Table 7: Summary of the evidence supporting the PC-PBN pathway rather than the PC-LC pathway in our experimental results.
It was important to consider whether the PC to LC pathway could contribute to some of our experimental findings in light of the close proximity of the LC to the PBN, and a previous report of a PC to LC pathway 15. Our experiments were performed with that possibility in mind, and as summarized in this table, our results establish the importance of the PC-PBN pathway.
| PCPBN pathway | PCLC pathway | |
|---|---|---|
| Fig. 2 g, h | High density of PC-PBN synapses (40X as many PC-PBN synapses as PC-LC synapses) | Very low density of PC synapses |
| Fig. 2 d-f, i | Shows extensive PC synapses throughout the PBN. | - |
| Fig. 2 k. | Optogenetic suppression of PC firing led to short latency firing in 50% of PBN neurons in vivo. This shows that PCs in the posterior vermis inhibit a large fraction of PBN neurons. | - |
| Fig. 2 l, m. | Optogenetic stimulation of PC fibers in brain slice evoked large IPSCs in 75% of PBN neurons with a latency of 2.3 ms. This establishes that PCs directly inhibit a large fraction of neurons in the posterior PBN. | - |
| Fig. 3 c-f. | PBN neurons are selectively activated in behavior studies targeting the PBN pathway. | LC cells are not activated in behavioral experiments targeting the PBN pathway. |
| Extended Data Fig. 7. | Shows labeled neurons for the 3 mice used for anatomical studies. Fluorescence is localized to the PBN, indicating that axonal labelling in the rest of the panel and in Fig. 4 is due to the PBN | Shows labeled neurons for the 3 mice used for anatomical studies. There are no fluorescently labelled cells in the LC for any of the mice, indicating that the fluorescence labelling in Figure 4 is not a result of LC labelling. |
| Fig. 5 a-c | Identification of multiple types of PBN neurons targeted by PCs. Readily interpretable because the PBN does not project back to the cerebellum. 3,11 | AAV1-Syn-Cre labelling of LC neurons is not interpretable because LC neurons project extensively to the cerebellum and could be retrogradely labelled13-15. This is supported by the observation that many of the neurons labelled by AAV1-Syn-Cre in the LC are not located in the vicinity of PC fibers (Extended Data Fig. 10e-g). |
| Fig. 5e. | Shows that trans-synaptically labelled neurons within the PBN are in the vicinity of PC fibers. | |
| Fig. 5g | Shows that there are neurons within the PBN that are directly inhibited by PCs that in turn project to the amygdala. | |
| Fig. 5 h-j. | Establishes the identity of some types of PBN neurons targeted by PCs. These neurons have been studied previously. There projection pattern overlaps with the projection pattern in our trans-synaptic studies, and place preference studies indicate that they are aversive, as in our behavioral studies. | - |
|
Schwarz et al., Nature 2015. Fig. S615 |
- | This figure shows that PC fibers are only present at the very posterior/dorsal edges of the LC, and that PC synapses within the LC are rare. |