Selective neurotoxic lesions of basolateral and central nuclei of the amygdala produce differential effects on fear conditioning

Abstract

In the fear conditioning literature, it is generally hypothesized that neurons in the basolateral amygdalar complex (BLA) (lateral and basal nuclei) support the formation of conditioned fear memory and project to neurons in the central nucleus (CeA) for the expression of conditioned fear responses. According to this serial processing-transmission view, damage to either BLA or CeA would comparably disrupt the expression of a variety of conditioned fear responses. In the present study, we further investigated the roles of BLA and CeA in fear conditioning by concurrently assessing freezing and 22 kHz ultrasonic vocalization (USV) as dependent measures of fear in rats. Selective neurotoxins, NMDA for the BLA and ibotenic acid for the CeA, were used to destroy intrinsic neurons [evidenced by thionin dye and NeuN (neuronal nuclei) antibody stainings] without damaging the fibers of passage (confirmed by myelin staining). During the 10 tone-footshock paired training, postshock freezing and USV responses were significantly impaired in BLA-lesioned animals, whereas CeA-lesioned animals exhibited only mild deficits. Similarly, conditioned fear responses assessed 24 hr after training were severely reduced in BLA-lesioned animals but not in CeA-lesioned animals. In contrast to ibotenic lesions of the CeA, small electrolytic lesions of the CeA strongly affected both postshock and conditioned freezing and USV. Together, these results do not support the currently espoused BLA-to-CeA serial processing-transmission view of fear conditioning. Instead, the expression of conditioned fear appears to primarily involve BLA projections that course through the CeA en route to downstream fear response structures.

Figure 1.

Photomicrographs of thionin-stained (left), NeuN-immunopositive (middle), and gold chloride myelin-stained (right) coronal sections from a vehicle-injected control (A-C), an NMDA-induced BLA-lesioned (D-F), an ibotenic acid-induced CeA-lesioned (G-I), and an electrolytic CeA-lesioned (J-L) brain. Note that the neurotoxic lesions of the BLA and the CeA show heavy gliosis (D, G) and neuron loss (E, H) in the lesion area. Spared neurons are evident in parts of the target nucleus distant from the cannula and micropipette tip. However, myelin is intact in the lesion area (F, I). In contrast, the electrolytic lesion of the CeA induces damage to fibers of passage (L), as well as neuron loss (K) in the lesion area.

Figure 2.

Schematic representation of the minimum (black) and maximum (gray) extent of damage from an NMDA-induced BLA-lesioned (left), an ibotenate-induced CeA-lesioned (middle), and an electrolytic CeA-lesioned (right) brain. Numbers represent distance in millimeters posterior to bregma. Coronal brain section images adapted from Paxinos and Watson (1998).

Figure 3.

Effects of neurotoxic and electrolytic lesions of the amygdala on conditioned freezing. A, Mean ± SE (error bars) percentage of freezing during the 1 min baseline and during the intervening 10 tone-shock pairings of the 1 min ITIs in context A from sham control (n = 12), NMDA-induced BLA lesion (n = 9), ibotenate-induced CeA lesion (n = 10), and eCeA (n = 10) animals. B, Mean ± SE percentage of freezing during the 8 min context-retention testing in context A. C, Mean ± SE percentage of freezing during the 1 min baseline and the 8 min tone-retention testing in context B.

Figure 4.

Effects of neurotoxic and electrolytic lesions of the amygdala on conditioned USV. A, Mean ± SE (error bars) percentage of USV during the 1 min baseline and during the intervening 10 tone-shock pairings of the 1 min ITIs in context A from CTRL, cBLA, cCeA, and eCeA animals (n = 10). B, Mean ± SE percentage of USV during the 8 min context-retention testing in context A. C, Mean ± SE percentage of USV during the 1 min baseline and the 8 min tone-retention testing in context B.

Figure 5.

Representative samples of conditioned freezing for cBLA (A-E), cCeA (F-J), and eCeA (K-O) rats with large or small lesions. Photomicrographs of NeuN-immunopositive sections from representative animals with small lesion (A, F, K) or large lesion (B, G, L). C, H, M, Typical changes in percentage of freezing and USV during the 1 min baseline and during the intervening 10 tone-shock pairings of the 1 min ITIs. D, I, N, Mean ± SE (error bars) percentage of freezing and USV during the 8 min context-retention testing. E, J, O, Mean ± SE percentage of freezing and USV during the 8 min tone-retention testing. Filled circles, triangles, and bars indicate representative rats with small lesions, and open circles, triangles, and bars indicate rats with large lesions from each group.

Figure 6.

Anatomical model of the amygdala circuitry for fear conditioning. Contextual CS converges with footshock US in LA and/or B (dotted thin lines). Contextual CS-US information courses through CeA en route to downstream fear response structures by way of B for freezing (dotted thick line). Auditory CS comes into association with the US in LA (solid thin lines). Auditory CS-US information is relayed to CeA directly (solid thick lines), as well as to the pathway that travels through CeA via B. The direct pathway to CeA neurons from LA partially mediates USV responses regardless of CS modality.