Amygdala intercalated neurons are required for expression of fear extinction

Abstract

Congruent findings from studies of fear learning in animals and humans indicate that research on the circuits mediating fear constitutes our best hope of understanding human anxiety disorders. In mammals, repeated presentations of a conditioned stimulus that was previously paired to a noxious stimulus leads to the gradual disappearance of conditioned fear responses. Although much evidence suggests that this extinction process depends on plastic events in the amygdala, the underlying mechanisms remain unclear. Intercalated (ITC) amygdala neurons constitute probable mediators of extinction because they receive information about the conditioned stimulus from the basolateral amygdala (BLA), and contribute inhibitory projections to the central nucleus (CEA), the main output station of the amygdala for conditioned fear responses. Thus, after extinction training, ITC cells could reduce the impact of conditioned-stimulus-related BLA inputs to the CEA by means of feed-forward inhibition. Here we test the hypothesis that ITC neurons mediate extinction by lesioning them with a toxin that selectively targets cells expressing micro-opioid receptors (microORs). Electron microscopic observations revealed that the incidence of microOR-immunoreactive synapses is much higher in ITC cell clusters than in the BLA or CEA and that microORs typically have a post-synaptic location in ITC cells. In keeping with this, bilateral infusions of the microOR agonist dermorphin conjugated to the toxin saporin in the vicinity of ITC neurons caused a 34% reduction in the number of ITC cells but no significant cell loss in surrounding nuclei. Moreover, ITC lesions caused a marked deficit in the expression of extinction that correlated negatively with the number of surviving ITC neurons but not CEA cells. Because ITC cells exhibit an unusual pattern of receptor expression, these findings open new avenues for the treatment of anxiety disorders.

Fig. 1

μOR immunoreactivity in the amygdala. (a) Coronal section processed to reveal μOR immunoreactivity (brown) and counterstained with cresyl violet (blue). μOR immunoreactivity is much higher in ITC cell clusters than surrounding nuclei. (b) Electron micrograph showing examples of μOR+ synapses in the ITC region. (c) Proportion of synapses (mean ± s.e.m.) where μOR immunoreactivity was founds in the post- (gray) or presynaptic element (black) in the ITC, BLA, or CEA.

Fig. 2

D-Sap infusions at BLA-CEA border cause a spatially circumscribed loss of μOR immunoreactivity. (a-f) Coronal sections obtained from rats that received either U-Sap (Control, a-c) or D-Sap (Experimental, d-f) infusions at BLA-CEA border. Two left-most sections obtained near the infusion site (white arrow); right section, near the caudal pole of the amygdala. Arrowheads and black arrows point to lateral and medial ITC clusters, respectively. Dashed lines indicate trajectory of infusion cannulas. (g) Unbiased stereological estimates of cell numbers (mean ± s.e.m.) in the medial ITC clusters and CEA in control (black) vs. experimental (red) animals. (h-i) D-sap infusion sites (white arrows) in the CEA (h) and BLA (i).

Fig. 3

D-Sap induced ITC lesions cause an extinction deficit. (a) Percent time freezing (y-axis; mean ± s.e.m.) in ITC-lesioned rats (red; n=8) vs. control rats that received U-Sap infusions at the same site (empty black circles; n=11) or D-Sap infusions in the BLA or CEA (n=15; filled black circles). Data obtained in the D-Sap BLA and CEA animals was pooled because the behavior of these two rat subsets was statistically undistinguishable (ANOVA F_(1,108)=0.847, p=0.38). (b) Relationship between number of ITC (bottom) or CEA (top) neurons (y-axis) vs. percent time freezing (x-axis) during extinction testing session (average of first three trials).