Although DA innervation at the CeA has been largely studied [9, 18–20], it remained unclear whether inputs arriving from the vPAG/DR and the VTA/SNc converge into the same CeA regions or rather constitute separate pathways that regulate the CeA with distinct properties. In this work, we demonstrate that the vPAG/DR and the VTA/SNc pathways assemble two different, mostly non-overlapping, circuits (Fig. 3). Furthermore, the topographical organization of DA fibers impinging the CeA allowed us to distinguish several regions among the three classical divisions of the CeA, each with specific reactivity to different DA agonists and antagonists (Figs. 4 and 5). Together, these results suggest that vPAG/DR and VTA/SNc modulate distinct subsets of topographically segregated CeA neurons which are likely involved in different, although not necessarily unrelated, functions.
Classically, it has been established that DA reuptake by DAT is a fundamental mechanism for terminating DAergic neurotransmission and, accordingly, the presence of DAT has been largely recognized as a marker of DAergic neurons. However, here we have demonstrated that DAergic vPAG/DR neurons have atypically low DAT levels compared to those found in VTA/SNc neurons. Similarly, limited DAT expression has been reported in VTA neurons projecting to the prefrontal cortex, nucleus accumbens medial shell and core and basolateral amygdala [25]. The lower levels of DAT observed in the PAG/DR is likely the consequence of an all or nothing Dat expression level in each individual neuron, as we observed in Dat+/IIRES−Cre.Ai14 mice in which only half of TH positive neurons express Dat. However, the lower intensity of DAT immunostaining found in the CeL (Fig. 2A and Fig. 3B) compared with that shown by the reporter tdTomato in Dat+/IRESCre.Ai14 mice (Supp. Figure 2) suggests that even neurons with transcriptionally active Dat express this gene at much lower levels than canonical DAergic neurons. The functional meaning of low DAT levels in the time-course of DAergic neurotransmission in the CeA should be further investigated in future studies, together with the effects of psychostimulants such as amphetamine and cocaine [26, 27].
The comparison of different Cre-driver mouse lines capable of targeting DA neurons is of high interest for a wide community of neuroscientists. In general, DAT-Cre mice have shown to be more selective than TH-Cre mouse lines to target DA neurons in the VTA [28–30] and in the vPAG/DR [31]. However, DAT-Cre lines were reported to have reduced penetrance in the VTA [30] due to reduced Dat expression in specific populations of VTA DAergic neurons [25, 32]. In agreement with the heterogeneous presence of DAT in VTA neurons, our results also show that DAT is present in ~ 50% of DAergic neurons of the vPAG/DR, indicating a limitation of DAT-Cre lines to target DAergic neurons. Of note, given that we used a Cre-reporter mouse line [22] instead of viral vectors-mediated gene delivery, the incomplete penetrance we show in our study cannot be attributed to a partially efficient transduction rate. In addition, we also noticed a population of Dat expressing neurons with undetectable TH, which were labeled with retrobeads injected in the CeA (Fig. 2B-E). Those DAT + and TH- neurons were small sized, rounded/oval shaped and located near the aqueduct of Sylvius, as previously described [33]. Of note, neurons with those characteristics have been proposed to be DAergic and to express low Th levels, as found in Th-GFP and Pitx3-GFP mouse lines [34].
The detailed molecular neuroanatomical study performed here sheds light on the intricate organization of the CeA, the complexity of the DAergic innervation that each CeA subdivision receives and their heterogeneous responses to DA. Although the CeA is classically divided in CeC, CeL and CeM, the segregated DAergic innervation and the effect of DA compounds revealed a compartmentalized organization in which three regions in the CeC (frontodorsal, frontoventral and caudal) and two in the CeL (frontal and caudal) are distinguished (Fig. 5D). This finding is particularly relevant because most functional studies performed to date have focused in caudal regions of the CeA and studied together the CeC and CeL, as if they were a single nucleus. This simplification has limited the study of amygdalar circuits and their implications in emotional behaviors. For example, the role of D2Rs in the CeA seems to be controversial, with reports suggesting both anxiogenic [12, 14] as well as anxiolytic [8, 16, 17] effects. Interestingly, our results show that frontal and caudal CeL display opposite responses to D2R stimulation and blockade (Fig. 4E, CeL), suggesting that small variations when placing stereotaxic-guided cannulas into the CeA may lead to different behavioral results, and therefore, seemingly diverging conclusions about the role of D2R in the CeA. Thus, this work highlights the need for more precise approaches to dissect the functional role of regions and particular neuronal populations of the CeA and also calls for a cautious interpretation of the current available data.