Abstract
Functional neuroanatomy of Pavlovian fear has identified neuronal circuits and synapses associating conditioned stimuli with aversive events. Hebbian plasticity within these networks requires additional reinforcement to store particularly salient experiences into long-term memory. Here we have identified a circuit that reciprocally connects the ventral periaqueductal gray and dorsal raphe region with the central amygdala and that gates fear learning. We found that ventral periaqueductal gray and dorsal raphe dopaminergic (vPdRD) neurons encode a positive prediction error in response to unpredicted shocks and may reshape intra-amygdala connectivity via a dopamine-dependent form of long-term potentiation. Negative feedback from the central amygdala to vPdRD neurons might limit reinforcement to events that have not been predicted. These findings add a new module to the midbrain dopaminergic circuit architecture underlying associative reinforcement learning and identify vPdRD neurons as a critical component of Pavlovian fear conditioning. We propose that dysregulation of vPdRD neuronal activity may contribute to fear-related psychiatric disorders.
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Acknowledgements
We thank B. Werner, N. Kaouane, and the Next Generation Sequencing (NGS) Core at Vienna Biocenter Core Facilities GmbH (VBCF) for neuronal population sequencing and S. Rumpel for scientific discussion and advice. We thank M. Pasieka of the Scientific Computing Unit at the Vienna Bio Campus (VBC), the Facility for Advanced Microscopy at the Vienna Bio Campus (VBC), and in particular, P. Pasierbek and T. Lendl for help with confocal microscopy. We further thank the Facility for Preclinical Phenotyping at the Vienna Biocenter Core Facilities GmbH (VBCF), M. al Banchaabouchi, the IMP animal facility, and A. Stepanek for help with behavioral assays and animal research. We thank HistoPathology at the VBCF for expertise and histological services. B. Ferger (Boehringer Ingelheim, Germany) and G. Filk (Brains On-Line LLC, San Francisco, USA) provided valuable discussions and microdialysis data, and L. Piszczek set up and analyzed FACS control experiments for D1R knockdown. We thank M. Roth and J. Jude for advice in RNAi experiments. W.H. was supported by a grant from the European Community’s Seventh Framework Programme (FP/2007-2013)/ERC grant agreement no. 311701, the Research Institute of Molecular Pathology (IMP), Boehringer Ingelheim, and the Austrian Research Promotion Agency (FFG). S.M., T.M., and V.L. were supported by the DFG (TP B06 of SFB 779). The Vienna Biocenter Core Facilities GmgH (VBCF) Preclinical Phenotyping Facility acknowledges funding from the Austrian Federal Ministry of Science, Research & Economy and from the City of Vienna.
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F.G. conceived, designed, performed, and analyzed most of the experiments and wrote the manuscript. T.M. and S.M. performed whole-cell patch-clamp and LFP recordings for LTP experiments. J.K. and P.P. performed and analyzed Ca2+ imaging experiments. J.G. performed mouse surgeries. D.K performed anatomical tracings and designed and tested AAVs for optogenetics, DREADDs, and GCaMP6m. A.R. designed, performed, and analyzed microdialysis experiments. S.B. and K.K. performed and analyzed SCH injections, fear conditioning, acute anxiety assays, and pain tests. J.Z. designed RNAi viral vectors and supervised knockdown experiments. V.L. co-supervised experiments and wrote the manuscript. W.H. initiated and conceived the project; designed, analyzed, and supervised experiments; and wrote the manuscript. All authors contributed to the experimental design and interpretation and commented on the manuscript.
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Integrated supplementary information
Supplementary Figure 1 Projections of vPdRD neurons.
(Top) AAV::DIO-EGFP injections in the vPAG/DR of TH::Cre and DAT::Cre mice. Data from Allen Mouse Brain Connectivity Atlas; TH::Cre: Experiment number: 272699357; DAT::Cre: Experiment number: 272699357. Note that vPdRD neurons have only two major targets in the brain: Bed nucleus of the stria terminalis (BNST) and CE. (Bottom) Functional roles of vPdRD neuron projection targets. Note that of the major projection targets, the CE is the target primarily associated with associative fear learning; thus, the most likely explanation for the observed effects of vPdRD neurons on Pavlovian conditioning is that they are mediated through their projections to the CE.
Supplementary Figure 2 Amygdala projections of midbrain DA regions.
(Top) AAV::DIO-EGFP injections in the vPAG/DR of TH::Cre mice. Data from Allen Mouse Brain Connectivity Atlas; vPAG/DR: Experiment number: 272699357; SN: Experiment number: 304761539; SN: Experiment number: 304337288. Note that vPdRD neurons provide dense innervation to the CE (left), whereas amygdala projections from either SN (middle) or VTA (right) DA neurons appear rather sparse; thus, DA in CE seems to predominantly originate from vPdRD neurons.
Supplementary Figure 3 DA is required for successful LTP at BLA–CEl synapses.
(a) Left Representative images of histological identification of biocytin labelled cells after LTP recordings of CEl neurons in aCSF with electric HFS stimulation in the BLA. Right Quantification of immunohistochemically identified SST+/PKCδ-, PKCδ+/SST- and PKCδ+/SST+ cells (n = 16 animals; values from one section per animal) (b). Both cell types failed to undergo LTP without DA (n = 4 SST+ cells and 5 PKCδ+ cells; tests for LTP: RM two-way ANOVA Finteraction (1, 7) = 0.2202 P = 0.6532, Ftime (1, 7) = 1.235, P = 0.3031; Fgroups (1, 7) = 0.004483, P = 0.9485; Holm-Sidak post-hoc tests). Representative images are derived from experiments that have been repeated 5 times independently. Bars are means ± s.e.m.
Supplementary Figure 4 GCaMP6 expression in vPAG/DR and vPdRD neurons.
(a, left) Injection of AAV::GCaMP6m in the vPAG/DR of wild-type animals. (Middle) Representative IHC for extent and reliability of GCaMP6m expression in the vPAG/DR. (Right) Quantification over all animals within this cohort (n = 2 animals, values from 3 sections per animal). (b, left) Injection of AAV::DIO-GCaMP6f in the vPAG/DR of TH-Cre animals. (Middle) Representative IHC for extent and reliability of GCaMP6f expression in vPdRD neurons. (Right) Quantification over all animals within this cohort (n = 4 animals, values from 3 sections per animal). Representative images from two independent experiments (animals). Bars are means ± s.e.m.
Supplementary Figure 5 Location of microdialysis probes and injection cannulas.
(a, left) Implantation of microdialysis probes in the amygdala. (Right) Approximate position of the 2 mm lateral probe membranes (green) in and adjacent to the CEl. This configuration samples extracellular release from CEl and BLA. (b, left) Implantation of infusion cannulas over the CEl. (Right) Locations of the implantation sites (tip of the cannula) in the SCH23390 cohort. (x) indicates an implantation site anterior to that section.
Supplementary Figure 6 Asymmetric expression of D1R in CEl neurons.
(a) CEl neuronal types from SST::Cre or PKCδ::Cre crosses to Cre-dependent Rosa::td-Tomato reporter mice were FACS sorted. (b) Expression levels (Fragments per kilobase of transcript per million reads mapped, FPKM) of SST and PKCδ marker genes and D1R from combined deep sequencing results (n = 4 animals) (a). (c) Row-normalized expression values. Bars are means ± s.e.m.
Supplementary Figure 7 vPAG/DR–CE circuit manipulations.
(a, left) Injection of AAV::DIO-M4 in the PAG of TH-Cre animals for selective expression in vPdRD neurons. Middle, Representative IHC for extent and reliability of M4-expression in TH+ neurons. Right, Quantification over all animals within this cohort (n = 11 animals, values from 3 sections per animal). (b, left) Injection of 6-OHDA in the vPAG/DR of SST-tdTomato animals. (Middle) Representative IHC for extent and reliability of vPdRD neuron lesioning by 6-OHDA. (Right) Quantification over all animals within this cohort (control group: n = 4, TH+ lesion group: n = 7; values from 3 sections per animal). (c, left) Injection of AAV::DIO-Arch in the PAG of TH-Cre animals for selective expression in vPdRD neurons. (Middle) Representative IHC for extent and reliability of Arch-expression in TH+ neurons. (Right) Quantification over all animals within this cohort (n = 7 animals; values from 3 sections per animal). (d, left) Injection of AAV::DIO- ChR2 in the PAG of TH-Cre animals for selective expression in vPdRD neurons. (Middle) Representative IHC for extent and reliability of M4-expression in TH+ neurons. (Right) Quantification over all animals within this cohort (n = 10 animals; values from 3 sections per animal). Representative images from at least 4 experiments (animals). Bars are means ± s.e.m.
Supplementary Figure 8 Modulation of vPdRD neuronal activity or CEl D1R signalling does not affect anxiety or pain sensitivity.
(a) Light/Dark transition test (control group: n = 11 animals, M4 group: n = 11 animals; RM two-way ANOVALight/Dark Finteraction (1, 20) = 0.09238, P = 0.7643; Ftime (1, 20) = 0.3528, P = 0.5592; Fgroups (1, 20) = 0.5466, P = 0.4683) and (b) Elevated plus maze (EPM) (control group: n = 11 animals, M4 group: n = 11 animals; RM two-way ANOVAEPM Finteraction (2, 40) = 2.728, P = 0.0775; Ftime (2, 40) = 1402, P < 0.0001; Fgroups (1, 20) = 0, P > 0.9999; Holm-Sidak post-hoc tests) during M4 vPdRD neuron inactivation. (c) Light/Dark transition test (control group: n = 11 animals, SCH23390 group: n = 11 animals; RM two-way ANOVALight/Dark Finteraction (1, 20) = 0.08953, P = 0.7679; Ftime (1, 20) = 37.73, P < 0.0001; Fgroups (1, 20) = 0.7573, P = 0.3945) and (d) EPM (control group: n = 11 animals, SCH23390 group: n = 11 animals; RM two-way ANOVAEPM Finteraction (2, 32) = 0.2519, P = 0.7787; Ftime (2, 34) = 131.8, P < 0.0001; F groups(1, 17) = 0.6561, P = 0.4291; Holm-Sidak post-hoc tests) during blocking of D1R signaling in the CEl by infusion of SCH 23390. (e) Hot Plate (control group: n = 8 animals, Arch group: n = 5 animals; RM two-way ANOVAHotPlate Finteraction (1, 11) = 0.2614, P = 0.6192; Ftime (1, 11) = 43.14, P < 0.0001; Fgroups (1, 11) = 1.005, P = 0.3376; Holm-Sidak post-hoc tests) and (f) von Frey Filament test (control group: n = 8 animals, Arch group: n = 7 animals; unpaired t-test, two-sided, t(13) = 0.3113, P Control vs ARCH Force = 0.7605, t(13) = 0.3382, P Control vs ARCH Latency = 0.7406) during ARCH mediated silencing of vPdRD neurons. (g) Hot Plate (control group: n = 10 animals, M4 group: n = 10 animals; RM two-way ANOVAHotPlate Finteraction (1, 18) = 0.1407, P = 0.7120; Ftime (1, 18) = 110.9, P < 0.0001; Fgroups (1, 18) = 1.254, P = 0.2774; Holm-Sidak post-hoc tests) and (h) von Frey filament test (control group: n = 11 animals, M4 group: n = 11 animals; unpaired t-test, two-sided, t(20) = 0.5277, P Control vs M4 Force = 0.6035; t(20) = 0.4701, P Control vs M4 Latency = 0.6433) during M4 mediated silencing of vPdRD neurons. Bars are means ± s.e.m.
Supplementary Figure 9 6-OHDA lesion of vPdRD neurons affects CEl plasticity.
(Left) Fear conditioning increases sEPSC amplitude of SST+ neurons vs PKCδ+ neurons (FC n = 16 cell pairs, FC lesion n = 19 cell pairs and HC n = 23 cell pairs; RM two-way Finteraction(2, 110) = 1.939, P = 0.1487; Ftime (2, 110) = 0.1784, P = 0.8369; Fgroups(1, 110) = 9.12, P=0.0031 Holm-Sidak post-hoc tests) in comparison to fear conditioned (FC) animals that underwent vPdRD neuron 6-OHDA lesioning as well as homecage (HC) animals. (right) Fear conditioning increases sEPSC frequency of SST+ neurons vs PKCδ+ neurons (FC n = 16 cell pairs, FC lesion n = 19 cell pairs and HC n = 22 cell pairs; RM two-way Finteraction (3, 108) = 1.982, P = 0.1210; Ftime (3, 108) = 0.5143, P = 0.6733 Fcolumns (1, 108) = 49.18, P=0.0001; Holm-Sidak post-hoc tests). Significance levels between groups (*) at * P<0.05, ** P<0.01 and **** P<0.0001. Bars are means ± s.e.m.
Supplementary Figure 10 RNAi-mediated knockdown of D1R.
(a) constructs of shRNAs and sensor cell line used in RNAi mediated D1R knock-down in the CEl (b) Reporter-based evaluation of shRNA knockdown potency in NIH-3T3 cells expressing an expressing tdTomato transgene harboring shRNA target sites in the 3’UTR. Histograms depict tdTomato fluorescence intensity in reporter cells expressing control (green, left panel) or D1R shRNAs (green, right panel) compared to cells that were not transduced with shRNA (red). (c) Quantification of triplicate experiments in (b) (RM one-way ANOVA F (3, 8) = 8.05, P = 0.0084); (d) Representative IHC showing delivery and expression of GFP-shRNA transcripts in the CEl. (e) Quantification of D1R shRNA expression in SST+PKCδ- and PKCδ+ SST- neurons in the CEl (n = 3 animals, values are from 2 sections per animal). (f) Freezing responses to CS presentations during conditioning (RM two-way ANOVAConditioning Finteraction (4, 64) = 0.9245, P = 0.4554; Ftime (4, 64) = 24.99, P < 0.0001, Fgroups (1, 16) = 0.5751, P = 0.4593) and recall sessions (RM two-way ANOVARecall Finteraction (1, 16) = 1.444, P = 0.2470; Ftime (1, 16) = 14.92, P = 0.0014; Fgroups F (1, 16) = 0.2322, P = 0.6364; Holm-Sidak post-hoc tests). Quantification of (c) over all animals within this cohort. Representative images from two independent experiments (animals). Significance levels between groups (*) and to baseline (BL) (#) at */# P<0.05, **/## P<0.01, Bars are means ± s.e.m.
Supplementary Figure 11 Heterogeneity of vPAG/DR-projecting CE output.
(a) Representative image of CTB (injected into the vPAG/DR) retrogradely labelled neurons in the CEl and CEm. (b) Distribution of CTB retrogradely labelled CE neurons (n = 2 animals, values are from 3 sections per animal). (c) Representative image of cell-type specific projections (green) from CEl to TH+ neurons in the vPAG/DR (red) in SST::Cre and PKCδ::Cre mice injected in CEl with AAV for Cre-dependent expression of GFP. (d) Density of projecting fibers originating from CEl PKCδ+ or SST+ cells. (n = 3 animals, values are from 2 sections per animal; Unpaired t-test, two-sided, t(4) = 8.746, PSST+ vs PKCδ+ = 0.0009). Representative images from at least two independent experiments (animals). Significance levels between groups (*) at *** P<0.001. Bars are means ± s.e.m.
Supplementary Figure 12 Modulation of vPAG/DR and vPdRD neuronal activity during associative learning.
(a) Bulk Ca2+ signals of vPAG/DR neuronal activity of freely moving animals (b) during different phases of fear conditioning. Each trial within every session (conditioning day 1, conditioning day 2, recall day) consisted of 60s continuous Ca2+ imaging (20s pre-CS, 20s CS co-terminating into 1s foot shock and 20s post-CS, n = ROIs from 2 animals)(dashed line indicates CS response). (b) Freezing of Ca2+ imaged mice (n = 2 animals) during different phases of fear conditioning (c) Example traces of vPAG/ DR neuron bulk shown in c. Ca2+ signals of individual trials (top) and their trial averages (bottom). (d) Example traces of single vPAG/DR neuronal units during different phases of fear conditioning. Ca2+ signals of individual trials (middle), their trial averages (bottom) and Ca2+ events during individual trials (top). (e) Cell-type specific bulk Ca2+ imaging of vPdRD neurons of freely moving animals during different phases of fear conditioning as shown in Fig. 4f-i. Ca2+ signals of individual trials (top) and their trial averages (bottom). (f) Example traces of single vPdRD neuronal units from Ca2+ imaging shown in Fig. 4f-i. Ca2+ signals of individual trials (middle), their trial averages (bottom) and Ca2+ events during individual trials (top). Lines with shaded regions represent means ± upper and lower bounds. Ca2+ signals and event amplitudes are derived from per ROI (a, c, e) or per cell (d, f) dF/F values, standardized over the whole experiment and given as units S.D.
Supplementary Figure 13 Optogenetic activation of vPdRD neurons alone is not sufficient for fear conditioning.
(a) Motor parameter mean velocity is unchanged during optogenetic activation of vPdRD neurons in TH::Cre mice injected with AAV for Cre-dependent expression of ChR2 (control group: n = 10 animals, ChR2 group: n = 9 animals; RM two-way Finteraction (4, 68) = 1.781, P = 0.1428; Ftime (4, 68) = 5.01, P = 0.0013; Fgroups (1, 17) = 0.5832, P = 0.4555 Holm-Sidak post-hoc tests). (b) Freezing responses to CS presentations during conditioning (control group: n = 10 animals, ChR2 group: n = 9 animals; RM two-way ANOVAConditioning Finteraction (4, 52) = 0.3696, P = 0.8292; Ftime (4, 52) = 2.691, P = 0.0410, Fgroups (1, 13) = 2.066, P = 0.1743) and recall sessions (RM two-way ANOVARecall Finteraction (1, 13) = 0.0001412, P = 0.9907; Ftime (1, 13) = 17.2, P = 0.0011; Fgroups (1, 13) = 2.966, P = 0.1087; Holm-Sidak post-hoc tests). The shock-US was replaced with optogenetic activation of vPdRD neurons during second half of the 20s CS presentation. Bars are means ± s.e.m. BL, Baseline.
Supplementary Figure 14 Model of vPdRD neuron–amygdala circuit interactions during associative learning.
Unpredicted US (shock) activates vPdRD neurons, which co-release glutamate and DA in the CEl. While glutamate activates both CEl PKCδ+ and SST+ neurons, asymmetric D1R expression predominately reinforces CS (tone) signals at BLA-CEl SST+ synapses. This potentiates CS-evoked responses of CEl SST+ neurons (synergizing with D2R activity on PKCδ+ cells43) which together with CEm neurons jointly facilitate CS specific freezing. Of note, additional presynaptic mechanisms in BLA-CEl circuitry might contribute. Reinforcement signals from vPdRD neurons inversely correlate with stimulus (initially US, later CS) expectancy, linking the formation of associative memory traces in amygdala to PEs. Feedback inhibition from CEl SST+ and CEm neurons controls activity of, and potentially encoding of PE in, vPdRD cells.
Supplementary Figure 15 Viral constructs and subject history.
(a) Viral constructs, short names, manufacturer and titer as used in different experiments. (b) Subject history of different mouse cohorts.
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Supplementary Text and Figures
Supplementary Figures 1–15
Supplementary Video 1
Ca2+ signaling of vPdRD neurons during first round of fear conditioning. Representative video of cell-type specific fiber-endomicroscopic Ca2+ imaging from vPdRD neurons of a freely moving TH-Cre mouse, that received a vPAG/DR targeted injection of AAV-DIO expressing GCaMP6f. This is the first session of a series comprising of two reinforced and one non-reinforced fear conditioning session on three consecutive days. The movie features three elements in a timely synchronized manner: The behavior of the mouse, Ca2+ signaling of vPdRD neurons and activity traces (grey mark: CS = 20s continuous 3 kHz tone, red mark: US = 1s foot shock). The Ca2+ signaling panel provides numbered and circled vPdRD neurons. Their automatically detected activity and corresponding events are shown in the activity trace panel. Note how vPdRD neurons fire strongly to the US right from the beginning, whereas responses to the CS become visible after three CS-US pairings, when the mouse has learned that the CS predicts the shock. A quantification of neurons from the entire mouse cohort is shown in Fig. 4f–j
Supplementary Video 2
Ca2+ signaling of vPdRD neurons during second round of fear conditioning. Representative video of Ca2+ activity from vPdRD neurons of a TH-Cre mouse. Identical setup to Supplementary Video 1 showing the second reinforced fear conditioning session on the consecutive day after the first reinforced fear conditioning session showed in Supplementary Video 1. In comparison to the first fear conditioning session, vPdRD neurons show higher activity during the CS right from the beginning as the mouse has already learned that the CS predicts the US during day 1. A quantification of neurons from the entire mouse cohort is shown in Fig. 4f–j
Supplementary Video 3 - Ca signaling of vPdRD neurons during fear recall.
Representative video of Ca2+ activity from vPdRD neurons of a TH-Cre mouse. Identical setup to Supplementary Video 1 and 2 showing the non-reinforced recall session on day 3 following the two reinforced fear conditioning sessions on day 1 and 2. vPdRD neurons respond to the presentation of a now emotionally relevant CS, notably, not only restricted to the onset of the tone. A quantification of neurons from the entire mouse cohort is shown in Fig. 4f–j
Supplementary Video 4 - Optogenetic activation of vPdRD neurons induces slow motion.
Optogenetic activation of vPdRD neurons induces constant slow movement (Fig. 5c). Notably, this ‘slow motion’ behavior is able to override freezing behavior (Fig. 5d)
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Groessl, F., Munsch, T., Meis, S. et al. Dorsal tegmental dopamine neurons gate associative learning of fear. Nat Neurosci 21, 952–962 (2018). https://doi.org/10.1038/s41593-018-0174-5
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DOI: https://doi.org/10.1038/s41593-018-0174-5
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