Article Figures & Data
Figures
- Figure 1.
Comparative distribution of mPFC projections to the SI/VP and BLA across cortical layers. A,B, Examples of CTB injections in the SI/VP and BLA. C,D, Mapping of the full extent of CTB injections in the SI/VP and BLA. E, Example of CTB and cFos labeling in the mPFC. Scale bar, 100 μm. Insets, Left, Example of CTB-488 labeling an IL→BLA projector, which was also cFos+. Right, An example of CTB-647 labeling of an IL→SI/VP projector, which was also cFos+. Scale bar, 10 μm. F, Comparative density mapping shows a significantly denser IL output to the SI/VP than BLA (post hoc comparison of cortical region × subcortical target; p < 0.001). G, Comparative density mapping shows a significantly larger PL output to the SI/VP than BLA (post hoc comparison of cortical region × subcortical target; p < 0.001). H, Mixed-model comparing density of SI/VP and BLA output shows denser PL and IL projections to the SI/VP from both L2/3 and L5. I, Comparative density mapping shows that PL and IL projections to the BLA are similarly dense, with BLA projections from both peaking in L2/3 but spreading to deeper layers as well. J, Comparative density mapping showing IL and PL output to the SI/VP. Although L5 IL→SI/VP projections appear to be more numerous than L5 PL→SI/VP projections, the three-way comparison of cortical region × subcortical target × layer was not significant (F(1,109) = 1.007; p > 0.05). K, Mixed-model comparing densities of PL and IL output across layers. Post hoc comparisons show that there are no differences in PL versus IL outputs to the BLA across layers. However, there is a significantly denser projection from L5 than L2/3 PL and IL to the SI/VP (post hoc, p < 0.001). Note that mPFC-BLA projections are illustrated with green (CTB-488), and mPFC→SI/VP projections are illustrated with magenta (CTB-647) for visualization purposes only. During experiments, CTB-488 and CTB-647 injections were counterbalanced between SI/VP and BLA. Main effects, *p < 0.05; **p < 0.01; ***p < 0.001. All data are shown as mean ± SEM. Abbreviations: BL, basolateral nucleus; BLA, basolateral amygdala; Ce, central nucleus; HDB, horizontal limb of the diagonal band of Broca; IL, infralimbic cortex; LA, lateral nucleus; PL, prelimbic cortex; SI, substantia innominata; VP, ventral pallidum.
- Figure 2.
Global PL activity is higher in L2/3 than L5, whereas global IL activity is similar across layers during behavior. A, Timeline of CTB injection surgeries and behavioral paradigm tailored for cFos expression. One week after CTB injections in the VP/SI and BLA, mice were randomly assigned to one of the three groups. The tone control group [Tone Ctrl. (low fear), gray] was exposed to unpaired tones across Days 1–3 and thus was in a low fear state when sampled for cFos. The extinction learning group [Ext Learn. (high fear), orange] was fear conditioned with five CS–US pairings on Day 1. Then, to control for tone exposure, this group received 20 trials of a new, unpaired 8 kHz tone on Day 2 and then, on Day 3, another five trials of the unpaired 8 kHz tone, followed by five trials of the fear-conditioned CS for extinction learning. This group was in a relatively high fear state when sampled for cFos. The extinction retrieval group [Ext Ret. (low fear), purple] was fear conditioned with five CS–US pairings on Day 1, extinguished with 20 CS trials on Day 2 and, on Day 3, underwent extinction retrieval with 10 CS trials. This group was in a relatively low fear state when sampled for cFos. All animals were perfused 90 min after the sixth tone on Day 3. B, Percentage defensive freezing in all groups throughout Days 1–3 of the behavioral paradigm. Day 3, Gray box highlights the trials for timing cFos capture, when the extinction learning group freezing was significantly higher than both in controls and extinction retrieval groups. C, Density mapping of PL cFos+ cells across all layers in all behavioral groups. D, The average number of PL cFos+ cells was higher in L2/3 than L5 for all behavioral groups. E, Density mapping of IL cFos+ cells across all layers in all behavioral groups. F, There were no differences in the average number of IL cFos+ cells across layers in all groups. *p < 0.05; **p < 0.01; ***p < 0.001. All data are shown as mean ± SEM.
- Figure 3.
The L5 IL→SI/VP pathway is more active during extinction learning, whereas the L2/3 PL→BLA and IL→BLA pathways are more active during extinction retrieval. A, Heatmap showing the anatomical distribution of PL→SI/VP projectors along the mediolateral and dorsoventral axes. B1, A density map of cFos+ cells in the PL→SI/VP pathway across cortical layers in all behavioral groups. B2, Quantification of PL→SI/VP pathway activity (cFos+ cells) in superficial and deep layers in all behavioral groups. The PL→SI/VP pathway does not show any differences in activity between groups. C, Heatmap showing the anatomical distribution of IL→SI/VP projectors along the mediolateral and dorsoventral axes. D1, A density map of cFos+ cells in the IL→SI/VP pathway across cortical layers in all behavioral groups. D2, Quantification of the IL→SI/VP pathway activity in superficial and deep layers in all behavioral groups. There are significantly more active L5 IL→SI/VP projectors in the extinction learning group than in controls. E, Heatmap showing the anatomical distribution of PL→BLA projectors along the mediolateral and dorsoventral axes. F1, A density map of cFos+ cells in the PL→BLA pathway across cortical layers in all behavioral groups. F2, Quantification of the PL→BLA pathway activity in superficial and deep layers in all behavioral groups. The L2/3 PL→BLA pathway is more active during extinction retrieval than control and extinction learning groups. G, Heatmap showing the anatomical distribution of IL→BLA projectors along the mediolateral and dorsoventral axes. H1, A density map of cFos+ cells in the IL→BLA pathway across cortical layers in all behavioral groups. H2, Quantification of the IL→BLA pathway activity in superficial and deep layers in all behavioral groups. The L2/3 IL→BLA pathway is more active during extinction retrieval than control and extinction learning groups. Mean and SEM are shown throughout. The color bar indicates the average number of CTB+ cells. *p < 0.05; **p < 0.01; ***p < 0.001.
- Figure 4.
Deep layer IL→SI/VP projectors are more excitable during extinction learning than retrieval. A, Timeline of injection surgeries and behavioral paradigm prior to in vitro recordings of IL projectors to the SI/VP. Example injection of rgAAV2-hSyn-eYFP in the SI/VP to identify IL→SI/VP projectors. After 2 weeks of expression, animals were randomly assigned to one of the four groups to probe IL→SI/VP projector excitability during early extinction when fear was high [Early Ext. (high fear), orange], late extinction when fear was low [Late Ext. (low fear), red], extinction retrieval [Ext. Retr. (low fear), purple], or tone control when fear was low, and no learning had occurred [Tone Ctrl. (low fear), gray]. Mice were killed for in vitro recordings 10 min after the last behavioral trial. B, Anatomical mapping of all viral injections in the SI/VP. C, The percentage defensive freezing for all behavioral groups. Vertical arrows mark the last two trials of behavior for each group, after which the mPFC was sliced for in vitro recordings. D, Average percentage freezing for each group in the last two trials before perfusion. E1, Left, Example traces showing IL→SI/VP projector excitability in each group at 40 and 50 pA current injection steps. Right, Excitability curves at RMP shown by the number of APs in response to increasing injections of current for each behavioral group. Significant post hoc tests are marked with their respective symbols shown in the key. E2, Replotting of excitability curves in subsets of groups for clarity. All significance testing was done on four groups. Left, Early versus late extinction excitability curves; post hoc tests that reached significance are marked with #, indicating increased excitability in late versus early extinction in IL→SI/VP projectors. Right, IL→SI/VP projectors in late extinction are significantly more excitable than in extinction retrieval in a wide range of testing conditions. F, Rheobase (pA), or the lowest level of current to evoke an AP, across groups. IL→SI/VP projectors show significantly lower rheobase in late extinction than extinction retrieval. G, IL→SI/VP RMP (mV) shows no difference across groups.
- Figure 5.
IL projections to the SI/VP constrain defensive freezing during extinction learning. A, Example of viral injection in the IL and fiber placement in the SI/VP and schematic showing the injections and fiber placement surgeries. B, Mapping of the viral spread of the virus in the IL (left) and fiber placements in the SI/VP (right). Gray, eYFP; green, eArch. C, Schematic of the behavioral paradigm. Mice were first habituated to the tone in fear conditioning Context A and to the laser in extinction Context B. The next day, mice were fear conditioned with five CS–US pairings. The next day, mice underwent fear extinction for 10 trials with laser light delivery during the tone, inhibiting IL inputs to the VP/SI. One day later, mice were re-exposed to the extinction context during a second 10-trial session of extinction, testing a mixture of extinction retrieval and re-extinction. D, Virus validation: recording setup during optogenetic manipulation. After IL injections of the AAV5-hsyn-eArch3.0-eYFP (n = 1) or the AAV5-hsyn-eYFP (n = 1) virus, an optrode, consisting of the optic fiber and stereotrode bundle, was placed in the SI/VP to record single units and MUA during a laser light-habituation session in Context B. Additional recordings showing the effects of IL→SI/VP inhibition on cell activity during extinction learning are shown in Extended Data Figure 5-1. E, A raster plot showing an example of single units and MUA firing for 10 s before light onset, during a 35 s light-on trial (green bar, ramp-on), and for 10 s after light offset (ramp-off). F, A peristimulus time histogram showing the average firing rate of all units shown individually in panel E. G, An average firing rate (spikes/sec) of three single units during five habituation trials during light-off (white) and light-on (green) periods. The average waveform of each unit is shown. Unit 1 has a firing rate of 6.1 spikes/sec, whereas Units 2 and 3 have higher firing rates (30 and 23 spikes/sec, respectively). None of the units change their firing rates during light-on periods. H, Average percentage change in firing (single units and MUA) during the light shows no change. Blue line, mean. I, Average percentage defensive freezing on each trial during habituation to tone presentations in Context A and optogenetic light stimulation in Context B in both groups. J, Average percentage defensive freezing on each trial in eYFP and eArch groups across fear conditioning, extinction learning, and Extinction 2. K, Average percentage defensive freezing during the fear acquisition session is similar in both groups. L, During extinction learning, left, average percentage defensive freezing is higher in the eArch than the eYFP group (unpaired t test; p = 0.019). Right, During extinction learning, average defensive freezing in Trials 1–2 and Trials 9–10 is higher in the eArch than eYFP group. M, Average percentage freezing during Extinction 2 is similar in both groups. All data, unless otherwise specified, are shown as mean ± SEM; *p < 0.05; #p < 0.07. In Extended Data Figure 5-2, we show that inhibition of the PL→SI/VP pathway during extinction learning does not affect behavior. Abbreviations: IL, infralimbic; MUA, multiunit activity; SI, substantia innominata; VP, ventral pallidum.
- Figure 6.
IL inputs to the SI/VP constrain freezing during active fear decrement during Extinction 2. A, Schematic showing the IL viral injections and SI/VP fiber placements and the mapping of the viral spread of the virus in the IL (left) and fiber placements in the SI/VP (right). Gray, eYFP; green, eArch. B, Schematic of behavioral paradigm. Mice were first habituated to the fear conditioning Context A and the tone, as well as to the extinction Context B and the laser. The next day, mice were fear conditioned with five CS–US pairings in Context A. The next day, mice underwent fear extinction learning for 10 trials in Context B. One day later, during Extinction 2, mice were re-exposed to the extinction Context B during a second 10-trial session of extinction with laser light delivery during the tones, inhibiting IL inputs to the SI/VP. C, Average percentage defensive freezing on each trial during habituation to tone presentations in Context A and optogenetic light stimulation in Context B in both groups. D, Average percentage defensive freezing on each trial in eYFP and eArch groups across fear acquisition, extinction learning, and Extinction 2. There were no significant differences in behavior when taking into account all trials in all conditions. E, Average percentage defensive freezing during the fear acquisition session is similar in both groups. F, Average percentage defensive freezing is also similar for both groups during extinction learning. G, Left, During Extinction 2, average percentage defensive freezing is similar across groups. Right, Average defensive freezing in Trials 1–2 is higher in the eArch than eYFP group, whereas both groups are at similarly low levels of freezing by Trials 9–10. All data are shown as mean ± SEM, *p < 0.05. Abbreviations: IL, infralimbic; SI, substantia innominata; VP, ventral pallidum.
- Figure 7.
A summary model. Schematic of the neuroanatomical distribution of IL projectors to the BLA (green circles) and to the SI/VP (pink circles). L5 IL→SI/VP projectors are more active (blue rim) during extinction learning, when fear is high and decreasing, whereas L2/3 IL→BLA projectors are more active (blue rim) during extinction retrieval when fear is suppressed.
Extended Data
Figure 5-1
Light alone doesn’t affect cell firing but light with tone during extinction cue affects cell firing. A) Schematic of surgery with injection and behavioral paradigm. Optogenetic light validation cell recordings was done during Extinction Learning. B) Recording setup during optogenetic manipulation. After IL injection of the AAV5-hsyn-eArch3.0-eYFP (n = 1) virus, an optrode, consisting of the optic fiber and stereotrode bundle, was placed in the SI/VP to record single units. C) Average firing rate (spikes/sec) of three single units during 10 extinction trials during 5 sec Pre-Light, 5 sec of Light alone and 30 sec of Light + Tone. The average waveform of each unit is shown. D) Percent change in firing during three periods (Pre-light, Light only, Light + tone) of each extinction trial from the average firing during all pre-light periods. A one-way RM-ANOVA showed a significant effect of period (F(2,58) = 7.084, p = 0.001). Pairwise RM t-tests with a Bonferroni correction showed no differences between Pre-light and Light firing (p = 0.82) but did show a significant decrease in firing rate during the Light + Tone period when compared to Pre-light (p = 0.018) and to Light alone (p = 0.019). Blue line, mean. All data is shown as means ± SEM. Abbreviations: IL, infralimbic; SI, substantia innominata; VP, ventral pallidum. Download Figure 5-1, TIF file.
Figure 5-2
PL projections to the SI/VP don’t affect defensive freezing during extinction learning. A) Example of viral injection in the PL, and fiber placement in the SI/VP, and schematic showing the injections and fiber placement surgeries. B) Mapping of the viral spread of the virus in the PL (Left), and fiber placements in the SI/VP (Right). Grey, eYFP; green, eArch. C) Schematic of behavioral paradigm. Mice were first habituated to the fear conditioning context and the tone, as well as to the extinction context and the laser. The next day, mice were fear conditioned with five CS-US pairings. The next day, mice underwent fear extinction for ten trials with laser light delivery during the tone, inhibiting IL inputs to the SI/VP. One day later, mice were re-exposed to the extinction context during a second 10-trial session of extinction, testing a mixture of extinction retrieval and re-extinction. D) Average percent defensive freezing on each trial during habituation to tone presentations in Context A and optogenetic light stimulation in Context B in both groups. E) Average percent defensive freezing on each trial in eYFP and eArch groups across fear acquisition, fear extinction, and extinction 2 sessions. F) Average percent defensive freezing during the Fear Acquisition is similar in both groups. G) Average percent defensive freezing is also similar for both groups during Extinction Learning. H) Average percent freezing during Extinction 2, is also similar across groups. All data is shown as means ± SEM. Abbreviations: PL, prelimbic; SI, substantia innominata; VP, ventral pallidum. Download Figure 5-2, TIF file.
