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. 2018 Nov 13;7:e38818. doi: 10.7554/eLife.38818

Figure 5. Inhibition of dopamine neuron firing at the time of the US omission impairs fear extinction learning.

(A) Schematic of the surgical procedure showing bilateral virus injection (left) and optical fiber implantation (right) in the VTA. (B) Example histological image showing Cre-dependent expression of NpHR-eYFP (green) along with immunostaining for tyrosine hydroxylase TH (red) and DAPI (blue) staining in the VTA. White vertical tracks indicate the bilateral optical fiber placements in the VTA. Scale bar: 0.5 mm. (C) Confocal images showing expression of NpHR-eYFP (left), TH (middle) and merged image (right) showing co-expression. Scale bar: 20 μm. (D) Schematic of the behavioral protocol. Fear Cond.: fear conditioning, Ext Recall: extinction recall. (E) Schematic of paired optogenetic inhibition of DA neurons at the time of the US omission. (F) Schematic of unpaired optogenetic inhibition during intertrial intervals. (G) Percent freezing to the CS during fear conditioning (FC), extinction and extinction recall sessions. The Paired-NpHR group showed impaired extinction learning and extinction recall. (**p<0.01, *p<0.05). (H) No difference in freezing to the CS between groups at the start of extinction (first CS). Ext: extinction. (I) Freezing levels during E-Ext (average of first 10 CSs) and L-Ext (average of last 10 CSs; two-way repeated measures ANOVA, main effect of group: F2,19 = 9.05, p = 0.0017; group × trial interaction: F2,19 = 7.38, p = 0.0043). The Paired-NpHR group (n = 7) showed significantly higher freezing to the CS compared to the Paired-eYFP (n = 7) and Unpaired-NpHR (n = 8) groups during L-Ext trials (***p<0.001). E-Ext: early extinction, L-Ext: late extinction. (J) Freezing levels during E-Ext Rec (average of first 10 CSs) and L-Ext Rec trials (average of last 10 CSs; two-way repeated measures ANOVA, main effect of group: F2,19 = 7.21, p = 0.0047). The Paired-NpHR group exhibited significantly higher freezing to the CS compared to the control groups during E-Ext Rec (***p<0.001). E-Ext Rec: early extinction recall, L-Ext Rec: late extinction recall. Error bars represent mean ± s.e.m. across animals.

Figure 5.

Figure 5—figure supplement 1. Placement of optical fibers and DA neuron-specific expression of NpHR-eYFP.

Figure 5—figure supplement 1.

(A) Schematic coronal sections showing the bilateral placement of the tips of the optical fibers for mice in Paired-NpHR (yellow) Paired-eYFP (black) and Unpaired-NpHR (gray) groups. Numbers represent distance posterior to the bregma. (B) Representative confocal images showing DAPI staining (nuclear marker, blue), immunostaining for TH (dopamine neurons, red) and expression of NpHR-eYFP (green). Scale bar: 20 μm. (C) Percentage of neurons that are eYFP+/TH+ (red) and eYFP+/TH- (gray) in NpHR-eYFP (n = 1464 neurons in four mice) and eYFP (n = 1589 neurons in four mice) groups. Both groups showed similarly high levels of specificity for DA neurons (NpHR-eYFP: 95.3%; eYFP: 95%).
Figure 5—figure supplement 2. Optical activation of NpHR inhibits DA neuron firing in awake behaving mice.

Figure 5—figure supplement 2.

(A) Left: histological example of a silicon probe track (red) in the VTA. Green: NpHR-eYFP expression, red: DiI, blue: DAPI staining. Right: Schematic coronal section showing the location of the opto-probe in the VTA. (B) A representative DA neuron showing inhibition of spiking in response to laser illumination. Left: Schematic of the laser illumination (top, laser was applied from 0 to 1 s); raster plot (middle) and spike rate plot (bottom). Middle: spike waveform (scale: 100 µV). Right: inter-spike interval distribution. (C) The same as (B), but for a representative DA neuron that shows rebound excitation at the laser offset. (D) The same DA neuron as in (C) in response to a laser pulse with a ramp-like reduction in light power (1 s ramp) similar to the ramp (1 s) used in optogenetic inhibition experiment in Figure 5. Note the reduced rebound excitation.