Serotonin modulates an inhibitory input to the central amygdala from the ventral periaqueductal gray

Abstract

Fear is an adaptive state that drives defensive behavioral responses to specific and imminent threats. The central nucleus of the amygdala (CeA) is a critical site of adaptations that are required for the acquisition and expression of fear, in part due to alterations in the activity of inputs to the CeA. Here, we characterize a novel GABAergic input to the CeA from the ventral periaqueductal gray (vPAG) using fiber photometry and ex vivo whole-cell slice electrophysiology combined with optogenetics and pharmacology. GABA transmission from this ascending vPAG-CeA input was enhanced by serotonin via activation of serotonin type 2 C (5HT_2C) receptors. Results suggest that these receptors are presynaptic. Interestingly, we found that GABA release from the vPAG-CeA input is enhanced following fear learning via activation of 5HT_2C receptors and that this pathway is dynamically engaged in response to aversive stimuli. Additionally, we characterized serotonin release in the CeA during fear learning and recall for the first time using fiber photometry coupled to a serotonin biosensor. Together, these findings describe a mechanism by which serotonin modulates GABA release from ascending vPAG GABA inputs to the CeA and characterize a role for this pathway in fear.

Fig. 1. vPAG GABA neurons have functional projections to the CeA.

A surgical schematic of channelrhodopsin infusion into the vPAG of Vgat-ires-Cre mice. B schematic of responses in vPAG and CeA upon blue light stimulation. C, D Representative image showing expression of channelrhodopsin (ChR2) in the vPAG of a Vgat-ires-Cre mouse. E, F Representative action potential firing at 10 Hz and 20 Hz. G Fidelity of action potential spiking in response to ChR2 activation by 1 ms pulses of blue 490 nM light in vPAG GABA neurons (c; n = 6 cells from 5 mice). H representative average trace of inhibitory post-synaptic potentials evoked by 1 ms pulses of blue 490 nM light (oIPSCs) recorded from a cell of the CeA proximal to ChR2 terminal expression in the absence and presence of the GABA-A ion channel blocker picrotoxin (25 μM). I Percent of cells recorded from in the CeA proximal to ChR2 terminal expression that showed oIPSCs in response to blue 490 nM light stimulation (n = 51 cells).

Fig. 2. 5HT is released in the CeA during fear conditioning.

A schematic of injections and fiber implant for iSeroSnFR recordings. B schematic of iSeroSnFR. C Schematic of tones and shocks presented during fear learning for fear and naïve groups. D, E Representative image of iSeroSnFR and GFP and optic fiber placement in CeA. F Freezing behavior during fear learning. Response to tone and shock presentations averaged across cohort for iSeroSnFR Fear (G), iSeroSnFR Naïve (H), and GFP Fear (I). Zoomed in traces show shock responses. J shock response averaged from t = 28–30 relative to tone onset across all trials. K post-shock response averaged from t = 33–60 relative to tone onset across all trials. Responses to first and last tone/shock presentations averaged across cohort for iSeroSnFR Fear (L), iSeroSnFR Naive (N), and GFP fear (P). Average post-shock response for first and last trials for iSeroSnFR Fear (M), iSeroSnFR Naive (O), and GFP fear (Q). n = 13 iSeroSnFR fear, 11 iSeroSnFR naïve, 6 GFP fear.

Fig. 3. The vPAG^Vgat-CeA pathway is modulated by serotonin directly and via presynaptic 5HT2C receptors.

A surgical schematic of channelrhodopsin infusion into the vPAG of Vgat-ires-Cre mice. B Representative traces showing oIPSCs at baseline and during serotonin bath application. C, D Time course and summary of the effects of bath application of serotonin (10 µM) on the amplitude of the first evoked peak relative to baseline values (n = 13 cells from 9 mice). E Summary of the effects of serotonin on the paired pulse ratio (PPR; amplitude of pulse 2/ amplitude of pulse 1) (n = 6 cells from 4 mice).Time course and summary of the effects of serotonin on the amplitude of the first evoked peak (F, G) and PPR (H, I) in the presence of 4-Aminopyridine (4 AP; 100 µM) and tetrodotoxin (TTX; 1 µM) (n = 4 cells from 2 mice). J–M Time course and summary of the effects of serotonin on the amplitude of the first evoked peak and PPR in the presence of the 5HT2C antagonist RS102221 (5 µM) (n = 5 cells from 4 mice). ^*p < 0.05; all data shown as means +/− SEM.

Fig. 4. Fear learning engages the vPAG^Vgat-CeA pathway.

A surgical schematic of channelrhodopsin infusion into the vPAG of Vgat-ires-Cre mice. B Experimental timeline for fear conditioning and electrophysiology experiments. C, D Time course and summary of the effect of bath application of serotonin (5HT; 10 µM) on the amplitude of the first evoked peak relative to baseline values in shock mice versus naïve mice (n = 7 cells per group from 3 mice per group), recordings in CeA. E Experimental timeline for fear conditioning and electrophysiology experiments with 5HT_2c antagonist pretreatment. Mice were administered the 5HT2C antagonist SB242084 (3 mg/kg, i.p.) or vehicle prior to the fear acquisition session (shock) or 45 min prior to sacrifice (naïve) and recordings were performed at terminals in the CeA. F Summary of the effects of serotonin on the paired pulse ratio (PPR; amplitude of pulse 2/amplitude of pulse 1) in shock or naive mice that did or did not receive drug (e; n = 7–13 cells per group from 3–5 mice per group). ^*p < 0.05; all data shown as means +/− SEM.

Fig. 5. vPAG^Vgat-CeA pathway is dynamically engaged during fear learning and responds to shock-predicting cues.

A surgical schematic of GCaMP7f infusion into the vPAG of Vgat-ires-Cre mice. B, C representative image of GCaMP expression and fiber placement in vPAG and CeA . D Representative trace showing vPAG and CeA signal during fear learning in a mouse that underwent fear conditioning. E vPAG and CeA responses to tone/shock presentation in fear conditioned mice averaged across cohort. F Tone response calculated as the average from t = 0–5, normalized to t = −5–0 relative to tone onset. G Shock response calculated as the average from t = 28–30, normalized to t = 23–28 relative to tone onset. H Representative trace showing vPAG and CeA signal during learning in a naïve mouse. I vPAG and CeA responses to tone presentation in naïve mice averaged across cohort. J Tone response calculated as the average from t = 0–5, normalized to t = −5–0 relative to tone onset. K schematic of analysis pipeline for alignment of freezing and photometry signal. L Freezing behavior during fear learning. M Representative trace showing alignment of freezing and vPAG signal with identified spikes classified as occurring during freezing (magenta) or mobility (purple). N vPAG spike frequency during freezing and mobility for fear mice (turquoise) and naïve (gray). O vPAG spike frequency during each epoch of fear learning, normalized to BL. P Representative trace showing alignment of freezing and CeA signal. Q average signal during freezing and mobility averaged across all bouts. n = 10 vPAG fear, 7 vPAG naïve, 8 CeA fear, 7 CeA naïve.