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Mapping thalamic innervation to individual L2/3 pyramidal neurons and modeling their ‘readout’ of visual input

Nature Neuroscience volume 26, pages 470–480 (2023)Cite this article

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Abstract

The thalamus is the main gateway for sensory information from the periphery to the mammalian cerebral cortex. A major conundrum has been the discrepancy between the thalamus’s central role as the primary feedforward projection system into the neocortex and the sparseness of thalamocortical synapses. Here we use new methods, combining genetic tools and scalable tissue expansion microscopy for whole-cell synaptic mapping, revealing the number, density and size of thalamic versus cortical excitatory synapses onto individual layer 2/3 (L2/3) pyramidal cells (PCs) of the mouse primary visual cortex. We find that thalamic inputs are not only sparse, but remarkably heterogeneous in number and density across individual dendrites and neurons. Most surprising, despite their sparseness, thalamic synapses onto L2/3 PCs are smaller than their cortical counterparts. Incorporating these findings into fine-scale, anatomically faithful biophysical models of L2/3 PCs reveals how individual neurons with sparse and weak thalamocortical synapses, embedded in small heterogeneous neuronal ensembles, may reliably ‘read out’ visually driven thalamic input.

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Fig. 1: Specific labeling of thalamic excitatory afferents onto the full dendritic arbors of L2/3 pyramidal cells.
Fig. 2: Magnified analysis of the proteome allows validation of two-photon scoring criteria for true thalamic boutons.
Fig. 3: Every PSD95+ spine receives either thalamic or cortical input, with thalamic inputs favoring smaller spines. Inhibitory synapses target spines receiving either thalamic or cortical input.
Fig. 4: Distribution of thalamic versus cortical synapses onto L2/3 neurons.
Fig. 5: Thalamocortical and corticocortical synaptic variability results in large response variability of L2/3 cells to visual input.
Fig. 6: Heterogenous synaptic density among L2/3 cells improves readout accuracy of visually driven thalamic input.

Data availability

Data necessary to interpret, verify or extend the research in the article are available upon request.

Code availability

Synapses were annotated using ObjectJ, a freely available ImageJ plugin (https://sites.imagej.net/Kpberry/).

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Acknowledgements

We thank K. Flavahan and K. Villa for technical support, and J. Boivin and M. Helmstaeder for comments on the manuscript. This work was supported by ONR grant N00014-19-1-2036 (to E.N. & I.S.), NEI grants RO1-EY011894 and RO1-EY034419 (to E.N.), JBP Foundation PNDRF award (to E.N. & K.C.), Research Supplement to Promote Re-Entry into Biomedical and Behavioral Research Careers award RO1-EY011894-S1 (to A.B.), JBP Foundation postdoctoral fellowship and NINDS grant K08-NS107591 (to R.G.), The Gatsby Charitable Foundation (to I. S.), and University of Virginia College of Arts and Sciences research funds (to A.E.). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. This work is dedicated to the memory of L. Safra, a great supporter of brain research.

Author information

Author notes

  1. Rebecca Gillani

    Present address: Department of Neurology, Massachusetts General Hospital, Boston, MA, USA

  2. Michael Doron

    Present address: Broad Institute of Harvard University and MIT, Cambridge, MA, USA

  3. Taeyun Ku

    Present address: Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology, Daejeon, Republic of Korea

Authors and Affiliations

  1. Picower Institute for Learning and Memory, Cambridge, MA, USA

    Aygul Balcioglu, Rebecca Gillani, Kendyll Burnell, Taeyun Ku, Kwanghun Chung & Elly Nedivi

  2. Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA, USA

    Aygul Balcioglu, Rebecca Gillani, Kendyll Burnell & Elly Nedivi

  3. The Edmond and Lily Safra Center for Brain Sciences, Jerusalem, Israel

    Michael Doron & Idan Segev

  4. Department of Neurobiology, The Hebrew University of Jerusalem, Jerusalem, Israel

    Michael Doron, Kwanghun Chung & Idan Segev

  5. Institute for Medical Engineering and Science, Cambridge, MA, USA

    Taeyun Ku & Kwanghun Chung

  6. Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA

    Taeyun Ku

  7. Department of Psychology, University of Virginia, Charlottesville, VA, USA

    Alev Erisir

  8. Broad Institute of Harvard University and MIT, Cambridge, MA, USA

    Kwanghun Chung

  9. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA

    Elly Nedivi

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Contributions

A.B. and E.N. designed the study. A.B. conducted all in vivo imaging experiments and data analysis. R.G. conducted all MAP experiments with T.K. and analyzed the data. R.G. developed two-photon scoring criteria based on MAP with A.B.’s help. T.K. established the MAP protocol under K.C.’s supervision. A.E. conducted all EM experiments and data analysis. K.B. contributed Extended Data Figs. 2 and 8. All modeling was done by M.D. with supervision by I.S. The manuscript was written by E.N. and I.S. with contributions from all authors.

Corresponding author

Correspondence to Elly Nedivi.

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Extended data

Extended Data Fig. 1 Crossing the Olig 3 Cre driver line to a Cre dependent Syn-Tdtomato reporter line faithfully and exclusively labels thalamic boutons from LGN but not LP.

Olig3/+ mice were crossed with Ai14 reporter mice expressing Cre-dependent Tdtomato. (a–c) Sections at different Bregma coordinates on the anterior posterior axis of the brain show cellular labeling exclusive to thalamic nuclei, with thalamic afferents visible in the internal capsule and layer 4 of primary sensory cortices. (d–f) Sections through visual cortex immunostained for Tdtomato (red) and VGlut2 (green) show perfect overlap in thalamic afferents in layers 4 and 1. g–j) Coronal sections at Bregma coordinates that include both LP and LGN show predominantly LGN labeling in Olig3−/+/Ai14 mice. Thal, thalamus; PoT, posterior thalamic nucleus; MGB, medial geniculate body; L4, cortical layer 4; L1, cortical layer1; BC, barrel cortex; LGN, lateral geniculate nucleus; IC, internal capsule; CP, caudate putamen; LV, lateral ventricle. (k–m) High magnification view in layer 2/3 of visual cortex showing beads-on-a-string appearance of thalamic boutons and specificity of VGlut2 staining to these boutons. (n-p) MAP processed sample labeled with n) anti-RFP to label genetically encoded tdTomato in thalamic boutons, and o) VGluT2. p) Composite image shows that all thalamic boutons genetically encoded with tdTomato, also are VGluT2 positive. q) Quantification of RFP and Vlut2 bouton co-labeling in MAP processed samples. Approximately 91% of Vglut2 boutons are RFP positive, with 9% potentially representing non-thalamic inputs. All (100%) RFP labeled boutons were Vlut2 positive. Brains from three animals were sectioned and imaged, and representative sections are shown in a–f, h, j, k–p). Scale bars: (a-c) 0.5 mm; (d–f) 100 µm; (h–j) 1 mm; (k–m) 2 µm; (n-p) 10 µm.

Extended Data Fig. 2 Two photon imaging system alignment is compatible with its theoretical diffraction limited resolution.

Two hundred nm fluorescent bead imaged on 2-photon system with a 1.0 NA objective at 915 nm with 100 nm/pixel XY sampling and 250 nm Z steps. a) Intensity plot on the XY axis (shown in red) with full width half maximum measured as 400 nm, as predicted by the Abbe limit. b) Intensity plot of the Z axis with full width half maximum measured as 1um.

Extended Data Fig. 3 Genetic background and plasmid system do not affect spine and synaptic densities, or spine volumes, on imaged neurons.

a) To confirm that the Olig3/Syntd genetic background and use of the Flp/FRT vs Cre/lox plasmid system does not influence synaptic densities, densities of all, PSD95 + , and PSD95- spines were compared with previously published data1. No difference was found between backgrounds and plasmid systems in any category of spines imaged imaged (Nested t test, t(4)= 0.2585, p = 0.808, n = 51 from 17 animals). b) Thalamic vs cortical dendritic spine volumes were compared in MAP images of L2/3 labeled neurons from B6 vs Olig3 mice, where thalamic synapses were identified by VGlut2 staining. Scatter plots of spine sizes shown per each MAP cell demonstrate that synaptophysin expression does not influence either thalamic (Nested t test, t(3) = 0.07544, p = 0.9446, n = 78 spines from 5 animals) or cortical dendritic spine volumes (Nested t test, t(3) = 0.6727, p = 0.5493, n = 721 spines from 5 animals).

Extended Data Fig. 4 Comparison of EM and MAP validates that MAP expands tissue uniformly.

a) Representative EM image showing a VGluT2+ terminal bouton (TC) that forms a perforated synapse on a spine (sp), and a small, non-perforated synapse on a dendrite shaft (d; identified by the presence of a mitochondria within). Yellow arrowheads mark synaptic zones from the postsynaptic side. An unlabeled terminal bouton (non-TC) that forms a perforated synapse on a spine is also marked. Scale bar = 250 nm. b) Magnification of 1 x-y plane of a MAP imaged dendritic spine, shown in white, with Vglut2 labeled terminal bouton (thalamic, magenta). Contours for measurement of spine area and terminal bouton area are shown. c) Same as b) but with a spine contacting a VGluT1 labeled bouton (cortical, green). Scale bars = 1 µm. d) Ratio of spine head area/terminal bouton area match for MAP and EM for thalamic synapses (Mann–Whitney U test, p = 0.9399, n = 76 for MAP and 33 for EM) and e) cortical synapses (Mann–Whitney U test, p = 0.1056, n = 69 for MAP and 61 for EM). Sp, dendritic spine; D, dendrite.

Extended Data Fig. 5 Distribution of thalamic vs cortical inputs onto L2/3 neurons.

Additional dendrograms showing the distribution of thalamic versus cortical inputs onto the dendritic arbors of four additional pyramidal neurons in L2/3 of binocular V1 not presented in Fig. 4. Apical and basal dendrites are shown on opposite sides of the cell body (marked as 0 on the scale). The scale illustrates the distance of each input from the cell body. Note that cells 5 & 3, are deeper, and shown on a different scale. Dashed lines depict branch segments that were traced, but not scored. Insets (top left) in each dendrogram show the traced skeleton of that cell with location of scored inputs.

Extended Data Fig. 6 Diverse morphology and statistics of the nine pyramidal cells analyzed.

Maximum intensity projections of cells imaged in vivo are presented in order of somal depth in relation to pia, with top left most superficial and bottom right deepest. Note that superficial cells have atypical apical dendrites as compared to deeper cells. They also tend towards more extensive basal dendrites (for example, Cell 1 has 13 basal dendrites while having only 2 apical dendrites. A similar morphology exists for Cell 2), while deeper cells have more apical and fewer basal dendrites (for example, Cell 9). Since distinction of basal vs apical is not always obvious in the maximum intensity projections, white arrows point to a basal dendrite for each cell, and an appended movie file shows a representative Z stack. A representative Supplementary video 1 shows a Z stack of Cell 7 encompassing both apical and basal dendrites. See Fig. 4c–d in main text for number of apical vs basal dendrites scored for each cell.

Extended Data Fig. 7 Thalamic inputs are heterogeneously distributed across individual branches of pyramidal cells, and across cells.

The overall percent of the thalamic vs. cortical synapses onto individual branches (marked as B followed by branch number) of all nine pyramidal cells imaged, with apical branches on the right and basal dendrites on the left for each cell. TA, TB, and Total summarize total apical and total basal, and total per cell, respectively.

Extended Data Fig. 8 Thalamic innervation is not correlated with dendritic branch depth or local bouton density.

a) Coronal section through visual cortex stained with DAPI (left) from Olig3/synTd mouse showing synTd labeling of thalamic pre-synaptic boutons (middle). SynTd boutons were counted per 100x25um bins and their density was plotted as a function of distance from the pial surface (right). Error bars: SEM n = 4 mice. Red triangles are a schematic representation of Cell 6 and Cell 9 with their respective % thalamic innervation. Their somal depth with basal and apical dendrite span are mapped in relation to the bouton density graph. While both Cell 6 & 9 are within L2/3, because of their different somal depth, their basal dendrites extend to to different depths. Although Cell 9 basal dendrites project much deeper than those of Cell 6 into a region with higher thalamic bouton density, Cell 6 has almost double the thalamic innervation. b) Diagram demonstrating how thalamic innervation onto specific dendritic branches was scored in relation to local thalamic bouton density, with 4x4um ROIs placed around each branch segment imaged in-vivo in the z-planes where it was brightest (green: synTd, cyan: PSD95-teal, red: YFP). c) Representative branches varying in depth were scored as shown in b, showing the number of thalamic synapses on each branch is unrelated branch depth or local thalamic bouton density. d) Linear regression comparing density of thalamic synapses on branches versus depth from pia (left) and local bouton density (right), including data from c, shows no correlation between branch depth, local thalamic bouton density, and number of thalamic contacts per branch.; r2 = 0.084 p = 0.449 and r2 = 0.209 p = 0.215 respectively.

Extended Data Fig. 9 Experimentally based model of L2/3 pyramidal cell in V1.

(a) Voltage response of typical cell (shown in inset) following a 3 msec and 0.6 nA subthreshold depolarizing step current. Blue, experimental trace; black, model. The morphology and physiology of this cell was taken from the Allen Institute Cell Atlas (see Methods). (b) Voltage response of the cell shown in a to three subthreshold long current pulses (0.01 nA, 0.03 nA and 0.05 nA, given for 1000 msecs). Blue, experimental trace; black, model. (c) Spiking response of the cell shown in a (in blue) to suprathreshold current injections (1.17 nA for 1000 msecs); model response is shown in black. (d) The response of a L4 cortical pyramidal cells model (inset, cell reconstructed by the Blue Brain Project, see Methods) to the activation of a single thalamo-cortical axon. The experimental somatic EPSP is shown in blue (courtesy of Randy Bruno, see Methods); thick black line shows the average EPSP in the biophysical model constructed for this L4 cell in response to the simultaneous activation of 7 synaptic contacts (red synapses). The gray area shows the standard deviation around the mean of the somatic EPSPs in repeated activation of 7 synaptic contacts that were randomly distributed repeatedly across the modelled dendritic tree. The best fit was achieved with peak conductance of 0.31 nS for both the AMPA- and the NMDA- components; see Methods for details of synapse and neuron models.

Supplementary information

Supplementary Information

Supplementary Tables 1–3

Reporting Summary

Supplementary Video

75-µm z-stack at 1-micron intervals of cell 7 showing both apical and basal dendrites.

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Balcioglu, A., Gillani, R., Doron, M. et al. Mapping thalamic innervation to individual L2/3 pyramidal neurons and modeling their ‘readout’ of visual input. Nat Neurosci 26, 470–480 (2023). https://doi.org/10.1038/s41593-022-01253-9

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