An intra-amygdala circuit specifically regulates social fear learning

Abstract

Adaptive social behavior requires transmission and reception of salient social information. Impairment of this reciprocity is a cardinal symptom of autism. The amygdala is a critical mediator of social behavior and is implicated in social symptoms of autism. Here we found that a specific amygdala circuit, from the lateral nucleus to the medial nucleus (LA–MeA), is required for using social cues to learn about environmental cues that signal imminent threats. Disruption of the LA–MeA circuit impaired valuation of these environmental cues and subsequent ability to use a cue to guide behavior. Rats with impaired social guidance of behavior due to knockout of Nrxn1, an analog of autism-associated gene NRXN, exhibited marked LA–MeA deficits. Chemogenetic activation of this circuit reversed these impaired social behaviors. These findings identify an amygdala circuit required to guide emotional responses to socially significant cues and identify an exploratory target for disorders associated with social impairments.

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Figure 1: Fear learning through social transmission.
Figure 2: Electrophysiological verification of DREADD-Gi effect on MeA and LA neuron firing in vivo.
Figure 3: Disruption of intra-amygdala path impairs social fear learning.
Figure 4: LA–MeA strength is associated with social fear learning.
Figure 5: Disruption of intra-amygdala path in Nrxn rats.
Figure 6: Intrinsic and synaptic properties of MeA neurons.
Figure 7: Social learning is impaired in neurexin rats.
Figure 8: DREADD-Gs activation of MeA partially rescues social learning deficits in Nrxn rats.

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Acknowledgements

The authors thank B. Roth (UNC School of Medicine) for making DREADD constructs available and for advice on immunohistological confirmation of expression. The authors would also like to thank B. Avonts for technical assistance mapping viral injection. Grant support was provided by Simons Foundation (SFARI Award 283746 to J.A.R.) and National Institutes of Health (R01MH084970 to J.A.R.).

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Authors

Contributions

J.E.V. designed experiments to measure mRNA and protein; R.C.T. and J.A.R. designed the remaining experiments. R.C.T., J.E.V., S.L., M.P. and J.A.R. performed all experiments. R.C.T., S.L., J.E.V. and J.A.R. performed statistical analyses. J.A.R. wrote the manuscript with input from all authors. R.C.T. and J.E.V. provided critical revisions.

Corresponding author

Correspondence to J Amiel Rosenkranz.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 DREADD-Gi expression location.

The expression of DREADD was mapped for all conditions. Expression sites in bilateral MeA (a) and LA (b) conditions. c) Expression sites in ipsilateral LA-MeA (left) and crossed LA-MeA conditions. LA-MeA sites were right-left hemisphere counterbalanced. For simplicity, these sites are shown with MeA on the right. Numbers denote the Bregma coordinates from the atlas of Paxinos and Watson.Paxinos, C.W.G. By George Paxinos - The Rat Brain in Stereotaxic Coordinates: 6th (sixth) Edition (Elsevier Science, 2009).

Supplementary Figure 2 MeA and LA recording sites.

The recording sites were histologically reconstructed and verified to lie within the MeA (a) or LA (b). Numbers denote the Bregma coordinates from the atlas of Paxinos and Watson.Paxinos, C.W.G. By George Paxinos - The Rat Brain in Stereotaxic Coordinates: 6th (sixth) Edition (Elsevier Science, 2009).

Supplementary Figure 3 Cre-DREADD-Gi expression location.

The expression of mCherry was mapped in conditions with CAV2-Cre infusion into the MeA and co-infusion of Cre-dependent DREADD-Gi-mCherry into the LA. Numbers denote the Bregma coordinates from the atlas of Paxinos and Watson.Paxinos, C.W.G. By George Paxinos - The Rat Brain in Stereotaxic Coordinates: 6th (sixth) Edition (Elsevier Science, 2009).

Supplementary Figure 4 Classical fear conditioning is not impaired by LA–MeA functional disconnection.

a) Bilateral inactivation of LA (n=6 rats) by CNO administered 40 minutes prior to conditioning impaired classical non-social fear conditioning compared to control (n=7 rats). This is demonstrated by decreased freezing during fear conditioning (left; main effect of inactivation p<0.0001, F(1,11)=52.01, η2=0.172; inactivation x trial interaction p=0.032, F(5,55)=2.66, η2=0.049, two-way RM-ANOVA) and decreased freezing to the CS+ tone in a novel context during testing after 48 hours (right; main effect of inactivation p<0.0001, F(1,11)=60.80, η2=0.267; inactivation x trial interaction p<0.0001, F(12,132)=11.43, η2=0.171, two-way RM-ANOVA). b) Bilateral inactivation of MeA (n=6 rats) or functional contralateral disconnection of LA and MeA (crossed, n=6 rats) by CNO administered 40 minutes prior to conditioning had no significant effect on freezing during fear conditioning compared to control (n=7 rats; left; main effect of inactivation p=0.096, F(2,16)=2.71, η2=0.028; inactivation x trial interaction p=0.337, F(10,80)=1.149, η2=0.020, two-way RM-ANOVA) and no significant effect on freezing to the CS+ tone in a novel context during testing after 48 hours (right; main effect of inactivation p=0.386, F(2,16)=1.011, η2=0.0068; inactivation x trial interaction p=0.252, F(24,192)=1.193, η2=0.024, two-way RM-ANOVA). Data shown here are mean ± 95% confidence intervals.

Supplementary Figure 5 Role of LA–MeA in unconditioned social and anxiety behavior.

Social behavior in the open field was measured. a) Bilateral inactivation of MeA (n=7 rats) or LA (n=7 rats) did not significantly decrease the amount of time engaged in social interaction with a novel rat in the open field (left; main effect of nucleus p=0.746, F(2,19)=0.297, η2=0.027; main effect of CNO p=0.786, F(1,19)=0.076, η2=0.0003; nucleus x CNO interaction p=0.063, F(2,19)=3.21, η2=0.025, two-way RM-ANOVA), nor the number of social interactions (right; main effect of nucleus p=0.902, F(2,19)=0.104, η2=0.01; main effect of CNO p=0.074, F(1,19)=3.58, η2=0.01; nucleus x CNO interaction p=0.709, F(2,19)=0.350, η2=0.002, two-way RM-ANOVA) compared to control (n=7 rats). b) Bilateral inactivation of the MeA, but not LA, decreased the duration of individual social interaction events (main effect of nucleus p=0.036, F(2,19)=3.97, η2=0.20; main effect of CNO p=0.0441, F(1,19)=4.647, η2=0.044; nucleus x CNO interaction p=0.0088, F(2,19)=6.142, η2=0.117, two-way RM-ANOVA; Holm-Sidak's multiple comparisons test p<0.05 control vs MeA inactivation, p>0.05 control vs LA inactivation), also displayed as the duration of interaction events after baseline correction (CNO duration – vehicle duration; p=0.0088, F(2,19)=6.142, η2=0.39, one-way ANOVA). c) Neither ipsilateral (n=7 rats) nor crossed (n=6 rats) inactivation of LA and MeA significantly reduced the amount of time engaged in social interaction (left; main effect of nucleus p=0.828, F(2,18)=0.828, η2=0.019; main effect of CNO p=0.154, F(1,18)=2.21, η2=0.008; nucleus x CNO interaction p=0.465, F(2,18)=0.799, η2=0.0056, two-way RM-ANOVA) compared to control rats (n=7 rats), nor the total number of interaction events (right; main effect of nucleus p=0.693, F(2,18)=0.375; main effect of CNO p=0.748, F(1,18)=0.106, η2=0.00045; nucleus x CNO interaction p=0.638, F(2,18)=0.460, η2=0.0004, two-way RM-ANOVA) compared to control (n=8, as above). d) Ipsilateral or crossed inactivation of LA and MeA did not significantly decrease the duration of interaction events compared to control rats (left; main effect of nucleus p=0.830, F(2,18)=0.188, η2=0.012; main effect of CNO p=0.654, F(1,18)=0.208, η2=0.0049; nucleus x CNO interaction p=0.964, F(2,18)=0.037, η2=0.0017, two-way RM-ANOVA), nor the duration of interaction events after baseline correction (right; p=0.964, F(2,18)=0.037, η2=0.0041, one-way ANOVA). e) Exploration in the open field, measured as time in the center area, was not significantly modified by bilateral inactivation of LA (n=6 rats), MeA (n=6 rats), or LA-MeA disconnection (n=6 rats), compared to control (n=7 rats) (p=0.921, F(3,21)=0.162, η2=0.023, one-way ANOVA). f) Behavior in the elevated plus maze was not significantly modified by bilateral inactivation of LA (n=6 rats), MeA (n=6 rats), or LA-MeA disconnection (n=6 rats), compared to control (n=7 rats) when exploration of the open arm (left; p=0.922, F(3,21)=0.159, η2=0.022, one-way ANOVA) or total number of arm entries was compared (right; p=0.879, F(3,21)=0.224, η2=0.031, one-way ANOVA). Data shown here are mean ± 95% confidence intervals.

Supplementary Figure 6 Neurexin-1α knockout (Nrxn) rats have reduced neurexin-1α expression.

Whole brain lysates from WT and Nrxn rats were used to verify neurexin-1α expression. a) RT-PCR was performed to quantify relative neurexin-1α mRNA normalized to GAPDH (WT n=5 rats, Nrxn n=4 rats; One sample from each rat was processed). PCR products were separated by agarose gel. Shown here are examples from different WT and Nrxn rats (cropped image). Depicted on the right is the uncropped film from the displayed example. b) Western blot analysis was used to quantify relative neurexin-1α protein expression normalized to α-tubulin (WT n=5 rats, Nrxn n=4 rats; One sample from each rat was processed). Shown here are examples from WT and Nrxn rats (cropped image). Depicted to the right are the uncropped films from the same blot probed for the neurexin antibody (top) and reprobed for the α-tubulin antibody (bottom). c) Both neurexin-1α mRNA (p=0.0001, t=7.828, df=7, Cohen's d=5.25, 95% C.I.=-1.21 to -0.65, two-tailed unpaired t-test) and protein levels were reduced (p=0.0016, t=4.982, df=7, Cohen's d=3.34, 95% C.I.=-1.23 to -0.44, two-tailed unpaired t-test) in KO rats compared to control. Data shown here are mean ± 95% confidence intervals. *p<0.05, two-tailed unpaired t-test.

Supplementary Figure 7 Excitatory response of MeA neurons to LA input.

Spontaneous firing was measured from neurons in the MeA (posterior) from WT (top; n=36 neurons) and Nrxn rats (bottom; n=59 neurons). Evoked firing was measured from neurons in the MeA (posterior) from WT (n=16 rats) and Nrxn rats (n=11 rats) upon stimulation of LA. All recording and stimulation sites were histologically verified. a) Map of the recording and stimulation sites. Coordinate in lower right of panels indicates the distance from Bregma as mapped onto the atlas of Paxinos and Watson. b) Stimulation of LA induced a short latency excitatory response in MeA neurons (LA→MeA; <8 ms, <3 ms variability, consistent with monosynaptic input) in most neurons that displayed a response (>30% change in firing, n=16/28 neurons, 57% of neurons). The remaining neurons displayed no response (n=8) or >30% inhibition (n=4 neurons). In contrast, few LA neurons displayed an excitatory response upon stimulation of MeA (MeA→LA; n=11/43 neurons, 26% of neurons). In neurons that were responsive, the LA→MeA response was significantly greater than the MeA→LA response (LA→MeA n=16 neurons, MeA→LA n=11 neurons; path x time interaction, p<0.0001, F(78,1482)=1.770, η2=0.045, two-way RM-ANOVA). Displayed here is the mean ± 95% C.I. of these response at 0.6 mA stimulation across time. c) There was a significant difference in the response probability across stimulation intensity between the LA→MeA and MeA→LA paths (main effect of path direction, p<0.0001, F(1,125)=81.87, η2=0.160; direction x intensity interaction, p<0.0001, F(4,125)=15.69, η2=0.123; two-way ANOVA). Data shown here are mean ± 95% confidence intervals except where noted. #p<0.05 two-way ANOVA.Paxinos, C.W.G. By George Paxinos - The Rat Brain in Stereotaxic Coordinates: 6th (sixth) Edition (Elsevier Science, 2009).

Supplementary Figure 8 LA neuron properties are not significantly different in Nrxn rats.

Properties of LA principal neurons (Lang and Pare, 1997; Faber et al, 2001; Hetzel and Rosenkranz, 2014) were measured in vitro. a) Input resistance (Rn) was equivalent in LA neurons from WT (n=14 neurons from 12 slices) and Nrxn rats (n=19 neurons from 12 slices) (p=0.177, t=1.383, df=31, Cohen's d=0.49, 95% C.I.=-37.34 to 7.25, two-tailed unpaired t-test). b) Resting membrane potential of these same LA neurons was similar between WT and Nrxn rats (p=0.605, t=0.522, df=31, Cohen's d=0.18, 95% C.I.=-3.45 to 2.04, two-tailed unpaired t-test). c) Excitability was measured as the number of action potentials evoked across a range of depolarizing current steps from a membrane potential of -70 mV. There was no significant difference in excitability in LA neurons from WT and Nrxn rats (n=14 neurons from 14 slices/group; main effect of genotype, p=0.615, F(1,26)=0.260, η2=0.0027; genotype x current amplitude interaction, p=0.830, F(5,130)=0.426, η2=0.0026, two-way RM-ANOVA). d) Miniature EPSCs (mEPSCs) were measured from LA neurons (left). There was no significant difference between WT (n=21 neurons from 14 slices) and Nrxn rats (n=25 neurons from 17 slices) in mEPSC frequency (p=0.314, t=1.019, df=44, Cohen's d=0.30, 95% C.I.=-1.56 to 0.51, two-tailed unpaired t-test) or mEPSC amplitude (p=0.483, t=0.707, df=44, Cohen's d=0.21, 95% C.I.=-2.42 to 1.16, two-tailed unpaired t-test). e) There was evidence for a subtle difference between WT (n=17 neurons from 12 slices) and Nrxn rats (n=20 neurons from 14 slices) in paired-pulse ratio (p=0.0005, t=3.818, df=35, Cohen's d=1.26, 95% C.I.=-0.32 to -0.097, two-tailed unpaired t-test) and CV of locally-evoked EPSCs (p=0.027, t=2.336, df=28, Cohen's d=0.86, 95% C.I.=0.0086 to 0.13, two-tailed unpaired t-test; WT n=14 neurons from 12 slices, Nrxn n=16 neurons from 12 slices). The nature of the differences in mEPSC and evoked EPSCs suggests that Nrxn rats may have a specific deficit in regulation of evoked glutamate release. Data shown here are mean ± 95% confidence intervals.Faber, E.S., Callister, R.J. & Sah, P. Morphological and electrophysiological properties of principal neurons in the rat lateral amygdala in vitro. J Neurophysiol 85, 714-723 (2001)Hetzel, A. & Rosenkranz, J.A. Distinct effects of repeated restraint stress on basolateral amygdala neuronal membrane properties in resilient adolescent and adult rats. Neuropsychopharmacology 39, 2114-2130 (2014)Lang, E.J. & Pare, D. Similar inhibitory processes dominate the responses of cat lateral amygdaloid projection neurons to their various afferents. J Neurophysiol 77, 341-352 (1997)

Supplementary Figure 9 Nonspecific factors do not underlie the social learning impairment in Nrxn rats.

a) Demonstrators for WT and Nrxn rats displayed similar freezing during conditioning (n=14 rats/group; no main effect of observer type, p=0.16, F(1,26)=2.1, η2=0.0296; no observer type x trial interaction p=0.93, F(6,156)=0.3, η2=0.004, two-way RM-ANOVA). Demonstrators for WT and Nrxn rats displayed similar approach behavior (right), quantified as time nose exploring through the divider (p=0.712, t=0.374, df=26, Cohen's d=0.14, 95% C.I.=-10.2 to 7.1, two-tailed unpaired t-test). b) Auditory capability was tested by measuring freezing in response to a loud tone (95 dB, 1.5 kHz, 1 s). All WT and Nrxn rats (10 out of 10 rats/group) displayed freezing in the 5 s immediately following the tone. There was no significant difference in the amount of freezing displayed between WT and Nrxn rats (p=0.51, t=0.679, df=26, Cohen's d=0.30, 95% C.I.=-0.84 to 1.64, two-tailed unpaired t-test). c) Olfaction was tested by measurement of the latency to uncover grape-flavored sucrose pellets that were buried under bedding. There was no significant difference between WT and Nrxn rats in the latency to find these sucrose pellets (n=10 rats/group, p=0.74, t=0.338, df=18, Cohen's d=0.15, 95% C.I.=-13.7 to 9.9, two-tailed unpaired t-test). d) To test olfaction for social cues, exploration of bedding from soiled female cages was measured and compared to bedding from soiled male cages. There was a significant preference for the bedding from female cages in both WT and Nrxn rats (n=8 rats/group, main effect of bedding type p=0.0008, F(1,14)=18.290, η2=0.469, two-way RM-ANOVA; p<0.05 Holm-Sidak's multiple comparisons test for bedding type in WT and in Nrxn rats), indicating that Nrxn rats have the ability to use olfactory cues to differentiate the bedding. However, Nrxn rats displayed a weaker preference for female bedding (genotype x bedding type interaction, p=0.0417, F(1,14)=5.026, η2=0.123, two-way RM-ANOVA; p=0.042, t=2.237, df=18, Cohen’s d=1.12, 95%C.I.=-1.09 to -0.02, two-tailed unpaired t-test of preference score). e) Classical fear conditioning was measured to test whether Nrxn rats have the capability to acquire associative memories. A sub-maximal conditioning procedure was utilized to avoid ceiling effects. Nrxn rats (n=6 rats) displayed significantly greater freezing during classical fear conditioning (main effect of genotype, p=0.044, F(1,11)=5.15, η2=0.058; genotype x trial interaction, p=0.049, F(4,44)=2.599, η2=0.048, two-way RM-ANOVA) compared to WT rats (n=7). f) There was no significant difference between these same WT and Nrxn rats in conditioned freezing to the CS+ when tested in a novel context after 48 hours (main effect of genotype, p=0.104, F(1,11)=3.149, η2=0.063; genotype x trial interaction, p=0.987, F(12,132)=0.309, η2=0.0093, two-way RM-ANOVA). g) There was no difference in the amplitude of foot shock required to induce forepaw withdrawal between WT and Nrxn rats (n=18/group; main effect of genotype, p=0.715, F(1,34)=0.136, η2=0.0040; genotype by trial interaction, p>0.999, F(3,102)=0, η2=0, two-way RM-ANOVA; displayed are number of rats), measured over 4 trials, although Nrxn rats tended to display increased behavioral response to the foot shock (e.g. time freezing after foot shock). h) Exploratory behavior was measured as time in the center of the open field under dim light conditions (20-25 lux). No significant difference between WT (n=16 rats) and Nrxn rats (n=17 rats) emerged (main effect of genotype, p=0.46, F(1,31)=0.563, η2=0.014, two-way RM-ANOVA). To test whether Nrxn and WT rats were similar because open field conditions were not anxiogenic enough, light levels were increased to promote the anxiogenic nature of the task (200-220 lux). This decreased exploration in the center of the open field (main effect of light, p<0.0001, F(1,31)=81.92, η2=0.172), but there was still no difference between WT and Nrxn rats in the time spent in the center of the open field (genotype x light interaction, p=0.786, F(1,31)=0.075, η2=0.0002). Nrxn rats displayed hyperactivity in the open field (right), measured as total distance traveled over the 5 min test (WT n=16 rats. Nrxn n=17 rats; p<0.0001, t=5.582, df=31, Cohen's d=1.94, 95% C.I.=7.27 to 15.63, two-tailed unpaired t-test). i) Nrxn rats (n=17 rats) did not display a significant difference in exploration of the open arms of the elevated plus maze (left; proportion of time in open arms, p=0.685, t=0.410, df=28, Cohen's d=0.15, 95% C.I.=-17.4 to 11.6, two-tailed unpaired t-test) compared to WT rats (n=13). There was also no significant difference in the total number of arm entries (right; p=0.336, t=0.978, df=28, Cohen's d=0.36, 95% C.I.=-1.75 to 4.94, two-tailed unpaired t-test). Data shown here are mean ± 95% confidence intervals. *p<0.05 two-tailed unpaired t-test or post-hoc Holm-Sidak comparison after ANOVA, #p<0.05, main effect of group in two-way RM-ANOVA.

Supplementary Figure 10 Abnormal mounting behavior in Nrxn rats.

a) A proportion of Nrxn rats (5/16 rats) displayed a mounting behavior directed towards the male conspecific during social interaction tests in the open field. None of the WT rats displayed this behavior (0/16 rats; p=0.0149, Chi square=5.926, z=2.434, two-tailed Chi square comparison between WT and Nrxn rats; shown here is the number of rats). b) The mounting behavior of cohabitating WT pairs and Nrxn pairs was also assessed in their home cages (10 pairs/group, 15 min observation, each pair observed 4 times). In WT rats, mounting was rarely observed in the home cage (1/10 pairs). Mounting was observed in Nrxn rats more frequently (left; 8/10 pairs displaying mounting at least during one observation; p=0.0017, Chi square=9.899, z=3.146, two-tailed Chi square comparison between WT and Nrxn rats; shown here is the number of pairs). There was an average of 2.6 ± 0.6 mounting episodes/pair of Nrxn pairs, but 0.1 ± 0.1 mounting episodes/pair of WT pairs (right; p=0.001, t=3.883, df=19, Cohen's d=1.74, 95% C.I.=1.147 to 3.853, two-tailed unpaired t-test between WT and Nrxn rats). Play-like behavior was observed in all of the WT pairs (10/10 pairs) and most of the Nrxn pairs (9/10 pairs). The number of episodes of play behavior/observation did not significantly differ between groups (WT 7.3 ± 1.4 episodes/pair, Nrxn 4.6 ± 1.3 episodes/pair; p=0.1696, t=1.431, df=19, Cohen's d=0.64, 95% C.I.=-6.67 to1.27, two-tailed unpaired t-test between WT and Nrxn rats; Data shown here are mean ± 95% confidence intervals). *p<0.05, two-tailed unpaired t-test.

Supplementary Figure 11 Map of recording sites of MeA neurons from DREADD-Gs-injected rats.

MeA (posterior) neurons were recorded after vehicle (2 tracks/rat) or CNO (2 tracks/rat) was administered in WT (a) or Nrxn rats (b). Coordinate in lower right of panels indicates the distance from Bregma as mapped onto the atlas of Paxinos and Watson c) CNO administration (1 mg/kg i.p.) increased the firing rate of MeA posterior neurons from WT and Nrxn rats (main effect of CNO, p=0.0021, F(1,80)=10.14, η2=0.112, two-way ANOVA; CNO x genotype interaction, p=0.600, F(1,80)=0.277, η2=0.0039), compared to vehicle (p<0.05, Holm-Sidak's multiple comparisons test between CNO (WT n=30 neurons/4 rats; Nrxn n=24 neurons/4 rats) and vehicle (WT n=19 neurons/4 rats; Nrxn n=11 neurons/4 rats)) and increased the number of active MeA posterior neurons from WT and Nrxn rats, measured as number of neurons/track (n=8 tracks/group, 2 tracks/rat; main effect of CNO, p<0.0001, F(1,28)=22.21, η2=0.381, two-way ANOVA; CNO x genotype interaction, p=0.852, F(1,28)=0.036, η2=0.006) compared to vehicle (p<0.05, Holm-Sidak's multiple comparisons test between CNO and vehicle). Data shown here are mean ± 95% confidence intervals. d) Expression of DREADD-Gs was targeted to the MeA (posterior). Ovals indicate location of the most-dense labeling of HA-tag-positive neurons. *p<0.05 post-hoc Holm-Sidak comparison after ANOVA.Paxinos, C.W.G. By George Paxinos - The Rat Brain in Stereotaxic Coordinates: 6th (sixth) Edition (Elsevier Science, 2009).

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Twining, R., Vantrease, J., Love, S. et al. An intra-amygdala circuit specifically regulates social fear learning. Nat Neurosci 20, 459–469 (2017). https://doi.org/10.1038/nn.4481

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