Recently in our journal club, we discussed a paper by Garfield et al from the Lowell lab. In discussion, some unusually interesting points were raised, and I'd like to think about them here.
I've written about this before, but here is a quick refresher on hunger in the brain. Many types of neurons control metabolism, but the most famous are AgRP neurons in the arcuate nucleus of the hypothalamus. If you stimulate AgRP neurons with channelrhodopsin, or chemogenetically, you can make mice "voraciously feed," aka stuff food in their face. Notably, AgRP neurons are GABAergic, which means they are shutting down neurons in other areas.
While I've written about AgRP neurons before, I've generally ignored what AgRP itself is: a neuropeptide. AgRP does not bind to AgRP receptors, but is actually an endogenous antagonist for the melanocortin-4 receptor (MC4R), whose principle ligand is α-MSH. AgRP neurons cohabitate in the arcuate nucleus with POMC neurons that produce α-MSH; stimulation of POMC neurons can reduce feeding (albeit on a longer timescale than AgRP neurons).
AgRP neurons project to a lot of different brain areas. To see whether all of these projections can induce feeding, the Sternson lab stimulated axon terminals in a bunch of different brain regions, and found that stimulation of only some of these terminals (namely the PVH, aBNST, and LH) could induce feeding. One question Garfield et al wanted to answer was, "what is the molecular identity of these downstream targets?"
Is occlusion anything?
Since AgRP does not have any agonistic receptor, Garfield and colleagues investigated neurons expressing MC4R in various brain regions. They started by performing channelrhodopsin assisted circuit mapping (CRACM!) to see if AgRP neurons connect to MC4R neuons. To do this, they infected AgRPCre :: MC4RCre mice with channelrhodopsin in the AgRP neurons, and GFP in the MC4R neurons in the PVH. They sliced brains, patched onto PVHMC4R neurons, and photostimulated the AgRP axon terminals (see diagram below). They found that 25/30 MC4R neurons received IPSCs following photostimulation, showing they were connected to AgRP neurons (panel A). They also patched onto non-MC4R neurons in the PVH, and found that only 2/10 neurons received AgRP input, showing that the AgRP-> PVH connection was fairly specific for MC4R neurons (panel B).
After showing the connection in-vitro, they wanted to show it functionally in-vivo using behaviour. They performed an occlusion study where they infected both AgRP and PVHMC4R neurons with ChR2, then put an optic fibre over the PVH to stimulate the AgRP fibre terminals simultaneously alongside PVHMC4R cell bodies (panel g, below). When they did this, they found the food intake was lower than AgRP neuron stimulation alone (panel h).
I originally liked the idea of behavioural occlusion, perhaps because it reminded me of LTP occlusion. However, I'm not sure that it's informative in circuit mapping. First, my intuition is that direct excitation beats indirect inhibition. So if you stimulate AgRP terminals and MC4R neurons at same time, and direct excitation wins, it doesn't really tell you anything. Second, if you know two brain regions oppositely modulate behaviour, stimulating both of them does not tell you whether they directly interact. For example, stimulation of
In fact, I think the term "occlussion" is misleading, and not used in the same way as it was in LTP. In LTP, two protocols "occlude" each other if they both induce LTP alone, but stimulating both of them does not yield additional LTP. They are said to occlude each other because they use the same signaling pathway. In behaviour, "occlusion" has referred to stimulation of opposing pathways where one wins out. This experimental paradigm seems to be catching on, but I'm not sure it actually means anything.
Do all neuropeptides synapse on receptors?
Garfield next started looking for other AgRP-MC4R connections in other brain regions. As before, they infected AgRP neurons with ChR2-mCherry, and patched onto GFP-infected MC4R neurons in the anterior BNST, and the lateral hypothalamus (LH). Unlike before, there were no connections between AgRP neurons and MC4R neurons in these other brain regions.
|Whole-cell patch onto neurons MC4R downstream from AgRP neurons expressing ChR2. No neurons, either in the aBNST or LH, were connected to AgRP neurons. Note the n's are not connected.
All neuropeptide neurons express multiple neurotransmitters, including classic ones and neuropeptides, and it's possible each transmitter work at different temporal and spatial scales. Fast neurotransmitters like glutamate or GABA are reuptaken quickly, and so cannot diffuse; in contrast, neuropeptides can last for minutes or hours. This would allow, for example, AgRP neurons to form GABAergic synapses on specific targets, and let their neuropeptides diffuse and act as paracrine [?] signaling.
In any case, I hope that as people explore these systems, they verify that the neurons we assume are connected, in fact are.
Once they identified PVHMC4R neurons as important for feeding, they wanted to know where they projected, and specifically if PVHMC4R projected to multiple targets or single ones. They identified PBN as a major target of PVHMC4R neurons, and used rabies virus to retrogradely label PVHMC4R that project to the PBN. When they investigated other brain regions that PVHMC4R projects to, they did not see axons, showing these neurons do not send collaterals.
recent paper from Luqin Luo's lab mapped axons of locus coeruleus norepinephrine neurons, and found that those neurons do send collaterals widely, albeit biased towards specific areas. As we get more information about cell types, and perform more extensive tracing studies, we will get a better sense of what parts of the brain have discrete pathways versus overlapping projections.