The hypothesis that ITC cells are involved in extinction13
has not been tested yet because ITC cells occur as small, distributed cell clusters, making selective electrolytic or excitotoxic ITC lesions impossible. Here, we circumvent this difficulty by exploiting the fact that ITC neurons express high levels of μORs ()14, 15
allowing selective ITC lesions with a peptide-toxin conjugate that only targets μOR-expressing cells. Indeed, targeted toxins take advantage of receptor-mediated endocytosis to deliver cytotoxins to specific types of neurons16
. Here, the peptide dermorphin, an agonist with a high affinity and selectivity for μORs17
, was conjugated to the ribosome inactivating protein saporin (D-Sap).
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 (more ...)
For this lesion method to be effective, μORs must be located postsynaptically in ITC cells, not in their afferents. Thus, we first used electron microscopy to determine their subcellular location. In the electron microscope, μOR-immunoreactivity was more concentrated in ITC cell clusters than BLA or CEA (see Fig. S1
) and it was generally found postsynaptically (; Fig. S2
). Indeed, the proportion of synapses that displayed postsynaptic μOR immunolabeling was ≈3-6 times higher in the ITC cell clusters than in CEA (p=0.009) or BLA (p=0.0001; X2
Having established that postsynaptic μOR expression is much higher in the ITC cell clusters than neighboring nuclei, we tested the feasibility of obtaining selective ITC lesions with the targeted toxin D-Sap and examined their impact on extinction. Briefly, 58 rats were habituated to the training chamber (Day-1) and fear conditioned to a tone (Day-2). Then, in a different context, they were trained on extinction (Day-3). On Day-4, they received bilateral infusions of either D-Sap (Experimental group) or the same volume and concentration of a scrambled peptide conjugated to saporin (U-Sap, Control group) aimed at ITC cells. On Day-11, extinction recall was tested. The conditioned response we monitored was behavioral freezing, quantified by an observer blind to the rats’ condition.
To maximize lesion specificity, we only considered rats in which the cannula tips were at the BLA-CEA border (i.e. where CEA-inhibiting ITC cells are located), without knowledge of the behavioral data. In the control U-Sap and experimental D-Sap groups, respectively 11 and 8 rats met this criterion. shows coronal sections obtained from such control () and experimental () rats. Compared to control (U-Sap) cases, D-Sap infusions caused a marked but spatially circumscribed reduction in OR staining restricted to the region adjacent to the infusion site (white arrows), where peri-CEA ITC clusters are normally found (black arrows). More distant ITC clusters such as those bordering the external capsule (arrowheads) or at the posterior pole of the amygdala () were not affected. Importantly, no difference in OR labeling was seen in CEA or BLA between experimental and control rats. Nevertheless, to control for possible effects of cell loss due to unintended D-Sap diffusion, we also analyzed the behavior of rats where histological controls revealed that D-Sap infusions missed the BLA-CEA border but instead ended in CEA (n=8; ) or BLA (n=7; ).
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 (more ...)
To evaluate the neuronal loss caused by D-Sap infusions in ITC clusters, we performed unbiased stereological estimates of ITC and CEA neuronal numbers on sections counterstained with cresyl violet. The observer was blind to the rats’ condition. Compared to U-Sap-treated rats, a significant reduction in the number of ITC cells was found in rats that received D-Sap infusions in ITC clusters (; U-Sap, 18,499 ± 1,406, n=8; D-Sap, 12,197 ± 1,523, n=6; -34%, t-test, p=0.01). In contrast, neuronal counts in CEA were nearly identical in the two groups (U-Sap, 37,949 ± 3,516, n=4; D-Sap, 40,471 ± 2,328, n=5; t-test, p=0.55).
shows percent time freezing (y-axis) during the different experimental phases in the three groups: the experimental group that received D-Sap infusions in the medial ITC clusters (red, n=8) and the two control groups that received either U-Sap infusions at the same site (empty black circles, n=11) or D-Sap infusions centered on BLA or CEA (filled black circles, n=15). Prior to D-Sap or U-Sap infusions, the three groups behaved similarly during habituation, fear conditioning, and extinction training (, Days 1-3). However, after ITC lesions (Day-4), their behavior differed significantly during recall of extinction (, Day-11) with ITC-lesioned rats showing impaired expression of extinction (two-way repeated measures ANOVA, F(2,270)=11.399, p=0.0002). Post-hoc t-tests with stepwise Bonferroni correction of the significance level (p<0.05) comparing the D-Sap ITC group vs. the average of the two control groups in blocks of two trials revealed that rats with ITC lesions had significantly higher freezing levels during the first four CSs. Moreover, a strong inverse correlation was found between freezing levels during extinction testing and the number of surviving ITC cells (, filled circles, r=-0.67, p<0.01, n=14). In contrast, no such relationship was seen with CEA cell counts (, empty circles; r=-0.13, p>0.05, n=9).
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 (more ...)
Inter-group differences were not attributable to non-specific increases in anxiety levels secondary to ITC lesions as exploratory behavior in a novel open field was indistinguishable between the three groups (ANOVAs, F(2,25): % time in center F=1.28, p=0.29, distance traveled F=1.11, p=0.34). Moreover, the three groups acquired conditioned fear responses to a different CS at the same rate (, Day-13; ANOVA, F(2,93)=0.007, p=0.9931). Finally, the extinction deficit seen in the experimental group did not result from a non-specific CEA disinhibition since freezing during the last CS of the first (Day-2) and second (Day-13) fear conditioning sessions was identical (paired t-test, p=0.61).
While ITC lesions produced a deficit in extinction expression, the difference between the three groups progressively decreased with repeated CS presentations. Several factors probably contributed to the development of extinction after ITC lesions. First, much evidence indicates that the gradual fear reduction seen within an extinction training session depends on different mechanisms than those underlying between-session extinction. Indeed, some lesions3
and pharmacological treatments6
leave within-session extinction intact or marginally reduced, yet severely reduce between-session extinction. This implies that extinction engages at least two parallel processes. A first process that develops rapidly but is short lasting, responsible for within-session extinction, and a second process that lasts longer and underlies between-session extinction. Although the mechanisms underlying within-session extinction remain unclear, a likely contributing factor is the progressive reduction in BLA unit responses as the CS is repeated. In addition, a participation of ITC cells to within-session extinction cannot be rule out. Indeed, because our ITC lesions were incomplete, surviving ITC neurons might have contributed to within-session extinction. However, since the deficit caused by ITC cells was most pronounced at the onset of the testing session, ITC cells are probably critical to between-session extinction, as described below.
Extinction is known to depend on the reinforcement of an active GABAergic process18
and on NMDA-dependent synaptic plasticity in the amygdala5-7, 19
. However, extinction training does not erase the initial fear memory because many BLA neurons maintain their increased CS responsiveness after extinction training20, 21
. Importantly, extinction training does not interfere with conditioned fear responses (CRs) to a different CS or with the subsequent acquisition of CRs to a different CS1
We propose that ITC neurons can account for these properties of extinction. Indeed, ITC neurons receive CS information from BLA8, 9
and send GABAergic projections to CEA10, 11
. Thus, they are in a perfect position to regulate the flow of CS information from the BLA to CEA9
. Importantly, ITC neurons receive a dense projection from the infralimbic (IL) cortex22
, whose stimulation accelerates extinction23
and inhibits CEA neurons24
. We hypothesize that the CS-specificity of extinction derives from the ability of BLA synapses onto ITC neurons to express activity- and NMDA-dependent LTP13, 25
. During extinction training, convergence of CS-related IL and BLA inputs onto ITC cells would facilitate induction of NMDA-dependent LTP, but only at those BLA to ITC synapses recruited by the CS. This CS-specific potentiation of BLA inputs onto ITC cells would enhance the depolarization produced by the CS in ITC cells. Consequently, the GABAergic output of ITC cells onto CEA neurons would be increased, ultimately leading to a CS-specific reduction of conditioned fear responses. In light of recent findings showing increased bursting and tone responsiveness of IL neurons after extinction training23, 26
, it is possible that IL facilitates extinction-related plasticity, during a consolidation phase.
Although the details of this model remain to be tested, the finding that ITC lesions produce a deficit in the expression of extinction that correlates negatively with the number of surviving ITC cells suggests that ITC cells are critically involved in extinction. The significance of this conclusion derives from earlier results suggesting that some human anxiety disorders reflect an extinction deficit3, 4, 27
and functional imaging evidence that the medial prefrontal cortex and amygdala are respectively hypo- and hyper-active in such disorders28, 29
. As a result, it might be possible to compensate for these abnormalities and facilitate extinction with pharmacological interventions that enhance the excitability of ITC cells by taking advantage of their unusual pattern of receptor expression14, 15,30