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. Author manuscript; available in PMC: 2017 Dec 4.
Published in final edited form as: Cell Rep. 2017 Nov 7;21(6):1426–1433. doi: 10.1016/j.celrep.2017.10.046

Prefrontal cortex drives distinct projection neurons in the basolateral amygdala

Laura M McGarry 1, Adam G Carter 1,*
PMCID: PMC5714295  NIHMSID: NIHMS917730  PMID: 29117549

SUMMARY

The prefrontal cortex (PFC) regulates emotional behavior via top-down control of the basolateral amygdala (BLA). However, the influence of PFC inputs on the different projection pathways within the BLA remains largely unexplored. Here we combine whole-cell recordings and optogenetics to study these cell-type specific connections in the mouse BLA. We characterize PFC inputs onto three distinct populations of BLA neurons that project to either the PFC, ventral hippocampus or Nucleus Accumbens. We find that PFC-evoked synaptic responses are strongest at amygdala-cortical and amygdala-hippocampal neurons, and much weaker at amygdala-striatal neurons. We assess the mechanisms for this targeting, and conclude that it reflects fewer connections onto amygdala-striatal neurons. Given the similar intrinsic properties of these cells, this connectivity allows the PFC to preferentially activate amygdala-cortical and amygdala-hippocampal neurons. Together, our findings reveal how PFC inputs to the BLA selectively drive feed-back projections to the PFC and feed-forward projections to the hippocampus.

Keywords: prefrontal cortex, basolateral amygdala, circuit, synapse, projection neuron

eTOC blurb

McGarry and Carter report that the prefrontal cortex (PFC) contacts three distinct populations of projection neurons in the BLA, with the strongest inputs onto neurons that project back to PFC and forward to hippocampus, and weakest inputs onto neurons that project to the striatum.

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INTRODUCTION

The basolateral amygdala (BLA) is responsible for directing adaptive behaviors in response to internal and external cues. The BLA exerts this influence via long-range projections to other brain regions, including the prefrontal cortex (PFC), ventral hippocampus (vHPC) and Nucleus Accumbens (NAc) (Pitkanen et al., 2000). Recent studies highlight distinct roles for these pathways in emotional and motivated behaviors (Janak and Tye, 2015). For example, projections to PFC and vHPC are important for fear (Herry et al., 2008; Senn et al., 2014) and anxiety (Felix-Ortiz et al., 2013; Felix-Ortiz et al., 2016), whereas projections to the NAc are involved in reward processing (Ambroggi et al., 2008; Beyeler et al., 2016; Namburi et al., 2015; Stuber et al., 2011).

Different output pathways ultimately derive from multiple populations of projection neurons located in the BLA. In the cerebral cortex, distinct projection neurons segregate into individual layers and often possess unique intrinsic and synaptic properties (Brown and Hestrin, 2009; Hattox and Nelson, 2007; Little and Carter, 2013; Morishima and Kawaguchi, 2006), but whether equivalent diversity exists for projection neurons in the BLA remains poorly understood. Recent studies indicate that different projection neurons can be distinctly regulated by local inhibitory circuits in the BLA (Vogel et al., 2016). However, it is not known whether projection neurons are also uniquely targeted by long-range excitatory inputs from other brain regions (Pitkanen et al., 2000). Consequently, it remains unclear how output pathways from the BLA can be selectively activated to guide different types of behaviors (Janak and Tye, 2015; LeDoux, 2000).

The PFC is a major input to the BLA, where it exerts powerful top-down control on the learning and expression of emotional behavior (Sotres-Bayon and Quirk, 2010). While the PFC engages inhibitory circuits in the amygdala (Arruda-Carvalho and Clem, 2014; Cho et al., 2013; Hubner et al., 2014; Quirk et al., 2003), it also makes direct, excitatory synapses onto projection neurons in the BLA (Arruda-Carvalho and Clem, 2014; Brinley-Reed et al., 1995; Cho et al., 2013; Hubner et al., 2014; Likhtik et al., 2005). Recently, inputs from the BLA have been shown to preferentially contact reciprocally connected neurons in the PFC (Little and Carter, 2013; McGarry and Carter, 2016). One untested possibility is that PFC inputs also selectively contact neurons in the BLA that project back to the PFC. This kind of preferential targeting could allow for the precise and direct routing of functional information between these two interconnected brain regions.

Here we investigate how PFC inputs engage different projection neurons in the BLA. We find that projections to the PFC, vHPC or NAc are mediated by distinct populations of neurons. In the dorsomedial BLA, cells projecting to PFC and vHPC receive strong PFC input, while those projecting to NAc receive only weak input. This targeting is primarily due to differences in the number of synaptic connections made onto the different cell types. Because these projection neurons have similar intrinsic properties, this connectivity allows the PFC to drive amygdala-cortical and amygdala-hippocampal circuits. Together, our findings reveal cell-type specific connectivity rules linking the PFC and BLA.

RESULTS

PFC contacts projection neurons in the dorsomedial BLA

We examined how PFC inputs engage different populations of projection neurons within the BLA. We first used anterograde and retrograde anatomy to define the connections between these brain regions. We injected AAV-mCherry into the BLA, and identified strong axonal projections to the prelimbic (PL) PFC (Fig. S1A–C). We then injected AAV-mCherry and Cholera Toxin B (CTB)-488 into the PL PFC, and found striking overlap of PFC axons and reciprocally connected amygdala-cortical (AC) neurons in the BLA (Fig. 1A). By examining fluorescence profiles along multiple axes within the BLA (Fig. S2A), we determined that both PFC axons and AC neurons are primarily concentrated in the anterior dorsomedial basal nucleus (Fig. 1A, S2B). These findings suggest that PFC axons are well positioned to contact neurons in the BLA that in turn project back to PFC.

Figure 1. PFC axons overlap with multiple projection neurons in the BLA.

Figure 1

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A) Left, Injections into the PL PFC to label axons and AC neurons in the BLA. Axes apply to all images. Right, Injection of AAV-mCherry (red) and CTB-488 (green) in the PL PFC (top left) labels PFC axons (red) and AC neurons (green) in the BLA (bottom middle), with expanded view (far right). DAPI staining in grey.

B) Average PFC-evoked AMPA-R EPSC at AC neurons before (green) and after (black) wash-in of NBQX. Arrowhead indicates time of light pulse.

C) Left, Dual injections of CTB into the PFC and NAc. Right, Labeling of AC (green) and AS (purple) neurons in the BLA.

D) Left, Dual injections of CTB into the PFC and vHPC. Right, Labeling of AC (green) and AH (orange) neurons in the BLA.

E) Left, Average PFC-evoked AMPA-R EPSC at pairs of AS neurons located in the medial (purple) and lateral (black) BLA. Right, Summary of EPSC amplitudes at AS neurons located in medial (M) or lateral (L) BLA.

F) Left, Average PFC-evoked AMPA-R EPSC at AH neurons located in the medial (orange) and lateral (black) BLA. Right, Summary of EPSC amplitudes at AH neurons located in medial (M) or lateral (L) BLA.

Our anatomy suggests that PFC axons may directly contact AC neurons, and we next used ex vivo physiology to explore the functional properties of these connections. We injected AAV-ChR2-eYFP and retrobeads into the PFC, waited for expression and transport, and prepared acute slices of the BLA, which severs axons but preserves their presynaptic terminals (Petreanu et al., 2007). We stimulated PFC inputs by activating ChR2 with wide-field illumination, isolating monosynaptic connections with TTX (1 mM), 4-AP (100 μM) and elevated external Ca2+ (4 mM) (Little and Carter, 2012; Petreanu et al., 2009). Recording at −70 mV, we observed short-latency EPSCs (170 ± 58 pA; n = 7 cells, 6 animals), which were completely blocked by the AMPA-R antagonist NBQX (10 μM) (3 ± 1 pA) (Fig. 1B). These results indicate that PFC axons make monosynaptic, excitatory connections onto AC neurons located within the dorsomedial BLA.

The BLA also sends long-range projections to other brain regions important for emotional and motivated behaviors, including the NAc and vHPC (McDonald, 1991; Pikkarainen et al., 1999) (Fig. S1A–C). To determine the locations of corresponding projection neurons in the BLA, and their overlap with AC neurons, we next performed dual-retrograde injections into two brain regions (Fig. 1C, 1D & S3). Injecting CTB into PFC and NAc, we found that amygdala-striatal (AS) neurons are distributed more widely in the amygdala, intermingled with AC neurons in the medial BLA, but also extending further into the lateral BLA, and dorsally into the lateral amygdala (LA) (Fig. 1C, S3A–B). Injecting CTB into PFC and vHPC, we found amygdala-hippocampal (AH) neurons also have distinct topography, intermingled with AC neurons in the medial BLA, and present in lateral BLA (Fig. 1D, S3C–D). We found minimal co-labeling of either AC and AS neurons (1.7 ± 0.2% co-labeled cells; n = 9 slices, 3 animals) or AC and AH neurons (1.4 ± 0.4% co-labeled cells; n = 9 slices, 3 animals) (Fig. S3G). In contrast, injection of two CTBs into a single brain region yielded a high degree of overlap (Fig. S3E–G) (PFC injection: 90.2 ± 1.1% co-labeled AC neurons; n = 9 slices, 3 animals), demonstrating high reliability of retrograde labeling.

While AC neurons overlap with PFC axons in the medial BLA, AS and AH neurons are also found in the lateral BLA, which is largely devoid of PFC axons. This anatomy suggests that AS and AH neurons in the medial BLA may be uniquely positioned to receive PFC inputs. To test this idea, we next injected retrobeads in the NAc or vHPC and AAV-ChR2-YFP in the PFC, and recorded PFC-evoked responses at pairs of AS or AH neurons in medial and lateral BLA. Wide-field illumination elicited pronounced AMPA-R EPSCs at AS neurons located close to the medial edge of the BLA (<400 μm), but not at those neurons located more laterally in the same slice (medial EPSC = 73 ± 34 pA; lateral EPSC = 6 ± 2 pA; p = 0.02; n = 7 pairs, 4 animals) (Fig. 1E). AH neurons showed a similar location dependence of PFC-evoked EPSCs (medial EPSC = 314 ± 90 pA; lateral EPSC = 8 ± 1 pA; p = 0.02; n = 7 pairs, 4 animals) (Fig. 1F). Together, these results indicate that PFC connections are primarily restricted to projection neurons located in the medial BLA.

Cell-type specific connectivity of PFC input onto BLA projection neurons

Our results show that AC, AS and AH neurons are found in the medial BLA, where they can be directly contacted by PFC inputs. We next asked if the PFC makes biased connections onto these three neighboring classes of BLA projection neurons. We first assessed dendritic morphology at these cells to determine if they have equal ability to sample PFC inputs. Targeted whole-cell recordings from projection neurons, combined with 2-photon reconstructions, indicated that AC, AS and AH neurons have spiny dendritic branches emerging from the soma and extending radially (Fig. 2A, S4A). These neurons have similar dendritic length distributions (effect of cell type p = 0.2, effect of Sholl radius distance p <0.0001, interaction p = 0.97; n = 7 neurons per group, 21 animals) (Fig. 2B) and total dendritic length (p = 0.1) (Fig. S4B) across projection class and region of the BLA. These anatomical findings suggest that AC, AS and AH neurons in the medial BLA have equivalent axo-dendritic overlap with PFC inputs.

Figure 2. PFC inputs are strongest onto AC and AH neurons.

Figure 2

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A) Dendritic reconstructions of AC (green), AS (purple) and AH (orange) neurons located in the medial BLA.

B) Summary of dendritic Sholl analysis for the five types of neurons.

C) Average PFC-evoked AMPA-R and NMDA-R EPSCs at pairs of neighboring AC (green) and AS (purple) neurons.

D) Summary of AS/AC amplitude ratio (left), and AMPA/NMDA ratio (right) for PFC-evoked EPSCs at AC and AS neurons.

E–F) Similar to (C–D) for AC (green) and AH (orange) neurons.

* = p < 0.05.

To test if the PFC makes selective connections onto AC, AS or AH neurons, we next recorded from pairs of neighboring AC:AS or AC:AH neurons in the medial BLA. In these experiments, we injected AAV-ChR2-eYFP in the PFC, and colored retrobeads in the PFC and either NAc or vHPC. In targeted recordings from labeled neurons, we measured both AMPA-R EPSCs at −70 mV and NMDA-R EPSCs at +40 mV. By comparing the EPSC amplitude ratios at pairs of cells, we were able to control for variability in viral injections and expression between slices and animals (Little and Carter, 2013; MacAskill et al., 2014; McGarry and Carter, 2016). We found that both AMPA-R and NMDA-R EPSCs are much larger at AC neurons than AS neurons (AS/AC ratio: AMPA-R = 0.29, 95% CI = 0.14–0.61, p = 0.004; NMDA-R = 0.38, 95% CI = 0.2–0.74, p = 0.008; n = 9 pairs, 7 animals) (Fig. 2C, D). However, the AMPA/NMDA ratio is similar at these two cell types (AC = 7.0 ± 0.9; AS = 5.6 ± 1.5; p = 0.16) (Fig. 2D). In contrast, we observed that AMPA-R and NMDA-R EPSCs were largely equivalent for AC and AH neurons (AH/AC ratio: AMPA-R EPSC = 0.84, 95% CI = 0.48–1.49, p = 0.8; NMDA-R EPSC = 0.72, 95% CI = 0.41–1.27, p = 0.6; n = 7 pairs, 5 animals) (Fig. 2E, F), as was their associated AMPA/NMDA ratio (AC = 6.2 ± 2.0; AH = 6.3 ± 1.3; p = 0.8) (Fig. 2F). Together, these results reveal that PFC inputs selectively target different populations of projection neurons in the medial BLA, making stronger connections onto AC and AH neurons than intermingled AS neurons.

Mechanisms underlying differential synaptic strength

In principle, both postsynaptic and presynaptic mechanisms could contribute to this cell-type specific connectivity (Gil et al., 1999; Zucker and Regehr, 2002). To assess these factors, we first recorded PFC-evoked asynchronous release by replacing extracellular calcium (Ca2+) with strontium (Sr2+) in the presence of TTX (1μM) (Fig. 3A). The resulting input-specific quantal EPSCs (qEPSCs) provide a direct measure of unitary synaptic strength (amplitude) and an indirect indication of number of connections and presynaptic properties (frequency) (Goda and Stevens, 1994; Little and Carter, 2013; MacAskill et al., 2014). In the absence of stimulation, baseline miniature EPSC (mEPSC) frequency was similar at AC, AS and AH neurons in medial BLA (AS = 2.0 ± 0.3 Hz, n = 8 cells; AC = 1.7 ± 0.2 Hz, n = 15 cells; AH = 1.8 ± 0.3 Hz, n =7 cells; p = 0.2). Optogenetic stimulation of PFC axons evoked asynchronous release, elevating the frequency of qEPSCs at each cell type (AS = 4.5 ± 0.6 Hz; AC = 6.5 ± 0.5 Hz; AH = 7.9 ± 1.1 Hz). However, the increase in qEPSC frequency was much lower at AS neurons compared to AC neurons (AS/AC ratio = 0.51, 95% CI = 0.3–0.89, p = 0.03; n = 8 pairs, 5 animals). In contrast, this change was similar at AH and AC neurons (AH/AC ratio = 1.14, 95% CI = 0.74–1.77, p = 0.4; n = 7 pairs, 5 animals) (Fig. 3B, C). Furthermore, we found no differences in the amplitude of PFC-evoked qEPSCs at pairs of either AC and AS neurons (AS/AC amplitude ratio = 0.94, 95% CI = 0.8–1.09, p = 0.3), or AC and AH neurons (AH/AC amplitude ratio = 1.04, 95% CI = 0.77–1.41, p = 0.5) (Fig. 3B, C). These findings indicate that PFC afferents make fewer connections onto AS neurons compared to nearby AC and AH neurons, whereas the unitary strength is broadly similar at these different cell types.

Figure 3. Differential synaptic strength reflects number of connections.

Figure 3

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A) Top, PFC-evoked qEPSCs recorded in the presences of strontium (Sr2+) at AS (purple), AC (green) and AH (orange) neurons, where asterisks indicate detected qEPSCs. Arrowheads indicate time of light pulses. Bottom, Histograms for example cells showing detected post-stimulation qEPSCs across all trials.

B) Summary of PFC-evoked qEPSC ∆ frequency (left) and amplitude (right) at AS, AC and AH neurons. Lines indicate pairs of recorded neurons.

C) Summary of AS/AC (left) and AH/AC (right) ratios for PFC-evoked qEPSC ∆ frequency and amplitude.

D) Left, Average PFC-evoked AMPA-R EPSCs evoked by trains of inputs (5 pulses at 20 Hz) at AC (green) and AS (purple) neurons. Right, Summary of paired pulse ratio (PPR = EPSCN/EPSC1) during trains.

E) Similar to (D) for AC (green) and AH (orange) neurons.

* = p < 0.05.

In addition to unitary strength and synapse number, presynaptic properties can contribute to differences in synaptic strength. We next used repetitive light-evoked stimulation of PFC inputs to study short-term synaptic plasticity at these neurons (Zucker and Regehr, 2002). We performed experiments in the absence of TTX and 4-AP, to preserve presynaptic action potentials (APs). In voltage-clamp recordings at −70 mV, we found that brief trains (5 pulses, 20 Hz) elicited depressing AMPA-R EPSCs at pairs of AC and AS neurons (n = 7 pairs, 5 animals) (Fig. 3D). Consistent with our findings in TTX and 4-AP, we again found that EPSCs were larger at AC neurons (Effect of cell type on amplitude, p = 0.02; post-test p < 0.0001 for each EPSC). Moreover, depression was equivalent at AC and AS neurons, yielding a constant AS/AC amplitude ratio throughout the duration of the trains (Effect of cell type on PPR, p = 0.48). We found that brief trains evoke similarly depressing AMPA-R EPSCs (Effect of cell type on PPR, p = 0.64) of equivalent amplitude (Effect of cell type on amplitude, p = 0.95) at AC and AH neurons (n = 8 pairs, 5 animals) (Fig. 3E). These results indicate that presynaptic properties are similar at these cells, supporting the idea that the number of connections is primarily responsible for differences in synaptic strength.

PFC inputs preferentially drive AC and AH neuron firing

Having assessed the mechanisms underlying targeting of PFC inputs, we next explored the consequences on AP firing at these projection neurons. As AP firing depends on both synaptic strength and cellular excitability, we first examined the intrinsic properties of AC, AS and AH neurons. In current-clamp recordings from all three cell types, we observed adapting trains of APs (Fig. 4A), as previously described for unspecified BLA pyramidal neurons (Washburn and Moises, 1992), with identical F–I curves (Fig. 4B). Overall, the active and passive properties are similar between cell types, with minimal differences across projection targets or locations in the BLA (Fig. S4C). Together, these results suggest that AC, AS and AH neurons can similarly transform synaptic input to AP firing.

Figure 4. PFC inputs preferentially drive firing of AC and AH neurons.

Figure 4

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A) AP firing and hyperpolarization of AC (green), AS (purple) and AH (orange) neurons located in the medial BLA in response to +200 pA and −50 pA current steps in the presence of synaptic blockers.

B) Firing vs current (F–I) curves for the five types of neurons.

C) Left, PFC-evoked EPSPs and APs at neighboring AC (green) and AS (purple) neurons. Example traces to light duration of 4 ms shown. Arrowhead indicates time of light pulse. Right, Probability of AP firing versus light duration.

D) Similar to (C) for AC (green) and AH (orange) neurons.

E) Left, PFC-evoked EPSPs and APs at neighboring AC (green) and AS (purple) neurons in response to trains (5 pulses at 20 Hz). Example traces to 4 ms light duration shown. Right, Probability of AP firing versus pulse number.

F) Similar to (E) for AC (green) and AH (orange) neurons.

* = p < 0.05.

To assess how PFC inputs drive firing at different projection neurons, we next combined optogenetic stimulation with current-clamp recordings in the absence of TTX and 4-AP. In pairs of nearby neurons in medial BLA, we found that PFC inputs readily drive AC neurons to fire APs, with greater probability at increased stimulation, whereas AS neurons remain sub-threshold, with a very low probability of AP firing even with maximum stimulation (Effect of cell type p = 0.0004; post-test: 0.5ms duration p = 0.7, 1ms duration p = 0.21, all other durations p < 0.0005; n = 7 pairs, 5 animals) (Fig. 4C). In contrast, we observed that PFC inputs readily drive both AC and AH neurons, with a similar probability of AP firing across a range of illumination (Effect of cell type p = 0.65; n = 7 pairs, 6 animals) (Fig. 4D). These findings confirm that AC and AH neurons in medial BLA receive stronger PFC input than AS neurons, and that PFC inputs are capable of driving firing of AC and AH neurons.

Our voltage-clamp recordings also indicated that PFC synapses exhibit robust short-term depression at all three projection neurons in the BLA. To study the impact of presynaptic dynamics on AP firing, we also measured responses to light-evoked trains of PFC input. In pairs of AC and AS neurons, we found that brief trains (5 pulses at 20Hz) elicit depressing EPSPs, which initially trigger AP firing in AC neurons, but rarely in AS neurons (Effect of cell type p = 0.0014; post-test: pulse 1–4 p < 0.0001, pulse 5 p = 0.002; n = 7 pairs, 5 animals) (Fig. 4E). In contrast, at pairs of AC and AH neurons, we found equivalent trains initially trigger AP firing in both neurons, with a similar decrease in probability throughout the train (Effect of cell type p = 0.2; n = 7 pairs, 6 animals) (Fig. 4F). Together, these results demonstrate that PFC inputs preferentially target reciprocal projections from the BLA as well as BLA projections to the vHPC, but minimally impact BLA projections to the NAc.

DISCUSSION

We have determined the properties of connections from the PFC onto different projection neurons in the BLA. We found that neurons projecting to the PFC, vHPC and NAc represent distinct populations with unique distributions in the BLA. All three cell types in the medial BLA receive excitatory, monosynaptic PFC input, but the input is much weaker at AS neurons. This difference is due to fewer connections onto AS neurons, while unitary strength and presynaptic properties are similar. Because the intrinsic excitability of these neurons is identical, PFC inputs can drive robust AP firing in AC and AH neurons but not AS neurons. Our findings describe cell-type specific connectivity for prefrontal control of the BLA, which is essential to understanding interactions between these brain regions.

The BLA is involved in diverse emotional and motivational behaviors due to its projections throughout the brain (Janak and Tye, 2015). Despite recent studies on the behavioral roles of BLA projections, it was unclear if they originated from distinct cell types. We examined the amount of overlap between projection neurons using a dual retrograde labeling approach (Little and Carter, 2013). Our findings demonstrate that AC, AS and AH neurons are three largely separate populations, which have indistinguishable physiological properties, but occupy unique locations in the BLA. While AC neurons are restricted to medial BLA, AH and AS neurons are present in both medial and lateral BLA, and AS neurons are also abundant in the LA. Interestingly, we also observed a small percentage of neurons that project to the PFC and either NAc or vHPC (<2%), consistent with findings of axon collateralization from AS and AH neurons (Beyeler et al., 2016). Further studies are needed to determine if these widely-projecting neurons have unique functional roles.

Projections from the PFC to the BLA participate in the top-down control of emotional behaviors (Sotres-Bayon and Quirk, 2010). We found that PFC axons are concentrated in the medial BLA, but largely restricted from the lateral BLA and the LA (McDonald et al., 1996). The distribution of PFC axons closely overlaps with AC neurons, onto whom they make strong connections. PFC inputs also make connections onto AH and AS neurons, but only in the medial BLA and not in the lateral BLA. Within the medial BLA, PFC inputs make cell-type specific connections, and are much stronger at AC and AH neurons. Importantly, this preferential connectivity was not predicted by anatomy, as PFC axons overlap with all three cell types. These results also demonstrate that the BLA consists of multiple zones, which receive unique inputs and make distinct outputs, akin to the different layers found in cerebral cortex. In the future, it will be interesting to examine how these zones map onto genetic cell types in the BLA (Kim et al., 2016; McCullough et al., 2016). It will also be important to compare inputs from more ventral, infralimbic (IL) PFC, which are known to have different functional roles in emotional behavior. Together, our results suggest the presence of a fine scale functional anatomy of the BLA, which is likely to be important for long-range and local circuits and their functional roles.

Several of our experiments provide mechanistic insight into why synaptic responses are smaller onto AS neurons compared to AC and AH neurons in the medial BLA. First, the frequency of PFC-evoked qEPSCs is lower at AS neurons, which could reflect fewer connections (N). Second, the PPR of PFC-evoked EPSCs is similar at all three cell types, suggesting equivalent probability of release (P). One caveat is that optogenetics can evoke non-physiological release (Zhang and Oertner, 2007), although this approach elicits similar short-term dynamics as electrical stimulation (Crandall et al., 2015), and has been used to demonstrate differences in short-term dynamics between different inputs to the same population of cells (Britt et al., 2012), as well as the same input to different cell types (Cruikshank et al., 2010; McGarry and Carter, 2016). Third, the amplitude of PFC-evoked qEPSCs is similar at the three cell types, indicating equivalent unitary strength (Q). Together, these results suggest that PFC-evoked EPSCs are smaller at AS neurons due to fewer connections, whereas other synaptic parameters are broadly the same. Importantly, both AMPA-R and NMDA-R EPSCs are smaller at AS neurons, such that the AMPA/NMDA ratio is equivalent at these cells. These results highlight that even large differences in synaptic strength can be missed when comparing AMPA/NMDA ratios.

Our results indicate that the PFC can drive firing of AC and AH neurons in the medial BLA, but not neighboring AS neurons. Interestingly, we previously found that the BLA preferentially contacts cortico-amygdala neurons in both the PL and IL PFC (Little and Carter, 2013; McGarry and Carter, 2016). Together, these studies suggest that the PFC and BLA can communicate in closed loops, with reciprocal interactions in both directions. In contrast, while the PFC receives input from the vHPC, it does not directly project to this brain region, and therefore cannot form a reciprocal circuit (Hoover and Vertes, 2007). Instead, the PFC can drive AH neurons in the BLA to form an indirect, multi-synaptic pathway to the vHPC. The vHPC also sends direct projections to the BLA (Canteras and Swanson, 1992; Hubner et al., 2014) and it will be important to determine whether the vHPC also makes selective connections onto different projection neurons.

While our study focused on different projection neurons, the PFC can also engage inhibitory networks in the amygdala. Classic work indicates that the PFC acts via the intercalated cell mass to inhibit the central amygdala (CeA) (Quirk et al., 2003). More recent studies show that the PFC also engages GABAergic interneurons in the BLA to evoke local inhibition (Arruda-Carvalho and Clem, 2014; Cho et al., 2013; Hubner et al., 2014). In the future, it will be important to assess whether these interneurons target specific projection neurons in the BLA. While this type of targeting has not been observed, inhibitory inputs display unique short-term plasticity onto particular projection neurons (Vogel et al., 2016). Ultimately, inhibition will strongly influence the ability of the PFC to drive AP firing in different projection neurons. However, in vivo recordings indicate that the PFC can overcome local inhibition to drive pyramidal cell AP firing (Likhtik et al., 2005). It is now important to assess how long-range excitatory inputs from the PFC and other areas interact with different forms of inhibition to sculpt specific BLA output pathways.

The selective activation of distinct projection neurons has important implications for the PFC-BLA circuit and its roles in regulating emotional behavior. By preferentially activating AC and AH neurons, the PFC may be able to rapidly regulate behaviors associated with fear and anxiety (Felix-Ortiz et al., 2013; Felix-Ortiz et al., 2016; Herry et al., 2008; Likhtik et al., 2014; Senn et al., 2014). In contrast, due to the limited effects on AS neurons, the PFC may have more modest regulation of reward processing (Ambroggi et al., 2008; Beyeler et al., 2016; Namburi et al., 2015; Stuber et al., 2011). However, a genetically defined subgroup of AS neurons are involved in aversive behaviors (Kim et al., 2016), and further studies are needed to determine if the synaptic inputs or intrinsic properties of this subgroup differs from the majority of AS neurons. Moreover, both AS and AH neurons in lateral BLA do not receive PFC input, suggesting they participate in distinct circuits driven by alternative inputs. In the future, it will be interesting to explore additional pathways in this circuit, including projections to the central amygdala (CeA), which are also known to regulate both aversive (Beyeler et al., 2016; Namburi et al., 2015; Quirk et al., 2003) and appetitive behaviors (Kim et al., 2017). Finally, it will be important to determine how fear conditioning and extinction influence cell-type specific connectivity in the BLA (Arruda-Carvalho and Clem, 2014; Cho et al., 2013). Together, our work provides a robust foundation for analysis of cell-type and input-specific synaptic connectivity within the BLA.

MATERIALS & METHODS

Experiments were performed using male mice in a C57 BL/6J background. All procedures followed guidelines approved by the New York University Animal Welfare Committee. Further details can be found in the Supplemental Experimental Procedures.

Stereotaxic Injections

Stereotaxic injections were performed on P28-P36 mice in the prelimbic PFC, NAc, BLA and vHPC. Anterograde viruses included AAV2/1-hSyn-GFP, AAV2/1-CB7-mCherry and AAV2/1-hSyn-hChR2-eYFP (UPenn Vector Core). Retrograde tracers were 0.2% dilutions of Alexa-conjugated Cholera Toxin subunit B (CTB-Alexa 488 or CTB-Alexa 647; Invitrogen) or undiluted retrobeads (green or red; Lumafluor). After injections, animals were returned to their home cages for 2–3 weeks before experiments.

Slice Physiology

Acute coronal sections (300 μm thick) were prepared from the BLA of P42–56 mice. Injection sites were always validated for accuracy. Targeted recordings were made from amygdala-cortical (AC), amygdala-hippocampal (AH) and amygdala-striatal (AS) neurons, identified by presence of red or green retrobeads. Unless otherwise noted, neurons were recorded in the medial BLA, where PFC inputs and all cell types are located. Recording order was alternated across experiments. Recordings were conducted at 31 – 34°C.

Glutamate release was triggered by illuminating channelrhodopsin-2 (ChR2) present in the presynaptic terminals of PFC inputs to the BLA, using 0.5–8 ms pulses of 473 nm light from a light-emitting diode (LED) delivered via a 10× objective (Olympus) at ∼3 mW.

Data Analysis

Imaging and physiology data were acquired with National Instruments boards using custom software in MATLAB (MathWorks). Image processing and analysis was performed using NIH ImageJ. Physiology analysis was performed using Igor Pro (Wavemetrics). Morphological reconstructions were performed using NeuronStudio (Wearne et al., 2005). Statistical analysis was performed using Prism 7.0 (GraphPad).

Summary comparisons are shown as arithmetic mean ± SEM, except for cell-type ratio (AS/AC and AH/AC) data, which are geometric mean ± 95% confidence interval. Comparisons of measured values between 2 groups were performed using the Mann-Whitney test if not acquired in pairs, or using the Wilcoxon Signed-Rank test if acquired in pairs. Comparisons of values between more than 2 groups were performed using the non-parametric Kruskal-Wallis test, followed by Dunn’s test for post-test evaluation of significant interactions. Comparisons of values across multiple variables (for example, AP firing over a range of current steps) were performed using two-way ANOVA, with Sidak’s test for multiple comparisons. Two-tailed p values < 0.05 were considered significant.

Supplementary Material

supplement

Highlights.

  • BLA neurons projecting to the PFC, vHPC or NAc are three distinct populations.

  • PFC input is much stronger at PFC and vHPC than NAc projecting BLA neurons.

  • Fewer PFC connections are made onto NAc projecting BLA neurons.

Acknowledgments

We thank members of the Carter lab for their helpful discussions and comments on the manuscript. This work was supported by NIH 1T32 NS086750 (LMM), a NARSAD Independent Investigator Award (AGC) and NIH R01 MH085974 (AGC).

Footnotes

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Author Contributions: L.M.M and A.G.C. designed experiments, L.M.M. performed experiments, L.M.M. analyzed data, L.M.M. and A.G.C. wrote the paper.

The authors have no financial conflicts of interest.

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supplement
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