Insular cortex mediates approach and avoidance responses to social affective stimuli

Abstract

Social animals detect the affective states of conspecifics and utilize this information to orchestrate social interactions. In a social affective preference text in which experimental adult male rats could interact with either naive or stressed conspecifics, the experimental rats either approached or avoided the stressed conspecific, depending upon the age of the conspecific. Specifically, experimental rats approached stressed juveniles but avoided stressed adults. Inhibition of insular cortex, which is implicated in social cognition, and blockade of insular oxytocin receptors disrupted the social affective behaviors. Oxytocin application increased intrinsic excitability and synaptic efficacy in acute insular cortex slices, and insular oxytocin administration recapitulated the behaviors observed toward stressed conspecifics. Network analysis of c-Fos immunoreactivity in 29 regions identified functional connectivity between insular cortex, prefrontal cortex, amygdala and the social decision-making network. These results implicate insular cortex as a key component in the circuit underlying age-dependent social responses to stressed conspecifics.

Figure 1. Social affective preference (SAP)

(A) The SAP arena containing juvenile conspecifics on the left and right with an experimental adult in the center. (B) Diagram of SAP test procedure. (C) Mean (individual replicates shown as connecting lines) time spent in social interaction with the naive or stressed conspecific by age (n = 8, PN 30; n = 12, PN 50). A bidirectional effect of age was apparent in time spent interacting with the stressed conspecifics (F_AGE(1, 18) = 27.93, p < 0.0001; F_AFFECT(1, 18) = 9.965, p = 0.006; F_AGE*AFFECT(1, 18) = 46.05, p < 0.0001). Experimental rats spent more time exploring the stressed PN 30 conspecific compared to the PN 30 naive, but spent less time exploring the stressed PN 50 conspecific compared to the PN 50 naive. (D) Mean (+ SEM with individual replicates) data in C expressed as the percent of time spent interacting with the stressed conspecifics relative to the total time spent interacting. Experimental adults showed a marked preference (values greater than 50%) for interaction with stressed conspecifics and avoidance (values less than 50%) of stressed adults (t(18) = 5.783, p < 0.0001, 2-tailed). (E) Mean (+ SEM with individual replicates, n = 51 for PN 30, n = 46 for PN 50) percent preference for interacting with the stressed conspecific pooled from all of the subjects in the experimental control groups included in the current report including vehicle, sham, and light OFF control groups of the later experiments. Percent preference scores for PN 30 and PN 50 interactions were significantly different t(95) = 7.66, p < 0.0001, 2-tailed) from each other, and in both conditions the mean percent preference differed from 50% (PN 30: t_one-sample(50) = 6.49, p < 0.0001, 2-tailed; PN 50: t_one-sample (45) = 4.39, p < 0.0001, 2-tailed). (F) Mean (+ S.E.M. with individual replicates) time spent in non-social behaviors during habituation tests and SAP tests (n = 10, PN 30; n = 7 PN 50). More investigation of the arena and time spent self-grooming was observed in the PN 50 rats (F_{AGE*BEHAVIOR*TEST}(4, 60) = 3.014, p = 0.025). (G) Diagram of 1-on-1 social interaction test and photo of typical adult-initiated interactions. (H) Mean (individual replicates, ns = 8/ age) time interacting with the naive or stressed conspecific in a 1-on-1 test shown as percent of test time. Experimental adults spent significantly more time interacting with the stressed PN 30 conspecific but significantly less time with the stressed PN 50 conspecific compared to the respective naive conspecific targets (F_AGE(1, 14) 103.10, p < 0.0001; F_AGE*AFFECT(1, 14) = 31.34, p < 0.0001). (I) Representative audio spectrograms depicting rising (top, white arrow), trills (top, black arrow) and 22kHz ultrasonic vocalizations (USVs). Scale bars indicate Y-axis ranges: 60–70kHz (top) and 30–35kHz (bottom). (J–K) Mean (+SEM with individual replicates, ns = 11/group) number of rising and trill USVs recorded during 5 min one-on-one social interactions. Fewer rising and trill calls were observed during interactions with stressed conspecifics with fewer rising calls observed in stressed adults compared to stressed juveniles but more 22kHz calls observed in stressed adults than stressed juveniles (F_STRESS(1, 40) = 26.16, p < 0.0001; F_{CALL TYPE}(2, 80) = 60.86, p < 0.0001; F_{STRESS*CALL TYPE}(2, 80) = 20.18, p < 0.0001; F_{CALL TYPE*AGE}(2, 80) = 3.43, p = 0.37). *p < 0.05, ** p < 0.01, *** p < 0.001 (Sidak).

Figure 2. Optogenetic silencing of insular cortex during SAP tests

(A) Representative digital photomicrograph containing insular cortex regions and Fos immunoreactive nuclei (black ovoid particles). Scale bar = 500µm. (B) Mean (+SEM, numbers indicate number of replicates) Fos immunoreactive nuclei by insular cortex subregion (AI = Agranular, DI = Dysgranular, GI = Granular) quantified 90 min after social interaction with a naive PN 30, naive PN 50, stressed PN 30 or stressed PN 50 conspecific (5 min test). Fos was found in all regions, but there was an effect of stress for interactions with PN 50 rats, such that less Fos was evident after interaction with PN 50 stressed conspecific interactions than after interaction with PN 50 naive conspecifics, and there was less Fos in the AI and DI after interactions with PN 50 Stressed rats than after interactions with PN 30 stressed rats (F_STRESS*AGE(1, 36) = 6.21, p = 0.017; F_SUBREGION(2, 72) = 7.90, p = 0.001). (C) Mean Fos immunoreactivity (pooled across insula) predicted time spent in social interaction. Regression analysis indicated strong prediction of social interaction by Fos level, Age, and modest interactions between Stress, Fos and Age (F_FOS(1, 34) = 19.72, p < 0.0001, F_AGE(1, 34) = 36.93, p < 0.0001; F_FOS*STRESS(1, 34) = 3.97, p = 0.055; F_STRESS*AGE(1, 34) = 4.09, p = 0.051). (D) Native mCherry expression in the insular cortex (false colored yellow) from a brain slice adjacent to one containing the cannula tract. (Scale bar = 500µm). (E) Diagram of SAP tests for optogenetic experiments. (F) Mean time spent interacting with PN 30 juvenile conspecifics (n = 9) or (G) PN 50 adult conspecifics (n = 12) on Days 3 and 4 of the SAP test. In the light OFF condition, the experimental adult spent significantly more time interacting with the stressed PN 30 conspecific, but this pattern was abolished in the light ON condition (F_{AGE*STRESS*LIGHT}(1, 19) = 41.31, p < 0.0001). In the light OFF condition, the experimental adult spent significantly less time interacting with the stressed PN 50 conspecific, but this pattern was reversed in the light ON condition. (H) Data from F and G converted to percent preference for interaction with stressed conspecifics. Here, a clear age by light interaction is apparent, with optogenetic silencing of insular cortex eliminating preference for interaction with the stressed juvenile and blocking the pattern of avoidance of stressed adult conspecifics (F_AGE*LIGHT(1, 19) = 23.53, p = 0.0001). No effect of optical stimulation was observed in sham transduced rats. * p < 0.05, **p < 0.01, *** p < 0.001 (Sidak). Brain Atlas illustrations were reproduced with permission as previously published in The Brain Atlas in Stereotaxic Coordinates, 4th Edition, Paxinos, G. & Watson, C. Pages 296, 303, 306–317 & 332. Copyright Elsevier (1998).

Figure 3. Intrinsic membrane properties in insular cortex pyramidal neurons are modulated by oxytocin and depend upon protein kinase C (PKC)

(A) Digital photomicrograph of a typical biocytin filled neuron, rf = rhinal fissure (Scale bar = 500µm). (B) Schematic diagram illustrating the region of interest for whole cell recordings (red shading). (C) Typical action potential before (black trace) and after bath application of 500nM OT (red). Inset: detail of the AP peak amplitude difference. Intrinsic properties were characterized from a sample of 27 neurons; the dependence of OT effects on PKC was determined by first characterizing the change in intrinsic properties from baseline followed by no treatment (aCSF) or OT (500nM) in either the presence of DMSO or DMSO and the pan-PKC inhibitor Gö 6983 (200nM, aCSF-DMSO n = 12; OT-DMSO; n = 12, aCSF-Gö; n = 11; OT-Gö, n = 12). (D) Mean action potential amplitude; OT significantly reduced amplitude. (E) Mean resting membrane potential. OT significantly depolarized the membrane at rest. (F) Mean action potential amplitude (+S.E.M. with individual replicates); OT significantly reduced spike amplitude but did not change in the presence of Gö 6983 (F_OT(1, 21) = 4.56, p = 0.044, aCSF vs. OT, p = 0.040). (G) Typical train of spikes evoked by 1 s 150pA current injection before (black) and after (red) OT. (H) Mean (+/− SEM) spikes evoked by increasing current injections; OT increased the spike frequency (F_OT(1, 279) = 10.42, p = 0.001). (I) Mean action potentials evoked by 250pA current; significantly more spikes were evoked after OT. (J) Mean (+ S.E.M. and individual replicates) spike frequency upon 250pA depolarization; OT increased spiking compared to aCSF and Gö 6983 reduced spiking on its own (F_OT*Go(1, 43) = 11.77, p = 0.0013, aCSF-OT vs. Gö-OT, p = 0.0007). (K) Membrane potentials evoked by subthreshold and hyperpolarizing current injections (left) and typical rectification curve (right) before (black traces) and after (red traces) OT. (L) Mean input resistance (R_input); OT significantly increased R_input. (M) Mean (+ S.E.M. and individual replicates) input resistance. OT increased input resistance (F_Go(2, 20) = 4.66, p = 0.043, OT-DMSO vs. OT-Gö, p = 0.03). Symbols and connecting lines indicate individual replicates. *p < 0.05, ** p < 0.01, *** p < 0.001 (Sidak). Brain Atlas illustrations were reproduced with permission as previously published in The Brain Atlas in Stereotaxic Coordinates, 4th Edition, Paxinos, G. & Watson, C. Pages 296, 303, 306–317 & 332. Copyright Elsevier (1998).

Figure 4. Oxytocin modulates excitatory synaptic transmission in the insular cortex

(A) Top view of 60 channel perforated MEA (left) for acute extracellular recordings of the insular cortex. Right depicts location of insular cortex slice during recording, rf = rhinal fissure. (B) Input/output curves for fEPSPs (Mean +/− SEM, OT n = 10, aCSF n = 9 slices) normalized to the peak amplitude observed in response to 5V stimulation under baseline conditions. OT significantly increased EPSP amplitude beginning at 2V with further enhancement during the washout (F_STIMULUS(10, 90) = 598.20, p < 0.0001; F_DRUG(2, 18) = 11.99, p < 0.001; F_{STIMULUS*DRUG}(20, 180) = 11.34, p < 0.0001). Without application of OT, EPSPs remain stable across the duration of the experiment (aCSF: F_STIMULUS(10, 80) = 385.90, p < 0.0001). ### p < 0.0001 OT vs. Baseline, $$$ p < 0.0001 Wash vs. Baseline, *** p < 0.0001 Wash vs. OT (Sidak). (C) Typical fEPSPs evoked by biphasic extracellular stimulation at baseline (blue) during application of 500nM OT (red) and after washout (green). Scale bar 500µV/ms. (D) Representative voltage clamp recordings of mEPSCs recorded before (aCSF; blue) and after OT (red). (E) Mean mEPSC amplitude before and after OT (n = 19 neurons); OT significantly reduced amplitude, **(paired t(18) = 3.29, p = 0.004, 2-tailed). (F) Mean mEPSC interval (n = 19 neurons); no effect of OT was apparent, (paired t(18) = 1.42, p = 0.17, 2-tailed). (G) Input/output curve for fEPSPs (Mean +/− S.E.M., n = 7 slices/condition) normalized to the peak amplitude observed in response to 5V stimulation under baseline conditions. OT (1µM) increased fEPSP at 4.5 and 5mV while no effect was observed in the presence of Gö 6983. *F_{STIMULUS*DRUG}(10, 240) = 3.40, p < 0.0003, OT vs. aCSF at 4.5 and 5V, p = 0.0306 and p = 0.0181, respectively (Sidak). (H) Mean (+ S.E.M. and individual replicates) fEPSP amplitude from (G) at 5V (F_OT(1, 24) = 5.076, p = 0.034). *OT-DMSO vs. aCSF-DMSO p = 0.018; vs. aCSF-Gö, p = 0.002; vs. OT-Gö, p < 0.0004 (Fisher’s LSD).

Figure 5. Social affective behaviors require insular cortex oxytocin

(A) Diagram of experimental design. (B) Mean (individual replicates) time spent exploring either PN 30 (n = 7) or PN 50 (n=7) conspecifics after bilateral intra-insula infusion of a selective OTR antagonist (OTRa, 20ng/side) in the SAP test. Vehicle-treated experimental adult rats spent more time interacting with the stressed than with the naïve PN 30 juvenile conspecifics and less time with the stressed than with the naïve PN 50 adult conspecifics. These effects were blocked and reversed, respectively, by infusion of OTRa (F_{AGE*DRUG*STRESS}(1, 12) = 31.843, p = 0.0001). (C) Data in (B) expressed as percent preference for interaction with the stressed conspecific (Mean with individual replicates). OTRa significantly reduced preference for the stressed PN 30 while increasing time spent with the stressed PN 50 conspecific (F_AGE*DRUG(1, 12) = 26.38, p < 0.0002). (D) Diagram of 1-on-1 social interaction tests with stressed conspecifics and pretreatment with either vehicle or OTRa. (E) Mean (with individual replicates, normalized as percent of 3 to 5 min long test, time spent interacting with the stressed conspecific in a 1-on-1 test; PN 30: n = 8, PN 50: n = 7). OTRa significantly reduced time interacting with the stressed PN 30 conspecific but increased time interacting with the stressed PN 50 conspecific (F_AGE(1, 13) = 28.66, p < 0.0001; F_AGE*DRUG(1, 13) = 32.56, p < 0.0001). (F) Mean (with individual replicates) time spent interacting with a naive conspecific after intra insular cortex OT (250pg/side) or vehicle administration in a 1-on-1 social interaction (PN 30: n = 15; PN 50: n = 15). OT caused a significant increase in social interaction with naive PN 30 juveniles but a significant decrease in interaction with naive PN 50 adults (F_AGE*DRUG(1, 28) = 30.08, p < 0.0001). (G) Mean (individual replicates) time spent exploring either PN 30 (n = 8) or PN 50 (n = 7) conspecifics after intra-insula infusion of Gö 6983 (0.5uL/side 200nM) or vehicle (10% DMSO in water) in the SAP test. Vehicle-treated experimental adult rats spent more time interacting with the stressed than with the naïve PN 30 juvenile conspecifics and less time with the stressed than with the naïve PN 50 adult conspecifics. These trends were blocked and reversed, respectively, by the PKC inhibitor (F_{STRESS*AGE*DRUG}(1, 13) = 63.75, p < 0.0001). (H) Data in (G) expressed as percent preference for interaction with the stressed conspecific (Mean with individual replicates). Gö 6983 significantly reduced preference for the stressed PN 30 while increasing time spent with the stressed PN 50 conspecific (F_AGE*DRUG(1, 13) = 141.10, p < 0.0001). *p < 0.05, **p < 0.01, ***p < 0.001 (Sidak).

Figure 6. Insular Cortex and the Social Decision-Making Network

Fos immunoreactivity was determined in 29 ROIs of the rats (N=44) used in the USV quantification experiment (abbreviations provided in Sup. Table 1 and representative images in Sup. Fig. 7). (A) Correlation matrix indicating functional correlations (Kendall’s tau) among ROIs. (B) Graph theory based community detection analyses identified two modules. Functional correlations within and between modules 1 and 2 were averaged across ROIs and shown for each group. (C) Network visualization of the nodes in modules 1 (green) and 2 (purple). Here an arbitrary threshold of 0.2 was applied to facilitate network visualization, such that only edges exceeding the threshold are shown, but note that all network analyses were based on the unthresholded network. Insular cortex nodes are outlined in black. (D) The degree to which each node is connected to multiple functional modules was estimated by computing the participation coefficient. Bars corresponding to insular cortex ROIs are outlined in black. Network analyses by treatment group provided in Sup. Fig. 8. NJ = naive juvenile, NA = naive adult, SJ = stressed juvenile, SA = stressed adult.