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Selective inhibitory control of pyramidal neuron ensembles and cortical subnetworks by chandelier cells

Nature Neuroscience volume 20, pages 1377–1383 (2017)Cite this article

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Abstract

The neocortex comprises multiple information processing streams mediated by subsets of glutamatergic pyramidal cells (PCs) that receive diverse inputs and project to distinct targets. How GABAergic interneurons regulate the segregation and communication among intermingled PC subsets that contribute to separate brain networks remains unclear. Here we demonstrate that a subset of GABAergic chandelier cells (ChCs) in the prelimbic cortex, which innervate PCs at spike initiation site, selectively control PCs projecting to the basolateral amygdala (BLAPC) compared to those projecting to contralateral cortex (CCPC). These ChCs in turn receive preferential input from local and contralateral CCPCs as opposed to BLAPCs and BLA neurons (the prelimbic cortex–BLA network). Accordingly, optogenetic activation of ChCs rapidly suppresses BLAPCs and BLA activity in freely behaving mice. Thus, the exquisite connectivity of ChCs not only mediates directional inhibition between local PC ensembles but may also shape communication hierarchies between global networks.

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Figure 1: L2 ChCs preferentially innervate BLAPCs over CCPCs in prelimbic cortex.
Figure 2: L2 ChCs receive strong input from CCPCs and weak input from BLAPCs.
Figure 3: Distinct local circuit connectivity of PVBCs and ChCs in PL upper layers.
Figure 4: Systematic tracing of local and long-range inputs reveal that L2 ChCs are preferentially recruited by bilateral CCPC input as opposed to BLA input.
Figure 5: Optogenetic activation of L2 ChCs in PL inhibits PL firing, including BLAPC firing, in freely behaving mice.

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Acknowledgements

We thank S. Schamiloglu for help with retrograde labeling of PL pyramidal neurons, B. Li, A. Kepecs and G. Buzsáki for comments on the manuscript. This work was supported in part by NIH R01 MH094705-05 and CSHL Robertson Neuroscience Fund to Z.J.H. and by NIH R01 MH081968 and the Hope for Depression Research Foundation to J.A.G. J.T. was supported by NRSA F30 Medical Scientist Predoctoral Fellowships. J.L. was supported by a NARSAD Postdoctoral Fellowship. N.P.C. was supported by the National Science Foundation. J.A.G. contributed to this article while at Columbia University, before joining the National Institute of Mental Health. The views expressed are the author's own and do not represent the views of the National Institutes of Health, the Department of Health and Human Services, or the United States government.

Author information

Author notes

  1. Miao He

    Present address: Institutes of Brain Science, State Key Laboratory of Medical Neurobiology, Collaborative Innovation Center for Brain Science, Fudan University, Shanghai, China

  2. Joshua A Gordon

    Present address: National Institute of Mental Health, Rockville, Maryland, USA

  3. Jiangteng Lu and Jason Tucciarone: These authors contributed equally to this work.

Authors and Affiliations

  1. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA

    Jiangteng Lu, Jason Tucciarone, Miao He & Z Josh Huang

  2. Program in Neuroscience and Medical Scientist Training Program, Stony Brook University, Stony Brook, New York, USA

    Jason Tucciarone

  3. Departments of Neuroscience and Psychiatry, Columbia University, New York, New York, USA

    Nancy Padilla-Coreano & Joshua A Gordon

  4. Institutes of Brain Science, State Key Laboratory of Medical Neurobiology, Collaborative Innovation Center for Brain Science, Fudan University, Shanghai, China

    Joshua A Gordon

  5. National Institute of Mental Health, Rockville, Maryland, USA

    Miao He

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Contributions

J.L. carried out electrophysiological and optogenetic studies of synaptic connectivity; J.T. performed anatomical and rabies synaptic tracing studies and contributed to in vivo and in vitro physiology studies and analyses; N.P.-C. performed in vivo electrophysiological recordings and data analysis; J.A.G. supervised in vivo electrophysiological recordings and data analysis; M.H. generated the LSL-Flp mouse line; Z.J.H. conceived and organized the study, and wrote the manuscript with contributions from all authors.

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Integrated supplementary information

Supplementary Figure 1 Spatial distribution of L2 ChCs in frontal cortex.

(a-b) ChCs were labeled by tomaxifen induction at E17.5 in a Nkx2.1-CreER;Ai14 mouse. Images were taken from 100 um coronal sections, and stereologically reconstructed across 1 mm thickness of cortical tissue. Green dots are in infralimbic cortex (IL), red dots in prelimbic cortex (PL), blue dots in anterior cingulate cortex and secondary motor cortex (AC/M2), and purple dots in orbitofrontal cortex (Orb). (c) Quantification of ChCs labeled in several subregions of frontal cortex in a single animal.

Supplementary Figure 2 Counting of axon cartridge number of individual L2 ChCs in PL.

(a) Left: a RFP (+) L2 ChC from sparse labeling with lose dose TM induction in Nkx2.1-CreER;Ai14 (arrowhead: soma, small arrow: dendrite, larger arrow: axon arbor). Middle: staining of PC AIS with phosphor-IkappB. Right: overlay. Arrows indicate soma and dendrite position. (b) The Cartridges revealed as string ChC axon buttons (arrowheads) along the AIS. Images are magnified from boxes in (a). (c) Example of counting cartridge number of a ChC.

Supplementary Figure 3 Spatial relationship between ChCs and PCs in PL and in paired patch recording experiments.

(a) RFP(+) L2 ChCs in PL were positioned alongside to CTB-488 labeled BLAPCs. Dendrites (short arrow), soma (arrowhead) and axons (long arrow) of ChCs are indicated. (b) An example of whole-cell patch recordings of a L2 ChC and an adjacent CTB labeled BLAPC in brain slice. Right panel of pseudocolored cells taken from boxed area of the left under fluorescent imaging. (c) Position of all recorded ChCs in brain slice with dimension axis as indicated in b. In the distance from their somata to pia (201±7 μm vs 196±5 μm, p=0.33, Mann-Whitney test). In the depth in the slice (z axis) (67±7 μm vs 72±5 μm, p=0.46, Mann-Whitney test). (d) Position of all recorded BLAPCs and CCPCs in the brain slice. In the depth (z axis) (62±3 μm vs 73±7 μm, p=0.33, Mann-Whitney test). Mann-Whitney test found a significant difference (p<0.01) in the distance from their somata to pia (238±9 μm vs 265±5 μm).

Supplementary Figure 4 CTB injection spread.

(a) (Left panel) Injection site of medial prefrontal cortex indicates spatial extent of CTB-488 spread. (right panel) Overlay of injection spread of individual animals (n=5) onto paxinos mouse atlas. A/P coordinates indicated. (b) (Top, bottom left figures) Injection site of individual BLA injections indicates spatial extent of CTB-488 spread. (lower right) Overlay of injection spread of individual animals (n=5) onto paxinos mouse atlas. A/P coordinates indicated.

Supplementary Figure 5 Intrinsic properties of 3 subsets of PCs in the upper layer of PL.

(a) An example of membrane resistance (Rm) measurement by a 5 mV hyperpolarization under voltage-clamp; summary in right panel. No significant difference (p=0.81, Mann-Whitney test) between BLAPCs (140±66 MΩ, n=13) and CCPCs (135±53 MΩ, n=16). (b) Examples of firing properties showing regular spiking (RS) and burst spiking (BS) in two PCs under current-clamp. Step-wise ramp depolarization currents were injected into the soma through the glass pipettes. BS was recognized as an interval (<10 ms) between initial two spikes. (c) The percentage of RS and BS in each subset of PCs. (d-h) Summaries of resting membrane potential, AP threshold, AP amplitude, AP half-width and after-hyperpolarization potential of 3 subsets of PCs. BLAPCs (n=13), CCPCs (n=16) and STPCs (n=18). Comparison of BLAPC vs CCPC by Mann-Whitney test showed no significant difference in resting membrane potential (-78.2±5.2 vs -78.7±3.9 mV, p=0.88), AP threshold (-34.7±3.6 vs -33.7±3.9 mV, p=0.74), AP amplitude (76.1±9.2 vs 74.6±6.7 mV, p=0.67), AP half-width (1.04±0.15 vs 0.97±0.17 ms, p=0.42), and after-hyperpolarization potential (7.9±3.0 vs 7.8±2.3 mV, p=0.71). Values are presented as mean±s.d.. Plots display median, mean, quartiles and range.

Supplementary Figure 6 Quantification of spatial relationship between L2 ChCs and PCs in paired recording experiments.

(a) 3D plots of all recorded PCs aligned to the recorded ChCs as origin (the red dot). Innervated (I) BLAPCs: green circle, n=20; innervated CCPCs: blue circle, n=7; non-innervated (N) BLAPCs: green diamond, n=3; non-innervated CCPCs: blue diamond, n=33. (b) Summarizes of soma distance to ChC for the four categories of PCs in (a). No significant difference was detected in distance to ChCs between I-BLAPCs vs N-BLAPCs (73±26 vs 92±24 μm, p=0.19), or between I-CCPCs vs N-CCPCs (85±20 vs 82±19 μm, p=0.70), or between I-BLAPC vs I-CCPC (p=0.19). (c) Summarizes of laminar distance to ChC for the four categories of PCs. There was no significant difference detected between I-BLAPCs vs N-BLAPCs (35±30 vs 54±26 μm, p=0.49) or between I-CCPCs vs N-CCPCs (69±22 vs 72±21 μm, p=0.72). In the laminar distance to ChCs, I-BLAPCs are significantly closer than I-CCPCs (p<0.01). Values are presented as mean±s.d. and Mann-Whitney test employed. (d-e) Summary showing that the connection probability (numbers in bars indicate connected / tested pairs) from ChCs to BLAPCs (d) and CCPCs (e) were the same whether PCs were located 0-60 μm (R1) or 60-120 μm (R2) from ChCs. Although the majority of BLAPCs were located in the upper L2 close to ChCs (R1: n=17 out of 19 pairs, 89.5 %), the more sparse BLAPCs in deeper L2/3 were innervated by L2 ChCs with similar probability (R2: n=3 out of 4 pairs, 75.0 %; Fisher exact test, p=0.45). L2 ChCs had similarly low innervation probability (Fisher exact test, p = 0.99) for CCPCs located in the upper (R1: n=2 out of 10 pairs, 20.0 %) or deeper (R2: n=5 out of 30 pairs, 16.7 %) L2/3. Plots display median, mean, quartiles and the range.

Supplementary Figure 7 Detection and quantification of PC to ChC connection by loose patch stimulation of PCs.

Loose patch was achieved by the tight seal (> 100 MΩ resistance) to the targeted PCs through the same pipette for the whole-cell patch recordings. Stimulation ranged from 0.1-1 V in 200 μs at 0.1Hz. Stimulation intensity was determined by the persistence of spikes in PCs after the stimulation artifact. (a) Example of lack of synaptic responses in ChCs (red) following loose patch stimulation of a BLAPC (green). Upper: schematic of loose patch stimulation in BLAPC and whole-cell patch recording in ChC. Lower: representative traces from paired recordings in ChCs; thick trace was averaged from 20 trials. (b) Summary of BLAPC to ChC connection probability (numbers in bar graph indicate connected / tested pairs). Fisher exact test revealed no significant difference (p=0.38) in the connection probability by loose patch stimulation of BLAPCs (n=0 out of 37 pairs, 0.0 %) compared to that by whole cell patch stimulation of BLAPCs (n=1 out of 23 pairs, 4.3 %). Thus, we pooled two sets of data for the analysis in Fig. 2c. (c) Same arrangement as in a showing example of synaptic responses in ChCs (red) following loose patch stimulation in CCPC (blue). (d) Same arrangement as in b showing summaries of CCPC to ChC connection probability (numbers in bar graph indicate connected / tested pairs). Fisher exact test revealed no significant difference (p=0.72) in the connection probability by loose patch stimulation of CCPCs (n=4 out of 31 pairs, 12.6 %) compared to that by whole cell patch stimulation (n = 4 out of 40 pairs, 10.0 %). Thus, we pooled two sets of data for the analysis in Fig. 2c. Mann-Whitney test revealed no significant difference (p=0.49) in the synaptic strength evoked by loose patch stimulation (83.9±31.7 pA, n=4) and by whole cell patch stimulation (42.5±16.3 pA, n= 4). Thus, we pooled two sets of experiments together for the analysis in Fig. 2c. Plots display median, mean, quartiles and the range.

Supplementary Figure 8 Reliable light-induced spiking in ChR2(+) CCPCs and postsynaptic responses in adjacent ChR2(-) PCs.

(a) Schematic CCPCs infection by dual viral injections first with HSV-Flp in cPL and then with AAV-FD-ChR2-YFP into PL. (b) Example paired recording of YFP(+) PC and adjacent YFP(-) PC in cortical slice under fluorescent microscope. Red: RFP(+) L2 ChCs; Green: ChR2(+) CCPCs in PL. (c) Example spikes in a ChR2(+) evoked by step-wise increase of blue laser intensity (2 ms duration) and postsynaptic potentials in a ChR2(-) PC. (d) Example of high fidelity of spiking in a ChR2(+) PC and synaptic potential in a ChR2(-) PC (black trace averaged from 15 trials of gray traces) evoked by laser flash of 2 ms duration at 10 Hz with a saturation intensity based on step-wise measurement in c. (e) and (f) were magnified from the corresponding events indicated in d.

Supplementary Figure 9 Identification of GABAergic inputs to L2 ChCs revealed by rabies tracing.

(a) Left: local EnvA-dG-GFP infected cells. Middle: GAD67 immunohistochemistry. Right: overlay. (b) Incidence of colocalized inputs with GAD67. Indicated by the arrow as in a. (c) Left: local EnvA-dG-GFP infection RFP+ starter ChCs are designated by white arrow head. Middle: PV immunohistochemistry. Right: overlay. (d) Incidence of colocalized inputs with PV as indicated by arrows. (e, f) Same as in (c, d) instead stained for somastain (SOM), note absence of colocalized input. (g, h) Same as in (c, d) instead stained for vasoactive intestinal peptide (VIP). Scale bar: 100 μm.

Supplementary Figure 10 Identification of diagonal band (DB) input to L2 ChCs revealed by rabies tracing.

(a) Left: 3D reconstruction for total basal forebrain inputs from a single animal color-coded by individual nuclei. Right: 45 degree rotation zoomed image of that basal forebrain. (b) Basal forebrain immunostained for PV (blue) and ChAT (red), overlaid with EnvA-dG-GFP rabies labeled inputs to L2 ChCs in PL. (c) Incidence of inputs colocalized with ChAT (arrowheads) but negative for PV.

Supplementary Figure 11 The Specificity of mPFC ChCs labeling by injection of AAV-FD-ChR2-YFP in Nkx2.1CreER;LSL-Flp mice induced at E17.5.

(a) Injection site following injection with AAV-FD-ChR2-YFP into mPFC, amplified and co-stained with antibodies against YFP tag (dash line indicates midline pia surface; arrow indicates L2 ChCs). (b) Zoomed in injection site reveals several morphologically identified ChCs (indicated by arrows). (c) Morphologically distinct and homogenous population ChCs can be identified via dendritic trees in L1, somata on the L1/2 border (large white arrows) and extensive axonal cartridge plexus in L2/3 (small white arrows). Of total virally labeled cells, 88.6±5.6% cells were ChCs and in L2/3 specifically 94.8±3.6% of cells were ChCs (n=5 mice). No incidences of virally labeled pyramidal cells were seen.

Supplementary Figure 12 Waveform and baseline firing of single units in in vivo recordings.

(a) Plot showing hyperpolarization, spike width and baseline firing for all recorded units identified by their effects in firing by ChC activation. The subpopulation of units indicated by colors as showing in b-d. Differences in spike width (b), average waveform (c) and baseline firing (d) for the different subpopulations of single units recorded. (excited n=3; inhibited n=13; unchanged n=63; Mann Whitney test inhibited vs unchanged: ***p<.001; z-score: 4.2626). (e) Example trial-by-trial correlation between baseline firing and ChC activation induced changes in firing in one single unit. (f-g) Across the inhibited, excited and unchanged populations at different time windows post laser: 0-5 ms (f), and 0-15 ms (g) on the left, the percent of cells with a significant correlation between the baseline firing rate and the firing rate effect of ChC activation (no proportion is statistically different from eYFP group) and on the right, the correlation coefficients for the inhibited and unchange subpopulations are not different than those for control group (inhibited n=13; no change n=63; eYFP n=65; Mann Whitney test, inhibited vs eYFP p=0.32; unchange vs eYFP p=0.85, in 0-15 ms window).

Supplementary Figure 13 Assessing the identity of inhibited units by phase locking analysis.

(a) Phase-locking between PL single units and BLA LFP, depicted in the left schematic. Top: An example of phases locking: spikes from a PL unit appeared to occur at or near the peaks of the raw (gray) and filtered (black) BLA LFP (top); bottom: PSTH and phase distributions of the representative PL single units. Bin size in all PSTHs is 2 ms. (b) Summary of the percentage of phase-locked units separated into light-inhibited and unchanged groups (numbers in bars indicate phase-locked / total units in the group). Significance of phase-locking was determined by Rayleigh’s test. The dashed lines indicated the amount of phase locking with shuffled data. The probability of obtaining as many phase-locked units as were seen in the actual dataset were p<0.01 for the inhibited units, and p=0.17 for the non-inhibited units (p values were obtained with bootstrapping methods). Light-inhibited units were preferentially synchronized to BLA LFP. (c-d) Assessing PL-BLA directionality with a lag analysis. Top, phase-locking strength to BLA LFP 3-6 Hz measured as normalized mean resultant length (MRL) as a function of lag for inhibited units (c) and other L2/3 units (d) aligned by peak lag. Bottom, distributions of lags at which peak phase-locking occurred, for inhibited units (c) and for other L2/3 units (d). Inset pie charts show in gray units with negative peak lags, in red units with positive peak lags, and in white units with a lag of zero. Units had to have at least 50 spikes to be included in these analyses.

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Lu, J., Tucciarone, J., Padilla-Coreano, N. et al. Selective inhibitory control of pyramidal neuron ensembles and cortical subnetworks by chandelier cells. Nat Neurosci 20, 1377–1383 (2017). https://doi.org/10.1038/nn.4624

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