Backward conditioning reveals flexibility in infralimbic cortex inhibitory memories
- School of Psychology, UNSW Sydney, Australia
- Department of Psychological Science, University of Vermont, United States
Figures
Figure 1
Ex-vivo cell recordings demonstrate successful optical infralimbic cortex (IL) stimulation.
(A) Wild-type Long-Evans rats (n=2 females and 2 males) were bilaterally infused in the IL with a channelrhodopsin (ChR2) virus. (B) The top representative raw trace is a cell-attached recording from a transfected IL neuron with LED train stimulations. In the same neuron under whole-cell current clamp, LED pulses excited the IL neuron as evidenced by enhanced action potentials (AP) from resting state baseline (bottom raw trace). (C) AP frequency was enhanced during LED illumination compared to their baseline state (n=10 cells). (D) Micrographs showing ChR2-transfected IL neurons that was labeled with biocytin.
Figure 2 with 1 supplement
Prior experience with fear extinction enables infralimbic cortex (IL) stimulation to facilitate subsequent fear extinction.
(A) Schematic representation of the behavioral design used in Experiment 1. The LEDs were activated in the Ext-ON and ReExt-ON groups during the second fear extinction session. Ext-OFF: n=3 females and 7 males; Ext-ON: n=4 females and 5 males; ReExt-OFF: n=5 females and 6 males; ReExt-ON: n=2 females and 6 males. (B) Mean percent freezing during all experimental stages. Data are shown as mean ± SEM. Test data include individual data points for female (filled circle) and male (open circle) rats. Asterisks denote significant effect (**p<0.01).
Figure 2—figure supplement 1
Histological and behavioral data related to Figure 2.
(A) Data for Experiment 1. Micrographs showing channelrhodopsin (ChR2) expression in infralimbic cortex (IL) neurons. Minimal (light green) and maximal (darker green) extent of IL viral expression. The location of fiber-optic cannulas is also represented (black circles). From left to right: +3.72; +3.24; +3.00; +2.76 mm from bregma. (B) Freezing across trials during the test presented in Figure 2B. Freezing decreased across trials (Trial: F(1,34) = 24.20; p<0.001; η2=0.42), but this decrease did not interact with the planned orthogonal contrasts reported in the manuscript (lowest p=0.77). Data are shown as mean ± SEM.
Figure 3 with 1 supplement
Prior experience with backward fear conditioning enables infralimbic cortex (IL) stimulation to facilitate subsequent fear extinction.
(A) Schematic representation of the behavioral design used in Experiment 2. The LEDs were activated in all groups during the fear extinction session. No-EYFP: n=5 females and 5 males; No-ChR2: n=3 females and 4 males; Back-EYFP: n=5 females and 5 males; Back-ChR2: n=4 females and 5 males. (B) Mean percent freezing during all experimental stages. Baseline freezing (before first tone presentation) at test is provided due to significant differences. Data are shown as mean ± SEM. Test data include individual data points for female (filled circle) and male (open circle) rats. Asterisks denote significant effect (**p<0.01; ***p<0.001).
Figure 3—figure supplement 1
Histological and behavioral data related to Figure 3.
(A) Data for Experiment 2. Micrographs showing EYFP and channelrhodopsin (ChR2) expression in infralimbic cortex (IL) neurons. Minimal (light gray for EYFP and light green for ChR2) and maximal (dark gray for EYFP and dark green for ChR2) extent of IL viral expression. The location of fiber-optic cannulas is also represented (black circles). From left to right: +3.72; +3.24; +3.00; +2.76 mm from bregma. (B) Freezing across trials during the test presented in Figure 3B. Freezing decreased across trials (Trial: F(1,32) = 31.53; p<0.001; η2=0.50), but this decrease did not interact with the planned orthogonal contrasts reported in the main manuscript (lowest p=0.42). Data are shown as mean ± SEM.
Figure 4 with 1 supplement
Backward fear conditioning produces an IL memory that is specific to the backward trained stimulus and is inhibitory.
(A) Schematic representation of the behavioral design used in Experiment 3. The LEDs were activated in all groups during the fear extinction session. Diff-EYFP: n=4 females and 3 males; Diff-ChR2: n=2 females and 4 males; Same-EYFP: n=4 females and 3 males; Same-ChR2: n=2 females and 4 males. (B) Mean percent freezing during all experimental stages. Baseline freezing (before first tone presentation) at test is provided to compare with Experiment 3. (C) Schematic representation of the behavioral design used in Experiment 4. No: n=12 females and 4 males; Back: n=8 females and 8 males. (D) Mean percent freezing during all experimental stages. Data are shown as mean ± SEM. Test data include individual data points for female (filled circle) and male (open circle) rats. Asterisks denote significant effect (*p<0.05; ***p<0.001; n.s., nonsignificant).
Figure 4—figure supplement 1
Histological and behavioral data related to Figure 4.
(A) Data for Experiment 3. Micrographs showing EYFP and channelrhodopsin (ChR2) expression in infralimbic cortex (IL) neurons. Minimal (light gray for EYFP and light green for ChR2) and maximal (dark gray for EYFP and dark green for ChR2) extent of IL viral expression. The location of fiber-optic cannulas is also represented (black circles). From left to right: +3.72; +3.24; +3.00; +2.76 mm from bregma. (B) Freezing across trials during the test presented in Figure 4B. Freezing decreased across trials (Trial: F(1,22) = 64.00; p<0.001; η2=0.74), but this decrease did not interact with the planned orthogonal contrasts reported in the main manuscript (lowest p=0.39). Data are shown as mean ± SEM.
Figure 5 with 1 supplement
Inhibitory memories encoded within the infralimbic cortex (IL) lack motivational information.
(A) Schematic representation of the behavioral design used in Experiment 5. The LEDs were activated in all groups during the fear extinction session. Diff-EYFP: n=4 females and 5 males; Diff-ChR2: n=4 females and 5 males; Same-EYFP: n=6 females and 3 males; Same-ChR2: n=6 females and 2 males. (B) Number of magazine entries during backward appetitive conditioning. (C) Mean percent freezing during remaining experimental stages. Data are shown as mean ± SEM. Test data include individual data points for female (filled circle) and male (open circle) rats. Asterisks denote significant effect (*p<0.05).
Figure 5—figure supplement 1
Histological and behavioral data related to Figure 5.
(A) Data for Experiment 5. Micrographs showing EYFP and channelrhodopsin (ChR2) expression in infralimbic cortex (IL) neurons. Minimal (light gray for EYFP and light green for ChR2) and maximal (dark graey for EYFP and dark green for ChR2) extent of IL viral expression. The location of fiber-optic cannulas is also represented (black circles). From left to right: +3.72; +3.24; +3.00; +2.76 mm from bregma. (B) Freezing across trials during the test presented in Figure 5C. Freezing decreased across trials (Trial: F(1,31) = 25.12; p<0.001; η2=0.45), but this decrease did not interact with the planned orthogonal contrasts reported in the main manuscript (lowest p=0.23). Data are shown as mean ± SEM.
Figure 6 with 1 supplement
Stimulus familiarity does not enable infralimbic cortex (IL) stimulation to facilitate fear extinction.
(A) Schematic representation of the behavioral design used in Experiment 6. The LEDs were activated in all groups during the fear extinction session. For-EYFP: n=3 females and 4 males; For-ChR2: n=4 females and 4 males; Diff-EYFP: n=2 females and 4 males; Diff-ChR2: n=2 females and 4 males. (B) Number of magazine entries during forward and backward appetitive conditioning. (C) Mean percent freezing during remaining experimental stages. Data are shown as mean ± SEM. Test data include individual data points for female (filled circle) and male (open circle) rats. Asterisks denote significant effect (**p<0.01).
Figure 6—figure supplement 1
Histological and behavioral data related to Figure 6.
(A) Data for Experiment 6. Micrographs showing EYFP and channelrhodopsin (ChR2) expression in infralimbic cortex (IL) neurons. Minimal (light gray for EYFP and light green for ChR2) and maximal (dark gray for EYFP and dark green for ChR2) extent of IL viral expression. The location of fiber-optic cannulas is also represented (black circles). From left to right: +3.72; +3.24; +3.00; +2.76 mm from bregma. (B) Freezing across trials during the test presented in Figure 6C. Freezing decreased across trials (Trial: F(1,23) = 52.61; p<0.001; η2=0.70), but this decrease did not interact with the planned orthogonal contrasts reported in the main manuscript (lowest p=0.34). Data are shown as mean ± SEM.
Figure 7 with 1 supplement
Prior experience with appetitive extinction enables infralimbic cortex (IL) stimulation to facilitate subsequent backward fear conditioning.
(A) Schematic representation of the behavioral design used in Experiment 7. The LEDs were activated in all groups during backward fear conditioning. EYFP: n=5 females and 5 males; ChR2: n=5 females and 4 males. (B) Number of magazine entries during forward appetitive conditioning and appetitive extinction. (C) Mean percent freezing during remaining experimental stages. Data are shown as mean ± SEM. Test data include individual data points for female (filled circle) and male (open circle) rats. Asterisks denote significant effect (*p<0.05).
Figure 7—figure supplement 1
Histological and behavioral data related to Figure 7.
(A) Data for Experiment 7. Micrographs showing EYFP and channelrhodopsin (ChR2) expression in infralimbic cortex (IL) neurons. Minimal (light gray for EYFP and light green for ChR2) and maximal (dark gray for EYFP and dark green for ChR2) extent of IL viral expression. The location of fiber-optic cannulas is also represented (black circles). From left to right: +3.72; +3.24; +3.00; +2.76 mm from bregma. (B) Freezing across trials during the test presented in Figure 7C. Freezing remained stable across trials (Trial: p=0.07) and the Trial factor did not interact with the planned orthogonal contrast reported in the main manuscript (p=0.84). Data are shown as mean ± SEM.
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Backward conditioning reveals flexibility in infralimbic cortex inhibitory memories
eLife 14:RP108719.
https://doi.org/10.7554/eLife.108719.3
