Stimulus processing in fear conditioning is constrained by parvalbumin interneurons (PV-INs) through inhibition of principal excitatory neurons. However, the contributions of PV-IN microcircuits to input gating and long-term plasticity in the fear system remain unknown. Here we interrogate synaptic connections between afferent pathways, PV-INs, and principal excitatory neurons in the basolateral amygdala. We find that subnuclei of this region are populated two functionally distinct PV-IN networks. PV-INs in the lateral (LA), but not the basal (BA), amygdala possess complex dendritic arborizations, receive potent excitatory drive, and mediate feedforward inhibition onto principal neurons. After fear conditioning, PV-INs exhibit nucleus- and target-selective plasticity, resulting in persistent reduction of their excitatory input and inhibitory output in LA but not BA. These data reveal previously overlooked specializations of amygdala PV-INs and indicate specific circuit mechanisms for inhibitory plasticity during the encoding of associative fear memories.

We utilized parvalbumin-specific Cre driver mice as well as optogenetic-assisted electrophysiology to investigate the properties and experience-dependent plasticity of PV-IN microcircuits. We report that function and plasticity of PV-INs varies by nucleus location within basolateral amygdala and that fear conditioning leads to downregulation of PV-IN transmission predominantly within microcircuits that mediate feedforward inhibition from sensory afferent pathways.

The majority of GABAergic synaptic inhibition throughout the forebrain is thought to originate from a heterogeneous population of locally projecting interneurons. Within the basolateral amygdala, more than half of inhibitory synapses formed onto principal excitatory neurons are associated with PV-INs (), which are considered to exert powerful control over the firing of these cells through dense somatic and axo-axonic synaptic terminals (). Recently, in vivo manipulations within the basolateral amygdala () as well as neocortical regions () have implicated PV-INs in fear acquisition and expression through cue-related inhibition and disinhibition of principal excitatory neurons. Therefore, it is important to understand the circuit mechanisms underlying PV-IN recruitment and resulting excitatory neuronal inhibition as well as to determine whether fear conditioning generates persistent alterations in PV-IN function.

Aversive memories acquired by classical conditioning provide insight into emotional learning under normal conditions as well as pathological states, such as posttraumatic stress disorder (PTSD) (). Cellular models of fear learning place a great deal of emphasis on amygdala excitatory neuronal plasticity (). However, many studies posit that co-regulation of excitation and inhibition may be important for network stability and that excitation:inhibition (E:I) imbalance may be a factor in some psychiatric conditions (). Notably, decreased GABA levels as well as GABA receptor binding and polymorphisms have been associated with PTSD (), and reduced GABA levels are predictive of disease progression (). Changes in inhibitory synaptic markers suggest that plasticity of GABAergic transmission in the basolateral amygdala may also be a feature of aversive memory formation under normal conditions (). Although ex vivo stimulation of amygdala brain slices has been shown to induce long-term plasticity in undefined GABAergic populations (), it remains unknown whether specific GABAergic cell types exhibit plasticity associated with emotional learning.

GABAA receptor endocytosis in the basolateral amygdala is critical to the reinstatement of fear memory measured by fear-potentiated startle.

Training-induced changes in the expression of GABAA-associated genes in the amygdala after the acquisition and extinction of Pavlovian fear.

Regulation of gephyrin and GABAA receptor binding within the amygdala after fear acquisition and extinction.

While synapse-selective changes can alter the relationship of PV-INs to specific presynaptic and postsynaptic targets, firing rate plasticity could render PV-INs more or less excitable and thus impact their general integration. To evaluate this possibility, we examined action potential discharge in PV-INs in response to somatic current injections after fear conditioning. No experience-dependent differences in firing frequency were observed in LA or BA ( Figures 7 A–7F). In naive animals, however, PV-INs in BA were more excitable than those in LA ( Figure 7 G), exhibiting reduced rheobase current (independent samples t test, t= 2.19, p = 0.04) and increased firing frequency (repeated-measure ANOVA, main effect of nucleus, F= 17.81, p = 0.0003). This nucleus-specific difference is likely attributable to increased cell size, as inferred from a difference in online capacitance measurements from PV-INs (LA 103.00 ± 32.48 pF; BA 69.17 ± 24.13 pF; independent-samples t test, t= 3.12, p = 0.004). No other differences in passive membrane properties were observed. Likewise, no differences were observed in individual spike characteristics (including threshold, amplitude, duration, or afterhyperpolization; Figures 7 H–7K).

(H–K) Individual spike characteristics were quantified in LA (H) and BA (I) at rheobase. Overlays of representative traces are shown. Scales: 10 pA × 2 ms. No differences were observed among groups or between nuclei in any spike parameter, including spike duration (J) and afterhyperpolarization (AHP) amplitude (K). n/group indicated on graphs.

(G) In naive animals, PV-INs in LA were less excitable than PV-INs in BA, requiring more current to fire a single action potential (rheobase current; inset) and firing fewer action potentials to current injections greater than 150 pA.

(E and F) No differences in intrinsic excitability were observed among groups in BA PV-INs ([E], representative traces at the 200 pA current injection in [F]). Scales: 50 pA × 25 ms.

(B and C) No differences in intrinsic excitability were observed among groups in LA PV-INs ([B], representative traces at the 200 pA current injection in [C]).

Action potentials were elicited in PV-INs in LA ([A]–[C]) and BA ([D]–[F]) by somatic current injections of increasing amplitude.

A previous report found that fear conditioning induces alterations in GABA receptor expression in the lateral amygdala that are reversible by extinction (). Therefore, we postulated that GABA release properties might exhibit similar bidirectional regulation. However, increased PPR of PV-IN IPSCs was not reversed by extinction training ( Figure S7 ).

GABAA receptor endocytosis in the basolateral amygdala is critical to the reinstatement of fear memory measured by fear-potentiated startle.

Given the dense innervation of PV-INs by other PV-INs in the BA (), we next questioned whether the observed changes in GABA release are specific to glutamatergic targets, in particular because fear conditioning altered mIPSC frequency in BA PV-INs ( Figure 3 L). To facilitate fluorescence-based targeting of PV-INs without activating ChR2, we injected Cre-dependent AAV-ChR2 into the amygdala of PV-IRES-Cre:Ai9 double transgenic mice ( Figure 6 K). This strategy allowed us to use non-overlapping LED spectra for PV-IN visualization and ChR2 excitation ( Figure 6 L). PPR analysis of PV-IN→PV-IN oIPSCs revealed no learning-induced differences in GABA release in LA or BA ( Figures 6 M–6P), indicating that, in addition to being nucleus-specific, learning-dependent changes in PV-IN inhibition exhibit target selectivity.

To determine whether PV-IN-specific plasticity corresponds to an overall reduction of inhibition onto LA principal neurons, we collected spontaneous IPSCs (sIPSCs) from these cells after fear conditioning. Consistent with a previous report (), both sIPSC frequency (one-way ANOVA, F (2,51) = 11.17, p = 0.0001) and amplitude (one-way ANOVA, F(2,51) = 6.80, p = 0.003) was decreased in LA of animals that received paired training ( Figures S6 D–S6G). However, no changes in sIPSC properties were observed in BA principal neurons ( Figures S6 H–S6K). While our data suggest that decreased GABA release from PV-INs may contribute to reduced sIPSC frequency in LA, it is important to consider that these events reflect the cumulative action of GABA transmission not only from PV-INs but also from other inhibitory cell types.

GABAA receptor endocytosis in the basolateral amygdala is critical to the reinstatement of fear memory measured by fear-potentiated startle.

Previous studies suggest that changes in inhibitory transmission may occur after fear conditioning, but this work relied mainly on analysis of GABA receptors from amygdala lysates (). It therefore remains unclear whether fear encoding alters GABA transmission within specific microcircuits. To enable stimulation of synapses formed by PV-INs, we injected Cre-inducible AAV vectors encoding ChR2-eYFP into PV-IRES-Cre mice, resulting in somal and axonal eYFP expression in PV-INs ( Figures 6 A and 6B ) and blue-light-driven action potential generation ( Figure 6 C). Consistent with selective recombination in PV-INs ( Figures 1 A and S1 ), we verified that optic stimulation in these mice did not result in non-GABAergic transmission ( Figures 6 D–6F). Nevertheless, subsequent experiments were conducted in the presence of glutamate receptor antagonists. We used paired-pulse optic stimulation (λ = 470 nm) to determine whether fear conditioning leads to changes in GABA release from PV-INs onto neighboring principal neurons. Repeated-measures ANOVA revealed that PPR differed between conditions in the LA (repeated-measures ANOVA, main effect of group, F= 26.13, p < 0.001) but not in the BA. In LA, PPR was increased in paired, but not unpaired, animals relative to naive controls. This decrease in GABA release was at least partly specific to PV-IN inputs because IPSCs evoked by local field stimulation were not modulated by training ( Figures S6 A–S6C).

(G, I, M, and O) n/group indicated in graphs. Repeated-measures ANOVA followed by planned comparisons with Holm-Bonferroni. ∗ p < 0.05 naive versus paired, @ p < 0.05 paired versus unpaired. Data presented as mean ± SEM.

(M–P) PPR of PV-IN→PV-IN IPSCs was unaffected by training in the LA ([M], representative traces in [N]) and BA ([O], representative traces in [P]). IPSC recordings were conducted at 0 mV in order to exclude postsynaptic ChR2 currents based on their ionic reversal. Scales: ([N], [P]) 50 pA × 50 ms.

(L) Schematic of (M)—(P). Release probability at PV-IN→PV-IN synapses was assayed by paired-pulse optical stimulation (λ = 470 nm) during recording from tdTomato-positive PV-INs in LA ([M], [N]) and BA ([O], [P]).

(K) AAV-DIO-ChR2-eYFP was injected into the basolateral amygdala of PV-IRES-Cre mice crossed to ROSA-tdTomato reporter mice to target PV-INs for electrophysiological recordings without gating ChR2. Scale: 50 μm.

(I and J) PPRs were unaltered in BA (I). Representative traces in (J). Representative traces are shown at the 50 ms interstimulus interval. Scales: (H) 50 pA × 100 ms; (J) 60 pA × 100 ms.

(G) Paired pulse ratio (PPR) of PV-IN→PN IPSCs in LA was increased in mice that received paired training compared to unpaired and naive conditions. Representative traces in (H).

(E and F) Light-evoked currents were unaffected by the glutamate receptor antagonists APV and CNQX ([E], scale: 20 pA × 25 ms) and abolished by the GABA A receptor antagonist PTX ([F], scale: 100 pA × 50 ms). Data are normalized to pre-antagonist amplitudes.

(D) Recording configuration in (E)–(J). Release probability at PV-IN→principal neuron (PN) synapses was assayed by paired-pulse optic stimulation (λ = 470 nm) in LA ([G], [H]) and BA ([I], [J]).

(A) AAV-DIO-ChR2-eYFP was injected into the basolateral amygdala of PV-IRES-Cre mice for optogenetic-assisted in vitro slice electrophysiology. Scale: 100 μm.

GABAA receptor endocytosis in the basolateral amygdala is critical to the reinstatement of fear memory measured by fear-potentiated startle.

Training-induced changes in the expression of GABAA-associated genes in the amygdala after the acquisition and extinction of Pavlovian fear.

Regulation of gephyrin and GABAA receptor binding within the amygdala after fear acquisition and extinction.

We next measured PPR of oEPSCs to determine whether MGN and TeA afferent plasticity could account for learning-dependent changes in glutamate release ( Figure 4 ). Surprisingly, this revealed no effect of fear conditioning on PPR at MGN synapses onto LA PV-INs ( Figures 5 I and 5J). However, consistent with the results of electrical stimulation ( Figures 4 D–4I), PPR of TeA oEPSCs was increased in paired animals relative to both naive and unpaired controls in LA (repeated-measures ANOVA, main effect of group, F= 12.03, p = 0.0002) but not BA PV-INs ( Figures 5 K–5N). These data confirm the nucleus specificity of PV-IN afferent plasticity in the auditory cortical pathway but suggest that plasticity of internal capsule responses is not attributable to auditory thalamic inputs.

To investigate functional synaptic connectivity between MGN/TeA and amygdala PV-INs, we targeted these areas with injections of an AAV encoding eYFP-tagged channelrhodopsin 2 (ChR2-eYFP) driven by the CaMKII promoter in PV-IRES-Cre:Ai9 double transgenic mice, in which tdTomato expression is localized to PV-INs. Following these injections, eYFP+ terminals were observed in the basolateral amygdala complex. However, MGN terminals were largely restricted to LA, whereas TeA terminals could be observed in both LA and BA ( Figures 5 D and 5E). Terminal stimulation evoked polysynaptic EPSCs in PV-INs ( Figure 5 F), likely the result of strong local excitatory connections to PV-INs. To isolate monosynaptic currents, we blocked NMDA receptors with saturating CPP (10 μM) and AMPA receptors with subsaturating CNQX (1 μM). Resulting optic-evoked EPSCs (oEPSCs) could be completely abolished with saturating CNQX (10 μM; Figure 5 F). Consistent with anatomical labeling, blue-light stimulation of MGN terminals resulted in monosynaptic oEPSCs in the vast majority of LA PV-INs (36/41), but no responses were detected in BA PV-INs (0/10). In contrast, oEPSCs were observed in both LA (28/35) and BA (27/30) PV-INs during TeA terminal stimulation ( Figure 5 G). Onset latencies of oEPSCs were consistent with monosynaptic transmission ( Figure 5 H).

Although electrical stimulation of the internal and external capsules is presumed to recruit glutamatergic axons from sensory thalamus and cortex, respectively, we sought to characterize this circuitry using region- and cell-selective tools. To determine the presynaptic origins of PV-IN innervation, we conducted unbiased Cre-dependent monosynaptic circuit tracing (). AAVs encoding conditional expression of the TVA receptor and rabies glycoprotein (G) were unilaterally injected into the basolateral amygdala of PV-IRES-Cre mice, restricting subsequent infection of G-deleted rabies virus (RVdG) pseudotyped with the envelop protein A (EnvA) to PV-INs and their retrograde monosynaptic contacts ( Figure 5 A). In addition to other subcortical and cortical regions ( Figure S5 ), amygdala PV-INs received robust innervation from the medial geniculate nucleus (MGN) and the temporal association cortex (TeA) ( Figures 5 B and 5C). These regions are known to convey auditory input to amygdala, and their axon terminals are presumed to be involved in plasticity of internal and external capsule synaptic responses in amygdala excitatory neurons ().

(I, K, and M) Repeated-measures ANOVA followed by Holm-Bonferroni. n/group indicated on graphs. Data presented as mean ± SEM.

(K–N) PPR of oEPSCs at TeA→PV-IN synapses was increased in paired compared to unpaired and naive mice in LA ([K], representative traces in [L]) but not BA ([M], representative traces in [N]).

(G) Terminal stimulation elicited oEPSCs from both pathways onto LA PV-INs; only TeA oEPSCs were observed in BA PV-INs.

(F) Optic-evoked EPSCs (oEPSCs) were conducted in the presence of 10 μM CPP and 1 μM CNQX to prevent polysynaptic activity and isolate monosynaptic currents. oEPSCs were abolished by saturating CNQX (10 μM). Scales: 10 pA × 10 ms.

(D and E) AAV-CaMKII-ChR2-eYFP was injected into the MGN (D) and TeA (E) of PV-IRES-Cre mice crossed to ROSA-tdTomato reporter mice to target PV-INs for electrophysiological recordings without exciting ChR2. eYFP-positive terminal expression is shown in center panels with the boxed portion representing the enlarged images of basolateral amygdala to the right. Scales: low magnification, 500 μm; enlargements, 100 μm.

(B and C) eGFP-positive cell bodies were observed in temporal association cortex (TeA; [B], [C]) and the medial portion of the medial geniculate nucleus (MGN; [B]). The boxed portion of the MGN on left is enlarged in the center panel. MG dorsal (d), ventral (v), and medial (m); suprageniculate nucleus (SG); posterior limitans nucleus (PLi); posterior intralaminar nucleus (PIN). Scales: low magnification, 500 μm; enlargement, 100 μm.

(A) AAV-FLEX-TVA-mCherry and AAV-FLEX-RG (rabies glycoprotein) was unilaterally injected into the basolateral amygdala of PV-IRES-Cre mice to restrict subsequent expression of EnvA+RVdG-eGFP to PV-INs and their monosynaptic retrograde partners. Confocal images of the injection site (boxed portion on left) are enlarged in the right panels. Retrograde labeling only occurred from PV-INs that coexpressed mCherry (red) and eGFP (green; circles). Scales: low magnification, 500 μm; enlargements, 100 μm.

The above changes suggest that PV-INs are modulated by a nucleus-specific adjustment of their presynaptic input. Given robust excitation of PV-INs by amygdala afferents ( Figure 1 ), we questioned whether excitatory presynaptic plasticity could be attributed to these pathways. As an assay of glutamate release probability, we measured the paired pulse ratio (PPR) of EPSCs evoked by subcortical and cortical stimulation. Consistent with decreased release probability, PPR was increased at both subcortical (repeated-measures ANOVA, main effect of group, F= 26.23, p < 0.001) and cortical (repeated-measures ANOVA, main effect of group, F= 41.73, p < 0.001) synapses in LA of mice that received paired training compared to unpaired and naive controls ( Figures 4 A–4F). Interestingly, PPR was also decreased at cortical synapses after unpaired training ( Figure 4 E), but evidently this was not sufficient to enhance overall excitation onto LA PV-INs ( Figure 3 B). To determine whether presynaptic plasticity was nucleus specific, we measured PPR of cortical EPSCs in BA PV-INs. This revealed no change in glutamate release in trained animals ( Figures 4 G–4I), consistent with a lack of modulation of mEPSC frequency by learning in BA PV-INs ( Figure 3 I).

(G–I) PPR of cortical EPSCs was not altered by fear conditioning in BA PV-INs. Representative traces in (I). Scales: 80 pA × 100 ms. n/group indicated on graphs. Repeated-measures ANOVA followed by Holm-Bonferroni, ∗ p < 0.05 naive versus paired, @ p < 0.05 paired versus unpaired, and # p < 0.05 naive versus unpaired. Data presented as mean ± SEM.

(D–F) PPR of cortical EPSCs was increased in LA PV-INs in mice that received paired training compared to both naive and unpaired conditions. Representative traces in (F).

(A–C) Paired-pulse ratio (PPR; EPSC 2 /EPSC 1 ) of subcortical EPSCs was increased in LA PV-INs from mice that received paired training compared to both unpaired and naive conditions. Representative traces in (C).

Release probability of excitatory subcortical and cortical inputs to PV-INs was assayed by paired-pulse stimulation of the internal (A) and external ([D], [G]) capsules, respectively.

To determine whether fear learning alters PV-IN properties, we conducted whole-cell recordings in PV-INs after auditory fear conditioning, which entailed paired or unpaired presentations of an auditory tone (CS) and footshock (US). We confirmed that auditory fear was specific to the paired condition, since CS-evoked freezing was observed 24 hr later in mice that received paired but not unpaired training ( Figure S3 ). All electrophysiological recordings were obtained 24 hr after training from a different set of animals in which retrieval was omitted in order to exclude the possibility of memory extinction or reconsolidation. An analysis of miniature (m) EPSCs and mIPSCs from paired, unpaired, and naive mice at this time point revealed nucleus-specific alterations in PV-INs that were correlated with auditory fear encoding ( Figure 3 ). The frequency of mEPSCs differed between conditions in LA (one-way ANOVA, F= 3.78, p = 0.04) but not in BA (one-way ANOVA, F= 2.34, p = 0.11; Figures 3 B–3D and 3I–3K). In LA PV-INs, mEPSC frequency was reduced in paired (p = 0.008), but not in unpaired (p = 0.74), mice compared to naive controls. In contrast, the frequency of mIPSCs differed between conditions in BA (one-way ANOVA, F= 4.42, p = 0.02) but not in LA (one-way ANOVA, F= 0.31, p = 0.74; Figures 3 E–3G and 3L–3N). In BA PV-INs, mIPSC frequency was increased in paired (p = 0.03), but not unpaired (p = 0.48), mice relative to naive controls. Fear conditioning did not affect the amplitude or kinetics of mEPSCs or mIPSCs. However, independent of fear conditioning, these properties were strongly affected by nucleus location of PV-INs ( Figure S4 ).

(L–N) The frequency (L) but not amplitude (M) of mIPSCs (representative traces in [N]) was increased after paired, but not unpaired, training compared to the naive condition. Scales: ([D], [G], [K]) 10 pA × 100 ms; [N] 20 pA × 100 ms. n/group indicated on bar histograms. One-way ANOVA followed by Fisher’s LSD, ∗ p < 0.05 naive versus paired, @ p < 0.05 paired versus unpaired. Data presented as mean ± SEM.

(I–K) The frequency (I) and amplitude (J) of mEPSCs (representative traces in K) in BA PV-INs were unaffected by training.

(B–D) Frequency (B) but not amplitude (C) of mEPSCs (representative traces in [D]) was reduced in LA PV-INs of mice that received paired, but not unpaired, training as compared to naive mice.

Miniature postsynaptic currents were recorded in PV-INs with a low chloride internal solution, allowing for isolation of EPSCs and IPSCs at −60 mV and 0 mV, respectively.

Consistent with our observation of rebound action currents in eArch3.0-expressing cells ( Figure S2 C), we also observed corresponding rebound IPSCs in principal neurons at LED offset ( Figures 2 E, 2G, and 2J). Interestingly, we found that peak rebound amplitude positively correlated with reduction of feedforward IPSC amplitude by LED illumination ( Figure 2 H; linear regression, F= 29.09, p < 0.0001). Since rebound amplitude partly reflects the number of viable eArch3.0-infected PV-INs in our recording field, this further indicates that PV-INs are readily engaged in feedforward inhibition in the LA.

Having validated our approach, we patched onto principal neurons in mice with conditional eArch3.0 expression in PV-INs and recorded feedforward IPSCs evoked by subcortical or cortical afferent stimulation in the presence or absence of yellow light ( Figure 2 A). Feedforward IPSCs from both pathways were attenuated by silencing PV-INs in LA ( Figures 2 D–2G; paired samples t test, subcortical: t= 3.59, p = 0.003; cortical: t= 2.57, p = 0.03). Consistent with low cortical excitatory drive onto BA PV-INs ( Figure 1 L), PV-IN silencing did not affect the amplitude of feedforward IPSCs in this nucleus ( Figures 2 I and 2J; paired samples t test, t= 0.05, p = 0.96).

To evoke monosynaptic EPSCs and feedforward IPSCs, principal neurons were clamped at membrane potentials of −70 mV and 0 mV, respectively, during stimulation of subcortical and cortical afferents. Feedforward IPSCs were abolished by the GABAreceptor antagonist picrotoxin as well as the AMPA/kainate receptor antagonist CNQX ( Figure 2 B). In addition, the onset latency of feedforward IPSCs was delayed relative to EPSCs (independent samples t test, t= 11.01, p < 0.0001), consistent with a disynaptic circuit mechanism ( Figure 2 C).

(H) Linear regression. ∗ p < 0.05, ∗∗ p < 0.005, and ∗∗∗ p < 0.0005. Data in (D), (F), and (I) are normalized to IPSC amplitude in the absence of LED stimulation. Data presented as mean ± SEM.

(I) PV-IN silencing did not affect the amplitude of disynaptic IPSCs evoked from cortical afferent stimulation in BA, representative traces in (J). Scales in (E), (G), and (J): 50 pA × 100 ms. n/group indicated on graphs.

(H) Reduction of disynaptic IPSC peak amplitude is correlated with the rebound IPSC amplitude generated at LED offset in LA.

(D–G) Silencing PV-INs attenuated the amplitude of disynaptic IPSCs evoked from subcortical ([D], representative traces in [E]) and cortical ([F], representative traces in [G]) afferent stimulation. Evoked IPSCs were blocked by PTX.

(B) Example traces of monosynaptic excitatory (red trace; −70 mV) and disynaptic inhibitory (black trace; 0 mV) postsynaptic currents evoked from cortical afferents. Disynaptic IPSCs are blocked by the GABA A receptor antagonist picrotoxin (PTX; purple trace, left) and the AMPA/kainate receptor antagonist CNQX (blue trace, right). Scales: 70 pA × 20 ms.

(A) Experimental design in (D)–(J). Subcortical ([D], [E]) and cortical (LA: [F], [G]; BA: [I], [J]) afferents were electrically stimulated to obtain monosynaptic EPSCs and disynaptic IPSCs in LA PNs during voltage clamp at −70 mV and 0 mV, respectively. Electrical stimulation alternated between light-off (n = 3) and light-on (n = 2) epochs (n = 10 sweeps per epoch).

Given the potent excitatory drive onto LA PV-INs, we hypothesized that PV-INs may generate greater feedforward inhibition onto principal neurons in LA compared to BA. To test this hypothesis, we harnessed the power of optogenetics to silence PV-INs during afferent stimulation while recording feedforward IPSCs. Conditional expression of the GFP-tagged enhanced light-driven inhibitory proton pump Archaerhodopsin-3.0 (eArch3.0;) in PV-IRES-Cre mice resulted in GFP expression in both the soma and axonal arbors of PV-INs ( Figures S2 A and S2B). Whole-cell recordings in eArch3.0-expressing cells confirmed yellow light-induced (λ = 590 nm) neuronal silencing ( Figures S2 C–S2E). Notably, PV-INs escaped voltage clamp and generated rebound action currents following light offset at every stimulus intensity ( Figure S2 C).

To determine whether differences in PV-IN anatomy could account for nucleus-specific excitatory drive, we performed morphological reconstructions of biocytin-filled PV-INs ( Figure 1 N). While soma and primary dendrite characteristics were comparable between nuclei, PV-INs in LA possessed twice as many dendritic branches (t= 3.09, p = 0.006) as those in BA, resulting in increased dendritic length (t= 2.27, p = 0.04; Figure 1 O). Furthermore, truncated dendrites were very infrequent and the total number of such artifacts was similar between nuclei (LA = 4; BA = 3), indicating that the observed differences in physiology and morphology were not attributable to slice preparation.

During classical conditioning, a benign auditory conditioned stimulus (CS) is paired with a naturally aversive unconditioned stimulus (US), forming an associative memory that links the CS and US. In vivo studies have found that amygdala PV-INs respond differentially to sensory stimulation with increased firing to auditory stimuli () and decreased firing to footshock () and other noxious stimuli (). However, it is unknown whether PV-INs are directly modulated by afferent pathways conveying CS activity. To address this question, we performed whole-cell recordings from PV-INs and neighboring principal neurons during stimulation of subcortical and cortical sensory afferent pathways, which traverse the internal and external capsules, respectively. We first investigated the input/output (I/O) relationship of compound postsynaptic currents consisting of monosynaptic excitatory postsynaptic currents (EPSCs) and disynaptic feedforward inhibitory postsynaptic currents (IPSCs) at increasing stimulus intensities ( Figures 1 B–1K). While there was no difference in the I/O slope of IPSCs between populations, the I/O relation of monosynaptic EPSCs from both subcortical and cortical afferents was far steeper in LA PV-INs compared to their glutamatergic neighbors ( Figure 1 G; two-way ANOVA, main effect of neuron type, F= 30.89, p < 0.0001), indicating far greater excitatory drive from these pathways in PV-INs. While converging excitation from the subcortical and cortical pathways to the LA is hypothesized to be a key mediator of learning-induced synaptic plasticity (), cortical neurons also send axon collaterals to BA (). To determine whether these inputs exhibit similar potency, we investigated the I/O relationship of BA cortical synaptic currents ( Figures 1 H–1K). Unlike in LA, PV-INs and principal neurons in BA exhibited similar I/O slope of both monosynaptic EPSCs and feedforward IPSCs ( Figure 1 K). We then calculated an index of I/O slopes (calculated as I/O slope/ I/O slope) as a measure of E:I balance for LA and BA neurons. PV-INs in LA exhibited greater E:I index compared to PV-INs in BA as well as to principal neurons in both nuclei ( Figure 1 L; two-way ANOVA, main effect of neuron type, F= 27.13, p < 0.0001; main effect of nucleus, F= 3.76, p = 0.03; interaction, F= 6.47, p = 0.004). Importantly, EPSC onset latency was similar across cells and nuclei ( Figure 1 M), indicating that these responses shared a monosynaptic mechanism.

To selectively target PV-INs for in vitro electrophysiology, we crossed R26-STOP-eYFP reporter mice to the PV-IRES-Cre driver line to selectively express enhanced yellow fluorescent protein (eYFP) in PV-INs. Given a previous report that only ∼60% of PV-positive cells in the basolateral amygdala co-express GABA (), we sought to determine the specificity of Cre-mediated recombination by quantifying double immunofluorescence staining with antibodies against parvalbumin and GABA ( Figures 1 A and S1 ). More than 90% of eYFP-positive cells in the basolateral amygdala co-expressed both PV and GABA ( Figure S1 ), demonstrating that this Cre line is highly selective for GABAergic PV-INs in this brain region.

(B–M) Membrane potential was clamped at −50 mV for all recordings. n/group indicated on bar histogram in (M) ([C]–[M]) and (O).

(B–D) Lateral amygdala PV-INs and neighboring principal neurons (PNs) respond to subcortical (C) and cortical (D) afferent stimulation with distinct biphasic synaptic responses, corresponding to monosynaptic EPSCs followed by disynaptic IPSCs. Scales: 500 pA × 40 ms.

(A) To identify PV-INs for electrophysiological targeting, we crossed PV-IRES-Cre mice to R26-stop-eYFP reporter mice to express eYFP specifically in PV-INs. Double immunofluorescence staining with antibodies against PV (blue) and GABA (red) revealed that Cre-dependent eYFP (green) expression selectively labels PV-positive GABAergic neurons in the basolateral complex. Scales: left, 100 μm; right, 20 μm.

Discussion

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Lüthi A. Dopamine gates LTP induction in lateral amygdala by suppressing feedforward inhibition. On the other hand, recently described effects of neuromodulators on PV-IN release properties suggest their potential involvement in fear-related inhibitory plasticity. Dopaminergic terminals form dense perisomatic synapses onto amygdala PV-INs (), and ex vivo dopamine application acts through D2 receptors to reduce GABA release from PV-INs (). Similar to fear conditioning ( Figure 6 ), dopamine effects are manifested at PV-IN synapses onto principal cells but not interneurons. In addition, D2 receptor activation reduces the frequency of sIPSCs as well as the magnitude of principal neuron feedforward inhibition in LA (). Thus, dopamine release may be a key mechanism leading to PV-IN plasticity and corresponding amygdala disinhibition.