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Developmentally defined forebrain circuits regulate appetitive and aversive olfactory learning

Abstract

Postnatal and adult neurogenesis are region- and modality-specific, but the significance of developmentally distinct neuronal populations remains unclear. We demonstrate that chemogenetic inactivation of a subset of forebrain and olfactory neurons generated at birth disrupts responses to an aversive odor. In contrast, novel appetitive odor learning is sensitive to inactivation of adult-born neurons, revealing that developmentally defined sets of neurons may differentially participate in hedonic aspects of sensory learning.

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Figure 1: Targeting DREADDs to adult-born neurons in the OB.
Figure 2: Inhibition of young-adult-born neurons disrupts responses to novel appetitive, but not aversive odors.
Figure 3: Inhibition of perinatally born neurons disrupts responses to novel aversive but not novel appetitive odors.

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Acknowledgements

We thank D. Dorman and M. Foster for sharing their startle response system, and B. Roth for discussions at the onset of the study. H.T.G. is supported by grants from the National Institutes of Health (R01NS098370 and R01NS089795).

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Authors and Affiliations

Authors

Contributions

H.T.G. conceived and designed the study. P.N.Y. produced AAVs at the initial stages of the project. N.M. and X.Z. performed all experiments and collected most data. C.A.J. collected some data and performed all statistical analyses. N.M., X.Z., C.A.J. and H.T.G. analyzed data. N.M., C.A.J. and H.T.G. wrote the manuscript.

Corresponding author

Correspondence to H Troy Ghashghaei.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Characterization of DREADD expression and induction in P42-Gi OB.

(a) Strategy to inhibit new neurons in P42-Gi mice via CNO administration followed by 90 minutes of survival, perfusion, and tissue processing for c-Fos analysis. (b) High-power confocal images of EGFP+ cells in a P42-Gi OB coexpressing HA or mCherry (red as indicated) following co-injection of AAV2-Gi tagged with either HA or mCherry and AAV2-pSYN::EGFP. Scale bar, 10 μm. (c) Percentages of AAV2 transduced cells in the OB double labeled for tagged markers (HA or mCherry) and EGFP as well as tag only and EGFP only cells. Data are mean ± s.e.m; n = 3 animals/group, 100 EGFP+ cells/animal. (d) Low power confocal image of a sagittal forebrain section of a P42-Gi mouse (blue, DAPI). EGFP expression was confined to a subset of neurons in the OB (Inset: EGFP+ and NeuN+ (red) neurons). Scale bar, 100 μm; Inset scale bar, 10 μm. (e) The pSYN promoter in AAV2-Gi vectors prevents expression in the SEZ or RMS (No EGFP+ cells in Dcx+ domains, red; DAPI, blue; V, ventricle). Scale bar, 25 μm. (f) High-power confocal images of a P42-Gi OB section containing EGFP+ neurons coimmunostained for c-Fos (i; Scale bar, 50 μm); layers of the OB: granule cell layer (GCL), external plexiform layer (EPL), periglomerular layer (PGL). Representative EGFP+ granule neurons with dendrites arborizing in the EPL (ii). Blanes- and Golgi-like neurons in the deep layers of the GCL (iii). Scale bars in ii and iii, 25 μm.

Supplementary Figure 2 CNO-induced inhibition of neurons generated in P42-Gi forebrains has no effect on basal behaviors in a novel environment.

(a) Flow diagram of testing of basal behaviors of vehicle- and CNO-administered P42-Gi mice during a single 45 minute period of exploring a test cage. (b) Dot plots are percent time spent on each behavior by individual animals; lines are mean ± s.e.m. Significance determined by two-tailed Mann-Whitney test: Mobile, U=12.0, P=0.39; Rearing, U=10.0, P=0.23; Grooming, U=14.5, P=0.63; Digging, U=18.0, P=1.0; n=6 animals/group; n.s., not significant.

Supplementary Figure 3 CNO administration has no effect on responses to an appetitive odor in naive and P42-Gi mice trained with the odor.

(a) Flowchart illustrating strategy to test the effects of CNO administration on responses to a novel appetitive odor (Treat A, peanut butter cookie) in P70 naïve mice (not injected with AAV2-Gi at P42). (b) Proportions of successful and failed trials in P70 Nestin-cre mice administered vehicle or CNO over seven days (Fisher’s Exact Test; Number of successful trials, vehicle, 35/42; CNO, 37/42; P=0.76; n=6 animals/group). (c) Temporal plot of responses by all vehicle and CNO treated naïve mice within the 10 minute test period (left y-axis) over the seven days (x-axis). FT, failed trial. Dotted lines represent trendlines calculated from proportions of failed trials (right y-axis) over seven days (shaded regions represent 95% confidence intervals, CI). Significance was tested by Hypothesis test of Linear Regression [Slope (vehicle): -7.14% ± 2.72%, CI: -12.64, -1.64; slope (CNO): -6.55% ± 2.34%/day, CI: -11.28, -1.82; H0: slope (vehicle) = slope (CNO); F(1,80)=0.027, P=0.87]. Fisher’s Exact Test was used to compare proportions of successful trials each day (days 1 to 7 in order: P=1.0, 0.55, 1.0, 1.0, 1.0, 1.0, 1.0). (d) Flowchart illustrating approach to test the effects of training P42-Gi mice with Treats A and B over seven days prior to CNO testing over a second seven day period. Control mice received vehicle injections in place of CNO. (e) Proportions of successful and failed trials in vehicle and CNO-administered P42-Gi mice during the second seven day test period following seven training days with Treats A and B during which CNO was withheld. There were no failed trials recorded in trained animals; n=6 animals/group.

Supplementary Figure 4 CNO administration has no effect on responses to an aversive odor in naive Nestin-cre mice.

(a) Flowchart illustrating strategy to test the effects of CNO administration on responses to an aversive odor (TMT) in P70 Nestin-cre mice. (b) Bar chart illustrates no significant difference in proportions of Freezing and No Freezing responses in vehicle- and CNO-administered control mice over seven days (Fisher’s Exact Test; Number of Freezing trials, vehicle, 35/42; CNO, 36/42; P=0.99; n=6 animals/group). (c) Temporal plot of responses by all vehicle and CNO treated Nestin-cre mice within the 10 minute test period (left y-axis) over the seven days (x-axis). FT, failed trial. Dotted lines represent trendlines calculated from proportions of failed trials (right y-axis) over seven days (shaded regions represent 95% confidence intervals, CI). Significance was tested by Hypothesis test of Linear Regression [Slope (vehicle): 8.93% ± 2.59%/day, CI: 3.7, 14.15; slope (CNO): 7.74% ± 2.48%/day, CI: 2.72, 12.75; H0: slope (vehicle) = slope (CNO); F(1,80)=0.11, P=0.74.]. Fisher’s Exact Test was used to compare proportions of successful trials each day (days 1 to 7 in order: P=1.0, 1.0, 1.0, 1.0, 1.0, 1.0, 1.0).

Supplementary Figure 5 AAV2-mediated targeting of perinatally born neurons of P0-Gi mice in the rostral forebrain.

Confocal tile scan of the rostral forebrain in a sagittal section from a mouse injected with AAV2-Gi at P0 and analyzed at P70 (P0-Gi; blue, DAPI). EGFP expression (green) was robust in OB neurons, but also found in various other forebrain regions (e.g., anterior olfactory nuclei, AON; accessory olfactory bulb, AOB) as expected from targeting of neonatal progenitors. Scale bar, 100 μm. Inset: EGFP expressing cells are mature neurons expressing NeuN (red). Inset scale bar, 5 μm. Abbreviations: subependymal zone (SEZ), rostral migratory stream (RMS), cortex (Ctx); Layers of the OB: periglomerular (PGL), external plexiform (EPL), mitral (ML), internal plexiform (IPL), superficial granule (GCLs), deep granule (GCLd).

Supplementary Figure 6 AAV2-mediated targeting of perinatally born neurons in P0-Gi mice following injections into the lateral ventricles (LV) or olfactory ventricles (OV).

Tracings of representative medial and lateral sagittal forebrain sections of P0-Gi mice with areal and nuclear boundaries demarcated by dashed lines. Green dots represent 1-3 EGFP+ neurons found in the sections. Scale bar, 1 mm. Olfactory bulb (OB), anterior olfactory nuclei (AON), accessory olfactory bulb (AOB), olfactory tubercle (OT), Piriform cortex (Pir), cortical amygdala (CoA), basolateral nucleus (BLA), basomedial nucleus (BM), central nucleus (CeL), anterior amygdalar area (AAA), Cornu Ammonis (CA1, CA2, CA3), dentate gyrus (DG), subiculum (S), presubiculum (PrS), entorhinal cortex (Ent), nucleus Accumbens (Acb), diagonal band nucleus (NDB), lateral septal nucleus (LS), Hypothalamus (Hypo), Thalamus (Thal), cerebral cortices (Ctx), Striatum (St), globus pallidus (GP), Substantia Innominata (SI).

Supplementary Figure 7 CNO-induced inhibition of neurons generated in P0-Gi forebrains has no effect on basal behaviors in a novel environment.

(a) Flow diagram of testing for basal behaviors of vehicle- and CNO-administered P0-Gi mice during a single 45 minute period of exploring a test cage. (b) Dot plots are percent time spent on each behavior by individual animals; lines are mean ± s.e.m. Significance determined by two-tailed Mann-Whitney test: Mobile, U=16.0, P=0.82; Rearing, U=17.0, P=0.94; Grooming, U=11.0, P=0.30; Digging, U=17.0, P=0.94; n=6 animals/group; n.s., not significant.

Supplementary Figure 8 Acoustic startle responses are intact in CNO-induced P0-Gi mice.

(a) Flowchart for acoustic startle testing of vehicle- and CNO- administered P0-Gi mice. (b) Dot plots of mean responses over seven days from 6 mice/group combined during pulse and prepulse noise presentations (26 recordings over 15 minutes; shaded areas define the pulse noise periods). (c) Dot plots of startle response presented as percent initial responses to train of Pulse Noises revealing that prepulse inhibition in both CNO- and vehicle-administered P0-Gi mice is intact. Lines are mean ± s.e.m. Significance determined by Mann-Whitney U test; mean ± s.e.m: vehicle, 72.4% ± 3.8%; CNO, 70.8% ± 7.4%; U=23.0, P=0.90; n=6 mice/group; n.s., not significant.

Supplementary Figure 9 AAV2-mediated targeting of new neurons in P0-Gi and P42-Gi mice in the olfactory bulbs.

(a) Low power micrographs illustrating medial to lateral distribution of differentiated neurons in the OB generated and transduced with AAV2-Gi (EGFP+, false-colored black) in P0-Gi and P42-Gi mice. Scale bar, 500 μm. (b) Distribution of neurons in the dorsal and ventral domains of the OB, as well as the accessory olfactory bulb (AOB). Scale bar, 50 μm. (c) Dot plots are percentages of EGFP+ neurons distributed in distinct regions of the OB calculated as a fraction of the entire EGFP+ population of cells counted in the forebrain. Lines are mean ± s.e.m; n=3 mice/group. Layers of the OB: periglomerular (PGL), external plexiform (EPL), mitral (ML), internal plexiform (IPL), superficial granule (GCLs), deep granule (GCLd). Note, while the vast majority of EGFP+ neurons in the OB of P42-Gi mice were situated in GCLs and GCLd, P0-Gi mice contained broadly distributed neurons in nearly all olfactory related nuclei, the AOB, and cell layers in the dorsal and ventral aspects of the OB.

Supplementary Figure 10 Characterization of BrdU+ and AAV2-EGFP cells in the OB.

(a) Experimental flow chart of BrdU and AAV injections in P0-Gi and P42-Gi mice. (b) Confocal micrographs of representative neurons in the OB labeled with EGFP (green) and BrdU (red). Arrows point to double labeled neurons. Scale bar, 50 μm. (c) Dot plots are percentages of double labeled cells as a fraction of total EGFP+ and total BrdU+ cells sampled in the OB. Lines are mean ± s.e.m of percentages; n= 3 mice/group. A total of 100 EGFP+ cells were counted together with single labeled BrdU+ cells in the same captured image for calculation of percentages of total EGFP and BrdU populations (see online methods for details).

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Muthusamy, N., Zhang, X., Johnson, C. et al. Developmentally defined forebrain circuits regulate appetitive and aversive olfactory learning. Nat Neurosci 20, 20–23 (2017). https://doi.org/10.1038/nn.4452

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