Article Figures & Data
Figures
- Figure 1.
Expression of BLA axons in PL and IL regions of the mPFC. A, Schematic of AAV-ChR2-YFP injection into the BLA. B, A representative coronal brain section (left, bright-field; right, epifluorescence) showing AAV-ChR2-YFP injection into the BLA. C, Bottom, Schematic of the rostral-caudal (R ↔ C) location of coronal slice containing the mPFC. C, Top, Schematic of a coronal brain section oriented so that the pia of both the PL and IL cortex was horizontal in the imaging chamber. D, A representative image of a coronal brain section containing PL and IL cortex (top, bright-field; bottom, epifluorescence) in which BLA axons were anterogradely labeled with YFP from AAV-ChR2-YFP. E, F, Bright-field and epifluorescence images showing laminar distribution of BLA expression in PL and IL. G, Normalized fluorescence intensity (mean ± SEM) of BLA axons (n = 5; 5 animals) as a function of normalized cortical distance (where pia = 0 and white matter = 1) in PL (black) and IL (blue). Dorsal-ventral, D↔V; lateral-medial, L↔M.
- Figure 2.
CP neurons in L5 of the PL and IL cortex receive monosynaptic and polysynaptic input from the BLA. A, Injection of AAV-ChR2-YFP into the BLA. B, Injection of retrograde tracer into the PAG. C, Epifluorescence image overlay of brain slice showing regional and laminar distribution of YFP positive BLA axons (green) and red beads illuminated CP neurons (red) in the PL and IL cortical regions (outlined with dotted lines). Layers 2 and 5 are marked as L2 and L5, respectively (dorsal-ventral, D↔V; lateral-medial, L↔M). D, Schematic showing how synaptic input was measured in whole-cell recordings of retrogradely labeled L5 CP neurons in IL cortex during wide-field activation of ChR2+ BLA axons via blue LED stimulation. E, Examples of EPSC (black trace; holding at −70 mV) and IPSC (red trace; holding at +10 mV) recorded from L5 IL-CP neurons before and after addition of polysynaptic blockers (TTX 0.5 µm, 4-AP 0.1 mm, CPP 5 µm: dashed traces). F, Paired comparison of recorded EPSC values in IL-CP neurons before (black) and after addition of polysynaptic blockers (gray). G, Paired comparison of recorded IPSC values for IL-CP neurons before (black) and after addition of polysynaptic blockers (gray). H, Schematic showing how synaptic input was measured in whole-cell recordings of retrogradely labeled L5 CP neurons in PL cortex during wide-field activation of ChR2+ BLA axons via blue LED stimulation. I, Examples of a BLA evoked EPSC (black trace; holding at −70 mV) and IPSC (red trace; holding at +10 mV) recorded from L5 PL-CP neurons before and after addition of polysynaptic blockers (TTX 0.5 µm, 4-AP 0.1 mm, CPP 5 µm: dashed traces). J, K, Paired comparison showing a significant decrease in BLA evoked EPSCs and IPSCs recorded from PL-CP neurons after addition of polysynaptic blockers. * = p < 0.05; error bars = SEM.
- Figure 3.
The BLA differentially targets CP neurons in PL and IL cortex. A, Comparison of the percentage EPSC (peak ± 1 ms) remaining following application of polysynaptic blockers for PL-CP and IL-CP neurons. B, Experimental paradigm for comparing monosynaptic input (in TTX, 4AP, CPP) to retrogradely labeled L5 CP neurons in PL and IL during wide-field activation of ChR2+ BLA axons via blue LED stimulation. C, Examples of monosynaptic EPSCs for a PL-CP neuron (black trace) and IL-CP neuron (gray trace) in the same slice following blue LED stimulation (stim) of ChR2+ BLA axons. D, Paired comparison of EPSC values for PL-CP and IL-CP neurons. E, Schematic representation for the 16 × 16 stimulation grid (75 µm spacing) using for local circuit mapping of retrogradely labeled PL-CP neurons using UV-assisted glutamate uncaging (L, layer; wm, white matter). F, Examples of an EPSC (black trace; holding at −70 mV) and an IPSC (red trace; holding at +10 mV) recorded from L5 PL-CP neurons following UV activation of caged glutamate in L2 of the PL cortex. G, Average of excitatory local circuit maps showing robust L2 input to PL-CP neurons. H, Average of excitatory inhibitory circuit maps showing robust inhibitory input to PL-CP neurons following activation of both L2 and L5. I, Coinjection of AAV-ChR2-YFP and CTB-AlexaFluor 647 (cholera toxin β-subunits conjugated with AlexaFluor 647) into the BLA. J, Experimental paradigm for comparing monosynaptic input to retrogradely labeled L2 PL-CA and L2 IL-CA neurons during wide-field activation of ChR2+ BLA axons via blue LED stimulation. K, Examples of monosynaptic EPSCs for a L2 IL-CA neuron (gray trace) and a L2 PL-CA neuron (black trace) in the same slice following blue LED stimulation (stim) of ChR2+ BLA axons. K, Inset, Action potential firing in response to blue LED stimulation recorded in cell-attached mode from a L2 PL-CA neuron. Scale bars: 5 mV, 20 ms. L, Paired comparison of EPSC values for L2 IL-CA and L2 PL-CA neurons. *p < 0.05; error bars = SEM.
- Figure 4.
Targeting of BLA axons in the IL cortex is projection and laminar specific. A, Coinjection of AAV-ChR2-YFP and CTB-AlexaFluor 647 into the BLA. B, Injection of red Retrobeads IX into the PAG. C, Epifluorescence image overlay of the mPFC showing regional and laminar distribution of CA neurons (purple), BLA axons (green), and CP neurons (red) in the PL and IL cortical regions of the mPFC. D, Experimental paradigm for comparing monosynaptic input to L5 CP and CA neurons in IL during wide-field activation of ChR2+ BLA axons via blue LED stimulation. E, Examples of monosynaptic EPSCs for a L5 IL-CP neuron (gray trace) and a L5 IL-CA neuron (black trace) in the same slice following blue LED stimulation (stim) of ChR2+ BLA axons. F, Paired comparison of monosynaptic EPSC values for L5 IL-CA and L5 IL-CP neurons. G, Experimental paradigm for comparing monosynaptic input to retrogradely labeled L2 and L5 CA neurons in IL during wide-field activation of ChR2+ BLA axons via blue LED stimulation. H, Examples of monosynaptic EPSCs for a L2 IL-CA neuron (black trace) and a L5 IL-CA neuron (gray trace) in the same slice following blue LED stimulation (stim.) of ChR2+ BLA axons. I, Paired comparison of monosynaptic EPSC values for L2 IL-CA and L5 IL-CA neurons. * = p < 0.05; error bars = SEM.
- Figure 5.
Working circuit diagram for BLA targeting of CP and CA neurons in PL and IL cortex. In PL cortex, the BLA sends robust projections to L2 PL-CA and weaker projections L5 PL-CP neurons. A local L2 CA → L5 CP pathway (a) and targeting of the BLA to apical dendrites of L5 PL-CP neurons (b) remain unresolved. In IL cortex, the BLA preferentially targets L5 IL-CP neurons over L3/5 IL-CA neurons. The BLA also preferentially targets L3/5 IL-CA neurons over L2 IL-CA neurons. Local L2 CA → L5 CP and L2 CA → L3/5 CA pathways (c) and targeting of L3/5 CA neurons specifically to the central lateral amygdala (CeL) and intercalated (ITC) regions (d) remain unresolved. Thickness of green arrows = relative strength of connection; dashed lines = unresolved connections.
Tables
Data Test Data Power, % Effect size a Unknown Paired t test IL-CP excitatory 14.3 0.33 b Normal Paired t test IL-CP inhibitory 97.3 1.62 c Normal Paired t test PL-CP excitatory 77.9 0.97 d Normal Paired t test PL-CP inhibitory 98.9 1.52 e Normal Unpaired t test PL-CP vs IL-CP 91.8 1.45 f Unknown Paired t test PL-CP vs IL-CP 77.2 1.03 g Normal Paired t test PL-CA vs IL-CA 67.6 1.09 h Unknown Paired t test IL-CP vs IL-CA 63.6 1.04 i Normal Paired t test L2 vs L5 IL-CA 89.4 1.33 Data normality was determined using a Lilliefors test. Nonparametric statistical analysis produced similar results. Post hoc power analysis was performed using G*Power 3(v3.1.9.2, www.Gpower.hhu.de; Faul et al., 2007).
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