lohmann, C. Activity-dependent clustering of functional synaptic inputs on developing hippocampal dendrites. Neuron 72, 1012–1024 (2011).
Wilson, D. E., Whitney, D. E., Scholl, B. & Fitzpatrick, D. Orientation selectivity and the functional clustering of synaptic inputs in primary visual cortex. Nat. Neurosci. 19, 1003–1009 (2016).
Iacaruso, M. F., Gasler, I. T. & Hofer, S. B. Synaptic organization of visual space in primary visual cortex. Nature 547, 449–452 (2017).
Chen, X., Leischner, U., Rochefort, N. L., Nelken, I. & Konnerth, A. Functional mapping of single spines in cortical neurons in vivo. Nature 475, 501–505 (2011).
Chen, T. W. et al. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499, 295–300 (2013).
Kasthuri, N. et al. Saturated reconstruction of a volume of neocortex. Cell 162, 648–661 (2015).
Bartol, T. M. et al. Nanoconnectomic upper bound on the variability of synaptic plasticity. Elife 4, e10778 (2015).
Sorra, K. E. & Harris, K. M. Occurrence and three-dimensional structure of multiple synapses between individual radiatum axons and their target pyramidal cells in hippocampal area CA1. J. Neurosci. 13, 3736–3748 (1993).
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).
Fiala, J. C., Allwardt, B. & Harris, K. M. Dendritic spines do not split during hippocampal LTP or maturation. Nat. Neurosci. 5, 297–298 (2002).
Schmidt, H. et al. Axonal synapse sorting in medial entorhinal cortex. Nature 549, 469–475 (2017).
Kasai, H., Matsuzaki, M., Noguchi, J., Yasumatsu, N. & Nakahara, H. Structure-stability-function relationships of dendritic spines. Trends Neurosci. 26, 360–368 (2003).
Vlachos, A. et al. Synaptopodin regulates plasticity of dendritic spines in hippocampal neurons. J. Neurosci. 29, 1017–1033 (2009).
Holderith, N. et al. Release probability of hippocampal glutamatergic terminals scales with the size of the active zone. Nat. Neurosci. 15, 988–997 (2012).
Sheng, Z. H. & Cai, Q. Mitochondrial transport in neurons: impact on synaptic homeostasis and neurodegeneration. Nat. Rev. Neurosci. 13, 77–93 (2012).
Wouterlood, F. G., Saldana, E. & Witter, M. P. Projection from the nucleus reuniens thalami to the hippocampal region: light and electron microscopic tracing study in the rat with the anterograde tracer Phaseolus vulgaris-leucoagglutinin. J. Comp. Neurol. 296, 179–203 (1990).
Micheva, K. D. & Smith, S. J. Array tomography: a new tool for imaging the molecular architecture and ultrastructure of neural circuits. Neuron 55, 25–36 (2007).
Rah, J. C. et al. Thalamocortical input onto layer 5 pyramidal neurons measured using quantitative large-scale array tomography. Front. Neural Circuits 7, 177 (2013).
Bloss, E. B. et al. Structured dendritic inhibition supports branch-selective integration in CA1 pyramidal cells. Neuron 89, 1016–1030 (2016).
Viswanathan, S. et al. High-performance probes for light and electron microscopy. Nat. Methods 12, 568–576 (2015).
Tervo, D. G. et al. A designer AAV variant permits efficient retrograde access to projection neurons. Neuron 92, 372–382 (2016).
Jasnow, A. M. et al. Thy1-expressing neurons in the basolateral amygdala may mediate fear inhibition. J. Neurosci. 33, 10396–10404 (2013).
Harnett, M. T., Makara, J. K., Spruston, N., Kath, W. L. & Magee, J. C. Synaptic amplification by dendritic spines enhances input cooperativity. Nature 491, 599–602 (2012).
Harvey, C. D., Yasuda, R., Zhong, H. & Svoboda, K. The spread of Ras activity triggered by activation of a single dendritic spine. Science 321, 136–140 (2008).
Spruston, N., Schiller, Y., Stuart, G. & Sakmann, B. Activity-dependent action potential invasion and calcium influx into hippocampal CA1 dendrites. Science 268, 297–300 (1995).
Kim, Y., Hsu, C. L., Cembrowski, M. S., Mensh, B. D. & Spruston, N. Dendritic sodium spikes are required for long-term potentiation at distal synapses on hippocampal pyramidal neurons. Elife 4, e06414 (2015).
Golding, N. L., Staff, N. P. & Spruston, N. Dendritic spikes as a mechanism for cooperative long-term potentiation. Nature 418, 326–331 (2002).
Remy, S. & Spruston, N. Dendritic spikes induce single-burst long-term potentiation. Proc. Natl. Acad. Sci. USA 104, 17192–17197 (2007).
Druckmann, S. et al. Structured synaptic connectivity between hippocampal regions. Neuron 81, 629–640 (2014).
Feng, G. et al. Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP. Neuron 28, 41–51 (2000).
Knott, G. W., Holtmaat, A., Trachtenberg, J. T., Svoboda, K. & Welker, E. A protocol for preparing GFP-labeled neurons previously imaged in vivo and in slice preparations for light and electron microscopic analysis. Nat. Protoc. 4, 1145–1156 (2009).
Sato, T. A modified method for lead staining of thin sections. J. Electron Microsc. (Tokyo) 17, 158–159 (1968).
Fiala, J. C. Reconstruct: a free editor for serial section microscopy. J. Microsc. 218, 52–61 (2005).
Royer, S. et al. Control of timing, rate and bursts of hippocampal place cells by dendritic and somatic inhibition. Nat. Neurosci. 15, 769–775 (2012).
Hines, M. L. & Carnevale, N. T. The NEURON simulation environment. Neural Comput. 9, 1179–1209 (1997).
Acknowledgements
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.
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E.B.B. conceived the project and designed the experiments in consultation with N.S. E.B.B. performed the experiments and analyzed the experimental data. M.S.C. performed the computer simulations and analyzed the simulation data. B.K. performed the image alignment for all experiments. J.C. built the array tomography microscope and assisted with imaging. R.D.F. advised on tissue preparation and ssTEM imaging. E.B.B. and N.S. wrote the paper with input from all coauthors.
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Supplementary Figure 1 Excitatory synaptic connections on SR and SLM dendrites; related to Fig. 1.
(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.
Supplementary Figure 2 Single and compound connections on SR and SLM dendrites; related to Fig. 2.
(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).
Supplementary Figure 3 SLM single and compound spine morphology; related to Fig. 3.
(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.
Supplementary Figure 5 Reconstructions of axon segments from SR and SLM; related to Fig. 5.
(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).
Supplementary Figure 7 Contralateral labeling of CA1-targeting EC projections; related to Fig. 7.
(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).
Supplementary Figure 8 Electrical advantages of compound connectivity; related to Fig. 8.
(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.
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Serial section TEM of area CA1
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Bloss, E.B., Cembrowski, M.S., Karsh, B. et al. Single excitatory axons form clustered synapses onto CA1 pyramidal cell dendrites. Nat Neurosci 21, 353–363 (2018). https://doi.org/10.1038/s41593-018-0084-6
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DOI: https://doi.org/10.1038/s41593-018-0084-6