Control of innate olfactory valence by segregated cortical amygdala circuits
- Department of Neurobiology, University of California, San Diego, United States
- Neurosciences Graduate Program, University of California, San Diego, United States
- Department of Neurosciences, University of California, San Diego, United States
- Center for Circadian Biology, University of California, San Diego, United States
- Zuckerman Mind Brain Behavior Institute, Columbia University, United States
- Salk Institute for Biological Sciences, United States
- Department of Pathology, University of California, San Diego, United States
- Howard Hughes Medical Institute, United States
Figures
Figure 1 with 2 supplements
The plCoA encodes odors of innate-valence using a population code for odor identity.
(A) Schematic representation of virus injection and GRIN lens implantation into aplCoA or pplCoA for two-photon microscopy. (B–C) Representative images (B) and traces (C) of fluorescence changes in individual neurons over an approximately 20-min period that includes periods of odor stimulation. Scale bar in (B), 100µm. (D) Schematic of odor exposure paradigm. Each trial presented 5 seconds of odor followed by a variable inter-trial interval (20–30 s). Odors were present in blocks of 20 trials per odor, with 2 counterbalanced block schedules (1 and 2). Six odors were used: the appetitive odors 2-phenylethanol (2PE) and peanut oil (Peanut), the neutral odors heptanol (HEP) and isoamyl acetate (IAA), and the aversive odors trimethylthiazoline (TMT) and 4-methylthiazoline (4MT). (E) Heatmap of trial-averaged and Z-scored odor-evoked activity over time from pooled plCoA neurons. Responses are grouped by hierarchical clustering, with the dendrogram (right). Odor delivery marked by vertical red lines. (F) Average of trial-averaged and Z-scored odor-evoked activity for each cluster concatenated. The order of color-coded blocks corresponds to the order of clusters in (E). (G) Proportion of neurons responsive to different numbers of odors. Bars represent the mean across 13 animals, and the error bars show SEM. (H) Proportion responsive to each odor for aplCoA (red) or pplCoA (blue). (I) Valence scores of individual neurons. White circles show the median of each distribution, whereas the gray rectangle shows the 25th-75th percentile range. (J) Proportion of neurons with significant valence scores calculated as a function of trial number. Calculated with a 10-trial moving window. Top half shows those with significant positive valence scores, the bottom half shows those with significant negative valence scores. (K) The percentage of neurons with responses (Z>2 for at least 5 frames) as a function of trial number for each odor. (L) Left, MNR accuracies for all pooled plCoA neurons (data) and a control distribution where the training labels are shuffled (shuffled) in a violin plot. Right, proportion of neurons in each animal that have MNR accuracy greater than the 95th percentile of the shuffled MNRs. (M) Cross-validated average accuracies of multinomial SVMs plotted as a function of the number of neurons used for training during the odor period. Circles represent the mean across 100 iterations of random sampling of neurons and error bars show the standard deviation. (N) Cross-validated accuracy of ecoc-SVM classifiers for a six-odor classification task trained using 200 neurons as a function of time. Lines indicate means and shaded areas show the standard deviation across 100 random samplings of 200 neurons from the pooled data and shuffled training controls where the label vectors are randomly shuffled. (O) An example confusion matrix for a multinomial SVM trained with 200 neurons. (O) Comparison of inter-valence and intra-valence confusion across number of neurons used in training the classifiers. Filled circles show the average of the data across 100 iterations, open circles show shuffled controls. (Q) The normalized average distance between odor pairs that have different valence (inter) or same valence (intra). Across panels, ns, not significant. Additional specific details of statistical tests can be found in Supplementary file 1.
Figure 1—figure supplement 1
Additional information for imaging experiments.
Related to Figure 1. (A) Histologically verified placements for GRIN lenses (bars) in wild type animals infected with AAV-hSyn-GCaMP8s for calcium imaging experiments. Horizontal lines indicate locations where lens placements were visible in coronal sections at various positions in the plCoA anterior-posterior axis. Different color bars represent placements in each of 13 animals. Those scored as anterior are outlined in red, and those in posterior are outlined in blue. (B) Histology for anterior plCoA (left) and posterior plCoA (right) lens implant and jGCaMP8s (green) viral injection sites in representative animals. Scale bar for full size images, 500 µm; scale bar for magnified images, 200 µm. (C) PID recordings across 20 repeated presentations of each odor. Colored traces represent individual trials (every other trial shown for clarity). Horizontal black bars indicate 5 second odor deliveries. (D) Mean PID amplitude during the odor presentation period plotted across trials for each odor. For each trace, the baseline was shifted to approximately zero to compensate for drift in recordings over time. (E) Representative peristimulus time heatmaps showing single-neuron responses across repeated odor presentations. Each panel displays activity from one neuron to one odor. The odor delivery window (0–5 s) is indicated by white dashed lines. (F) Histograms reporting the distribution for the number of trials neurons respond to each odor. Neurons were classified as responsive on any trial with a Z>2. (G) Ranked sub-accuracies for single-neuron MNR classifiers (Figure 1L) in violin plots for those trained on real (top) or shuffled (bottom) data. Black lines connect the median across each rank. (H) Scatter plot showing proportion of sub-accuracies exceeding the 95th percentile of the shuffled controls for each animal, ordered by sub-accuracy rank. Red and blue lines show the linear and exponential fit, respectively, and a gray dashed line connects data averages. (I) Heatmap of the normalized average pairwise Euclidean distance between odor response vectors across biological replicates.
Figure 1—figure supplement 2
Analysis of odor-evoked calcium activity with respect to walking behavior during imaging trials.
Related to Figure 1. (A) Heatmap of normalized walking velocity in head-fixed mice across imaging trials. Trials grouped by hierarchical clustering of velocity during and after odor exposure, with the dendrogram (left). Odor delivery indicated by vertical red lines. (B) Mean of trial-averaged and z-scored velocity for each cluster, without respect to odor identity. (C) Percentage of trials for each cluster separated by odor. (D) Percentage of trials for each cluster as a function of trial number, plotted for each odor. (E) Heatmap of trial averaged and z-scored odor-evoked activity clustered according to the walking velocity cluster (A–D). (F) Mean of trial-averaged and Z-scored calcium activity for each velocity cluster, concatenated. The order of color-coded blocks corresponds to the order of clusters in (E).
Figure 2 with 3 supplements
The plCoA has a topographic organization capable of driving approach and avoidance behaviors.
(A) Schematic of plCoA domains divided into anterior (aplCoA), middle (mplCoA), and posterior (pplCoA) regions based on histology, positioning, and gradients observed in past observations (Root et al., 2014). (B) Strategy to activate anterior-posterior topographical ensembles via optogenetics. (C) Representative histology and fiber/virus placement for aplCoA and pplCoA ChR2 animals. Scale bar, 500 µm. (D) Schematic of four-quadrant open field behavioral assay with closed-loop photostimulation. (E–F) Linear fit of change in performance index (E) or mean port distance (F) as a function of anterior-posterior position along plCoA for optical stimulation. (G) Paths traveled during the stimulus period for a representative mouse (left) and baseline-normalized collective heatmaps (right) from both the ChR2- and eYFP-infected groups with aplCoA-localized fiber implants. Lower right stimulus quadrant indicated in blue. (H–I) Mean effect of Photostimulation of aplCoA neurons on time spent in stimulated quadrant (performance index) (K) and distance from the corner (port distance) (I). (J) Paths traveled during the treatment period for a representative mouse (left) and baseline-normalized collective heatmaps (right) from both the ChR2- and eYFP-infected groups with pplCoA-localized fiber implants. Lower right stimulus quadrant marked in blue. (K–L) Mean effect of photostimulation of pplCoA neurons infected with ChR2, but not eYFP, is sufficient to increase time spent in the stimulation quadrant (K) and reduce its average distance from the stimulation port during the stimulation period (L). Abbreviations: aplCoA, anterior zone of posterolateral cortical amygdala; mplCoA, middle zone of posterolateral cortical amygdala; pplCoA, posterior zone of posterolateral cortical amygdala. Across panels, ns, not significant; * p<0.05; ** p<0.01; *** p<0.001. Specific details of statistical tests can be found in Supplementary file 1.
Figure 2—figure supplement 1
Targeting of plCoA neurons for optogenetic stimulation.
Related to Figures 2, 5 and 7 (A) Histologically verified placements for optic fiber implants (bars) and viral injection sites (circles) in wild type animals infected with AAV-hSyn-ChR2-mCherry (blue) or AAV-hSyn-eYFP (green) in topographic optogenetic cell body stimulation experiments in Figure 1. (B) Same as (A), but for cell-type-specific optogenetic cell body stimulation experiments for VGluT1-Cre (top) and VGluT2-Cre (bottom) animals infected with AAV-DIO-EF1A-ChR2-eYFP (blue) or AAV-EF1A-DIO-eYFP (green) in Figure 5. (C) Respective placements for fiber implants (bars) and injection sites (plCoA-NAc, circles; plCoA-MeA, diamonds) in wild-type animals infected with AAV-hSyn-ChR2-mCherry (blue) or AAV-hSyn-eYFP (green) in projection-specific optogenetic axon terminal stimulation experiments in Figure 7. n denotes the number of mice per group batched across 4-quad, elevated plus maze, and open field test experiments, exceeding n values for individual experiments due to behavioral cohort design (see STAR Methods). Relevant regions are highlighted in gray and outlined: plCoA (red), NAc (purple, only in C), and MeA (pink, only in C). All mouse brain sections reproduced from Paxinos and Franklin, 5th Edition, and numbers below all images denote their anterior-posterior distance from bregma in this atlas (Paxinos and Franklin, 2019). All scale bars, 500 µm.
Figure 2—figure supplement 2
Activation of sparse ensembles in aplCoA and pplCoA elicits avoidance and approach.
Related to Figure 2. The Arc-creERT2 mouse (Root et al., 2014) in combination with a cre-dependent AAV was used to express ChR2-eYFP in either aplCoA or pplCoA. Mice were administered tamoxifen and exposed to TMT. (A) Representative images of ChR2-eYFP in the aplCoA (top) or pplCoA (bottom). (B) Optogenetic stimulation-induced change in performance index for aplCoA- (left) or pplCoA-injected (right) mice. Photostimulation induces aversive responses in aplCoA and approach responses in pplCoA. * p<0.05; ** p<0.01; Additional specific details of statistical tests can be found in Supplementary file 1.
Figure 2—figure supplement 3
Behavioral effects of topographic plCoA stimulation are limited to valence alone.
Related to Figure 2. (A) Behavioral paradigm for optogenetic stimulation in the open field test. (B–D) Optogenetic stimulation-induced change in time spent (B) and number of entries (C) into the open arms, as well as distance traveled (D) in the elevated plus maze is not correlated to anteroposterior axis position in plCoA. (E–G) Effects of optogenetic stimulation of aplCoA neurons in the elevated plus maze in 1 min bins over time (left) and during off and on periods (right). Photostimulation of aplCoA neurons does not induce a significant change in time spent (E) and number of entries (F) into the open arms, as well as distance traveled (G) in the elevated plus maze. (H–J) Effects of optogenetic stimulation of pplCoA neurons in the elevated plus maze in 1 min bins over time (left) and during off and on periods (right). Photostimulation of pplCoA neurons does not induce a significant change in time spent (H) and number of entries (I) into the open arms, as well as distance traveled (J) in the elevated plus maze. (K) Behavioral paradigm for optogenetic stimulation in the open field test. (L–N) Optogenetic stimulation-induced change in time spent in the center (L) and corners (M), as well as distance traveled (N) in the open field test is not correlated to anteroposterior axis position in plCoA. (O–Q) Effects of optogenetic stimulation of aplCoA neurons in the open field test in 1 min time bins (left) and during off and on periods (right). Photostimulation of aplCoA neurons does not induce a significant change in time spent in the center (O) and corners (P), as well as distance traveled (Q) in the open field test. (R–T) Effects of optogenetic stimulation of pplCoA neurons in the open field test in 1 min time bins (left) and during off and on periods (right). Photostimulation of pplCoA neurons does not induce a significant change in time spent in the center (R) and corners (S), as well as distance traveled (T) in the open field test. All ‘ON’ and ‘OFF’ comparisons in bar graphs and linear regressions are on a per 5 min basis. (B–D, L–M) Least-squares linear regression ±95% confidence interval. Across panels: ns, not significant. Additional specific details of statistical tests can be found in Supplementary file 1.
Figure 3 with 2 supplements
Transcriptomic heterogeneity of plCoA molecular cell types.
(A) Schematic of freeze-and-re-pool strategy for snRNA-seq. (B) Two-dimensional UMAP (n=47,132 nuclei, see also Figure 3—figure supplement 1), colored by broad cellular identity assigned by graph-based clustering of neuronal and non-neuronal nuclei. (C) Cell-type-specific expression of canonical marker genes indicating broad cellular identity in the brain. Dot size is proportional to percentage of nuclei expressing the marker, with color scale representing normalized expression level. (D) Total proportion of cells of each identified type in each domain of plCoA. (E) Two-dimensional UMAP of glutamatergic neurons, colored by molecular cell type. (F) Clustered heatmap showing Euclidean distance between averages of each subtype positioned based on hierarchical clustering (left), and dot plot of marker genes for all glutamatergic subtypes (right). (G) Two-dimensional UMAP of GABAergic neurons, colored by molecular cell type, like in (E). (H) Clustered heatmap showing Euclidean distance between averages of each subtype positioned based on hierarchical clustering (left), and dot plot of marker genes for all GABAergic subtypes (right), like in (F).
Figure 3—figure supplement 1
Additional information and quality control for single-nucleus sequencing experiments.
Related to Figure 3. (A) Representative images of tissue microdissection sites from aplCoA and pplCoA following extraction and DAPI staining (blue). Scale bars, 500 µm. (B) Location of all tissue sample sites used for snRNA-seq, color coded by plCoA zone (n=3 pools per zone, 4–11 sections per pool). Scale bars, 500 µm. (C) Validation of nuclear enrichment after FANS. Ethidium homodimer-1 (EthD-1, red) labels nuclei on a hemocytometer after sorting, with an absence of non-nuclear, EthD-1-negative debris. Scale bar, 100 µm. (D) Common gating strategy for FANS sorts for snRNA-seq in a representative sample. Far left, morphology gate on forward and side scatter area excludes likely debris. Middle left, forward scatter gate excludes nuclear doublets with high forward scatter width. Middle right, side scatter gate excludes nuclear multiplets with high side scatter width. Far right, stoichiometric DRAQ7+ fluorescence allows enrichment of single nuclei and exclusion of debris and multiplets. (E) Absolute number and proportion of snRNA-seq nuclei passing quality control filters from each replicate in each plCoA zone (n=27,726 in aplCoA, 19,406 in pplCoA, 3 libraries/batches each). (F) Violin plot of UMIs detected per snRNA-seq nucleus for each replicate, filtered at the median per library +five times the median absolute deviation within each library (median 6081 UMIs/nucleus). (G) Violin plot of genes detected per nucleus from each replicate, filtered at a minimum of 1000 features per nucleus (median 2547 genes/nucleus). (H) Percent mitochondrial gene UMIs per snRNA-seq nucleus, filtered at median +five times the median absolute deviation per library (median 0.02% mitochondrial UMIs/nucleus). (I) Percent ribosomal gene UMIs per snRNA-seq nucleus, filtered at median +five times the median absolute deviation per library (median 0.17% ribosomal UMIs/nucleus). (J) Principal component analysis of pseudobulk snRNA-seq samples created from each batch, colored based on their combination of zone and batch identity. (K) Evaluation of transcriptomic homology between batches, where the distance matrix is based on Spearman correlation between median expression of highly variable features for the whole dataset, and the dendrogram was created via hierarchical clustering of batches on this correlation matrix. (L) UMAP of all snRNA-seq nuclei colored by both target region and batch identity. (M) Relative proportion of nuclei of each type for all snRNA-seq batches. Brain diagrams were reproduced from Paxinos and Franklin, 2019.
Figure 3—figure supplement 2
Additional information and quality control for spatial gene expression.
Related to Figure 4. (A–C) Allen ISH data for marker genes from molecular cell types adjacent to, but not within plCoA. Sim1 (A) marks cells in the NLOT, Etv1 (B) marks cells in the BLA and posterior basomedial amygdala, and Fign (C) marks cells in the cortex-amygdalar transition area, but none of these mark cells in the plCoA. Arrow points to respective clusters of cells marked with these genes. (D) UMAP of all plCoA GABAergic neurons, colored by domain of origin. (E) Relative proportion of molecular subtype nuclei from each domain within GABAergic neurons. Dotted line indicates chance level for plCoA GABAergic neuron nuclei. (F) UMAP of all plCoA OPCs, colored by domain of origin. (G) Two-dimensional UMAP of astrocytes, colored by molecular cell type. (H) Heatmap of astrocyte subtype marker genes. (I) UMAP of all plCoA astrocytes, colored by domain of origin. (J) Left, relative proportion of molecular subtype nuclei from each domain within astrocytes. A dotted line indicates chance level for plCoA astrocyte nuclei. Right, relative abundance of each astrocyte subtype within plCoA. (K) Two-dimensional UMAP of immune cells, colored by molecular cell type. (L) Heatmap of immune cell subtype marker genes. (M) UMAP of all plCoA immune cells, colored by domain of origin. (N) Left, relative proportion of molecular subtype nuclei from each domain within immune cells. A dotted line indicates chance level for plCoA immune cell nuclei. Right, relative abundance of each immune cell type within plCoA. (O) Two-dimensional UMAP of VLMC nuclei, colored by molecular cell type. (P) Heatmap of VLMC subtype marker genes. (Q) UMAP of all plCoA VLMC nuclei, colored by domain of origin. (R) Left, relative proportion of molecular subtype nuclei from each domain within VLMCs. Dotted line indicates chance level for plCoA VLMC nuclei. Right, relative abundance of each VLMC subtype within plCoA. (S) Left, representative section on a Visium slide capture area stained with hematoxylin and eosin. Right, representative section with capture spots overlaid (gray) and plCoA-overlapping spots highlighted (red). (T) Violin plots of quality metrics for individual Visium sections on a per-spot basis in plCoA-overlapping spots (N=21 sections). Upper left, UMIs per spot; upper right, features per spot; lower left, proportion mitochondrial UMIs per spot; lower right, proportion ribosomal UMIs per spot. (U) Number of plCoA-overlapping capture spots per section (n=3,616 total spots). (V) Two-dimensional UMAP of all plCoA-overlapping capture spots, colored by section of origin. (W) Evaluation of transcriptomic homology between sections, where the distance matrix is based on Spearman correlation between median expression of highly variable features for the whole dataset, and the dendrogram was created via hierarchical clustering of sections on this correlation matrix. Across panels: ** p<0.01; *** p<0.001; ns, not significant. Additional specific details of statistical tests can be found in Supplementary file 1.
Figure 4
Glutamatergic neuron subtypes in plCoA are spatially distributed along an anteroposterior molecular gradient.
(A) UMAP of all plCoA nuclei colored by zone of origin, with dotted outlines and labels denoting the major cell types. (B) Relative proportion of nuclei from each domain within each broad identity class. Dotted line indicates chance level for all plCoA nuclei. (C) Top, abundance of domain-specific DEGs for each major cell type, either enriched in aplCoA nuclei (top) or pplCoA nuclei (bottom). Bottom, volcano plots for domain-specific DEGs for glutamatergic (left) and GABAergic neurons (right), the two cell types with the greatest degree of domain-specific gene expression, where negative log-fold changes indicate enrichment in pplCoA and positive log-fold changes indicate enrichment in aplCoA. (D) UMAP of plCoA glutamatergic neurons colored by domain of origin, with dotted outlines and labels denoting the subtypes on the graph. Groups of glutamatergic neuron types identified previously via Euclidean distance and hierarchical clustering are overlaid on top of the neuron types of interest. (E) Relative proportion of molecular subtypes from each domain within glutamatergic neurons, where relevant subtypes are outlined according to their glutamatergic neuron group. Dotted line indicates chance level for plCoA glutamatergic neuron nuclei. (F) UMAP of all glutamatergic neuron nuclei, colored by expression levels of VGluT2 (top) or VGluT1 (bottom). (G) Left, representative images of in situ RNAscope labeling of VGluT2 RNA (red) and VGluT1 RNA (green) across plCoA domains. Right, proportions of glutamatergic neurons expressing VGluT2, VGluT1, or both. Scale bars, 500 µm (main image), 50 µm (inset). (H) UMAP of all plCoA-overlapping Visium capture spots, colored by cluster. Broad spatial position of groups of clusters is overlaid on top of the capture spots of interest. (I) UMAP of all plCoA-overlapping Visium capture spots, colored by expression levels of VGluT2 (top) or VGluT1 (bottom). (J) Representative plCoA-overlapping region of one section on a Visium slide capture area, with capture spots colored by cluster. (K) Representative plCoA-overlapping region of one section on a Visium slide capture area, with capture spots colored by expression levels of VGluT2 (top) or VGluT1 (bottom). (L) Prediction scores for representative glutamatergic neuron subtypes within Group 1 (left) and Group 2 (right), shown on a UMAP of all plCoA-overlapping capture spots across all sections (top) and on a representative plCoA-overlapping region of one section (bottom). (M) Prediction scores for a representative GABAergic neuron subtype, shown on a UMAP of all plCoA-overlapping capture spots across all sections (top) and on a representative plCoA-overlapping region of one section (bottom). Across panels: * p<0.05; ** p<0.01; *** p<0.001; ns, not significant. Additional specific details of statistical tests can be found in Supplementary file 1.
Figure 5 with 1 supplement
Glutamatergic plCoAVGluT2+ and plCoAVGluT1+ neurons contribute to innate approach and avoidance behaviors.
(A) Schematic for selective photostimulation of distinct glutamatergic cell type. VGluT2-Cre and VGluT1-Cre animals were injected with Cre-dependent viral vectors into mplCoA with a fiber optic implant placed just above the injection site. (B) Representative histology from ChR2 viral injection and fiber implantation site in a VGluT2-Cre animal (top) and a VGluT1-Cre animal (bottom). Scale bar, 500 µm. (C) Baseline-normalized collective heatmaps from both the ChR2- and eYFP-infected groups in VGluT2-Cre and VGluT1-Cre animals with plCoA-localized fiber implants. Lower right stimulus quadrant marked in blue. (D–G) Effect of photostimulation of plCoAVGluT2+ neurons (D–E) or plCoAVGluT1+ neurons (F–G) on time spent in the stimulation quadrant (D, F) and distance from the corner (E, G). (H) Behavioral paradigm to assess innate valence responses to odor. Left, schematic of four-quadrant open field behavioral assay for spatially specific odor delivery. Upper right, within-trial timeline. Lower right, odors delivered and their associated innate valence. (I) Schematic of strategy for selective chemoinhibition of molecularly defined glutamatergic plCoA neurons. (J–M) Effect of chemoinhibition of plCoA VGluT1+ neurons on time spent in the odor quadrant (J, L) or decrease in mean port distance (K, M) in response to 2PE (J–K) or TMT (L–M). (N–Q) Effect of chemoinhibition of plCoA VGluT2+ neurons on time spent in the odor quadrant (N, P) or decrease in mean port distance (O, Q) in response to 2PE (N–O) or TMT (P–Q). Across panels, ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001, **** p < 0.0001. Additional specific details of statistical tests can be found in Supplementary file 1.
Figure 5—figure supplement 1
Additional information for Cre-dependent molecularly targeted chemogenetic inhibition experiments.
Related to Figure 5. (A) Representative histology for inhibition experiments for AAV-hM4D(Gi)-mCherry-infected VGluT2-Cre (left) or VGluT1-Cre (right) animals. (B) Strategy for chemogenetic inhibition in the open field test and elevated plus maze. (C–E) Effect of chemogenetic inhibition of plCoAVGluT2+ neurons in the elevated plus maze. Inhibition does not induce a significant change in time spent (C) or number of entries (D) into the open arms, as well as distance traveled (E) in the elevated plus maze. (F–H) Effect of chemogenetic inhibition of plCoAVGluT2+ neurons in the open field test. Inhibition does not induce a significant change in time spent in the center (F) or corners (G), as well as distance traveled (H) in the open field test. (I–K) Effect of chemogenetic inhibition of plCoAVGluT1+ neurons in the elevated plus maze. Inhibition does not induce a significant change in time spent (I) and number of entries (J) into the open arms, as well as distance traveled (K) in the elevated plus maze. (L–N) Effect of chemogenetic inhibition of plCoAVGluT1+ neurons in the open field test. Inhibition does not induce a significant change in time spent in the center (L) or corners (M), as well as distance traveled (N) in the open field test. (O) Histologically verified placements for viral injection sites in VGluT2-Cre animals infected with AAV-DIO-hSyn-mCherry (red) or AAV-DIO-hSyn-hM4D(Gi) (light orange) in molecularly targeted chemogenetic inhibition experiments. (P) Same as (O), but in VGluT1-Cre animals. n denotes number of mice per group batched across 4-quad, elevated plus maze, and open field test experiments, exceeding n values for individual experiments due to behavioral cohort design (see STAR Methods). All mouse brain sections reproduced from Paxinos and Franklin, 5th Edition, with plCoA highlighted in gray and outlined in purple, and numbers below all images denote its anterior-posterior distance from bregma in this atlas (Paxinos and Franklin, 2019). All scale bars, 500 µm. Across panels: ns, not significant. Additional specific details of statistical tests can be found in Supplementary file 1.
Figure 6 with 2 supplements
Projections to MeA and NAc from plCoA are topographically organized.
(A) Left, whole-hemisphere view at AP = 0.98 mm from bregma. Scale bar, 500 µm. Right, Magnified images of the areas highlighted inside white dashed lines. Scale bar, 200 µm. (B) Left, whole-hemisphere view at AP = –1.06 mm from bregma. Scale bar, 500 µm. Right, Magnified images of the areas highlighted inside white dashed lines. Scale bar, 200 µm. (C) Other plCoA projections not found in cross-sections of the brain found in (A) and (B). Scale bar, 200 µm. (D) Magnitude of anterograde synaptophysin-eYFP fluorescence in primary downstream targets of plCoA projection neurons ordered by total output strength, colored based on each region’s function. (E) Schematic for topographic retrograde mapping strategy from MeA and NAc into plCoA. Red retrobeads are injected into MeA or NAc and topographical projection bias is examined along the anterior-posterior axis. (F) Representative images (top) for injection into MeA (left) or NAc (right) and number of neurons labeled along the anterior-posterior axis as distance (mm) from bregma (bottom). Gray lines denote individual replicates, where colored lines indicate mean ± s.e.m. (G) Proportion of retrobead-labeled neurons projecting to MeA or NAc for each 100 µm segment as a function of distance from bregma. Dashed line indicates overall balance of all retrobead-labeled neurons across entire plCoA. (H) Proportion of retrobead-labeled neurons from either target within each plCoA zone. MeA-labeled neurons are significantly enriched in aplCoA compared to NAc-labeled neurons, while NAc-labeled neurons are significantly enriched in pplCoA compared to those labeled from MeA. (I) Representative histological images for the injection sites in aplCoA (left) and pplCoA (right) from a representative animal. Scale bar, 200 µm. (J) Representative histological images for MeA from the animal in (J). Scale bar, 200 µm. (K). Representative histological images for NAc from the animal in (J). Scale bar, 200 µm. (L) Output strength as a proportion of total fluorescence from aplCoA and pplCoA to MeA and NAc. (M) Representative histological images for the injection site in plCoA from a representative VGluT1-Cre and VGluT2-Cre animal. Scale bar, 200 µm. (N) Representative histological images from MeA and NAc from a representative animal of either genotype. Scale bar, 200 µm. (O) Left, output strength as a proportion of total fluorescence from plCoAVGluT2+ and plCoAVGluT1+ neurons to MeA and NAc. Right, comparison of same data, but by target region within genotype. (P) Same data as (O), but by target region within genotype. (Q–T) Mapping collateral projections from NAc- and MeA-projecting neurons. (Q) Representative histological images for the injection site in plCoA from a representative animal receiving retrograde virus into MeA or NAc. Scale bar, 200 µm. (R) Representative histological images of NAc and MeA retro-Cre targeting (red) and outputs (green). (S) Comparison of absolute integrated fluorescence intensities in MeA and NAc when retroAAV was injected into NAc (top) or MeA (bottom). (T) Quantification of fluorescence in selected downstream brain regions from plCoA originating from plCoA-NAc neurons proportional to eYFP fluorescence in NAc (top) or MeA (bottom). Abbreviations: NAc, nucleus accumbens; BNST, bed nucleus of stria terminalis; MeA, medial amygdala; Pir, piriform cortex; BLA, basolateral amygdala; Ahi, amygdalo-hippocampal transition area; pmCoA, posteromedial cortical amygdala; Str, striatum; OT, olfactory tubercle; EA, extended amygdala; IPAC, inferior peduncle of the anterior commissure; AA, anterior amygdala; LA, lateral amygdala; HDB, horizontal limb of the diagonal band; VP, ventral pallidum; AIC, anterior insular cortex; mfb, medial forebrain bundle; MO, medial orbitofrontal cortex; LOT, lateral olfactory tract; ACo, anterior cortical amygdala; AOA, anterior olfactory area; DG, dentate gyrus; Rt, reticular nucleus; LPO, lateral preoptic area; VMH, ventromedial hypothalamus; DEn, dorsal endopiriform claustrum; LH, lateral hypothalamus; IL, infralimbic cortex; DP, dorsal peduncular cortex; LS, lateral septum; CxA, cortex-amygdala transition area; sox, supraoptic decussation; StHy, striohypothalamic nucleus; GP, globus pallidus; PLH, perirhinal cortex; ZI, zona incerta. Across panels, ns, not significant; * p<0.05; ** p<0.01; **** p<0.0001. Additional specific details of statistical tests can be found in Supplementary file 1.
Figure 6—figure supplement 1
Additional information for plCoA anterograde tracing experiments.
Related to Figure 6. (A) Schematic for general anterograde output mapping strategy, where a virus labeling cell bodies in plCoA with mRuby and presynaptic terminals in downstream regions with synaptophysin-bound eYFP. (B) Histological image of plCoA injection site in a representative animal. Scale bar, 200 µm. (C) Histologically verified placements for viral injection sites in initial anterograde tracing experiments. (D) Schematic for dual-color topographic anterograde tracing: two counterbalanced fluorophores were injected into aplCoA and pplCoA, and each color was quantified in major projection targets of plCoA. (E) Histologically verified placements for viral injection sites in dual-color anterograde tracing experiments. (F) Schematic for Cre-dependent anterograde output mapping strategy: a Cre-dependent virus expressing eYFP was injected into either a VGluT2-Cre or VGluT1-Cre animal to determine relative output enrichment for either broad cell type. (G) Histologically verified placements for viral injection sites in Cre-dependent molecular anterograde tracing experiments. (H) Schematic of anterograde viral strategy to explore collateralization of plCoA MeA and NAc projection neurons to other regions. (I) Histologically verified placements for viral injection sites in collateralization experiments. Relevant regions are highlighted in gray and outlined: plCoA (red), NAc (purple, only in I), and MeA (pink, only in I). All mouse brain sections reproduced from Paxinos and Franklin, 5th Edition, and numbers below all images denote their anterior-posterior distance from bregma in this atlas (Paxinos and Franklin, 2019). All scale bars 500 μm unless noted elsewhere.
Figure 6—figure supplement 2
Analysis of VGluT expression in MeA- and NAc-projecting neurons.
Related to Figure 6. (A) Representative mplCoA images of in situ RNAscope labeling of VGluT2 (red) and VGluT1 (green) in animals with Retrobeads (blue) injected into the MeA (top) or NAc (bottom). The RNAscope and Retrobead signals are overlaid on DAPI staining (gray). White arrow in merged image points to a Retrobead+ and VGluT2+ cell. (B) Quantification of the percentage of Retrobead labeled neurons positive for VGluT1, VGluT2, or both for MeA- and NAc-projecting neurons (top and bottom, respectively) in mplCoA. White arrow in merged image points to a Retrobead+ and VGluT1+ cell.
Figure 7 with 1 supplement
Divergent neurons projecting from plCoA to NAc and MeA control innate olfactory attraction and aversion.
(A) Schematic for optogenetic MeA terminal stimulation in plCoA neurons. Strategy to activate MeA-projecting plCoA neuron terminals via optogenetics (top) and representative histology from ChR2 viral injection and fiber implantation site (bottom). Scale bars, 200 µm. (B) Baseline-normalized collective heatmaps from both the ChR2- and eYFP-infected plCoA groups with MeA- and NAc-localized fiber implants. Lower right stimulus quadrant marked in blue. (C–D) Optogenetic MeA terminal stimulation of plCoA neurons infected with ChR2, but not eYFP, is sufficient to reduce time spent in the stimulation quadrant (C) and increase its average distance from the port (D) during the stimulation period. (E–F) Optogenetic NAc terminal stimulation of plCoA neurons infected with ChR2, but not eYFP, is sufficient to increase time spent in the stimulation quadrant (E) and decrease its average distance. from the stimulation port (F) during the stimulation period. (G) Viral strategy for selective retrograde chemoinhibition of projection-defined plCoA neurons. (H) Schematic for selective retrograde chemoinhibition of projection-defined plCoA neurons. (I–L) Chemoinhibition of NAc-projecting plCoA neurons significantly eliminates the 2PE-evoked increase in time spent in the odor quadrant (I) and decreases in mean port distance (J). The response to TMT is unaffected in time spent in odor quadrant (K) or port distance (L). (M–P) Chemoinhibition of MeA-projecting plCoA neurons does not affect 2PE-evoked increase in time spent in the odor quadrant (M) or decrease in mean port distance (N) significantly decreases the TMT-evoked reduction in time spent in the odor quadrant (O) or increase in mean port distance (P). Across panels, ns, not significant; * p<0.05; ** p<0.01; *** p<0.001. Additional specific details of statistical tests can be found in Supplementary file 1.
Figure 7—figure supplement 1
Manipulation of plCoA projections to MeA or NAc in either direction does not change features of behavior unrelated to innate valence.
Related to Figure 7. (A–C) Effects of optogenetic stimulation of plCoA terminals in MeA in the elevated plus maze in time bins of 1 min (left) and during off and on periods (right). Photostimulation does not induce a significant change in time spent (A) and number of entries (B) into the open arms, as well as distance traveled (C) in the elevated plus maze. (D–F) Effects of optogenetic stimulation of plCoA terminals in NAc in the elevated plus maze in time bins of 1 min (left) and during off and on periods (right). Photostimulation does not induce a significant change in time spent (H) and number of entries (I) into the open arms, as well as distance traveled (J) in the elevated plus maze. (G–I) Effects of optogenetic stimulation of plCoA terminals in MeA in the open field test in time bins of 1 min (left) and during off and on periods (right). Photostimulation does not induce a significant change in time spent in the center (G) and corners (H), as well as distance traveled (I) in the open field test. (J–L) Effects of optogenetic stimulation of plCoA terminals in NAc in the open field test in time bins of 1 min (left) and during off and on periods (right). Photostimulation does not induce a significant change in time spent in the center (G) and corners (H), as well as distance traveled (I) in the open field test. (M–O) Effects of chemogenetic inhibition of plCoA-MeA projection neurons in the elevated plus maze. Inhibition does not induce a significant change in time spent (M) and number of entries (N) into the open arms, as well as distance traveled (O) in the elevated plus maze. (P–R) Effects of chemogenetic inhibition of plCoA-MeA projection neurons in the open field test. Inhibition does not induce a significant change in time spent in the center (P) and corners (Q), as well as distance traveled (R) in the open field test. (S–U) Effects of chemogenetic inhibition of plCoA-NAc projection neurons in the elevated plus maze. Inhibition does not induce a significant change in time spent (S) and number of entries (T) into the open arms, as well as distance traveled (U) in the elevated plus maze. (V–X) Effects of chemogenetic inhibition of plCoA-NAc projection neurons in the open field test. Inhibition does not induce a significant change in time spent in the center (V) and corners (W), as well as distance traveled (X) in the open field test. Across panels: ns, not significant. Additional specific details of statistical tests can be found in Supplementary file 1.
Figure 8
Models that could support innate odor-evoked attraction and avoidance.
Two potential models that could support valence responses to odor with a population code imposed on divergent circuitry. (A) Balance of activation between MeA- and NAc-projecting neurons determines the valence. In this model, an odor may activate a different proportion of these projection-defined neurons, and the valence is determined by the balance. For example, activation of more NAc-projecting neurons should cause attraction, and activation of more MeA-projecting neurons should cause aversion. (B) Dynamic activity evolves over time due to recurrent processing or integration of behavioral state variables, in an attractor-like network. In this model, the activity may evolve from an initial broad population code (T0) towards preferential activation of one output population over time (Tn).
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mouse) | C57BL/6 J | Jackson labs | RRID:MGI:3028467 | |
| Strain, strain background (Mouse) | VGlut2-cre | Jackson labs | RRID:IMSR_JAX:028863 | |
| Strain, strain background (Mouse) | VGlut1-cre | Jackson labs | RRID:IMSR_JAX:023527 | |
| Strain, strain background (Mouse) | Arc-CreERT2 | Jackson labs | RRID:IMSR_JAX:022357 | |
| Transfected construct (AAV) | AAV5-hSyn-eYFP | UNC Vector Core | RRID:SCR_023280 | |
| Transfected construct (AAV) | AAV5-hSyn-ChR2-mCherry-WPRE-PA | UNC Vector Core | RRID:SCR_023280 | |
| Transfected construct (AAV) | AAV5-EF1A-DIO-hChR2(H134R)-eYFP | UNC Vector Core | RRID:SCR_023280 | |
| Transfected construct (AAV) | AAV5-EF1A-DIO-eYFP | UNC Vector Core | RRID:SCR_023280 | |
| Transfected construct (AAV) | AAV9-hSyn-jGCaMP8s-WPRE | Addgene | RRID:Addgene_162374 | |
| Transfected construct (AAV) | AAV2-hSyn-DIO-hM4D(Gi)-mCherry | Addgene | RRID:Addgene_44362 | |
| Transfected construct (AAV) | AAVretro-hSyn-EGFP-Cre | Addgene | RRID:Addgene_105540 | |
| Transfected construct (AAV) | AAVDJ-hSyn-FLEX-mRuby-T2A-SynEGFP | Provided as a gift From Byungkook Lim | PMID:28689640 | |
| Transfected construct (AAV) | AAV5-EF1A-mCherry-IRES-Cre-WPRE | Addgene | RRID:Addgene_55632 |
Additional files
-
MDAR checklist
- https://cdn.elifesciences.org/articles/104677/elife-104677-mdarchecklist1-v1.pdf
-
Supplementary file 1
Statistical analysis details for all figures.
- https://cdn.elifesciences.org/articles/104677/elife-104677-supp1-v1.xlsx
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Control of innate olfactory valence by segregated cortical amygdala circuits
eLife 14:RP104677.
https://doi.org/10.7554/eLife.104677.3
