Inhibitory Gating of Basolateral Amygdala Inputs to the Prefrontal Cortex

Abstract

Interactions between the prefrontal cortex (PFC) and basolateral amygdala (BLA) regulate emotional behaviors. However, a circuit-level understanding of functional connections between these brain regions remains incomplete. The BLA sends prominent glutamatergic projections to the PFC, but the overall influence of these inputs is predominantly inhibitory. Here we combine targeted recordings and optogenetics to examine the synaptic underpinnings of this inhibition in the mouse infralimbic PFC. We find that BLA inputs preferentially target layer 2 corticoamygdala over neighboring corticostriatal neurons. However, these inputs make even stronger connections onto neighboring parvalbumin and somatostatin expressing interneurons. Inhibitory connections from these two populations of interneurons are also much stronger onto corticoamygdala neurons. Consequently, BLA inputs are able to drive robust feedforward inhibition via two parallel interneuron pathways. Moreover, the contributions of these interneurons shift during repetitive activity, due to differences in short-term synaptic dynamics. Thus, parvalbumin interneurons are activated at the start of stimulus trains, whereas somatostatin interneuron activation builds during these trains. Together, these results reveal how the BLA impacts the PFC through a complex interplay of direct excitation and feedforward inhibition. They also highlight the roles of targeted connections onto multiple projection neurons and interneurons in this cortical circuit. Our findings provide a mechanistic understanding for how the BLA can influence the PFC circuit, with important implications for how this circuit participates in the regulation of emotion.

Significance statement: The prefrontal cortex (PFC) and basolateral amygdala (BLA) interact to control emotional behaviors. Here we show that BLA inputs elicit direct excitation and feedforward inhibition of layer 2 projection neurons in infralimbic PFC. BLA inputs are much stronger at corticoamygdala neurons compared with nearby corticostriatal neurons. However, these inputs are even more powerful at parvalbumin and somatostatin expressing interneurons. BLA inputs thus activate two parallel inhibitory networks, whose contributions change during repetitive activity. Finally, connections from these interneurons are also more powerful at corticoamygdala neurons compared with corticostriatal neurons. Together, our results demonstrate how the BLA predominantly inhibits the PFC via a complex sequence involving multiple cell-type and input-specific connections.

Keywords: basolateral amygdala; inhibition; interneuron; prefrontal cortex; projection neuron; synapse.

Figure 1.

Properties of CA and CS neurons in the infralimbic PFC. A, Schematic of injecting CTB-Alexa-647 into the NAc and CTB-Alexa-488 into the BLA of wild-type mice, to label neurons in the infralimbic PFC. Cardinal axes pictured to right for injection schematics apply to all figures. B, Left, CA (green) and CS (purple) neurons in the PFC, with DAPI staining in gray. White lines indicate laminar borders. Right, Quantification of CA, CS, and colabeled (red) cells in the PFC. C, Two-photon image of neighboring CA and CS neurons. D, AP firing and hyperpolarization in response to 250 pA and −50 pA current injections in the presence of synaptic blockers. E, Summary of AP firing over a range of current injections. F, Summary of resting membrane potential, input resistance, adaptation ratio, and voltage sag.

Figure 2.

BLA-evoked excitation and inhibition. A, Schematic of injecting AAV-mCherry into the BLA of wild-type mice, to label BLA axons in the infralimbic PFC. B, Left, BLA axons in a coronal section. White box indicates analyzed portion of PFC. Middle, BLA axons in the PFC. White lines indicate laminar borders. Right, Distribution of BLA axons in the PFC, with fluorescence normalized to an unlabeled portion of the slice. C, Schematic of injecting red retrobeads into the NAc and green retrobeads and AAV-ChR2-eYFP into the BLA of wild-type mice, to label CS and CA neurons and express ChR2 in BLA axons in the infralimbic PFC. D, Average BLA-evoked AMPA-R EPSCs at −70 mV and GABA_A-R IPSCs at 10 mV in the presence of CPP. Green arrowhead indicates time of LED pulse. E, Summary of amplitudes of BLA-evoked EPSCs (left) and IPSCs (middle) at CA and CS neurons. Lines connect pairs of recorded neurons. Summary of CS/CA amplitude ratio for EPSCs and IPSCs (right). F, Summary of EPSC/IPSC amplitude ratio at CA and CS neurons. *p < 0.05.

Figure 3.

BLA inputs are stronger onto CA neurons. A, Schematic of injecting red retrobeads into the NAc and green retrobeads and AAV-ChR2-eYFP into the BLA of wild-type mice, to label CA and CS neurons and express ChR2 in BLA axons in the infralimbic PFC. B, Average BLA-evoked AMPA-R EPSCs at −70 mV and NMDA-R EPSCs at 40 mV in the presence of TTX, 4-AP, high Ca²⁺, and gabazine. Green arrowhead indicates time of LED pulse. C, Summary of amplitudes of BLA-evoked AMPA (left) and NMDA (middle) EPSCs at CA and CS neurons. Lines connect pairs of recorded neurons. Summary of CS/CA amplitude ratio (right). D, Summary of AMPA/NMDA ratio at CA and CS neurons. E, Average AMPA-R EPSCs evoked by trains of BLA inputs (5 pulses at 20 Hz) in the presence of CPP and gabazine. Green arrowheads indicate timing of LED pulses. F, Summary of CS/CA amplitude ratio (left) and PPR (right) during trains. *p < 0.05.

Figure 4.

PV and SOM interneurons in the infralimbic PFC. A, Labeling of PV interneurons in the PFC of a PV-Cre mouse paired with Ai9 tdTomato reporter mouse, pseudo-colored blue. Left, Coronal section. Right, PV interneurons in different layers of infralimbic PFC, with DAPI labeling in gray. B, Similar to A for SOM interneurons in the PFC of a SOM-Cre mouse paired with Ai9 tdTomato reporter mouse, pseudo-colored orange. C, Two-photon images of PV and SOM interneurons. D, AP firing and hyperpolarization in response to 200 pA and −50 pA current injections in the presence of synaptic blockers. E, Summary of resting membrane potential, input resistance, adaptation ratio, and voltage sag. *p < 0.05.

Figure 5.

PV and SOM inhibition is stronger at CA neurons. A, Schematic of injecting AAV-DIO-ChR2-eYFP into the infralimbic PFC, red retrobeads into the NAc, and green retrobeads into the BLA of either PV-Cre or SOM-Cre mice, to express ChR2 in either PV or SOM interneurons and label CS and CA neurons in the PFC. B, Light-evoked AP firing in PV (blue) and SOM (orange) interneurons expressing ChR2 in the presence of NBQX and CPP, showing multiple trials at a 1 ms LED duration. Arrowheads indicate time of LED pulse. C, Summary of AP firing over range of LED durations for PV and SOM interneurons. D, Average GABA_A-R IPSCs evoked by PV interneurons (blue arrowhead) in the presence of NBQX and CPP. E, Summary of amplitudes of PV-evoked IPSCs at CA and CS neurons (left). Lines connect pairs of recorded neurons. Summary of CS/CA amplitude ratio in the absence (−) or presence (+) of TTX and 4-AP (right). F, Average GABA_A-R IPSCs evoked by SOM interneurons (orange arrowhead) in the presence of NBQX and CPP. G, Summary of amplitudes of SOM-evoked IPSCs at CA and CS neurons (left). Lines connect pairs of recorded neurons. Summary of CS/CA amplitude ratio in the absence (−) or presence (+) of TTX and 4-AP (right). *p < 0.05.

Figure 6.

BLA inputs are strongest at PV interneurons. A, Schematic of injecting AAV-FLEX-RG, AAV-FLEX-TVA-mCherry, and SADΔG-GFP(EnvA) into the PFC of PV-Cre mice, to retrogradely label monosynaptically connected cells in the BLA. B, Left, Coronal image showing the injection site in the PFC. Right, GFP-expressing neurons in the BLA that project to PV interneurons. C, Schematic of injecting AAV-FLEX-tdTomato into the PFC and green beads and AAV-ChR2-eYFP into the BLA of PV-Cre mice, to label PV interneurons and CA neurons and express ChR2 in BLA axons in the infralimbic PFC. D, Average BLA-evoked AMPA-R and NMDA-R EPSCs in the presence of TTX, 4-AP, high Ca²⁺, and gabazine. Green arrowhead indicates time of LED pulse. E, Summary of amplitudes of BLA-evoked AMPA (left) and NMDA (middle) EPSCs at CA and PV neurons. Lines connect pairs of recorded neurons. Summary of PV/CA amplitude ratio (right). F, Summary of AMPA/NMDA ratio at CA and PV neurons. G, Average AMPA-R EPSCs evoked by trains of BLA inputs (5 pulses at 20 Hz) in the presence of CPP and gabazine. Green arrowheads indicate timing of LED pulses. H, Summary of PV/CA amplitude ratio (left) and PPR (right) during trains. *p < 0.05.

Figure 7.

Facilitating BLA inputs onto SOM interneurons. A, Schematic of injecting AAV-FLEX-RG, AAV-FLEX-TVA-mCherry, and SADΔG-GFP(EnvA) into the PFC of SOM-Cre mice, to retrogradely label monosynaptically connected cells in the BLA. B, Left, Coronal image showing the injection site in the PFC. Right, GFP-expressing neurons in the BLA that project to SOM interneurons. C, Schematic of injecting AAV-FLEX-tdTomato into the PFC and green beads and AAV-ChR2-eYFP into the BLA of SOM-Cre mice, to label SOM interneurons and CA neurons and express ChR2 in BLA axons in the infralimbic PFC. D, Average BLA-evoked AMPA-R and NMDA-R EPSCs in the presence of TTX, 4-AP, high Ca²⁺, and gabazine. Green arrowhead indicates time of LED pulse. E, Summary of amplitudes of BLA-evoked AMPA (left) and NMDA (middle) EPSCs at CA and SOM neurons. Lines connect pairs of recorded neurons. Right, Summary of SOM/CA amplitude ratio. F, Summary of AMPA/NMDA ratio at CA and SOM neurons. G, Average AMPA-R EPSCs evoked by trains of BLA inputs (5 pulses at 20 Hz) in the presence of CPP and gabazine. Green arrowheads indicate timing of LED pulses. H, Summary of SOM/CA amplitude ratio (left) and PPR (right) during trains. *p < 0.05.

Figure 8.

BLA inputs preferentially activate interneurons. A, Average experimentally recorded BLA-evoked EPSCs scaled relative to CA neuron response. Black arrowhead indicates time of current injection. B, Left, CA neuron EPSCs over scale factors 1–10 used for current injections. Black arrowhead indicates time of current injection. Right, Peak current of injected EPSC over scale factors 1–10 for each cell type. C, EPSPs and APs elicited by EPSC current injections over scale factors 1–10 in CA (green), CS (purple), PV (blue), and SOM (orange) neurons, in the presence of synaptic blockers. Black arrowhead indicates time of current injection. D, Summary of probability of AP firing (left) and number of evoked APs (right) as a function of scale factor. E, Left, BLA-evoked EPSPs and APs at neighboring CA neurons (green) and PV interneurons (blue), in the presence of CPP and gabazine. Responses to LED duration of 4 ms shown. Green arrowhead indicates time of LED pulse. Right, Probability of AP firing versus LED duration. F, Same as in E for CA neurons (green) and SOM interneurons (orange).

Figure 9.

Unique activation during trains of BLA inputs. A, Average experimentally recorded BLA-evoked EPSC trains scaled relative to CA neuron response. Black arrowheads indicate timing of current injections. B, Left, CA neuron EPSC trains over scale factors 1–10 used for current injections. Black arrowheads indicate timing of current injections. Right, Peak current of injected EPSCs within a train at scale factor of 4 for each cell type. C, EPSPs and APs elicited by EPSC train current injections (5 pulses at 20 Hz) in CA (green), CS (purple), PV (blue), and SOM (orange) neurons, in the presence of synaptic blockers. Responses to scale factor of 4 shown. Black arrowheads indicate timing of current injections. D, Summary of probability of AP firing as a function of pulse number, with low (2), medium (4), and high (8) scale factors shown in light to dark shades. E, Left, EPSPs and APs evoked at neighboring CA neurons (green) and PV interneurons (blue) in response to train of BLA inputs (5 pulses at 20 Hz), in the presence of CPP and gabazine. Responses to LED duration of 4 ms shown. Green arrowheads indicate timing of LED pulses. Right, Probability of AP firing versus pulse number, with LED durations of 2, 4, and 8 ms shown in light to dark shades. F, Same as in E for CA neurons (green) and SOM interneurons (orange).