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. 2022 Jan 7;375(6576):eabl5981.
doi: 10.1126/science.abl5981. Epub 2022 Jan 7.

Dense functional and molecular readout of a circuit hub in sensory cortex

Cameron Condylis  1   2 , Abed Ghanbari  3 , Nikita Manjrekar  3 , Karina Bistrong  3 , Shenqin Yao  4 , Zizhen Yao  4 , Thuc Nghi Nguyen  4 , Hongkui Zeng  4 , Bosiljka Tasic  4 , Jerry L Chen  1   2   3   5
Affiliations

Dense functional and molecular readout of a circuit hub in sensory cortex

Cameron Condylis et al. Science. .

Abstract

Although single-cell transcriptomics of the neocortex has uncovered more than 300 putative cell types, whether this molecular classification predicts distinct functional roles is unclear. We combined two-photon calcium imaging with spatial transcriptomics to functionally and molecularly investigate cortical circuits. We characterized behavior-related responses across major neuronal subclasses in layers 2 or 3 of the primary somatosensory cortex as mice performed a tactile working memory task. We identified an excitatory intratelencephalic cell type, Baz1a, that exhibits high tactile feature selectivity. Baz1a neurons homeostatically maintain stimulus responsiveness during altered experience and show persistent enrichment of subsets of immediately early genes. Functional and anatomical connectivity reveals that Baz1a neurons residing in upper portions of layers 2 or 3 preferentially innervate somatostatin-expressing inhibitory neurons. This motif defines a circuit hub that orchestrates local sensory processing in superficial layers of the neocortex.

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Conflict of interest statement

Competing interests: T.N.N. is currently employed at Cajal Neuroscience.

Figures

Fig. 1.
Fig. 1.. Multiplexed identification of transcriptomic cell subclasses and types in functionally imaged neurons.
(A) Schematic of the CRACK platform. (B) Expression patterns of genes selected to identify L2/3 S1 excitatory (blue) and inhibitory (green) cell subclasses and types. (C) Barcode scheme for multiplexed HCR-FISH of selected genes. (D) Registration of in vivo calcium-imaged neurons to ex vivo tissue section across multiple rounds of HCR-FISH. (Top left) In vivo two-photon images of RCaMP1.07+ neurons. (Top right) Ex vivo confocal images of reidentified RCaMP1.07+ neurons showing endogeneous protein (green) followed by HCR-FISH staining transcripts (magenta). (Bottom) Overlays of (left) B2-488 and (right) B1-647 readout channels across all HCR-FISH barcode rounds. (E) Decoding of in vivo imaged neuron [(D), dotted rectangle] identified as an Adamts2 cell type expressing Fst and Slc17a7. Positive readouts are identified with green rectangles. Scale bars, (D) 50 μm; (E) 20 μm.
Fig. 2.
Fig. 2.. Task encoding across L2/3 excitatory cell types.
(A) Schematic of whisker-based delayed nonmatch to sample behavioral task. (B) Encoding of task-related activity in individual neurons using a GLM. (C) Encoding of task factors determined by comparing full and partial GLM fits (ΔAIC). (D to G) Cumulative probability distributions of (D) full model deviance explained, (E) encoding strength of stimulus direction, (F) encoding strength of whisker kinematics, and (G) estimated event rate across the three excitatory cell types. [(D) to (F)] Mann Whitney U test; (G) one-tailed Student’s t test. In (E) and (F), solid and dotted lines indicate significant (P < 0.01) and nonsignificant encoding strengths, respectively, by means of χ2 test. n = 1107 neurons from seven animals.
Fig. 3.
Fig. 3.. Persistent IEG expression and homeostatic plasticity in Baz1a neurons.
(A) Examples of selectively (top) enriched and (bottom) not enriched immediate early genes in Baz1a cells. (B) Time course of bilateral whisker deprivation (BD) experiment. (C) (Top) Example of Baz1a neuron with stable high fosGFP expression across in vivo imaging sessions. (Middle) Average stimulus responses during calcium imaging. (Bottom) Post hoc identification of neuron and HCR-FISH for select genes. (D) HCR-FISH Fos spot density in high (1.2-fold above background) and low fluorescent fosGFP cells (two-tailed Student’s t test). (E) Mean stimulus-evoked activity before and after BD across functionally imaged neurons (one-way ANOVA with post hoc multiple comparison test, n = 2569 cells from three animals). (F) Change in stimulus-evoked responses before BD versus at 1 day or 5 days BD across neurons with stable low, dynamic, and stable high fosGFP expression (two-tailed Student’s t test, n = 790 cells from three animals). (G) Change in stimulus-evoked responses before BD versus at 1 day or 5 days BD across excitatory cell types (χ2 test, n = 181 Adamts2, 136 Baz1a, and 153 Agmat cells from three animals). (H) Fraction of fosGFP neurons with stable high expression across all pre-BD sessions (days −2, −1, and 0) and across all BD sessions (days 1, 3, and 5) for excitatory cell types (two-tailed Student’s t test, n = 3753 cells from three animals). * P < 0.05, ** P < 0.005 in (E) to (G). Error bars = SEM; (H) SD from bootstrap analysis.
Fig. 4.
Fig. 4.. Task encoding across L2/3 inhibitory subclasses.
(A to C) Cumulative probability distributions for (A) full model deviance explained, (B) encoding strength of stimulus direction, and (C) encoding strength of whisker kinematics for three major inhibitory subclasses (Mann Whitney U Test). (D and E) Estimated event rate responses to preferred stimulus direction for (D) Sst subclasses and (E) Vip subclasses. (F and G) Cumulative probability distribution of ΔAIC for task factor encoding direction for (F) Sst subclasses and (G) Vip subclasses (Mann Whitney U Test). (H and I) Cumulative probability distribution of ΔAIC for (H) task factors encoding sampleEARLY DELAY and (I) touch onset for Vip subclasses (Mann Whitney U Test). (J) Estimated event rate for Vip subclasses along with mean whisking amplitude aligned to whisker-rotor touch onset preceding sample and test periods. (K to M) Cumulative probability distribution of ΔAIC for task factors encoding (K) free whisking amplitude, (L) angle, and (M) phase for Vip subclasses (Mann Whitney U Test). In (B), (C), (F) to (I), and (K) to (M), solid and dotted lines indicate significant (P < 0.01) and nonsignificant encoding strengths, respectively, by means of χ2 test. Shaded regions in (D) and (E) indicate SEM. n = 48 Pvalb cells, 47 Sst/Chodl+ cells, 88 Sst/Chodl− cells, 40 Vip/Pthlh+ cells, and 49 Vip/Pthlh− cells from seven animals.
Fig. 5.
Fig. 5.. Cell type functional connectivity across task networks.
(A) Strength of coupling factor encoding across varying coupling ranks. *P < 0.02, repeated measures ANOVA test, F2,6. (B) Schematic of network analysis for example neuron. (C) Task-specific networks generated by selecting for neurons with significant encoding for a given task factor in the task GLM. Networks are sorted according to average edge strength. (D) Network strength across task networks. The dotted line indicates strength of shuffled network. (E) Cell type and subclass differences in number of significant (left) input and (right) output nodes across task networks. (F) Strength and variability of functional connectivity in network edges across task networks. Network edges with significantly high strength and low variability are indicated with a box. P < 0.05, permutation test. Shaded region in (A) indicates SEM. n = 1996 neurons, direction; 1374 neurons, sampleEARLY DELAY; 1076 neurons sampleLATE DELAY; 360 neurons, category; 623 neurons, choiceTEST; 830 neurons, choiceREPORT; 898 neurons, touch onset; 1033 neurons, touch offset; 864 neurons, kinematics; and 273 neurons, noncoding from seven animals.
Fig. 6.
Fig. 6.. Upper layer Ba1za neurons target Sst neurons.
(A and B) Example of cell type–specific transmonosynaptic tracing in (A) Sst-IRES-Cre and (B) Vip-IRES-Cre mice. (Left) Confocal images of starter cells (magenta) and nGFP+ input neurons (green). (Right) Sublaminar distribution of input density from left images, along with injection scheme. (C) Average sublaminar somatic density distribution of inputs across L2/3 for Sst and VIP neurons. (D) Relative proportion of excitatory cell types and Gad2+ inhibitory neurons as a function of laminar depth for Sst and Vip input neurons. (E) Density of excitatory cell types as a function of laminar depth for Sst and Vip input neurons. (F) Circuit model of L2/3 illustrating cell type–specific connectivity between Vip, Sst, Baz1a, and other local excitatory neurons. Shaded regions in (C) and (E) indicate SEM. n = 4 Sst-IRES-Cre animals, 16 slices, 33,957 neurons; and 4 Vip-IRES-Cre animals, 14 slices, 35,926 neurons. Scale bars, 100 μm.
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