The subcellular organization of neocortical excitatory connections

Leopoldo Petreanu; Tianyi Mao; Scott Sternson; Karel Svoboda

doi:10.1038/nature07709

. Author manuscript; available in PMC: 2009 Sep 17.

Published in final edited form as: Nature. 2009 Feb 26;457(7233):1142–1145. doi: 10.1038/nature07709

The subcellular organization of neocortical excitatory connections

Leopoldo Petreanu ¹, Tianyi Mao ¹, Scott Sternson ¹, Karel Svoboda ¹

PMCID: PMC2745650 NIHMSID: NIHMS143295 PMID: 19151697

Abstract

Understanding cortical circuits will require mapping the connections between specific populations of neurons ¹, as well as determining the dendritic locations where the synapses occur ². The dendrites of individual cortical neurons overlap with numerous types of local and long-range excitatory axons, but axodendritic overlap is not always a good predictor of actual connection strength ³^-⁵. Here we developed an efficient Channelrhodopsin-2 (ChR2)-assisted method ⁶^-⁸ to map the spatial distribution of synaptic inputs, defined by presynaptic ChR2 expression, within the dendritic arbors of recorded neurons. We expressed ChR2 in two thalamic nuclei, the whisker motor cortex and local excitatory neurons and mapped their synapses with pyramidal neurons in layers (L) 3, 5A, and 5B in the mouse barrel cortex. Within the dendritic arbors of L3 cells, individual inputs impinged onto distinct single domains. These domains were arrayed in an orderly, monotonic pattern along the apical axis: axons from more central origins targeted progressively higher regions of the apical dendrites. In L5 arbors different inputs targeted separate basal and apical domains. Input to L3 and L5 dendrites in L1 was related to whisker movement and position, suggesting a role of these signals in controlling the gain of their target neurons ⁹. Our experiments reveal exquisite specificity in the subcellular organization of excitatory circuits.

We recorded from pyramidal neurons in neocortical brain slices containing ChR2-expressing axons ⁷(Fig. 1a). To map the dendritic locations of input from ChR2-positive axons (Fig. S1) we used a laser to depolarize these axons only in the vicinity of the laser beam (i.e. with action potentials blocked), triggering local glutamate release (subcellular Channelrhodopsin-2 Assisted Circuit Mapping, sCRACM). We blocked Na⁺ channels (1 μM tetrodotoxin,TTX ) and the K⁺ channels that are critical for repolarization of the axon (200 nM α-dendrotoxin or 100 μM 4-aminopyridine, 4-AP) (Fig. 1 b)¹⁰. Under these conditions, photostimulation with short (1 ms) light pulses (< 2 mW) triggered robust excitatory postsynaptic currents (EPSC_sCRACM) (Fig. 1 b). Higher light intensities caused larger EPSC_sCRACM amplitudes (Fig. 1 c) and shorter onsets (Fig. S2). As the cylindrical laser beam was scanned over the dendrites of a recorded neuron (map pattern: 12 × 24 grid; 50 μm spacing), EPSC_sCRACM were detected only when the laser beam overlapped with the dendritic arbor of the recorded cell and with ChR2-positive axons (Fig. 1 b, d-f), indicating that under these conditions light depolarizes ChR2-positive axons to cause local release of neurotransmitter. Converting EPSC_sCRACM into pixel values (EPSC_sCRACM averaged over a time window 0 – 75 ms post-stimulus) provides a two-dimensional ‘image’ of the distribution of specific input within the dendritic arbors of the recorded cell (Fig. 1 e,f). Measurements of the point-spread-function revealed that sCRACM maps specific types of input with ~ 60 μm spatial resolution (Fig. S3).

a, Confocal image showing L2/3 neurons expressing mCherry (red) and ChR2-Venus (green) in the barrel cortex. b, Left, Schematic of the photostimulation geometry. Right, EPSCs evoked by photostimuli corresponding to the locations indicated in the schematic. Blue ticks indicate the laser pulse. Laser power is indicated on top. c, EPSC_sCRACM evoked by photostimulation with increasing laser powers (right). d, Brightfield image of a brain slice showing the recording pipette and the photostimulation grid (blue dots). e, sCRACM map overlayed on a fluorescence image, showing ChR2-positive neurons, and the reconstructed dendrite of the recorded neuron (same experiment as d). Non-zero EPSC_sCRACM are color-coded to represent mean amplitude. f, EPSC_sCRACM recorded in the boxed regions in Panel e.

EPSC_sCRACM amplitudes depend on the density of ChR2-positive axons, the fraction of axons that make synapses with the recorded neuron, the strength of the synapses, and their electrotonic distance from the soma ¹¹ (see Methods). Since the density of ChR2-positive axons varies between preparations, sCRACM maps were normalized to the largest pixels within a map and thus represent the relative strength of input within the dendritic tree. Repeated sCRACM maps where reproducible at the level of single pixels (Fig. S4), and the structure of peak-normalized maps was similar across a large range of light intensities (Fig. S4).

Multiple types of axon overlap with the dendrites of cortical neurons ⁵^,¹²^-¹⁷. Some axons arise locally ⁵^,¹⁴^,¹⁸, whereas others ascend from the thalamus ¹⁵^,¹⁶ or descend from higher cortical areas ¹⁷. Which axons in a target region connect with a particular cell type? And what are the spatial distributions of input within the dendritic tree? To answer these questions we expressed ChR2 in five distinct axonal populations (in separate experiments) that overlap with pyramidal cell dendrites in the barrel cortex (Fig. S5). The spatial distribution of labeled axons in the barrel cortex was largely in agreement with previous anatomical studies (Fig. S5). Projections from the ventral posterior medial nucleus (VPM) of the thalamus were focused in L4 and at the border of L5 and L6 (Fig. 2 a and Fig. S5) ¹⁶, but diffuse axons were found throughout all cortical layers ¹⁵. L4 axons arborized in L4 and ascended into L2/3, up to the lower edge of L1 ¹⁸; a weaker projection descended into L5 and L6 ¹⁸. Axons from L2/3 pyramidal cells arborized within L2/3 and on the border of L5A and L5B ⁷^,¹⁴. Axons from the primary whisker motor cortex (M1) arborized densely in L1, and more diffusely in L5 and L6 ¹⁷. A dense bundle of ascending M1 axons was often apparent next to the most medial barrels (Fig. 2 a, arrow head). Axons from the posterior medial nucleus (POm) of the thalamus were focused in L5A, and more weakly in L1 ¹⁵.

a, Examples of sCRACM maps overlaid on reconstructed dendrites and fluorescence images showing ChR2-positive axons (VPM, M1 and POm) or axons and dendrites (L2/3 and L4). White arrow head, bundle of ascending axons from M1. b, Group averages aligned by pia position (White triangles, soma position). c, top: Group averages aligned by soma position ; bottom: vertical profiles of the distribution of synaptic input (red) and the dendritic length density (green; from d). d, Average normalized dendritic length density of L3 pyramidal neurons. Error bars, s.e.m.

We mapped specific types of input within the dendritic trees of individual L3 cells (Fig. 2a). Maps were then averaged across cells either aligned on the pia (Fig.2 b), to visualize the laminar location of the inputs, or aligned on the soma, to measure the location of the inputs relative to the soma (Fig.2 c). L3 cells received input from all five projections. Each input overlapped with a single contiguous dendritic subregion. Ascending VPM→L3 input was focused on the bottom part of the basal dendritic arbors. Input from ascending L4→L3 axons was centered on the soma and basal arbor, above the input from VPM. Input from recurrent L2/3→L3 axons was mostly in the upper basal dendrites and the apical oblique dendrites, above the input from L4 (see also Fig.S6). Feedback from M1 targeted the tuft branches in L1, above the input from L2/3. The positions of VPM, L4, L2/3, and M1 input along the apical axis of L3 neurons mirrors the flow of excitation within the cortical circuit: more peripheral (central) input impinges on lower (higher) parts of the dendritic arbor. During somatosensation L3 neurons thus receive an ascending wave of excitatory input. POm→L3 input was weighted towards L1, although it was distributed relatively broadly, spanning most of the dendritic arbor.

We next mapped the same group of five inputs within the dendrites of L5 pyramidal neurons. Both L5A and L5B pyramidal cells received input from L4, L2/3, M1 and VPM (Fig.3 a,b and S7) . To quantify the strength of input from defined axonal projections across postsynaptic cells in different layers, we recorded from pairs of cells in the same column with identical laser powers (Supplemental table 1). L5B cells received 62-fold less input from POm compared to L5A cells, despite pronounced overlap between L5B dendrites and POm axons (Fig. 3c,d, Fig. S7 and Supplemental table 1) (dendritic length: L5A pyramids (n=12), in L1, 871±546 μm , in L5A, 2158±899 μm; L5B pyramids (n=13), in L1, 1609 ±732 μm, in L5A, 761 ± 350 μm). L5A, but not L5B, pyramidal cells received significant input from POm. This confirms that average cortical connectivity between populations of neurons cannot always be deduced from the structure of axons and dendrites alone ³^,⁴^,⁵ . Because L5B pyramidal neurons constitute the main projection from barrel cortex to POm ¹⁹, there appears to be no disynaptic loop between these two areas.

a, Examples of sCRACM maps overlaid on reconstructed dendritic arbors and fluorescence images. b, Group averages aligned by pia position. White triangles, soma position. c, sCRACM map of POm input onto a L5A pyramidal cell (blue). No responses were detected on the L5B neuron (green). d, EPSC_sCRACM recorded on the L5A neuron (blue) or the L5B neuron (green) when photostimuating the boxed regions in Panel c . The stimulus occurred at the beginning of each trace.

In contrast to L3 cells, the inputs on L5 cells were not limited to a single compartment, but were split into basal and apical domains (Fig.3 and Fig. S7), reinforcing the view that large pyramidal neurons consist of multiple, weakly coupled compartments ²⁰. Here we describe the inputs to L5B neurons (Fig. 3), and then highlight the differences with L5A neurons (Fig. S7). VPM→L5B input was distributed along most of the dendritic arbor, but was most prominent on the basal dendrites and in L4. L4→L5B input was centered on the basal dendrites, overlapping with VPM input; weak input was also detected along the apical dendrite up to the edge of L1. L2/3→L5B input was focused on the upper basal and apical oblique dendrites, as well as on the apical tuft in L2. M1→L5B input was on the basal dendrites and on the apical tuft in L1.

Inputs to L5A pyramidal neurons similarly targeted dual dendritic compartments (Fig. S7), with some differences. POm →L5A input was prominent, both on the basal dendrites and the apical tuft in L1. Unlike for L2/3→L5B input, L2/3→L5A input was centered on the basal dendrites (Fig. 4 a and Fig. S7). For all L5 neurons taken together, there was a monotonic relationship between the laminar position of the recorded cell and the location of L2/3 input relative to the soma position (Fig. 4 b-d). The axodendritic overlap of L2/3 axons and L5 dendrites is likely an important factor determining the subcellular location of L2/3→L5 input.

a, Examples of the subcellular distribution of L2/3 input on superficial (L5A) (left) or deep (L5B) (right) pyramidal neurons. b, Vertical profiles of the subcellular distribution of L2/3→L5 inputs. Each column represents one cell, ordered by cortical depth. Cells were aligned by pia position. The relative density of L2/3 axons in the deep layers is indicated to the left of the panel. c, Average subcellular location of L2/3 input (aligned by soma position) of L5 cells grouped by increasing distance from the pia (groups correspond to the white lines in b).d, Plot of the vertical distance from the soma to the center of mass of L2/3 input on the perisomatic region ( < 285 μm from the soma) of L5 pyramidal neurons vs. cortical depth. The line is a regression fit.

sCRACM maps functional neural circuits with subcellular resolution. Since sCRACM maps are based on somatic measurements of synaptic currents generated in the dendrites signal attenuation due to dendritic filtering influences the structure of the maps. For example, input on the apical tufts of L5 neurons could be reduced several-fold compared to more proximal input ¹¹. sCRACM maps thus represent a “somatocentric” view of the dendritic distribution of synaptic input, where electrotonically distant synapses will appear relatively weak (Figure S8).

We mapped input from VPM, L4, L2/3, M1 and POm, within the dendritic arbors of L3 and L5 neocortical pyramidal cells. L3 and L5 cells received input from most axonal populations, with the exception of POm→L5B. Some connections (VPM→L3, VPM→L5A, VPM→L5B, POm→L5A, L4→L3, L4→L5A, L2/3→L3, L2/3→L5A, L2/3→L5B) have been previously characterized ³^,¹⁴^,¹⁸^,²¹^,²², whereas others (M1→L3, M1→L5A, M1→L5B, POm→L3, L4→L5B,) were previously unknown.

We identified several connections in L1: M1→L3, M1→L5A, M1→L5B, POm→L3 and POm→L5A. Axons from VPM, L2/3 and L4 did not contribute significantly to L1 input. POm neurons are thought to encode aspects of whisker position ²³ and whisker M1 carries signals related to voluntary whisker control ²⁴. Our findings suggest that synapses in L1 carry signals related to whisker movement and position.

The spatial segregation of specific types of input within dendritic arbors might subserve several functions. Segregated inputs are less likely to interact at the level of synaptic plasticity ²⁵. Spatially clustered coactive synapses are more efficacious in driving postsynaptic neurons than spatially distributed synapses ²⁶^,²⁷. For a fixed number of synapses, spatial segregation of different axonal populations within dendritic arbors might thus serve to strengthen the effective coupling between pre- and postsynaptic populations.

Supplementary Material

Supplementary Methods

In utero electroporation. Experimental protocols were conducted according to the National Institutes of Health guidelines for animal research and were approved by the Institutional Animal Care and Use Committee at Janelia Farm Research Campus. Venus ³⁴ (gift from A. Miyawaki) was fused to the C-terminus of the first 315 amino acids of channelrhodopsin-2 (gift from G. Nagel). The constructs were inserted into pCAGGS vector modified for in utero electroporation . DNA was purified and concentrated using Qiagen plasmid preparation kits and dissolved in 10 mM Tris–HCl (pH 8.0).

L2/3 progenitor cells were transfected via in utero electroporation ²⁸, as previously described ⁸. E16 timed-pregnant C57BL / 6J mice (Charles River, Wilmington, MA) were deeply anesthetized using an isoflurane-oxygen mixture (1% vol isoflurane / vol O₂) delivered by an anesthesia regulator (SurgiVet, Waukesha, Wisconsin). The uterine horns were exposed and ~ 1 μl of DNA solution with Fast Green (Sigma) was pressure injected (Picospritzer, General Valve) through a pulled glass capillary tube (Warner Instruments, Hamden, CT) into the right lateral ventricle of each embryo. The DNA solution contained a mixture of plasmids encoding ChR2-Venus and mCherry in a 3:1 molar ratio, at final concentration of 2 μg/μl. The head of each embryo was placed between custom-made tweezer-electrodes, with the positive plate contacting the right side of the head. Electroporation was achieved with 5 square pulses (duration = 50 ms, frequency = 1Hz, 40V). mCherry fluorescence was used to screen for positive animals under a fluorescence dissecting scope (MVX10, Olympus), 1 to 2 days after birth. The transfected cortical region in electroporated animals was always restricted to L2/3 in the electroporated hemisphere. It usually encompassed most of the barrel cortex and in some cases included parts of auditory, visual and secondary somatosensory areas.

Virus preparation and injections. The ChR2-Venus fusion was cloned into an adeno-associated viral cassette (serotype 2/1) containing the CAG promoter, a woodchuck posttranscriptional regulatory element (WPRE) and the SV40 polyadenylation site. Viral vectors were packaged and purified to 1.8×10¹² gc/ml (University of Pennsylvania Vector Core). C57BL / 6J mice (Charles River, Wilmington, MA) were deeply anesthetized using an isoflurane-oxygen mixture (1 % vol isoflurane / vol O₂) at postnatal day 12-15 and placed in a custom stereotactic apparatus. A small incision was made in the scalp to expose the skull. We injected bilaterally in the ventral posteriomedial nucleus (VPM) of the thalamus (1.45 mm posterior to bregma, 1.6 mm lateral ; 3.1 mm deep from pia), the posterior medial (POm) thalamus (1.45 mm posterior to bregma, 1.6 mm lateral; 2.9 mm deep), or unilaterally into vibrissal primary motor cortex (1 mm anterior to bregma, 0.6 mm lateral; 0.4 and 0.7 mm deep). 40 nl of viral suspension at 4.5×10¹¹ gc/ml were injected over ~ 1 min using a pulled glass micropipette (Drummond, Broomall, PA). To label L4 neurons, 40 nl of a Cre recombinase-dependent AAV virus encoding ChR2-H134R ³⁵ fused with mCherry (3.4×10¹³ gc/ml) ²⁹ was injected in the barrel cortex of P15 Six3-Cre#69 mice ³⁰, ³⁶. ChR2-mCherry expression was dense within a few barrels in L4 (Fig. 2,3 and Fig. S5). Only a few scattered cells were labeled in L3, L5 and L6 (laminar distribution of labeled cells; L3, 0.9 ± 0.8 %; L4, 96.6 ± 2.5 %; L5, 1.5 ± 0.9 %; L6, 1 ± 1.3 %; n=4) (Fig. S5). It is thus unlikely that these cells contribute significantly to our measurements.

Experiments started after 10 days of expression. To control for possible retrograde labeling we inspected labeled somata in barrel cortex after AAV virus injections in M1, POm and VPM. Even after immunohistochemical amplification we did not observe any labeled somata in barrel cortex.

Slice preparation. P26 to P34 mice were anesthetized with an intraperitoneal injection of a ketamine/xylazine mixture (0.13 mg ketamine/0.01 mg xylazine/g body weight) and perfused through the heart with a small volume (~ 5 ml) of ice cold ACSF containing in mM (in mM): 127 NaCl, 25 NaHCO₃, 25 D-glucose, 2.5 KCl, 1 MgCl₂, 2 CaCl₂, and 1.25 NaH₂PO₄, aerated with 95% O₂ / 5% CO₂. The brain was removed and placed into ice-cold cutting solution containing (in mM): 110 choline chloride, 25 NaHCO₃, 25 D-glucose, 11.6 sodium ascorbate, 7 MgCl₂, 3.1 sodium pyruvate, 2.5 KCl, 1.25 NaH₂PO₄, and 0.5 CaCl₂. 300 μm thick coronal slices of the right barrel cortex were cut with a vibrating slicer (Microm, Walldorf, Germany) and incubated in oxygenated ACSF for 45 min at 37°C before the recordings.

Identification and definition of cortical layers. L1 was identified by its low cell density. Based on functional mapping studies ²¹, L2 was defined as a 75 μm thick band immediately below L1. L3 was between L2 and the top of the barrels. L4 was identified by the presence of barrels (Fig. 1 d). L5A was identified as a light band under brightfield illumination (Fig. 1 d) below the barrels (~ 100 μm thick). L5B was a darker band immediately below L5A and contained the largest pyramidal cells (~ 200 μm thick). L6 extended from the bottom of L5B to the white matter.

Morphological reconstructions and analysis. During the recordings cells were loaded with biocytin (3mg/ml) and fixed overnight on 4% paraformaldehyde (PFA). For biocytin staining, after a quenching step ( 30 min in 1% H₂O₂ ), samples were incubated overnight in ABC solution (ABC kit, Vector Labs) in the presence of 4% Triton × ( Sigma). After rinsing for 8 hs in PBS, sections were reacted to DAB and mounted in AquaMount (Polysciences). Dendrites were reconstructed in Neurolucida (MicroBrightField) using a 40x objective and analyzed using custom Matlab (Mathworks ) routines. Neurolucida tracings were corrected for shrinkage. Dendritic length density ⁵ was calculated in 50 μm bins. For group averages, peak normalized dendritic length density maps were aligned to the soma (Fig. 2 and S7) or pia position (Fig. S6 and S8) and linearly interpolated for display.

Simulation of ChR2-mediated photoexcitation of axons. Non-myelinated axons (0.1 μm diameter) were simulated in NEURON 6.1³⁷. The passive membrane properties were: axial resistance, 150 Ω-cm; membrane capacitance, 0.75 μF/cm². The membrane conductances were: fast Na⁺ current (I_Na), voltage-activated K⁺ current (I_Kv) ²⁰, dendrotoxin-sensitive K⁺ current (I_Kd) ¹¹, leak current with a reversal potential at −70mV. The reversal potentials for Na⁺ and K⁺ were 60 mV and −90 mV respectively. Channel densities were homogeneous throughout the axon: I_Na, 1000 pS/μm²; I_Kv, 20 pS/μm²; I_Kd, 2500 pS/μm²; leak , 6.67e-5 S/cm². The ChR2 conductance was modeled as an alpha conductance with τ = 2.5 ms, gmax= 0.000154 μS and a reversal potential of 0 mV. The temperature of the model was 23 °C.

Supplementary discussion:

sCRACM compared to other methods for circuit mapping

Most cortical wiring diagrams describe the connectivity between populations of neurons, based on reconstructions of dendrites and axons and the supposition that synaptic connections are made in proportion to the overlap of dendrites and axons (i.e. ‘Peters’ rule’) ¹³^,¹⁵^,¹⁹, ³⁸. Peters’ rule ignores specificity in cortical connections beyond the overlap of axons and dendrites ³^-⁵, ^{39, 40}, necessitating functional measurements or measurements with synaptic resolution, such as electron microscopy.

sCRACM is a novel method for mapping functional neural circuits with subcellular resolution. sCRACM measures connections between presynaptic cells, defined by ChR2 expression, and postsynaptic cells, defined by intracellular recording. sCRACM is therefore well-poised to probe functional connections between genetically defined neurons. Because photostimulation is performed with action potentials blocked, sCRACM does not suffer from ambiguities due to excitation of polysynaptic pathways. Neuronal circuits have been functionally mapped in the past using laser scanning photostimulation (LSPS) of caged glutamate ⁴^,²¹^,³¹, ⁴¹ or paired recordings ¹⁵^,¹⁹^,²². These methods require the circuit studied to be preserved in the slice and are therefore limited to the study of local connections. In contrast, because ChR2-labeled axons are photoexcitable, even when severed from their parent somata, functional connectivity can be mapped across all spatial scales, including long-range connections ⁸.

Electron microscopy (EM) methods can map inputs within the dendritic arbors of postsynaptic neurons with submicrometer resolution ³^,¹⁵^,¹⁹, ⁴². However, EM is typically used to analyze only short dendritic segments. It is possible that recently developed super-resolution optical techniques might allow more efficient mapping of the spatial distribution of synapses ⁴³. However, structural techniques by themselves do not provide information about synaptic strength. sCRACM provides a lower resolution view of the distribution of functional input within entire dendritic arbors of single cells. sCRACM is therefore complementary to EM methods. sCRACM is applicable to any system in which afferent axons can be labeled.

The electrotonic spread of depolarization within photostimulated axons is likely a key determinant of sCRACM resolution. Simulations show that the length constant of thin axons (diameter, 0.1 μm) is ~ 70 μm, on the same order as the sCRACM resolution. The spread of excitation light within the tissue might also contribute, but likely plays only a minor role. Before entering the tissue, the diameter of the excitation beam was smaller than the sCRACM resolution. Furthermore, varying the beam diameter by almost a factor of three (from 6 to 16 μm) did not change sCRACM resolution. We conclude that the beam diameter by itself does not determine the sCRACM resolution. Within the tissue, the excitation light spreads due to light scattering. However, the EPSC_sCRACM amplitude is a steeply supralinear function of the intensity of the excitation light (Fig. 1c), suggesting that scattered light might be too dilute to contribute appreciably to the EPSC_sCRACM amplitude. The non-linear relationship between EPSC_sCRACM amplitude and light intensity suggests that three-dimensional sectioning in sCRACM mapping could be achieved without two-photon excitation.

Impact of filtering on sCRACM maps

sCRACM maps are based on somatic measurements of synaptic currents generated in the dendrites signal attenuation due to dendritic filtering influences the structure of the maps. For example, input on the apical tufts of L5 neurons could be reduced several-fold compared to more proximal input ¹². sCRACM maps thus represent a “somatocentric” view of the dendritic distribution of synaptic input, where electrotonically distant synapses will appear relatively weak (Fig. S8).

To approximate the relative input strength at the source (Fig. S8) we compensated the sCRACM maps for dendritic attenuation of synaptic charge. The dendritic attenuation, 1/g(r), was estimated as an exponentially increasing function with distance from the soma:

Q (x, y) ~ ρ_{a} (x, y) ρ_{b} (x, y) f q g (L (x, y)) .

where r(x,y) is the distance from the soma of the photostimulation location. We use λ = 270 μm ¹², measured in rat L5 pyramidal neurons with K⁺-based internal solution ¹². These measurements were obtained from the main apical tuft, whereas most synapses impinge on secondary and tertiary dendrites in the apical tuft. In addition, the measurements are from rat neurons with larger dendritic diameters compared to our mouse neurons. As a consequence, the attenuation-compensated maps (Fig. S8) likely still underestimate dendritic filtering by distant inputs.

Dendritic input domains

L3 pyramidal neurons received specific types of input on single, spatially distinct domains (Fig. 2). Remarkably, the position of VPM, L4, L2/3, and M1 input along the apical axis of L3 neurons mirrors the flow of excitation within the cortical circuit: more peripheral (central) input impinges on lower (higher) parts of the dendritic arbor. After stimulation of the principal whisker, lower basal dendrites are the first to receive input (directly from VPM, ~ 8 ms post-stimulus), followed by middle basal dendrites (from L4, ~ 10 ms post-stimulus), followed by upper basal and apical dendrites (from L2/3, ~13-16ms post-stimulus) ⁴⁴, followed by the apical tuft in L1 (feedback from M1, ~10 ms later). Input for POm is expected to activate synapses sparsely over most of the dendritic tree (~20 ms post-stimulus) ⁴⁵.

What determines the locations of the dendritic input domains? To asses the input from different presynaptic sources per unit length of dendrite we divided and peak-normalized vertical profiles of synaptic input by the mean vertical profile of dendritic length-density (Fig. S6a and Fig. S8). Unsurprisingly, the peaks in this profile overlapped roughly with axonal labeling (Fig. S5), with some exceptions (discussed below and in Fig. 3 c & d).

In some cases the overlap of axons with dendritic arbors seems to specify the location of the dendritic input domain. The axons of L2/3 neurons arborize in a thin (thickness ~ 200 um) lamina at the border of L5A and L5B (Fig. 4). The peak of the input from L2/3 onto L5 dendrites was closely aligned with the peak of axonal density (Fig. 4b). Cells with somata that were superficial to the band of L2/3 axons received L2/3 input on the bottom of the basal dendrites, whereas deep L5B cells received input on the upper half of the basal dendrites and in the apical oblique dendrites. In other cases, functional connections do not seem to be predicted by the product of axonal and dendritic density. The overlap of L2/3 axons and L3 dendrites is centered on the basal dendrites, dominated by the great abundance of dendritic branches ¹⁵. However, we found that the majority of L2/3→L3 input was above the soma and most of the basal dendrites (Fig. 2); this finding was independent of cortical depth of the postsynaptic neurons (Fig. S6). Thus, unlike L2/3→L5 input, the subcellular domain for L2/3→L3 input cannot be explained by the presence of presynaptic axons in a restricted lamina. L2/3→L3 inputs might avoid the denser basal branches, or get preferentially strengthened in the upper basal and apical dendrites. Since EPSC_sCRACM amplitudes depend on both the fraction of axons that make synapses with the recorded neuron and the strength of these synapses, sCRACM can not distinguish these two alternatives.

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Supplementary Table 1: Ratio of total sCRACM input per map in pairs of cells in different layers recorded in the same column with the same photostimulation power (mean ± S.D.). The total sCRACM input per map is a quantitative measure of the input from defined projections; it is defined as the sum of all sCRACM map pixels.

Figure S1: Summary of the main results. a, Schematic of the laminar position of the axonal arborizations from the five axonal projections studied. The sources of the projections are indicated by the boxes, stars and triangles. Horizontal bars indicate the layers of primary arborization. The relative thickness of the bars is a qualitative indication of the strength of the arborization. b, Schematic of the location of the different inputs (colors correspond to panel a) on the dendrites of L3, L5A and L5B pyramidal neurons. Vertical bars indicate the locations of functional input. The relative thickness of the bars is a measure of the strength of the input.

Figure S2: sCRACM responses as a function of power and distance to the soma. a, Onset of L2/3-->L5A EPSC_sCRACM for four different neurons as a function of laser power. b, Onsets of EPSC_sCRACM for three different axonal populations (from POm, L4 and L2/3) recorded in L5A pyramidal neurons. Each circle represents the mean value of the onset of the responses on the perisomatic area (<107 μm from the soma) for one cell.c Unitary EPSC_sCRACM traces obtained by repeatedly photostimulating a single spot at the minimal laser power eliciting a response. The blue tick indicates the laser pulse. d, Group average of the 10-90% rise time of EPSC_sCRACM responses of L2/3 inputs mapped onto L5 dendrites, aligned by soma position. e, Risetime vs. distance to the soma of the photostimulation site for basal dendrites. Responses at different distances from to soma in locations corresponding to the basal dendrites were binned and averaged (white semi-circles in d). f, Risetime vs. distance to the soma of the photostimulation site for apical dendrites. Pixels in the two columns above the soma (white box in d) were used, as they are more likely to correspond to the main trunk of the apical dendrite. Error bars, SD. (a,b) or s.e.m. (e,f).

Figure S3: sCRACM point-spread-function and resolution. a, Measurement of the sCRACM point-spread-function. Line scan over the apical dendrite of a L5A neuron receiving L2/3 input (mCherry fluorescence). Locations with non-zero EPSC_sCRACM are color coded to represent mean amplitude. b, Responses shown in Panel a (blue) and similar responses obtained with twice the laser power normally used for sCRACM mapping (red). c, Full-width-at-half-maximum (FWHM) of the Gaussian fits of the responses obtained on apical dendrites of different L5A pyramidal neurons. Experiments performed at the same locations with different laser powers are connected by lines. d, L2/3-->L5 input on an apical dendrite. e, Histogram of the distance (‘d’ in panel d) between the apical dendrite and locations where non-zero EPSC_sCRACM were evoked by photostimulation of L2/3 (n=6) , L4 (n=5) or VPM axons (n=3).

Figure S4: Reproducibility of sCRACM maps. a, Left, sCRACM map overlaid on a brain slice containing ChR2-Venus-positive L2/3 neurons. Right, four repetitive EPSC_sCRACM responses recorded from a L5A pyramidal neuron. The stimulus occurred at the beginning of each trace. Traces correspond to the pixels surrounded by the dashed boxes. b, sCRACM maps of L2/3 input on a L5A pyramidal neuron at different laser powers (white triangles, soma position). c, Same maps as in b, normalized to their peak. d, Four repetitions of individual maps and corresponding average map (same cell as in a). e, Two-dimensional correlation coefficients between map repetitions (n=8 cells, 3 repetitions per cell), and between peak-normalized maps obtained with different laser powers (n=6 cells, average of 3 repetition per power level). Error bars, SD.

Figure S5: Targeting ChR2 expression to specific neuronal populations. a, mCherry fluorescence from L2/3 cells transduced by in utero electroporation. b, Detail of the barrel cortex, showing mCherry and ChR2-Venus fluorescence. c, ChR2-mCherry expression after injection of Cre-recombinase-dependent ChR2-mCherry AAV in the barrel cortex of a Six3Cre mouse. d, Detail of mCherry expression in barrel cortex. e, AAV virus expressing ChR2-Venus in the VPM nucleus of the thalamus. f, Detail of the barrel cortex, showing ChR2-Venus labeled VPM axons.g, GFP-immunostained ChR2-Venus labeled VPM axons in layers 1-3 of barrel cortex. h, AAV virus expressing ChR2-Venus in the POm nucleus of the thalamus. i, Detail of the barrel cortex, showing ChR2-Venus labeled POm axons.j, GFP-immunostained ChR2-Venus labeled POm axons in layers 1-3 of barrel cortex. k, AAV virus expressing ChR2-Venus in M1. l, Detail of the barrel cortex showing ChR2-Venus positive axons. m, Confocal image of GFP-positive M1 axons in layers 1-3 of barrel cortex.

Figure S6: Inputs to L3 pyramidal neurons. a, Vertical profile of the laminar distribution of the dendritic input density obtained by dividing the pia-aligned synaptic input maps (from Fig 2a) by the dendritic length-density of L3 pyramidal neurons (right). Error bars, s.e.m. b, Vertical profiles of the relative subcellular distribution of L2/3-->L2/3 inputs. Each column represents an individual cell, ordered by increasing cortical depth. Cells were aligned by pia position. The white vertical lines indicate the boundaries of the three cortical depth bins used for averaging in c. c, Average relative subcellular location of L2/3 inputs, aligned by soma (top) or pia position (bottom) , of groups of L2/3 cells at increasing distances from the pia (corresponding to b). d, Vertical profiles of the relative subcellular distribution of L4-->L2/3 inputs. e, Average relative subcellular location of L4 inputs, aligned by soma(top) or pia position (bottom) , of groups of L2/3 cells at increasing distances from the pia (corresponding to d).

Figure S7: Subcellular distribution of specific inputs onto L5A pyramidal neurons. a, Examples of sCRACM maps overlaid on reconstructed dendritic arbors and fluorescence images. b, Group averages aligned by pia position. White triangles, soma position. c, top: Group averages aligned by soma position ; bottom: vertical profiles of the distribution of synaptic input (red) and the dendritic length density (green; from panel d). d, Average normalized dendritic length density of L5A pyramidal neurons. Error bars, s.e.m.

Figure S8: Inputs to L5 pyramidal neurons, corrected for dendritic attenuation and dendritic density. a, Top, Group averages corrected for the estimated dendritic attenuation of inputs onto L5B pyramidal neurons aligned by pia position. White triangles, soma position. Bottom, Vertical profiles of the distribution of synaptic input as recorded from the soma (red) or corrected for the estimated dendritic attenuation (blue). Vertical profiles of the dendritic length density (green; from panel b). Error bars, s.e.m. b, Average normalized dendritic length density of L5B pyramidal neurons. c Vertical profiles of the distribution of synaptic input divided by the dendritic length-density of L5B pyramidal neurons, as recorded from the soma (red), or corrected for the estimated dendritic attenuation (blue). d, Top, Group averages corrected for the estimated dendritic attenuation of inputs onto L5A pyramidal neurons aligned by pia position. White triangles, soma position. Bottom, Vertical profiles of the distribution of synaptic input as recorded from the soma (red) or corrected for the estimated dendritic attenuation (blue). Vertical profiles of the dendritic length density (green; from panel e). Error bars, s.e.m.e, Average normalized dendritic length density of L5A pyramidal neurons. f, Vertical profiles of the distribution of synaptic input divided by the dendritic length-density of L5A pyramidal neurons, as recorded from the soma (red), or corrected for the estimated dendritic attenuation (blue).

Figure S9: Simulations of Chr2-mediated photostimulation of axons. ChR2-mediated depolarization of the axons, with the transient Na+, fast voltage activated K+ and dendrotoxin (I_Kd) sensitive K⁺ currents (black), without the Na⁺ current (blue) or without the Na⁺ and I_Kd currents (red).

NIHMS143295-supplement-1.pdf^{(457.8KB, pdf)}

Acknowledgments

We thank A. Karpova for help with viral constructs, G. Oliver and B. Xu for the Six3Cre mouse line, D. Chklovskii, G. Shepherd, and Q. Wen for comments on the manuscript, Y. Yu for the model of the K_d channel, and T. O'Connor for software development.

Methods

Electrophysiology and photostimulation

Neurons were patched with borosilicate pipettes (resistance 4–6 MΩ). The intracellular solution contained in mM (in mM) 128 Kgluconate, 4 MgCl₂, 10 HEPES, 1EGTA, 4 Na₂ATP, 0.4 Na₂GTP, 10 sodium phosphocreatine, 3 sodium L-ascorbate and 0.015 Alexa-594 (Molecular Probes) (pH 7.25; 290 mOsm). Cells were recorded at depths of 50 to 95 μm in the brain slice. Data were acquired using custom programs (Ephus, available at https://openwiki.janelia.org/). Photostimulation was with a blue laser (473 nm; Crystal Laser). The beam's position was controlled with galvanometers (Cambridge Scanning, Inc.). The beam was delivered through an air immersion objective (4×; 0.16 NA; UPlanApo, Olympus). The optics were designed to generate a nearly cylindrical beam (~6-16 μm, full-width at half max at the focal plane). The duration and intensity of the light pulses were controlled with a Pockels cell (ConOptics) and a shutter (Uniblitz).

For sCRACM mapping we delivered light pulses (duration, 1ms; inter-stimulus interval, 400 ms) on a 12 × 24 grid with 50 μm spacing (Fig. 1 d). The grid area (0.6 × 1.2 mm²) included the entire thickness of the cortical grey matter. Stimuli were given in a spatial sequence pattern designed to maximize the time between stimuli to neighboring spots ³¹. To avoid sequence-specific responses during consecutive mapping we flipped and rotated the stimulus pattern between maps. TTX (1 μM), CPP (5 μM), and 4-AP (100 μM) were added to the bath. Without 4-AP (or α-dendrotoxin, Alomone Labs, Israel, 200 nM), TTX (1 μM) abolished 98 ± 1.9 % of the EPSCs evoked in the absence of drugs (6 cells, 573 sites), even at high light intensities (> 1 mW) (Fig. 1b). When mapping inputs from L4 axons we also added Bicuculline (10 μM ) to block contributions from GABAergic neurons in L4 ³⁰. EPSC_sCRACM were recorded in voltage clamp (−75 mV). Access resistances were < 40 MOhm and stable (< 20% change during the experiment); resting potentials were less than −55 mV.

EPSC_sCRACM have relatively long delays (mean, 10.4 ± 2.5 ms; L2/3→L5A perisomatic responses; < 110 μm from the soma; 146 sites; 18 cells) (Fig. S2), likely reflecting the slow charging and discharging of the axonal membrane (Fig. S9). The delays varied across photostimulation sites (range: 6.4 - 21 ms) and EPSC_sCRACM rise- (mean (10 – 90 %): 6.5 ± 3.1 ms; range: 2.4 - 18.3 ms) and decay-times (mean: 35 ± 28 ms; range: 6.2 - 160 ms) were long. EPSC_sCRACM on occasion displayed multiple peaks. Minimal stimulation experiments (Fig. S2c) revealed that unitary currents were slightly desynchronized at a single photostimulation location (latency jitter, 1.03±0.5 ms, n = 6) and highly desynchronized across different locations (latency range, 10.4-17.2 ms, mean 15.2±3 ms , n=6) (Fig. S2). The temporal smearing of the EPSC_sCRACM waveform is therefore dominated by differences in latencies across different synapses. The risetime of the responses increased with distance to the soma, both along the apical and basal dendrites, consistent with filtering expected from cable theory (Fig. S2) ³², ³³.

For each recorded cell, laser powers were adjusted to cause EPSC_sCRACM with peak amplitudes of approximately - 75 pA (L2/3 pyramidal neurons, −72±47 pA; L5 pyramidal neurons: −84±45 pA). The corresponding laser powers varied over one order of magnitude (120 μW-1.9 mW at the specimen plane), reflecting variations in the fraction of ChR2-positive axons and ChR2 expression levels across mice. sCRACM maps were repeated 2-5 times for each cell (Figure S4). After the recordings dendritic arbors were imaged using fluorescence microscopy (Qimaging, Surrey, Canada) and subsequently processed for biocytin staining and reconstructed. Only data from neurons where the apical dendrites ran parallel to the slice surface were included in the analysis.

Since photostimulation was with a cylindrical beam, sCRACM maps represent the 2-D projections of the 3-D distribution of inputs. As a consequence, the peak values of the distribution of inputs were sometimes centered on the somata, although somata are mostly devoid of excitatory synapses (Fig. 2 and Fig. 3). This is analogous to the 2-D projection of the density of basal dendrites, which also peaks on the soma (Fig. 2, 3 and S7). Furthermore, under our conditions the sCRACM resolution was ~ 60 μm, large compared to the diameters of most somata.

Data Analysis

Individual pixels of sCRACM maps at position x, y (Q(x, y)) were computed as the mean EPSC_sCRACM amplitude in a response window from 0 to 75 ms after the stimulus, and thus are a measure of charge. For consistency with previous studies, and because synaptic current is a more familiar unit, data are given in units of picoamperes (pA). Q is given by

1 ∕ g (r) = 1 - 2^{(- r ∕ λ)},

ρ_a and ρ_b are the density of axons and dendrites respectively. f is the filling fraction, defined as the fraction of axons making synapses with nearby dendrites ³⁴. q is the charge per synapse per light flash. g(L) is the dendritic attenuation as a function of electrotonic distance, L(x,y), between the site of photostimulation and the soma. Because of dendritic attenutation, Q provides a somatocentric view of the synaptic input (see Supplementary Discussion). Since ρ_a, and possibly q, depend on details of the gene transfer method, it is challenging to compare the strengths of different projections onto the same cell. Since ρ_a varies between preparations, sCRACM maps were normalized to the largest pixels within a map and thus represent the relative strength of input within the dendritic tree.

Averaged EPSC_sCRACM were scored as non-zero if their amplitudes (0-50 ms poststimulus) were larger than 5x the standard deviation of the baseline (Fig. 1 e, 2a, 3 a, 4 a, S3 and S7). Maps were either aligned on the soma or on the pia. In the case of alignment to the pia it was necessary to correct for variations in cortical thickness; individual maps were therefore morphed by linear interpolation to a template based on the average cortical thickness. Similarly, the cortical depth of individual neurons (Fig. 4) was also normalized to the average depth across slices. Individual maps are presented as raw pixel images, whereas group averages are linearly interpolated without smoothing (for display only). To measure the density of L2/3 axons (Fig. 4 b) we measured and peak normalized mCherry fluorescence along the cortical axis in L4 through L6 in in utero electroporated animals (n=5).

sCRACM Resolution

The effective resolution of sCRACM mapping can be inferred from the point-spread-function. We measured the point-spread-function from the spatial distribution of the photostimulation sites that produce detectable responses in the vicinity of isolated dendritic branches. L5A cells often received input from L2/3 neurons along a single unbranched apical dendrite within L2/3 (Fig. S3). To measure sCRACM resolution, we first identified the peak of L2/3 input on the apical dendrites of L5A cells within L2/3. We next photostimulated in a line across the apical dendrite, through the peak of L2/3 input (12 positions, 15 μm spacing between stimuli, inter-stimulus interval, 6 s ) (Fig. S3 a). Since the activated synapses were on a single dendritic branch in the vicinity of the photostimuli, the spatial distribution of responses represents a measure of the spatial resolution. After the experiment the dendritic arbor of the recorded neurons were reconstructed. Only cells where the apical dendrite did not ramify within 100 μm the photostimulation sites were included for analysis. For the light intensities used for sCRACM mapping, the full-width-at-half-max (FWHM) of the spatial profile of the responses was 59 ± 14 μm (n = 4) (Fig. S3 b-c). Higher laser intensities degraded the resolution slightly.

To verify this resolution estimate within our data set, we identified stretches of unbranched L5 apical dendrites that received input from ChR2-positive axons originating either in VPM, L4, or L2/3. Only inputs separated by at least 100 μm from branchpoints were used. In addition, to avoid over-representation of a subset of inputs, we only scored inputs that were at least 100 μm apart. Detectable EPSC_sCRACM were only evoked within 75 μm of the apical dendrite (Fig. S3 d,e). We conclude that sCRACM maps specific types of input with ~ 60 μm spatial resolution.

Data in the text are given as mean ± SD.

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Footnotes

Supplementary Information is linked to the online version of the paper at www.nature.com/nature.

Methods Summary

Specific neuronal populations were labeled with ChR2 in vivo either by in utero electroporation ⁷, ²⁸(L2/3 pyramidal cells), Adeno-Associated Virus (AAV) infection (VPM, POm, M1), or infection with a Cre recombinase-dependent AAV virus ²⁹ in mice expressing Cre (L4) ³⁰. Acute coronal slices were from young adult (P26-34) mice. Pyramidal neurons in L3 and L5 were recorded in voltage clamp at room temperature (22-24°C). Photostimulation was with blue (wavelength, 473nm) laser pulses (duration, 1ms; inter-stimulus interval, 400 ms; beam diameter 6-16 μm) in the presence of TTX (1 μM), 3-((R)-2-Carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP ,5 μM), and 4-AP (100 μM).

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Methods

Supplementary discussion:

sCRACM compared to other methods for circuit mapping

Impact of filtering on sCRACM maps

Q (x, y) ~ ρ_{a} (x, y) ρ_{b} (x, y) f q g (L (x, y)) .