Intrinsic Circuits in the Lateral Central Amygdala

Firing types of neurons in the central lateral amygdala are as follows: late-firing nonaccommodating and early-spiking accommodating. A, B, Example traces of the two main firing types recorded in the CeL: LF-NA (A) and ES-Ac (B) with example traces of current injections below. Calibration: 20 mV, 500 ms, 80 pA. The top two current injections shown are at threshold and twice threshold (2T). On average, LF-NA neurons displayed significantly longer onset to firing of the first AP (onset indicated by black arrowheads) when compared with ES-Ac neurons (LF-NA, 330 ± 25 ms, n = 80 neurons; ES-Ac, 209 ± 23, n = 59 neurons; p < 0.001, Mann–Whitney test) and little to no accommodation at 2T. To demonstrate accommodation, early (green lines) and late (red lines) interspike intervals are indicated. C, Whereas AP frequency over eight action potentials remained consistent for LF-NA neurons (n = 20; AP_1-2 frequency, 17 ± 1 Hz; AP_7-8 frequency, 16 ± 1 Hz; p = 0.6, Wilcoxon matched-pairs test), ES-Ac AP frequency gradually decreased (AP_1-2 frequency, 32 ± 4 Hz; AP_7-8 frequency, 13 ± 1 Hz; p < 0.001, Wilcoxon matched-pairs test).

, Stuttering neurons in the CeL. A, Example trace of firing of a stuttering (S) neuron at threshold, and twice and three times threshold. In addition to its fast AP kinetics (Table 1) and distinct firing pattern, large fast afterhyperpolarizations (as indicated by the red arrowhead) are also typical of this firing type. Inset shows a closeup of a spontaneous EPSP (sEPSP) in green. B, Overlay of the first AP of a stuttering (red), LF-NA (black), and ES-Ac (blue) neurons. The AP rise time and half-width of S neurons were significantly faster than those of LF-NA and ES-Ac neurons (Table 1). C, sEPSPs in S neurons were significantly more numerous than in LF-NA and ES-Ac neurons during the hyperpolarizing steps of this protocol. Numbers shown are the total counted over the −60, −40, and −20 pA current injections (B; S vs LF-NA: p = 0.001, unpaired t test; S vs ES-Ac: p < 0.0001, unpaired t test). D, Example biocytin recovery of an S neuron, which was PKCδ(−) (top inset, yellow arrowhead indicates the soma of the S neuron). Scale bars: 20 µm; top inset, 10 µm; bottom inset, 5 µm. This neuron displayed an extensive axon with inset showing a closeup of the axon in the dotted white square. E, Percentage of firing types for recovered neurons that were PKCδ(+) (n = 8) or PKCδ(−) (n = 17). F, Shows total percentage of each firing type.

Neurons in the central lateral amygdala form local connections. A, Paired recordings were performed in the CeL, the location of which is shown in a diagram of a coronal slice (left). Middle, A bright-field image (300 µm slice) of the area within the orange rectangle: the border of the CeL is clearly defined by visible fiber bundles, and the right panel shows the approximate outline of the three main amygdala regions: BLA, CeL, and CeM. In reality, the CeL extends slightly more ventrally than outlined here; however, we aimed to keep recordings within the outlined area to ensure that we did not mistakenly record from CeM neurons. B, C, Example traces of IPSCs, which were on average 20 ± 3 pA, from a unidirectional connection (B) and a bidirectional connection (C). In each case, “cell 1” was current clamped and given a short current injection (5 ms, 600–700 pA, illustrated in black directly under each current trace) to elicit one AP, while “cell 2” was voltage clamped at −40 mV. The protocol was then repeated in the opposite direction: from cell 2 to cell 1. Example average traces (black) and representative traces from single episodes (gray) are shown. D, Approximately 29% of paired recordings (n = 45 of 152) were connected, with the large majority of connected pairs being unidirectional connections (42 of 45) and the remainder being bidirectional connections. E, Biocytin recovery of the connected recorded pair in B, where a yellow arrowhead indicates the presynaptic cell and a white arrowhead indicates the postsynaptic cell.

Morphology and anatomic location of local connections within the central lateral amygdala. A, Example morphologic reconstruction (spines not depicted) of a connected pair with the presynaptic neuron in black and the postsynaptic neuron in gray. Blue arrowheads indicate where the presynaptic axon (red) crossed over a postsynaptic dendrite in the same z-plane, representing putative synapse locations. Inset shows average traces of this connection, with the presynaptic trace in black and the postsynaptic trace in gray (postsynaptic cell voltage clamped at −40 mV). B, Recovered neurons typically had a medium spiny morphology; spine counts of recovered connected neurons showed that the postsynaptic neuron was not significantly more spiny than its presynaptic neuron. Example images show closeups of secondary dendrites from a presynaptic (“Pre”) neuron and corresponding postsynaptic (“Post”) neuron from the pair shown in A. Scale bar, 5 µm. Bar graph shows mean spine densities (number of spines per micrometer) for presynaptic and postsynaptic neurons, with connected neurons joined by a dotted line (n = 3 connected pairs). Data points with red borders correspond to the Pre and Post closeups depicted in B. C, Image of biocytin recovery of the connected pair of neurons shown in A to show the location within the CeL. BA, Basal amygdala; D, dorsal; M, medial. Scale bar, 100 µm. D, Locations within the CeL (yellow; central medial amygdala is in white) of 35 recorded pairs that could be reliably located at different rostrocaudal locations (−1.22 to −1.70 mm from bregma; Di–Div). Presynaptic cells are represented by black circles, and postsynaptic cells are represented by solid gray circles. White circles indicate pairs where a connection was not detected.

Local connections in the CeL are inhibitory. A, Example traces of change in current to voltage in 10 mV steps (left, from −40 to −90 mV) and average current−voltage (I–V) curve of local IPSCs (right, n = 5 paired recordings). This I–V curve is typical of a chloride current: a linear I–V relationship (r ² = 0.98) that reverses here at −72 mV, close to the theoretical reversal potential (∼73 mV). B, Local IPSCs were also blocked by the GABA_A receptor antagonist bicuculline (10 µM); example traces with aCSF in black and bicuculline in red (left). IPSCs were completely blocked by bicuculline (right, mean IPSC aCSF: 24.7 ± 5.4 pA; mean IPSC bicuculline: 1.7 ± 0.5 pA; n = 5 paired recordings; p = 0.03, one-tailed Wilcoxon test; dotted line joins data points from the same neuron). C, Overlay of 10 example traces from a connected pair where a short positive current injection (5 ms, 600–700 pA) was applied to the presynaptic cell to fire one AP at t = 0 s (top trace). Meanwhile, the postsynaptic cell was also in current-clamp mode, and current was injected such that the cell fired continuously (bottom trace). A single AP in the presynaptic cell evoked an IPSP that was sufficient to stop the postsynaptic cell from firing. Bottom histogram shows the number of APs fired in the above trace over time, in 50 ms bins. D, The spike probability was significantly lower in the 200 ms following inhibition onset compared with preinhibition (mean spike probability before inhibition, 0.14 ± 0.02; mean spike probability during inhibition, 0.02 ± 0.01; p = 0.02, paired t test), and in most cases increased when the postsynaptic cells recommenced firing (mean spike probability before inhibition, 0.14 ± 0.02; mean spike probability after inhibition, 0.2 ± 0.02; p = 0.01, paired t test). Each color represents data points from the same neuron (n = 5 pairs).

PKCδ(+) and PKCδ(−) neurons form local connections in the CeL. A–C, Example images (left-hand panels; scale bars, 50 µm) of connected cells that were biocytin filled and recovered with a fluorescent streptavidin (red). Insets show closeups of each cell with PKCδ staining (green fluorescence; DAPI is shown in blue in B to help locate the postsynaptic neuron). Yellow arrowheads indicate the presynaptic neuron, and white arrowheads indicate the postsynaptic neuron. Example average traces for each recovered pair are shown in the right-hand panels, Calibration: 50 mV, 10 pA, 20 ms. A, PKCδ(−) → PKCδ(−) connection. B, PKCδ(−) → PKCδ(+) connection. C, PKCδ(+) → PKCδ(+) connection. D, Approximate locations of each successfully identified pair. E, Connected paired recordings were predominantly between PKCδ(−) cells (∼ 55%; 6 of 11 successfully recovered and stained connected paired recordings), whereas ∼27% of connections were PKCδ(−) → PKCδ(+) (3 of 11) and ∼18% (2 of 11) were PKCδ(+) → PKCδ(+). No PKCδ(+) → PKCδ(−) connections were observed in these experiments. IPSC amplitudes of each type were as follows: PKCδ(−) → PKCδ(−), 20.23 ± 5.6 pA; PKCδ(−) → PKCδ(+), 16.8 ± 8.7 pA; PKCδ(+) → PKCδ(+), 28.75 ± 0.7 pA. F, In terms of firing properties, the majority of connections occurred between LF-NA → LF-NA (∼26%, n = 5 of 19), LF-NA → ES-Ac (∼26%, n = 5 of 19), and ES-Ac → LF-NA (∼21%, 4 of 19) neurons. ES-Ac → ES-Ac connections were less common (∼11%, 2 of 19), and in all connections that involved a stuttering (S) neuron (∼16%, n = 3 of 19), the S neuron was the presynaptic cell.

Somatostatin-positive neurons form local connections in the central lateral amygdala of somatostatin-cre mice. The CeL of SOM-Cre C57BL/6J mice was injected with an AAV-DIO-tdtomato to fluorescently label SOM(+) cells. A, Subsections (50 µm thick) of injected CeL were stained with a NeuN antibody and a PKCδ antibody. Representative sections at bregma −1.60 mm are shown. B, NeuN-positive cells were counted for PKCδ and SOM labeling; 47 ± 3% (mean, n = 122 ± 27 neurons/1.3 × 10⁻³ mm³) of total counted neurons were PKCδ(+) but SOM(−), whereas 39 ± 1% (mean, n = 100 ± 16 neurons/1.3 × 10⁻³ mm³) of total neurons were SOM(+)/PKCδ(−), with very little overlap [i.e., SOM(+) and PKCδ(+): 2 ± 1% (mean, n = 3 ± 1 neurons/1.3 × 10⁻³ mm³) and 12 ± 1% negative for both (mean, n = 32 ± 6 neurons/1.3 × 10⁻³ mm³)]. C, Whole-cell recordings were performed and complete firing properties for 33 neurons were recorded from SOM(+) and SOM(−) neurons. As with wild-type mice LF-NA (∼55%), ES-Ac (∼42%) and stuttering (S; 3%) neurons were observed. SOM(−) neurons were mostly ES-Ac (∼65%, LF-NA 29%, S 6%, n = 17 neurons), whereas SOM(+) neurons were mostly LF-NA (∼81%, ES-Ac 19%, n = 16 neurons). D, ∼29% of paired recordings showed either a unidirectional (n = 8 paired recordings) or bidirectional (n = 1 paired recording) connection, whereas in 71% of recordings no connection was detected. E, Unidirectional connections were observed between different combinations of SOM(−) and SOM(+) neurons: SOM(−) → SOM(+) (n = 2); SOM(+) → SOM(+) (n = 4); SOM(−) → SOM(−) (n = 2); and one bidirectional connection was recorded that occurred between two SOM(+) neurons. Calibration: 50 mV, 20 pA, 20 ms. Current injection applied to the presynaptic cell is illustrated in black under each trace. F, Shows IPSC amplitudes for each connection type: SOM(−) → SOM(+) mean amplitude, 23.5 pA (n = 2 pairs); SOM(+) → SOM(+) mean amplitude, 24.9 ± 7.3 pA (n = 5 pairs – 4 unidirectional IPSCs, 2 bidirectional IPSCs); SOM(−) → SOM(−) mean amplitude, 6.6 pA (n = 2 pairs). Gray dots represent IPSCs from the bidirectional connection. G, Diagram showing the approximate location of connected paired recordings within the CeL. H, Shows the number of paired recordings where a connection either was or was not detected for each SOM(+) and SOM(−) combination. A connection was more likely to be observed when recording from two SOM(+) neurons (∼62% connection success rate) as opposed to a SOM(−) → SOM(+) (∼12% connection success rate) or a SOM(−) → SOM(−) combination (∼28% connection success rate).

Channelrhodopsin activation of SOM terminals in the central lateral amygdala. A, AAV-DIO-channelrhodopsin-mCherry was injected into the CeL of SOM-Cre C57BL/6J mice. B, Example image of fluorescence of injection site in the CeL (BA, basal amygdala; CeM). C, Using a KMeSO₄ internal solution (K-Me), we recorded responses from SOM(−) cells in response to a short light pulse (2 ms, 470 nm; blue rectangle; example voltage-clamp traces at −40 and −70 mV), resulting in an IPSC (mean amplitude: 162 ± 24 pA, n = 15 cells). D–F, To determine whether all cell types received inhibition from SOM(+) CeL neurons, we also used a cesium-based internal solution (Cs), allowing voltage clamping at 0 mV (ChR reversal potential); average traces are shown in black, and example individual traces are shown in gray. SOM(+) neurons responded with large IPSCs in response to light activation (D), as did SOM(−) cells (E). F, Light-activated IPSCs were detected in 100% of SOM(+) cells (n = 10 neurons) and 100% of SOM(−) cells (n = 22). The overall mean amplitude in SOM(+) neurons was 1358 ± 231 pA (n = 10 neurons; light pulse: 2 ms, 470 nm), and the mean amplitude in SOM(−) neurons was 609 ± 202 pA (n = 12, light pulse 2 ms, 470 ms; the remaining 10 neurons were tested with a 1 ms light pulse: mean amplitude 294 ± 70 pA). G, Bicuculline (10 μM) blocked SOM(+)-driven IPSCs (aCSF mean amplitude, 450 ± 206 pA; bicuculline mean amplitude, 11 ± 4 pA; p = 0.04, one-tailed paired t test). H, SOM(−) neurons that received SOM(+)-driven inhibition were recovered and stained for PKCδ (n = 10 neurons). Five of these neurons were PKCδ(+), while the remainder were PKCδ(−). Example images are shown with biocytin recovery shown in cyan (left), PKCδ staining shown in purple (middle), and the merge shown in the right-hand panel. The white arrowhead indicates one PKCδ(−) neuron, and the yellow arrowhead indicates one PKCδ(+) neuron across all three panels. I, To exclude variation in ChR2 infection and light intensity, and therefore to allow direct comparison of light-evoked IPSC amplitudes, we performed simultaneous recordings from one SOM(+) neuron and one neighboring SOM(−) neuron within the same slice (top diagram). SOM(−) cells typically had smaller IPSCs than their neighboring SOM(+) cell (SOM(+) mean amplitude, 1206 ± 188 pA; SOM(−) mean amplitude, 399 ± 64.8 pA; p = 0.01 unpaired t test, Welch’s correction; bottom graph, dotted lines join cells that were recorded at the same time, n = 5 paired recordings). J, In two cases, light stimulation of SOM(+) terminals during connected paired recordings was possible. Ji, Here, a connected SOM(−) → SOM(−) paired recording is shown with example traces of the connection. Jii, Both the SOM(−) presynaptic and postsynaptic cells of this pair also received SOM(+) inputs. These recordings were conducted using a KMeSO₄ internal solution.