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. 2021 Oct 5;118(40):e2111069118.
doi: 10.1073/pnas.2111069118.

Midbrain dopaminergic innervation of the hippocampus is sufficient to modulate formation of aversive memories

Theodoros Tsetsenis  1 , Julia K Badyna  2 , Julianne A Wilson  3 , Xiaowen Zhang  3 , Elizabeth N Krizman  3   4 , Manivannan Subramaniyan  2 , Kechun Yang  2 , Steven A Thomas  3 , John A Dani  1
Affiliations

Midbrain dopaminergic innervation of the hippocampus is sufficient to modulate formation of aversive memories

Theodoros Tsetsenis et al. Proc Natl Acad Sci U S A. .

Abstract

Aversive memories are important for survival, and dopaminergic signaling in the hippocampus has been implicated in aversive learning. However, the source and mode of action of hippocampal dopamine remain controversial. Here, we utilize anterograde and retrograde viral tracing methods to label midbrain dopaminergic projections to the dorsal hippocampus. We identify a population of midbrain dopaminergic neurons near the border of the substantia nigra pars compacta and the lateral ventral tegmental area that sends direct projections to the dorsal hippocampus. Using optogenetic manipulations and mutant mice to control dopamine transmission in the hippocampus, we show that midbrain dopamine potently modulates aversive memory formation during encoding of contextual fear. Moreover, we demonstrate that dopaminergic transmission in the dorsal CA1 is required for the acquisition of contextual fear memories, and that this acquisition is sustained in the absence of catecholamine release from noradrenergic terminals. Our findings identify a cluster of midbrain dopamine neurons that innervate the hippocampus and show that the midbrain dopamine neuromodulation in the dorsal hippocampus is sufficient to maintain aversive memory formation.

Keywords: fear conditioning; locus coeruleus; optogenetics; substantia nigra pars compacta; ventral tegmental area.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
Anterograde tracing of VTA/SNc dopaminergic projections to the CA1 hippocampal field. (A) An AAV allowing the conditional expression of a synaptophysin-mRuby fusion protein (AAV-DIO-Snpn-mRuby) was injected into the VTA/SNc of DAT-Cre mice to facilitate anterograde labeling of DA terminals originating from the midbrain. (B) Coronal midbrain section showing the infection in the VTA/SNc with Snpn-mRuby (red, Left), labeling of DA cells with TH (green, Middle), and a merged image showing the colocalization of Snpn-mRuby–infected cells with TH (orange/yellow, Right). (Scale bars, 100 µm.) (C) Representative low-magnification coronal section of dCA1 stained with DAPI. The boxed area corresponds to the high-magnification images of dCA1 in D. (Scale bar, 100 µm.) (D) Coronal section of dCA1, corresponding to the boxed area in C showing DA terminals expressing Snpn-mRuby (red, Left) immunostained with an antibody against TH (green, Middle) and a merged image showing the colocalization of the two markers. (Scale bars, 50 µm.) (E) Higher-magnification images of the boxed areas in D, showing that anterogradely labeled terminals coexpress TH (yellow, Right). (Scale bars, 5 μm.) (F) Percentage of Snpn-mRuby–positive puncta that are also positive for TH (Snpn-mRuby/TH double-positive: 94.6 ± 2.0% and n = 5 mice). Data represent means ± SEM. dDG, dorsal dentate gyrus.
Fig. 2.
Fig. 2.
Retrograde tracing identifies a population of DA neurons in the anterolateral VTA/SNc that directly projects to the hippocampus. (A) CAV2-Cre injected into the hippocampus of Ai14 reporter mice to allow the retrograde labeling of projection neurons. (B) Coronal section showing the infection of the dHip of Ai14 mouse with CAV-Cre and the induction of tdTomato expression in infected cells. (Scale bar, 100 µm.) (C) Coronal section of anterolateral VTA/SNc showing retrogradely labeled neurons expressing tdTomato (red) and immunostained with an antibody against TH (green). (Scale bar, 200 µm.) (D) Higher magnification of the boxed area in C, showing that retrogradely labeled cells coexpress TH. (Scale bar, 50 μm.) (E) CAV2-Flex-Flpo was injected in the dHip of DAT-Cre mice, and AAV-DIO-eYFP was injected in the anterior VTA/SNc. (F) Coronal section of anterolateral VTA/SNc showing retrogradely labeled DA neurons expressing YFP (green) and immunostained with an antibody against TH (red) to highlight the VTA/SNc region. As before in C and D, labeled neurons are located in the SNc and lateral VTA. (Scale bar, 200 µm.) The numbers on the top right corner represent the distance from bregma (65).
Fig. 3.
Fig. 3.
Optogenetic activation of midbrain dopaminergic terminals in dCA1 during cFC training causes an increase in freezing responses during recall in a context-specific manner. (A) An AAV encoding Cre-dependent ChR2-eYFP was bilaterally injected in the VTA/SNc of DAT-Cre mice, and optic fibers were implanted above the dCA1. (B) Coronal sections from the midbrain of a DAT-Cre mouse injected with AAV-DIO-ChR2-eYFP were immunostained with an antibody against GFP to enhance eYFP fluorescence to visualize ChR2 expression and DAPI. The numbers on the top left corner represent the distance from bregma (65). (Scale bar, 1 mm.) (C) Confocal image of the dCA1 from the same mouse as in B showing the expression of ChR2-positive fibers and optic fiber placement. (Scale bar, 100 µm.) (D) High magnification of dashed box in C showing the colocalization of ChR2-positive fibers with TH. (Scale bar, 50 µm.) (E) Schematic illustration of cFC procedure. A horizontal black line with light trains as vertical blue bars and shocks as lightning bolts represents the timeline of the exposure to each context. Blue light delivery and shocking occurred only during training. (F) Bar graph showing the freezing responses of ChR2-positive and YFP control mice after reexposure to context “A” 24 h after training. Unpaired Student’s t test, P = 0.0007; n = 12 and 11 for YFP and ChR2, respectively; ***P < 0.001; and data represent means ± SEM. (G) Graph showing freezing responses of ChR2-positive and YFP control mice in an altered context “B” 24 h after reexposure to context A. Unpaired Student’s t test, P = 0.270; n = 8 for YFP and ChR2; not significant (NS): P > 0.05; and data represent means ± SEM.
Fig. 4.
Fig. 4.
Inhibition of D1/D5 signaling in hippocampal dCA1 impairs the acquisition of cFC. (A) Illustration of the procedure for drug infusions and cFC. Mice were infused with drugs through bilateral cannulas implanted above the hippocampal dCA1. After the completion of infusions, animals were allowed to rest for 15 min and then subjected to cFC training. Around 24 h later, mice were reintroduced to the same chamber to test for freezing. (B) Image of a coronal brain slice showing the position of the bilateral cannulas and the graphic illustration of the cannulas and injectors for drug delivery. (C) Graph showing the freezing responses of mice treated with vehicle (saline, blue) and SCH (red) to inhibit D1/D5 receptors during cFC testing 24 h later. Unpaired Student’s t test, P = 0.0068; n = 12 and 11 for saline and SCH, respectively; **P < 0.01; and data represent means ± SEM.
Fig. 5.
Fig. 5.
Generation and characterization of LC TH-deficient mice (THLC KO). (A) A simplified graphical illustration of the breeding for the generation of THLC KO mice, resulting in the genetic ablation of DA and NE production in the LC (see Materials and Methods section for the detailed breeding information). (B) Coronal sections of the LC of THLC KO and a littermate control stained for DAPI (blue, Top) and TH (red, Bottom). 4V represents the fourth ventricle. (Scale bars, 200 µm.) (C) Bar graphs showing the integrated density (IntDen) of TH immunofluorescence in the LC of control and THLC KO mice. Unpaired Student’s t test, P = 0.0004; n = 5 for control and THLC KO; ***P < 0.001; and data represent means ± SEM. (D) Bar graphs showing the tissue content of NE in the brainstem of control and THLC KO mice. Unpaired Student’s t test, P < 0.0001; n = 5 for control and THLC KO; ****P < 0.0001; and data represent means ± SEM. (E) Bar graphs showing the tissue content of DA in the brainstem of control and THLC KO mice. Unpaired Student’s t test, P < 0.0001; n = 5 for control and THLC KO; ****P < 0.0001; and data represent means ± SEM. (F) Coronal midbrain sections of THLC KO and a littermate control stained for TH (red). (Scale bars, 100 µm.) (G) Bar graphs showing the IntDen of TH immunofluorescence in the midbrain of control and THLC KO mice. Unpaired Student’s t test, P = 0.6545; n = 5 for control and THLC KO; not significant (NS): P > 0.05; and data represent means ± SEM. A.U., arbitrary units.
Fig. 6.
Fig. 6.
Genetic ablation of catecholamine production in the LC does not affect normal aversive learning. (A) Coronal sections of the dCA1 hippocampal region from a THLC KO and a littermate control stained for TH (red). (Scale bars, 50 µm.) (B) Bar graphs showing the percent of the total pixel area containing TH immunofluorescence in the dCA1 of control and THLC KO mice. Unpaired Student’s t test, P < 0.0001; n = 5 for control and THLC KO; ****P < 0.0001; and data represent means ± SEM. (C) Bar graphs showing the tissue content of NE in the dHip of control and THLC KO mice. Unpaired Student’s t test, P < 0.0001; n = 6 for control and THLC KO; ***P < 0.0001; and data represent means ± SEM. (D) Bar graphs showing the tissue content of DA in the dHip of control and THLC KO mice. Unpaired Student’s t test, P = 0.3002; n = 6 for control and THLC KO; not significant (NS): P > 0.05; and data represent means ± SEM. (E) Graph showing the freezing responses of THLC KO and control mice after reexposure to the same context 24 h after training with a weak cFC protocol. Unpaired Student’s t test, P = 0.3839; n = 10 and 9 for control and THLC KO, respectively; NS: P > 0.05; and data represent means ± SEM. (F) Graph showing the freezing responses of THLC KO and control mice after reexposure to the same context 24 h after strong cFC training. Unpaired Student’s t test, P = 0.3175; n = 12 for control and THLC KO; NS: P > 0.05; and data represent means ± SEM.
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