Optogenetic modulation of descending prefrontocortical inputs to the dorsal raphe bidirectionally bias socioaffective choices after social defeat

Abstract

It has been well established that modulating serotonin (5-HT) levels in humans and animals affects perception and response to social threats, however the circuit mechanisms that control 5-HT output during social interaction are not well understood. A better understanding of these systems could provide groundwork for more precise and efficient therapeutic interventions. Here we examined the organization and plasticity of microcircuits implicated in top-down control of 5-HT neurons in the dorsal raphe nucleus (DRN) by excitatory inputs from the ventromedial prefrontal cortex (vmPFC) and their role in social approach-avoidance decisions. We did this in the context of a social defeat model that induces a long lasting form of social aversion that is reversible by antidepressants. We first used viral tracing and Cre-dependent genetic identification of vmPFC glutamatergic synapses in the DRN to determine their topographic distribution in relation to 5-HT and GABAergic subregions and found that excitatory vmPFC projections primarily localized to GABA-rich areas of the DRN. We then used optogenetics in combination with cFos mapping and slice electrophysiology to establish the functional effects of repeatedly driving vmPFC inputs in DRN. We provide the first direct evidence that vmPFC axons drive synaptic activity and immediate early gene expression in genetically identified DRN GABA neurons through an AMPA receptor-dependent mechanism. In contrast, we did not detect vmPFC-driven synaptic activity in 5-HT neurons and cFos induction in 5-HT neurons was limited. Finally we show that optogenetically increasing or decreasing excitatory vmPFC input to the DRN during sensory exposure to an aggressor's cues enhances or diminishes avoidance bias, respectively. These results clarify the functional organization of vmPFC-DRN pathways and identify GABAergic neurons as a key cellular element filtering top-down vmPFC influences on affect-regulating 5-HT output.

Keywords: depression and anxiety disorders; dorsal raphe; electrophysiology; optogenetics; serotonin; social defeat; social perception; ventromedial prefrontal cortex.

Figure 1

Visualization of vmPFC axon terminals in the DRN (A) An AAV vector coding for the Cre-dependent expression of the fluorescently tagged synaptic protein Synaptophysin (SynP-GFP) was injected in the vmPFC of CaMKIIa-Cre mice. Topographic distribution of vmPFC axon terminals in the DRN was visualized using confocal microscopy. (B) Distribution of vmPFC terminals as determined by SynP-GFP expression as reported in this study. Scale bar 50 μm. (C) Anterograde tracing using an AAV-GFP vector injected in the vmPFC as reported by the Allen Brain Connectivity Atlas. Picture is courtesy of the Allen Mouse Brain Atlas [Internet]. © 2012 Allen Institute for Brain Science (Seattle, WA). Available from: http://mouse.brain-map.org. Despite use of different tracing methods, the pattern of innervation revealed is strikingly similar, with sparse innervation of the midline area of the DRN, classically containing 5-HT neurons, and denser innervation of lateral portions of the DRN. Scale bar 50 μm.

Figure 2

Spatial organization of 5-HT and GABA neurons and vmPFC terminals in the DRN. Native fluorescence of GABA (GAD2-tdTomato, column 1) and 5-HT (Pet1-tdTomato, column 2) neurons as well as antibody enhanced glutamatergic vmPFC terminal fluorescence (CaMKIIa: SynP-GFP) is visualized in serial sections of the DRN. Individual cellular or synaptic localization was overlaid on a map using the aqueduct as a reference. Scale bar 50 μm.

Figure 3

Topographic distribution of vmPFC terminals correlates with GABAergic populations in the DRN. At each of the 6 rostrocaudal levels of the DRN, images of each fluorescent signal were divided into a 10 × 10 grid. Relative intensities were calculated for each of the SynP-GFP (vmPFC terminals), GAD2-tdTomato (GABA neurons) and Pet1-tdTomato (5-HT neurons) fluorescent signals for each box of the grid at every rostrocaudal level. (A) For example, depicted here are DRN slices at −4.60 mm from Bregma. Highlighted grid boxes depict areas that were calculated to have the highest (a) GAD2-tdTomato intensity, (b) Pet1-tdTomato intensity and (c) SynP-GFP intensity. Intensity values of SynP-GFP were correlated with that of (B) GAD2-tdTomato or (C) Pet1-tdTomato at each of the 6 rostrocaudal levels. These were graphed on scatter plots with (x,y) coordinates plotted as (SynP-GFP intensity, tdTomato intensity). Grid boxes highlighted in (A) are plotted in (B) and (C) as examples. Pearson's correlation coefficients (r) were calculated for GAD2-tdTomato or Pet1-tdTomato vs. SynP-GFP with r = 1 signifying strong correlation, r = 0 signifying no correlation and r = −1 signifying strong negative correlation. (D) Overall there is a stronger correlation of topographic distribution of GABA neurons with vmPFC terminals than 5-HT neurons except in the most caudal DRN. (Bars with asterisks indicate Pearson coefficients that are significantly non-zero; ^*p < 0.05, ^**p < 0.005, ^***p < 0.001).

Figure 4

Preferential cFos induction in GABA neurons after photostimulation of vmPFC terminals in the DRN. (A) CaMKIIa-driven ChR2-YFP was injected in the vmPFC of GAD2-tdTomato or Pet1-tdTomato mice. (B) After allowing 6 weeks, robust expression of ChR2-YFP was achieved. Scale bar 25 μm. (C) Fluorescent neurons in layer V of the vmPFC were recorded. Current-clamp traces of YFP⁺ vmPFC neurons demonstrate temporal fidelity of ChR2-mediated photocurrents at 5 and 25 Hz, and loss of precision at 100 Hz. Scale bar 40 pA, 0.2 s. (D) At the same time point post-injection (6 weeks), vmPFC terminals in the DRN were photostimulated for 20 min using 473 nm light (10 mW, 25 Hz, 10 ms pulse width). (E) cFos immunofluorescence was evaluated after photostimulation to indicate neuronal activation. Scale bar 50 μm. (F) Photostimulation resulted in a significant increase in total cFos expression with no difference between genotype (Student's t-test, ^***p < 0.001). (G) Quantification of cFos colocalization with native tdTomato fluorescence was used to determine the neuronal populations activated. Closed arrows indicate colocalized neurons. (H) Without laser stimulation there is minimal cFos expression. After photoactivation of vmPFC terminals there were increases in neuronal activation of both GABA and 5-HT neurons, however a much higher percentage of GAD2-tdTomato neurons colocalize with cFos (Two-Way ANOVA, Fisher post-hoc, ^***p < 0.001).

Figure 5

Photoactivation of vmPFC terminals results in time-locked EPSC events in DRN GABA neurons. (A) High magnification confocal image of a biocytin-filled recorded GAD2-tdTomato neuron (red) in close contact with axon terminals from vmPFC neurons (green) anterogradely traced using AAV-CaMKIIa-ChR2-YFP. Inset displays a schematic of DRN topography in recorded slices from GAD2-tdTomato mice. Recorded neurons were purposely selected in ventrolateral subregions of the DRN that are rich in afferents from the vmPFC while the DRN was photostimulated. Aq—aqueduct. (B) Average voltage-clamp traces of spontaneous and laser evoked EPSC events. Application of 20 μM DNQX prevented laser-evoked EPSC events. Scale bar 4 pA, 3 ms. (C) Raw voltage-clamp data trace recorded from a GAD2-tdTomato neuron during pulsed photoactivation of ChR2 containing vmPFC terminals (473 nm, 10 mW, 0.5 Hz, 10 ms pulse width). Blue circles mark laser pulses. Black triangles indicate spontaneous EPSC events. Scale bar 10 pA, 0.5 s. (D) EPSC events were time-locked to a 25 Hz laser stimulation train. Scale bar 5 pA, 0.1 s. (E) Recordings from Pet1-tdTomato neurons did not display EPSC events in response to laser stimulation. Scale bar 10 pA, 0.5 s.

Figure 6

Divergent effects of vmPFC terminal photostimulation and photoinhibition in the DRN. (A) CaMKIIa-Cre mice were injected in the vmPFC with Cre-dependent AAVs coding for the expression of ChR2-YFP, Arch-GFP or tdTomato. Cohorts of mice were exposed to 5 min of physical defeat followed by 20 min of sensory contact with either photoactivation by ChR2 (473 nm, 10 mW, 25 Hz, 10 ms pulse width) or photoinhibition by Arch (561 nm, 10 mW) of vmPFC terminals via implanted fiber optic targeting the DRN. Mice were then placed in homecages overnight. This was repeated with exposure to a novel aggressor each day. On day 11, mice were assessed for approach or avoidance in the social interaction test. IZ—interaction zone. (B) Heat maps depicting representative behavioral effects of photoactivating (ChR2) or photoinhibiting (Arch) vmPFC terminals during the sensory period of social defeat on interaction with a novel social target (orange box). Red and green areas depict areas where mice spent the most time. No effect was observed in mice injected with sham tdTomato virus. (C) In defeated mice, photosilencing of vmPFC terminals prevented a decrease in social interaction (Two-Way ANOVA, Fisher post-hoc, ^*p = 0.050) while photoactivation did not decrease interaction times significantly from ChR2-expressing mice that did not receive laser stimulation. Undefeated control mice whose vmPFC terminals in the DRN were stimulated displayed a significant decrease in social interaction compared to unstimulated counterparts (Two-Way ANOVA, Fisher post-hoc, ^*p = 0.049). No effect of virus of photostimulation was observed when a novel social target was not present. (D) With a novel social target present, mice whose vmPFC terminals were photoactivated via ChR2 during sensory contact displayed significant increases in latencies to first enter the IZ in both control (Two-Way ANOVA, Fisher post-hoc, ^*p = 0.033) and defeated conditions (^*p = 0.049). Defeated mice whose vmPFC terminals in the DRN were photosilenced via Arch during sensory contact displayed shorter latencies to first enter the IZ (^*p = 0.048). (E) Total number of entries into the IZ was decreased in both control and defeated mice whose vmPFC terminals in the DRN were photoactivated during sensory contact (Two-Way ANOVA, Fisher post-hoc, control ^*p = 0.042, defeated ^*p = 0.049).

Figure 7

Defeat and photoactivation of vmPFC terminals in the DRN bias social approach/avoidance choices. (A) Analysis was performed on 97 previously defeated and 20 undefeated mice. Defeated mice were characterized as resilient or susceptible based on previously described criteria (Golden et al., ; Challis et al., 2013). The cumulative time spent in the interaction zone (IZ) was plotted across the duration of the social interaction test. The cumulative interaction time of susceptible mice significantly diverged from that of both resilient and control mice at 4 s (Repeated measures ANOVA, Fisher post-hoc, p = 0.020 at 4 s). Data shown is average per group. Dashed lines represent the s.e.m. (B) Latencies to the first IZ entry were determined in 5-s bins. Plotted are the proportions of mice within each behavioral group to display first IZ entry within the given timeframe or less. A greater proportion of resilient mice first entered the IZ faster that of susceptible mice (Kolmogorov-Smirnov test, p < 0.001). The dashed vertical line represents the latency at which the proportion discrepancy between the two behavioral is the largest (0.36). (C) Cumulative time spent in the IZ is plotted across time in the social interaction test for undefeated CaMKIIa-Cre mice expressing ChR2 in the vmPFC with and without laser stimulation of vmPFC terminals in the DRN. Increase in cumulative interaction time from 0 s did not significantly differ until 60 s in laser-treated mice (Repeated measures ANOVA, Fisher post-hoc within Laser group, p = 0.045 at 60 s). (D) Cumulative time spent in the corners distal to the novel social target is plotted across time in the social interaction test. Increase in cumulative corner time from 0 s significantly differed at 14 s in laser-treated mice (Repeated measures ANOVA, Fisher post-hoc within Laser group, p = 0.047 at 14 s).