Mechanisms contributing to the induction and storage of Pavlovian fear memories in the lateral amygdala

Abstract

The relative contributions of plasticity in the amygdala vs. its afferent pathways to conditioned fear remain controversial. Some believe that thalamic and cortical neurons transmitting information about the conditioned stimulus (CS) to the lateral amygdala (LA) serve a relay function. Others maintain that thalamic and/or cortical plasticity is critically involved in fear conditioning. To address this question, we developed a large-scale biophysical model of the LA that could reproduce earlier findings regarding the cellular correlates of fear conditioning in LA. We then conducted model experiments that examined whether fear memories depend on (1) training-induced increases in the responsiveness of thalamic and cortical neurons projecting to LA, (2) plasticity at the synapses they form in LA, and/or (3) plasticity at synapses between LA neurons. These tests revealed that training-induced increases in the responsiveness of afferent neurons are required for fear memory formation. However, once the memory has been formed, this factor is no longer required because the efficacy of the synapses that thalamic and cortical neurons form with LA cells has augmented enough to maintain the memory. In contrast, our model experiments suggest that plasticity at synapses between LA neurons plays a minor role in maintaining the fear memory.

Figure 1.

Electroresponsive properties of model LA neurons. Voltage responses of model cells to intracellular current injection. (A) The responses of the three types of principal cells (types A, B, and C) to current injections (left, 400 pA; middle, 300 pA; right, −100 pA; duration 600 msec) are similar to those reported in Faber et al. (2001). (B) Voltage responses of the interneuron model to 200-msec current injections of the same magnitude as in A.

Figure 2.

Spatial structure, intrinsic connectivity, and fear conditioning protocol for the LAd network model. (A) The model consists of 800 principal cells (red and green dots, 400 each, represent principal cells in LAdd and LAdv, respectively) and 200 interneurons (black dots). The principal cells in the model were populated randomly in the horn-shaped tridimensional structure with dimensions of 800 μm in the rostral–caudal, 800 μm in the ventral–dorsal, and 400 μm in the medial–lateral directions. (B) Intrinsic connectivity of the model in the coronal and horizontal planes: glutamatergic connections to principal cells (blue) and GABAergic connections to principal cells (red). Excitatory connections to principal cells were different in shell and core regions of LAd. (1) Shell neurons in the dorsal region excited principal cells in the ventral region (e.g., for cells separated by 300–400 μm, the connectivity was 10%). (2) Principal cells in the ventral shell region are inhibited by interneurons in the dorsal region (for distances between 50 μm and 300 μm, the connectivity was 23%). (3) Within the core region, cells in the dorsolateral region excited principal cells in the ventromedial region (e.g., cells within a radius of 200-μm dorsal–lateral had 5% connection probability; see text). (4) Inhibitory connections were provided to all cells within a radius of 100 µm from the interneuron, with 20% connection probability. Inhibitory connectivity changed as a function of distance and direction (see text). (5) Excitatory connections in the horizontal plane prevalently ran in the lateromedial direction. The connection probability was a function of the distance between the cells (5%–17% connectivity was changed depending on the distance of 50–800 μm in lateromedial direction, and 2%–5% connectivity for those separated by 50–400 μm in mediolateral direction). (6) Interneuron–principal cell connections were higher in the mediolateral direction (8%–20% connectivity was assumed for those separated by 0–600 μm) than in the lateromedial direction (5%–20% connectivity was assumed for those separated by 0–600 μm). (C) Fear conditioning protocol. As in the experiments of Repa et al. (2001), the “behavioral” protocol included habituation, conditioning, and extinction phases, with eight, 16, and 20 trials, respectively.

Figure 3.

Plasticity of tone inputs is required to generate plastic cells in LAd. With no plasticity in thalamic and cortical inputs, i.e., frequency fixed to 20 Hz, the number of plastic cells decreased (N = 89/800) and their CS responsiveness also decreased by 61 ± 3% (triangles, P < 0.001), compared to experimental (black circles, N = 24/100; data adapted from Repa et al. 2001), and control model (gray squares, N = 198/800) values.

Figure 4.

Impact of various manipulations (A–C) on the tone responses of TP (A1–C1) and LP (A2–C2) cells. (A) Tone responses of LAd cells during the different phases of the behavioral protocol. (A1) Model (squares, N = 96/800) and experimental (black circles, N = 12/100; data adapted from Repa et al. 2001) tone responses of TP cells show a sudden increase during early conditioning, and then drop to habituation levels during late conditioning. (A2) Model (squares; N = 102/800) and experimental (filled circles; N = 12/100) tone responses of LP cells increase gradually with conditioning and persist during extinction. (B) Tone responsiveness of model TP (B1) or LP (B2) cells with plasticity (squares) or without plasticity (triangles) at tone inputs. Data shown include only cells where conditioning induced significant changes in CS responsiveness, using the criterion in Repa et al. (2001). (C) Tone responsiveness of model TP (C1) or LP (C2) cells in two conditions: with plasticity of tone inputs during conditioning and the recall test (squares, from 20 Hz to 40 Hz) or only during conditioning (circles). In the latter case, tone inputs were returned to habituation levels (20 Hz) during the recall test.

Figure 5.

Coronal view of LAd showing the location of TP cells (red) and LP cells (blue) in LAd. The distribution of TP and LP cells was similar to that seen experimentally (see Fig. 8 in Repa et al. 2001).

Figure 6.

(A) Cortical inputs are necessary for the formation of LP cells. Spikes per tone (mean ± SEM) of plastic LAd model cells. TP (A1) and LP (A2) cells, for the following cases: control (square) and no cortical input (diamond). (B) Intrinsic connectivity contributes to the generation of the two cell types in LAd. Spikes per tone (mean ± SEM) of LAd model cells. TP (B1) and LP (B2) cells, for the following cases: control (squares), uniform connectivity (circles, see below), and no LAdd to LAdv connectivity (triangles). Random uniform connectivity was implemented as follows: 3% excitatory connectivity within a 50- to 400-μm radius of a principal cell, and a 35% inhibitory connectivity within a 50- to 200-μm radius for an interneuron, resulting in average excitatory and inhibitory connections per principal cell of 21.25 and 20.05, respectively. This ensured that the average excitatory and inhibitory connections to principal cells matched the control case values of 21.4 and 22.2, respectively.

Figure 7.

(A) Clamping the tone responses of TP cells at habituation levels alters the impact of fear conditioning on LAd cells. Model tone responses in control conditions (squares), or when the tone responses of TP cells were maintained at habituation levels (circles). (B) Model tone responses decrease considerably when the tone-pyr weights are set to habituation levels during the recall test (circles). (C) Model tone responses are largely unaltered when the synaptic weights between neurons within LAd are set to habituation levels during recall (circles).