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Neuromics chicken anti cgrp
Chicken Anti Cgrp, supplied by Neuromics, used in various techniques. Bioz Stars score: 91/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/chicken+anti+cgrp/pmc06361136-340-187-191?v=Neuromics
Average 91 stars, based on 19 article reviews

chicken anti cgrp - by Bioz Stars, 2026-08

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a. Schematic of the CTA paradigm. b, Flavor preference across three consecutive daily retrieval tests for mice that consumed either a novel (top) or familiar (bottom) flavor and then were injected with either LiCl (red) or saline (black) on pairing day ( n = 8 mice per group). The specific flavor (sweetened grape kool-aid) and amount consumed (1.2 ml) was the same for all groups. The familiar group was pre-exposed to the flavor on four consecutive days before conditioning, whereas the novel group was completely naïve. c, Schematic and example Fos expression data (100-µm maximum intensity projection) for the brainwide light sheet imaging pipeline. d, Schematic of the Consumption Fos timepoint ( n = 12 mice per group). The line above “10 min” is a scale bar and the gray bar represents the 60-min wait before perfusion. e, Novel flavors preferentially activate sensory and amygdala regions. Left: Comparison of individual familiar (blue) and novel (red) flavor condition mice for every significantly novel flavor-activated brain region. Right: Visualization of the spatially resolved difference in Fos + cell density across flavor conditions with Allen CCF boundaries overlaid. f, Familiar flavors preferentially activate limbic regions. Panels are analogous to e . P -values in b are from GLMM marginal effect z -tests corrected for multiple comparisons across retrieval days within each flavor condition, P -values in e,f are from GLMM marginal effect z -tests corrected for multiple comparisons across timepoints within each brain region . * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. Error bars represent mean ± s.e.m. See for list of brain region abbreviations.

Journal: bioRxiv

Article Title: A neural mechanism for learning from delayed postingestive feedback

doi: 10.1101/2023.10.06.561214

Figure Lengend Snippet: a. Schematic of the CTA paradigm. b, Flavor preference across three consecutive daily retrieval tests for mice that consumed either a novel (top) or familiar (bottom) flavor and then were injected with either LiCl (red) or saline (black) on pairing day ( n = 8 mice per group). The specific flavor (sweetened grape kool-aid) and amount consumed (1.2 ml) was the same for all groups. The familiar group was pre-exposed to the flavor on four consecutive days before conditioning, whereas the novel group was completely naïve. c, Schematic and example Fos expression data (100-µm maximum intensity projection) for the brainwide light sheet imaging pipeline. d, Schematic of the Consumption Fos timepoint ( n = 12 mice per group). The line above “10 min” is a scale bar and the gray bar represents the 60-min wait before perfusion. e, Novel flavors preferentially activate sensory and amygdala regions. Left: Comparison of individual familiar (blue) and novel (red) flavor condition mice for every significantly novel flavor-activated brain region. Right: Visualization of the spatially resolved difference in Fos + cell density across flavor conditions with Allen CCF boundaries overlaid. f, Familiar flavors preferentially activate limbic regions. Panels are analogous to e . P -values in b are from GLMM marginal effect z -tests corrected for multiple comparisons across retrieval days within each flavor condition, P -values in e,f are from GLMM marginal effect z -tests corrected for multiple comparisons across timepoints within each brain region . * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. Error bars represent mean ± s.e.m. See for list of brain region abbreviations.

Article Snippet: CGRP stim timepoint samples also received primary (chicken anti-GFP, 1:500; Aves GFP-1020) and secondary (Alexa Fluor 594 donkey anti-chicken, 1:500; Jackson Immuno 703-585-155) antibodies for ChR2-YFP immunolabeling during the above protocol.

Techniques: Injection, Saline, Expressing, Imaging, Comparison

a, Map of average Fos + cell density across all mice for the Consumption timepoint ( n = 24 mice). The Allen CCF is overlaid. Coronal sections are spaced by 0.5 mm, the section corresponding to Bregma is marked with a *, and key brain regions are labeled. b, Map of the difference in average Fos + cell density across Novel versus Familiar flavor condition mice for the Consumption timepoint ( n = 12 mice per flavor condition). c, Map of average Fos + cell density across all mice for the Malaise timepoint ( n = 24 mice). d, Map of the difference in average Fos + cell density across Novel versus Familiar flavor condition mice for the Malaise timepoint ( n = 12 mice per flavor condition). e, Map of average Fos + cell density across all mice for the Retrieval timepoint ( n = 24 mice). f, Map of the difference in average Fos + cell density across Novel versus Familiar flavor condition mice for the Retrieval timepoint ( n = 12 mice per flavor condition). An interactive visualization of these Fos + cell density maps is available at https://www.brainsharer.org/ng/?id=872 . See for list of brain region abbreviations.

Journal: bioRxiv

Article Title: A neural mechanism for learning from delayed postingestive feedback

doi: 10.1101/2023.10.06.561214

Figure Lengend Snippet: a, Map of average Fos + cell density across all mice for the Consumption timepoint ( n = 24 mice). The Allen CCF is overlaid. Coronal sections are spaced by 0.5 mm, the section corresponding to Bregma is marked with a *, and key brain regions are labeled. b, Map of the difference in average Fos + cell density across Novel versus Familiar flavor condition mice for the Consumption timepoint ( n = 12 mice per flavor condition). c, Map of average Fos + cell density across all mice for the Malaise timepoint ( n = 24 mice). d, Map of the difference in average Fos + cell density across Novel versus Familiar flavor condition mice for the Malaise timepoint ( n = 12 mice per flavor condition). e, Map of average Fos + cell density across all mice for the Retrieval timepoint ( n = 24 mice). f, Map of the difference in average Fos + cell density across Novel versus Familiar flavor condition mice for the Retrieval timepoint ( n = 12 mice per flavor condition). An interactive visualization of these Fos + cell density maps is available at https://www.brainsharer.org/ng/?id=872 . See for list of brain region abbreviations.

Techniques: Labeling

Journal: bioRxiv

Article Title: A neural mechanism for learning from delayed postingestive feedback

doi: 10.1101/2023.10.06.561214

Figure Lengend Snippet:

Techniques:

a, Schematic of the Malaise and Retrieval Fos timepoints ( n = 12 mice per flavor condition per timepoint). There were six total groups of mice in the main Fos dataset: two flavor conditions (Novel, Familiar) × three timepoints (Consumption (from ), Malaise, Retrieval). The lines above “10 min” are scale bars for each experiment. b, Description of the GLMM for brainwide Fos data. Briefly, we modeled the number of Fos + neurons in each brain region ( Fos counts ) as a fixed effect interaction of flavor condition ( Novel ) and experimental timpeoint ( Timepoint ; in the formula, * represents all possible main effects and interactions) with additional contributions from sex (fixed effect: Sex ), technical batch (random effect: (1| Batch )), and total brainwide Fos + cell count (offset term: ln( Total Counts )) using a negative binomial link function. This model properly accounts for the statistical structure of brainwide Fos data as well as batch-to-batch variation in tissue clearing, immunolabeling, and imaging. We then used this model to calculate the average marginal effect (Novel – Familiar ΔFos, in standardized units) of flavor on Fos + cell counts for each brain region (see Methods and for more details). c, Novel – Familiar ΔFos effect distribution at each timepoint across brain regions, for all regions that were significantly modulated by Novel , Timepoint , or their interaction ( n = 130 brain regions). Each point represents a single brain region. d, Hierarchical clustering of Novel – Familiar ΔFos effects. See for an expanded version. e, Detail of the amygdala network (Cluster 1 from d ) that is preferentially activated by novel flavors at every stage of learning. The heatmap columns in e are in the same order as the rows in d (left to right: Consumption, Malaise, Retrieval). f, Visualization of the spatially resolved difference in Fos + cell density across flavor conditions with Allen CCF boundaries overlaid. g, Comparison of individual familiar (blue) and novel (red) flavor condition mice for the CEA at each timepoint. P -values in c are from Kolmogorov-Smirnov tests corrected for multiple comparisons across timepoints, P -values in g are from GLMM marginal effect z -tests corrected for multiple comparisons across timepoints . * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. Error bars represent mean ± s.e.m. See for list of brain region abbreviations and for GLMM statistics.

Journal: bioRxiv

Article Title: A neural mechanism for learning from delayed postingestive feedback

doi: 10.1101/2023.10.06.561214

Figure Lengend Snippet: a, Schematic of the Malaise and Retrieval Fos timepoints ( n = 12 mice per flavor condition per timepoint). There were six total groups of mice in the main Fos dataset: two flavor conditions (Novel, Familiar) × three timepoints (Consumption (from ), Malaise, Retrieval). The lines above “10 min” are scale bars for each experiment. b, Description of the GLMM for brainwide Fos data. Briefly, we modeled the number of Fos + neurons in each brain region ( Fos counts ) as a fixed effect interaction of flavor condition ( Novel ) and experimental timpeoint ( Timepoint ; in the formula, * represents all possible main effects and interactions) with additional contributions from sex (fixed effect: Sex ), technical batch (random effect: (1| Batch )), and total brainwide Fos + cell count (offset term: ln( Total Counts )) using a negative binomial link function. This model properly accounts for the statistical structure of brainwide Fos data as well as batch-to-batch variation in tissue clearing, immunolabeling, and imaging. We then used this model to calculate the average marginal effect (Novel – Familiar ΔFos, in standardized units) of flavor on Fos + cell counts for each brain region (see Methods and for more details). c, Novel – Familiar ΔFos effect distribution at each timepoint across brain regions, for all regions that were significantly modulated by Novel , Timepoint , or their interaction ( n = 130 brain regions). Each point represents a single brain region. d, Hierarchical clustering of Novel – Familiar ΔFos effects. See for an expanded version. e, Detail of the amygdala network (Cluster 1 from d ) that is preferentially activated by novel flavors at every stage of learning. The heatmap columns in e are in the same order as the rows in d (left to right: Consumption, Malaise, Retrieval). f, Visualization of the spatially resolved difference in Fos + cell density across flavor conditions with Allen CCF boundaries overlaid. g, Comparison of individual familiar (blue) and novel (red) flavor condition mice for the CEA at each timepoint. P -values in c are from Kolmogorov-Smirnov tests corrected for multiple comparisons across timepoints, P -values in g are from GLMM marginal effect z -tests corrected for multiple comparisons across timepoints . * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. Error bars represent mean ± s.e.m. See for list of brain region abbreviations and for GLMM statistics.

Techniques: Cell Counting, Immunolabeling, Imaging, Comparison

Panels a–c show that the brainwide shift towards activation by the novel flavor is primarily localized to subcortical regions. a, Novel – Familiar ΔFos effect distribution of all cortical regions (Cerebral Cortex in the Allen CCF) at each timepoint ( n = 38 brain regions). b, Novel – Familiar ΔFos effect distribution of all subcortical forebrain (Cerebral Nuclei, Thalamus, and Hypothalamus in the Allen CCF) regions at each timepoint ( n = 54 brain regions). c, Novel – Familiar ΔFos effect distribution of all midbrain and hindbrain regions (Midbrain, Pons, and Medulla in the Allen CCF) at each timepoint ( n = 38 brain regions). d, Hierarchical clustering of Novel – Familiar ΔFos effects. This is an expanded version showing all brain region names. e–n , Left: Illustration of the brain regions comprising each cluster from the hierarchical clustering analysis. Right: Summary of the Novel – Familiar ΔFos effect for each cluster at each timepoint, showing each brain region as an individual point. P -values in b,c are from Kolmogorov-Smirnov tests corrected for multiple comparisons across timepoints. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001, NS, not significant ( P > 0.05). Outlines in a–c represent kernel-density estimates of the empirical distributions. Error bars in e–n represent mean ± s.e.m. See for list of brain region abbreviations.

Journal: bioRxiv

Article Title: A neural mechanism for learning from delayed postingestive feedback

doi: 10.1101/2023.10.06.561214

Figure Lengend Snippet: Panels a–c show that the brainwide shift towards activation by the novel flavor is primarily localized to subcortical regions. a, Novel – Familiar ΔFos effect distribution of all cortical regions (Cerebral Cortex in the Allen CCF) at each timepoint ( n = 38 brain regions). b, Novel – Familiar ΔFos effect distribution of all subcortical forebrain (Cerebral Nuclei, Thalamus, and Hypothalamus in the Allen CCF) regions at each timepoint ( n = 54 brain regions). c, Novel – Familiar ΔFos effect distribution of all midbrain and hindbrain regions (Midbrain, Pons, and Medulla in the Allen CCF) at each timepoint ( n = 38 brain regions). d, Hierarchical clustering of Novel – Familiar ΔFos effects. This is an expanded version showing all brain region names. e–n , Left: Illustration of the brain regions comprising each cluster from the hierarchical clustering analysis. Right: Summary of the Novel – Familiar ΔFos effect for each cluster at each timepoint, showing each brain region as an individual point. P -values in b,c are from Kolmogorov-Smirnov tests corrected for multiple comparisons across timepoints. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001, NS, not significant ( P > 0.05). Outlines in a–c represent kernel-density estimates of the empirical distributions. Error bars in e–n represent mean ± s.e.m. See for list of brain region abbreviations.

Techniques: Activation Assay

a, Correlation matrices showing the animal-by-animal pairwise Fos correlation for every pair of brain regions during consumption (left), delayed malaise (middle), or memory retrieval (right). Brain regions are sorted using the hierarchical clustermap obtained from the Novel – Familiar ΔFos effects in . b, Summary of the average within-cluster Fos correlation for individual amygdala network (Cluster 1 from ) regions by timepoint ( n = 12 regions). The high animal-by-animal correlation among all of the regions in cluster 1 suggest that these regions form a functional network. Panels c,d show that activation of other clusters of brain regions is more correlated with amygdala network activation at experimental timepoints when those clusters are more strongly novel flavor-selective, including for clusters that were specifically engaged during the initial flavor consumption (comprising sensory cortices; cluster 2) or during retrieval (including the BST; cluster 6). This suggests that the amygdala network may play a role in orchestrating the brainwide response to novel flavors at different stages of learning. c, Summary of the average across-cluster Fos correlation between the amygdala network and every other cluster at each timepoint as a function of the other cluster’s standardized Novel–Familiar effect at that timepoint ( n = 9 clusters × 3 timepoints). d, Scatter plots showing the pairwise correlation between AIp (top; example Cluster 2 region) or BST (bottom; example Cluster 6 region) and the CEA ( n = 24 mice per timepoint). P -values in b are from Wilcoxon signed-rank tests corrected for multiple comparisons across timepoints, P -values in c,d are from Pearson correlation t -tests. Error bars represent mean ± s.e.m. Shaded areas in c,d represent 95% confidence interval for linear fit. *** P ≤ 0.001, **** P ≤ 0.0001, NS, not significant ( P > 0.05). See for list of brain region abbreviations.

Journal: bioRxiv

Article Title: A neural mechanism for learning from delayed postingestive feedback

doi: 10.1101/2023.10.06.561214

Figure Lengend Snippet: a, Correlation matrices showing the animal-by-animal pairwise Fos correlation for every pair of brain regions during consumption (left), delayed malaise (middle), or memory retrieval (right). Brain regions are sorted using the hierarchical clustermap obtained from the Novel – Familiar ΔFos effects in . b, Summary of the average within-cluster Fos correlation for individual amygdala network (Cluster 1 from ) regions by timepoint ( n = 12 regions). The high animal-by-animal correlation among all of the regions in cluster 1 suggest that these regions form a functional network. Panels c,d show that activation of other clusters of brain regions is more correlated with amygdala network activation at experimental timepoints when those clusters are more strongly novel flavor-selective, including for clusters that were specifically engaged during the initial flavor consumption (comprising sensory cortices; cluster 2) or during retrieval (including the BST; cluster 6). This suggests that the amygdala network may play a role in orchestrating the brainwide response to novel flavors at different stages of learning. c, Summary of the average across-cluster Fos correlation between the amygdala network and every other cluster at each timepoint as a function of the other cluster’s standardized Novel–Familiar effect at that timepoint ( n = 9 clusters × 3 timepoints). d, Scatter plots showing the pairwise correlation between AIp (top; example Cluster 2 region) or BST (bottom; example Cluster 6 region) and the CEA ( n = 24 mice per timepoint). P -values in b are from Wilcoxon signed-rank tests corrected for multiple comparisons across timepoints, P -values in c,d are from Pearson correlation t -tests. Error bars represent mean ± s.e.m. Shaded areas in c,d represent 95% confidence interval for linear fit. *** P ≤ 0.001, **** P ≤ 0.0001, NS, not significant ( P > 0.05). See for list of brain region abbreviations.

Techniques: Functional Assay, Activation Assay

a, Schematic of the neural pathway that conveys visceral malaise signals from the gut to the amygdala via the area postrema (AP) and parabrachial CGRP neurons – , , , . b, Fiber photometry recordings showing that CGRP neurons are activated in vivo by LiCl-induced malaise ( n = 5 mice). c, Top: Strategy for using ChR2-assisted circuit mapping to identify monosynaptic connections (optogenetically evoked excitatory postsynaptic currents (oEPSCs) in the presence of the voltage-gated sodium channel blocker tetrodotoxin (TTX) and the voltage-gated potassium channel blocker 4-aminopyridine (4AP)) between CGRP neurons and the CEA. Middle: Strong monosynaptic inputs from CGRP neurons to the CEAc/l (amplitude: –327.0 ± 136.3 pA; mean ± s.e.m.; n = 5/5 neurons from 3 mice). Bottom: Weaker monosynaptic inputs from CGRP neurons to the CEAm (amplitude: –15.6 ± 6.4 pA; mean ± s.e.m.; n = 4/5 neurons from 3 mice). The dark lines represent the average and the transparent lines represent individual trials for each example neuron. d, Top/Left: Schematic and example ChR2-YFP expression data for optogenetic CGRP neuron stimulation experiment. Bottom: Retrieval test flavor preference for the same experiment ( n = 6 mice per group). e, Schematic of CGRP stim Fos timepoint ( n = 14 novel flavor mice, 13 familiar flavor mice). f, Correlation between the average Fos + cell count across both flavor conditions in each brain region for the LiCl-induced malaise timepoint versus the CGRP stim timepoint for the amygdala network (Cluster 1 from ; top; n = 12 regions) and for all other regions (bottom; n = 117 regions). See also Extended Data Fig. 6c. g, Panels are analogous to f , but comparing the difference between Novel and Familiar flavor groups. See also h, Visualization of the spatially resolved difference in Fos + cell density across novel versus familiar flavor with Allen CCF boundaries overlaid. i, Comparison of individual familiar (blue) and novel (red) flavor condition mice for the CEA. P -value in d is from a Wilcoxon rank-sum test, P -value in i is from a GLMM marginal effect z -test, P -values in f,g are from Pearson correlation t -tests. Error bars represent mean ± s.e.m. Shaded areas in b represent mean ± s.e.m. and in f,g represent 95% confidence interval for linear fit. Units in i are % per mm . ** P ≤ 0.01, **** P ≤ 0.0001.

Journal: bioRxiv

Article Title: A neural mechanism for learning from delayed postingestive feedback

doi: 10.1101/2023.10.06.561214

Figure Lengend Snippet: a, Schematic of the neural pathway that conveys visceral malaise signals from the gut to the amygdala via the area postrema (AP) and parabrachial CGRP neurons – , , , . b, Fiber photometry recordings showing that CGRP neurons are activated in vivo by LiCl-induced malaise ( n = 5 mice). c, Top: Strategy for using ChR2-assisted circuit mapping to identify monosynaptic connections (optogenetically evoked excitatory postsynaptic currents (oEPSCs) in the presence of the voltage-gated sodium channel blocker tetrodotoxin (TTX) and the voltage-gated potassium channel blocker 4-aminopyridine (4AP)) between CGRP neurons and the CEA. Middle: Strong monosynaptic inputs from CGRP neurons to the CEAc/l (amplitude: –327.0 ± 136.3 pA; mean ± s.e.m.; n = 5/5 neurons from 3 mice). Bottom: Weaker monosynaptic inputs from CGRP neurons to the CEAm (amplitude: –15.6 ± 6.4 pA; mean ± s.e.m.; n = 4/5 neurons from 3 mice). The dark lines represent the average and the transparent lines represent individual trials for each example neuron. d, Top/Left: Schematic and example ChR2-YFP expression data for optogenetic CGRP neuron stimulation experiment. Bottom: Retrieval test flavor preference for the same experiment ( n = 6 mice per group). e, Schematic of CGRP stim Fos timepoint ( n = 14 novel flavor mice, 13 familiar flavor mice). f, Correlation between the average Fos + cell count across both flavor conditions in each brain region for the LiCl-induced malaise timepoint versus the CGRP stim timepoint for the amygdala network (Cluster 1 from ; top; n = 12 regions) and for all other regions (bottom; n = 117 regions). See also Extended Data Fig. 6c. g, Panels are analogous to f , but comparing the difference between Novel and Familiar flavor groups. See also h, Visualization of the spatially resolved difference in Fos + cell density across novel versus familiar flavor with Allen CCF boundaries overlaid. i, Comparison of individual familiar (blue) and novel (red) flavor condition mice for the CEA. P -value in d is from a Wilcoxon rank-sum test, P -value in i is from a GLMM marginal effect z -test, P -values in f,g are from Pearson correlation t -tests. Error bars represent mean ± s.e.m. Shaded areas in b represent mean ± s.e.m. and in f,g represent 95% confidence interval for linear fit. Units in i are % per mm . ** P ≤ 0.01, **** P ≤ 0.0001.

Techniques: In Vivo, Expressing, Cell Counting, Comparison

a, Schematic of the acute Neuropixels recording experiment. b, Reconstruction of recording trajectories registered to the Allen CCF. Each line represents one insertion of a single-shank Neuropixels 1.0 probe targeting the amygdala ( n = 24 insertions from 4 mice). c, Left: PETHs of neural activity time-locked to CGRP neuron stimulation trains ( n = 3,524 amygdala neurons from 24 insertions). Neurons were divided into four response types using a GMM model (see Methods): two CGRP neuron stimulation-activated response types (7.3% strongly activated, dark green; 22.4% weakly activated, light green), one CGRP neuron stimulation-inhibited response type (24.6%, purple), and one unmodulated response type (45.8%, gray). Right: Average PETHs for each GMM response type. d, Percentage of recorded neurons that were CGRP neuron stimulation-activated based on the GMM across amygdala subregions ( n = 339 CEAc, 272 CEAl, 717 CEAm, 526 BMAa, 129 COAa, 133 IA, 41 BLAp, 30 PAA, 182 MEA, 354 BLAa, 54 Other (AAA, LA, PA), 44 BLAv, 250 BMAp, and 58 COAp neurons). Regions in the CTA amygdala network (Cluster 1 from ) are shown in red, and other amygdala regions are shown in black. e, Anatomical distribution of all CGRP-activated (green), CGRP-inhibited (purple), and unmodulated (gray) neurons projected onto a single coronal or sagittal section of the Allen CCF. f, Left: Light sheet imaging data for each animal of Neuropixels probe trajectories aligned to the Allen CCF with amygdala subregions overlaid. Right: Reconstruction of recording trajectories in the amygdala for each animal. For each animal, a single sagittal section corresponding to the center-of-mass of all active recording sites is shown. The colormap for amygdala regions in b is also used in e and f . See for list of brain region abbreviations.

Journal: bioRxiv

Article Title: A neural mechanism for learning from delayed postingestive feedback

doi: 10.1101/2023.10.06.561214

Figure Lengend Snippet: a, Schematic of the acute Neuropixels recording experiment. b, Reconstruction of recording trajectories registered to the Allen CCF. Each line represents one insertion of a single-shank Neuropixels 1.0 probe targeting the amygdala ( n = 24 insertions from 4 mice). c, Left: PETHs of neural activity time-locked to CGRP neuron stimulation trains ( n = 3,524 amygdala neurons from 24 insertions). Neurons were divided into four response types using a GMM model (see Methods): two CGRP neuron stimulation-activated response types (7.3% strongly activated, dark green; 22.4% weakly activated, light green), one CGRP neuron stimulation-inhibited response type (24.6%, purple), and one unmodulated response type (45.8%, gray). Right: Average PETHs for each GMM response type. d, Percentage of recorded neurons that were CGRP neuron stimulation-activated based on the GMM across amygdala subregions ( n = 339 CEAc, 272 CEAl, 717 CEAm, 526 BMAa, 129 COAa, 133 IA, 41 BLAp, 30 PAA, 182 MEA, 354 BLAa, 54 Other (AAA, LA, PA), 44 BLAv, 250 BMAp, and 58 COAp neurons). Regions in the CTA amygdala network (Cluster 1 from ) are shown in red, and other amygdala regions are shown in black. e, Anatomical distribution of all CGRP-activated (green), CGRP-inhibited (purple), and unmodulated (gray) neurons projected onto a single coronal or sagittal section of the Allen CCF. f, Left: Light sheet imaging data for each animal of Neuropixels probe trajectories aligned to the Allen CCF with amygdala subregions overlaid. Right: Reconstruction of recording trajectories in the amygdala for each animal. For each animal, a single sagittal section corresponding to the center-of-mass of all active recording sites is shown. The colormap for amygdala regions in b is also used in e and f . See for list of brain region abbreviations.

Techniques: Activity Assay, Imaging

a, Example brainwide Fos imaging data (200-µm maximum intensity projections) for four example CGRP stim timepoint animals. Strong Fos expression in the PB driven by optogenetic stimulation of CGRP neurons is highlighted in green. b, Summary of Fos + cell counts in the PB at each experimental timpoint, confirming high levels of neural activation during both LiCl-induced malaise and CGRP neuron stimulation ( n = 24 mice for Consumption, 24 mice for Malaise, 27 mice for CGRP stim, and 24 mice for Retrieval). c, Analysis analogous to but using the Fos GLMM from . Here, the correlation among the standardized coefficients ( Z = estimate/standard error) for the main LiCl effect and main CGRP stim effect on Fos + cell counts from the regression Fos counts ∼ Timepoint + Sex + (1| Batch ) + ln( PB Counts ) are plotted ( n = 12 amygdala network regions, 117 other regions). d, Analysis analogous to but using the Fos GLMM from . Here, the correlation among the average marginal effect (Novel – Familiar ΔFos, in standardized units) of flavor on Fos + cell counts from the regression Fos counts ∼ Novel*Timepoint + Sex + (1| Batch ) + ln( PB Counts ) are plotted ( n = 12 amygdala network regions, 117 other regions). e, Map of average Fos + cell density across all mice for the CGRP stim timepoint ( n = 27 mice). The Allen CCF is overlaid. Coronal sections are spaced by 0.5 mm, the section corresponding to Bregma is marked with a *, and key brain regions are labeled. f, Map of the difference in average Fos + cell density across Novel versus Familiar flavor condition mice for the CGRP stim timepoint ( n = 14 novel flavor mice, 13 familiar flavor mice). P -values in c,d are from Pearson correlation t -tests. Error bars represent mean ± s.e.m. Shaded areas in c,d represent 95% confidence interval for linear fit. * P ≤ 0.05, ** P ≤ 0.01, **** P ≤ 0.0001, NS, not significant ( P > 0.05). See for list of brain region abbreviations and for GLMM statistics.

Journal: bioRxiv

Article Title: A neural mechanism for learning from delayed postingestive feedback

doi: 10.1101/2023.10.06.561214

Figure Lengend Snippet: a, Example brainwide Fos imaging data (200-µm maximum intensity projections) for four example CGRP stim timepoint animals. Strong Fos expression in the PB driven by optogenetic stimulation of CGRP neurons is highlighted in green. b, Summary of Fos + cell counts in the PB at each experimental timpoint, confirming high levels of neural activation during both LiCl-induced malaise and CGRP neuron stimulation ( n = 24 mice for Consumption, 24 mice for Malaise, 27 mice for CGRP stim, and 24 mice for Retrieval). c, Analysis analogous to but using the Fos GLMM from . Here, the correlation among the standardized coefficients ( Z = estimate/standard error) for the main LiCl effect and main CGRP stim effect on Fos + cell counts from the regression Fos counts ∼ Timepoint + Sex + (1| Batch ) + ln( PB Counts ) are plotted ( n = 12 amygdala network regions, 117 other regions). d, Analysis analogous to but using the Fos GLMM from . Here, the correlation among the average marginal effect (Novel – Familiar ΔFos, in standardized units) of flavor on Fos + cell counts from the regression Fos counts ∼ Novel*Timepoint + Sex + (1| Batch ) + ln( PB Counts ) are plotted ( n = 12 amygdala network regions, 117 other regions). e, Map of average Fos + cell density across all mice for the CGRP stim timepoint ( n = 27 mice). The Allen CCF is overlaid. Coronal sections are spaced by 0.5 mm, the section corresponding to Bregma is marked with a *, and key brain regions are labeled. f, Map of the difference in average Fos + cell density across Novel versus Familiar flavor condition mice for the CGRP stim timepoint ( n = 14 novel flavor mice, 13 familiar flavor mice). P -values in c,d are from Pearson correlation t -tests. Error bars represent mean ± s.e.m. Shaded areas in c,d represent 95% confidence interval for linear fit. * P ≤ 0.05, ** P ≤ 0.01, **** P ≤ 0.0001, NS, not significant ( P > 0.05). See for list of brain region abbreviations and for GLMM statistics.

Techniques: Imaging, Expressing, Activation Assay, Labeling

a, Hypotheses for how the amygdala associates temporally separated flavor and malaise signals to support CTA learning. Hypothesis 1: Individual novel flavor-coding neurons may be persistently activated long after a meal, either through cell-autonomous or circuit mechanisms, in a manner that provides passive overlap with delayed CGRP neuron malaise signals. Hypothesis 2: CGRP signals may specifically reactivate novel flavor-coding neurons. Hypothesis 3: CGRP signals may specifically activate a separate population of neurons that subsequently becomes incorporated into the novel flavor representation upon memory retrieval. Each of these hypotheses provides a plausible mechanism for linking flavors to malaise signals in the amygdala, but they make mutually exclusive predictions about single-neuron and populational-level activity across the stages of learning. b, Schematic of the CTA paradigm for chronic Neuropixels recordings and CGRP neuron stimulation. We trained mice to consume the water and novel flavor at relatively equal rates, and the total amount consumed was equal ( ; see Methods). c, Reconstruction of recording trajectories registered to the Allen CCF. Each line represents one shank of a four-shank Neuropixels 2.0 probe targeting CEA ( n = 32 shanks from 8 mice). d, Heatmap showing the trial-average spiking of all recorded CEA neurons ( n = 1,104 single- and multi-units from 8 mice) to novel flavor and water consumption during the consumption period (left) and during the delay and CGRP neuron stimulation periods (right). Neurons are grouped by their novel flavor/water preference and then sorted by consumption response magnitude. Consumption PETHs are time-locked to delivery of the flavor or water, which was triggered by the animal entering the port. e, Average spiking of the novel flavor-preferring (red; n = 373 neurons), water-preferring (blue; n = 121 neurons), and non-selective (black; n = 610 neurons) populations across the entire experiment. Inset: average response of each population during the entire 45-min CGRP neuron stimulation period. f, Left: Heatmap showing the trial-average spiking of all recorded neurons to individual 3-s bouts of 10-Hz CGRP neuron stimulation. Right: Average spiking of the novel flavor-preferring (red), water-preferring (blue), and non-selective (black) populations during CGRP neuron stimulation bouts. Inset: average response of each population within individual 3-s bouts of CGRP neuron stimulation. g, Example multinomial logistic regression decoder session. The top row shows the moment-by-moment decoder posterior for the novel flavor (red) and water (blue). The raster below shows time-locked neural activity for novel flavor-preferring, water-preferring, and non-selective neurons. The symbols in the legend represent the true event times (novel flavor delivery, water delivery, CGRP neuron stimulation), not decoder predictions. Only a subset of recorded neurons is shown for clarity (50 out of 90). h, Average decoder posterior time-locked to CGRP neuron stimulation for the example animal (top) and across all mice (bottom; n = 6). i, Average reactivation rate for the novel flavor and water across the delay and CGRP neuron stimulation periods ( n = 6). We defined a reactivation event as any peak in the decoder posterior trace that was > 0.5. j, Top: Schematic of the population activity dimensionality reduction analysis. Bottom: The first two principal components explained >70% of the variance in trial-average population dynamics during novel flavor and water consumption. k, Left: Neural trajectories for novel flavor consumption (red), water consumption (blue), and CGRP neuron stimulation in PC-space. Right: Time-courses along the PC1 and PC2 axes for the trajectories to left. l, Dimensionality reduction analysis performed separately for four individual example mice, rather than on all mice combined as in j,k . P -values in d,f are from Wilcoxon rank-sum tests corrected for multiple comparisons across neuron groups, P -values in e are from Pearson correlation t -tests. Error bars represent mean ± s.e.m. Shaded areas in d,f,i represent mean ± s.e.m. and in e represent 95% confidence interval for linear fit. *** P ≤ 0.001, **** P ≤ 0.0001, NS, not significant ( P > 0.05).

Journal: bioRxiv

Article Title: A neural mechanism for learning from delayed postingestive feedback

doi: 10.1101/2023.10.06.561214

Figure Lengend Snippet: a, Hypotheses for how the amygdala associates temporally separated flavor and malaise signals to support CTA learning. Hypothesis 1: Individual novel flavor-coding neurons may be persistently activated long after a meal, either through cell-autonomous or circuit mechanisms, in a manner that provides passive overlap with delayed CGRP neuron malaise signals. Hypothesis 2: CGRP signals may specifically reactivate novel flavor-coding neurons. Hypothesis 3: CGRP signals may specifically activate a separate population of neurons that subsequently becomes incorporated into the novel flavor representation upon memory retrieval. Each of these hypotheses provides a plausible mechanism for linking flavors to malaise signals in the amygdala, but they make mutually exclusive predictions about single-neuron and populational-level activity across the stages of learning. b, Schematic of the CTA paradigm for chronic Neuropixels recordings and CGRP neuron stimulation. We trained mice to consume the water and novel flavor at relatively equal rates, and the total amount consumed was equal ( ; see Methods). c, Reconstruction of recording trajectories registered to the Allen CCF. Each line represents one shank of a four-shank Neuropixels 2.0 probe targeting CEA ( n = 32 shanks from 8 mice). d, Heatmap showing the trial-average spiking of all recorded CEA neurons ( n = 1,104 single- and multi-units from 8 mice) to novel flavor and water consumption during the consumption period (left) and during the delay and CGRP neuron stimulation periods (right). Neurons are grouped by their novel flavor/water preference and then sorted by consumption response magnitude. Consumption PETHs are time-locked to delivery of the flavor or water, which was triggered by the animal entering the port. e, Average spiking of the novel flavor-preferring (red; n = 373 neurons), water-preferring (blue; n = 121 neurons), and non-selective (black; n = 610 neurons) populations across the entire experiment. Inset: average response of each population during the entire 45-min CGRP neuron stimulation period. f, Left: Heatmap showing the trial-average spiking of all recorded neurons to individual 3-s bouts of 10-Hz CGRP neuron stimulation. Right: Average spiking of the novel flavor-preferring (red), water-preferring (blue), and non-selective (black) populations during CGRP neuron stimulation bouts. Inset: average response of each population within individual 3-s bouts of CGRP neuron stimulation. g, Example multinomial logistic regression decoder session. The top row shows the moment-by-moment decoder posterior for the novel flavor (red) and water (blue). The raster below shows time-locked neural activity for novel flavor-preferring, water-preferring, and non-selective neurons. The symbols in the legend represent the true event times (novel flavor delivery, water delivery, CGRP neuron stimulation), not decoder predictions. Only a subset of recorded neurons is shown for clarity (50 out of 90). h, Average decoder posterior time-locked to CGRP neuron stimulation for the example animal (top) and across all mice (bottom; n = 6). i, Average reactivation rate for the novel flavor and water across the delay and CGRP neuron stimulation periods ( n = 6). We defined a reactivation event as any peak in the decoder posterior trace that was > 0.5. j, Top: Schematic of the population activity dimensionality reduction analysis. Bottom: The first two principal components explained >70% of the variance in trial-average population dynamics during novel flavor and water consumption. k, Left: Neural trajectories for novel flavor consumption (red), water consumption (blue), and CGRP neuron stimulation in PC-space. Right: Time-courses along the PC1 and PC2 axes for the trajectories to left. l, Dimensionality reduction analysis performed separately for four individual example mice, rather than on all mice combined as in j,k . P -values in d,f are from Wilcoxon rank-sum tests corrected for multiple comparisons across neuron groups, P -values in e are from Pearson correlation t -tests. Error bars represent mean ± s.e.m. Shaded areas in d,f,i represent mean ± s.e.m. and in e represent 95% confidence interval for linear fit. *** P ≤ 0.001, **** P ≤ 0.0001, NS, not significant ( P > 0.05).

Techniques: Activity Assay

a, Schematic of the CTA paradigm for chronic Neuropixels recordings and LiCl-induced malaise. b, Heatmap showing the trial-average spiking of all recorded amygdala neurons ( n = 449 neurons from 3 mice) to novel flavor and water consumption during the consumption period (left) and during the delay and malaise periods (right). Neurons are grouped by their novel flavor/water preference and then sorted by reward response magnitude. c, Average spiking of the novel flavor-preferring (red; n = 60 neurons), water-preferring (blue; n = 45 neurons), and non-selective (black; n = 344 neurons) populations across the entire experiment. Inset: average response of each population following LiCl injection. d–f, These panels show that individual CGRP neuron stimulation-activated amygdala neurons are also activated by LiCl-induced malaise, and that LiCl injection activates a broader population of amygdala neurons than CGRP neuron activation alone. d, Schematic of the paradigm for tracking neurons during CGRP neuron stimulation and then during LiCl-induced malaise. e, Heatmap showing the trial-average spiking of all recorded amygdala neurons ( n = 971 neurons from 4 mice) to CGRP neuron stimulation (left) and then during LiCl-induced malaise (right). Neurons were classified as CGRP neuron stimulation-activated using the GMM trained on the data in . f, Average spiking of the CGRP neuron stimulation-activated neurons (green; n = 200 neurons) and other neurons (black; n = 771 neurons) populations during LiCl-induced malaise. Inset: average response of each population following LiCl injection. P -values in c,f are from Wilcoxon rank-sum tests corrected for multiple comparisons across neuron groups. Shaded areas in c,f represent mean ± s.e.m. * P ≤ 0.05, ** P ≤ 0.01, NS, not significant ( P > 0.05).

Journal: bioRxiv

Article Title: A neural mechanism for learning from delayed postingestive feedback

doi: 10.1101/2023.10.06.561214

Figure Lengend Snippet: a, Schematic of the CTA paradigm for chronic Neuropixels recordings and LiCl-induced malaise. b, Heatmap showing the trial-average spiking of all recorded amygdala neurons ( n = 449 neurons from 3 mice) to novel flavor and water consumption during the consumption period (left) and during the delay and malaise periods (right). Neurons are grouped by their novel flavor/water preference and then sorted by reward response magnitude. c, Average spiking of the novel flavor-preferring (red; n = 60 neurons), water-preferring (blue; n = 45 neurons), and non-selective (black; n = 344 neurons) populations across the entire experiment. Inset: average response of each population following LiCl injection. d–f, These panels show that individual CGRP neuron stimulation-activated amygdala neurons are also activated by LiCl-induced malaise, and that LiCl injection activates a broader population of amygdala neurons than CGRP neuron activation alone. d, Schematic of the paradigm for tracking neurons during CGRP neuron stimulation and then during LiCl-induced malaise. e, Heatmap showing the trial-average spiking of all recorded amygdala neurons ( n = 971 neurons from 4 mice) to CGRP neuron stimulation (left) and then during LiCl-induced malaise (right). Neurons were classified as CGRP neuron stimulation-activated using the GMM trained on the data in . f, Average spiking of the CGRP neuron stimulation-activated neurons (green; n = 200 neurons) and other neurons (black; n = 771 neurons) populations during LiCl-induced malaise. Inset: average response of each population following LiCl injection. P -values in c,f are from Wilcoxon rank-sum tests corrected for multiple comparisons across neuron groups. Shaded areas in c,f represent mean ± s.e.m. * P ≤ 0.05, ** P ≤ 0.01, NS, not significant ( P > 0.05).

Techniques: Injection, Activation Assay

Journal: bioRxiv

Article Title: A neural mechanism for learning from delayed postingestive feedback

doi: 10.1101/2023.10.06.561214

Figure Lengend Snippet: a, Spike waveforms, autocorrelograms, and flavor response rasters for one example neuron tracked across conditioning and retrieval days. b, Heatmap showing the average spiking of all recorded neurons ( n = 939 neurons from 8 mice) to novel flavor and water consumption during the consumption period (left) and during the delay and CGRP neuron stimulation periods (middle) on conditioning day, and the responses of the same neurons to flavor and water consumption on retrieval day. Neurons are grouped by their novel flavor/water preference on pairing day and then sorted by CGRP response magnitude. c, Left: Trial-average spiking of the novel flavor-preferring population ( n = 265 neurons) during flavor consumption on conditioning day (black) and retrieval day (red). The inset quantifies the average response on each day. Middle/Right: Trial-average spiking of the novel-preferring neurons with the highest 10% of CGRP response magnitudes (High CGRP; middle) and of the remaining novel-preferring neurons (Low CGRP; right) on conditioning day and retrieval day. The insets show the average CGRP response profile of each subpopulation. d, Correlation between each neuron’s change (Retrieval – Conditioning) in flavor response (top) or selectivity (bottom) during the consumption period to its average response during the CGRP neuron stimulation period. Shown separately for the novel flavor-preferring (left; n = 265 neurons), water-preferring (middle; n = 123 neurons), and non-selective (right; n = 551 neurons) populations. e, Left: Schematic for the flavor familiarization experiment. Right: Trial-average spiking of the initially flavor-preferring population ( n = 201 neurons from 7 mice; classified on novel day) during flavor consumption on novel day (black) and familiar day (blue). f, Illustration of the neural mechanism for learning from delayed postingestive feedback using malaise-driven reactivation of amygdalar novel flavor representations. P -values in d are from Pearson correlation t -tests, P -value in e is from a Wilcoxon signed-rank test. Error bars represent mean ± s.e.m. Shaded areas in c,e represent mean ± s.e.m. and in d represent 95% confidence interval for linear fit. ** P ≤ 0.01, **** P ≤ 0.0001, NS, not significant ( P > 0.05).

Techniques:

a, Summary of the proportion of flavor-preferring units classified separately on conditioning day or on retrieval day for the CGRP neuron stimulation conditioning experiment in ( n = 8 mice). b, Neural trajectories for flavor consumption (red), water consumption (blue), and CGRP neuron stimulation in the PC-space on conditioning day (left) and retrieval day (right). Trajectories for both days were calculated using the PCA loadings from conditioning day, and the novel flavor trajectory remained largely stable. c, Heatmap showing the average spiking of all recorded neurons ( n = 924 neurons from 7 mice) to flavor and water consumption on novel day (left) and, two days later, on familiar day (right). Neurons are grouped by their novel flavor/water preference on novel day and then sorted by reward response magnitude. d, Summary of the proportion of flavor-preferring units classified separately on novel day or on familiar day for the familiarization experiment ( n = 7 mice). e, Trial-average spiking of the initially water-preferring population ( n = 160 neurons from 7 mice; classified on novel day) during flavor consumption on novel day (black) and familiar day (blue). f, Neural trajectories for flavor consumption (red) and water consumption (blue) in the PC-space on novel day (left) and familiar day (right). Trajectories for both days were calculated using the PCA loadings from the novel day, and the flavor trajectory was strongly degraded following familiarization. g, Comparison of the time-courses specifically along the PC2 axis during drinking for the conditioning (from the data in b ) and familiarization (from the data in f ) experiments. Top: The flavor drinking trajectory on conditioning day (“Novel”) is shown in grays and the stable trajectory on retrieval day (“CTA Retrieval”) is shown in reds. Bottom: The flavor drinking trajectory on novel day (“Novel”) is shown in grays and the degraded trajectory on familiar day (“Familiar”) is shown in blues. P -values in a,d,e are from Wilcoxon signed-rank tests. Error bars represent mean ± s.e.m. Shaded areas in e represent mean ± s.e.m. * P ≤ 0.05, ** P ≤ 0.01, NS, not significant ( P > 0.05).

Journal: bioRxiv

Article Title: A neural mechanism for learning from delayed postingestive feedback

doi: 10.1101/2023.10.06.561214

Figure Lengend Snippet: a, Summary of the proportion of flavor-preferring units classified separately on conditioning day or on retrieval day for the CGRP neuron stimulation conditioning experiment in ( n = 8 mice). b, Neural trajectories for flavor consumption (red), water consumption (blue), and CGRP neuron stimulation in the PC-space on conditioning day (left) and retrieval day (right). Trajectories for both days were calculated using the PCA loadings from conditioning day, and the novel flavor trajectory remained largely stable. c, Heatmap showing the average spiking of all recorded neurons ( n = 924 neurons from 7 mice) to flavor and water consumption on novel day (left) and, two days later, on familiar day (right). Neurons are grouped by their novel flavor/water preference on novel day and then sorted by reward response magnitude. d, Summary of the proportion of flavor-preferring units classified separately on novel day or on familiar day for the familiarization experiment ( n = 7 mice). e, Trial-average spiking of the initially water-preferring population ( n = 160 neurons from 7 mice; classified on novel day) during flavor consumption on novel day (black) and familiar day (blue). f, Neural trajectories for flavor consumption (red) and water consumption (blue) in the PC-space on novel day (left) and familiar day (right). Trajectories for both days were calculated using the PCA loadings from the novel day, and the flavor trajectory was strongly degraded following familiarization. g, Comparison of the time-courses specifically along the PC2 axis during drinking for the conditioning (from the data in b ) and familiarization (from the data in f ) experiments. Top: The flavor drinking trajectory on conditioning day (“Novel”) is shown in grays and the stable trajectory on retrieval day (“CTA Retrieval”) is shown in reds. Bottom: The flavor drinking trajectory on novel day (“Novel”) is shown in grays and the degraded trajectory on familiar day (“Familiar”) is shown in blues. P -values in a,d,e are from Wilcoxon signed-rank tests. Error bars represent mean ± s.e.m. Shaded areas in e represent mean ± s.e.m. * P ≤ 0.05, ** P ≤ 0.01, NS, not significant ( P > 0.05).

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