Walk Along the Paper Trail: Garfield Gap

It's been three years since I did a Walk Along paper summary! Wow!

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.

Background

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 AgRP^Cre :: MC4R^Cre mice with channelrhodopsin in the AgRP neurons, and GFP in the MC4R neurons in the PVH. They sliced brains, patched onto PVH^MC4R 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).

Patch recordings of MC4R neurons downstream from AgRP. AgRP neurons express ChR2-mCherry (red), and MC4R neurons express GFP (green). A. When you photostimulate AgRP neuron terminals, most MC4R neurons receive GABAergic inputs. B. When you photostimulate AgRP input to Non-MC4R neurons, most neurons do not receive input.

A previous paper by Atasoy and Sternson had claimed that AgRP neurons project to oxytocin or SIM1 neurons in the PVH, so Garfield investigated a few other neuron types in the PVH as well, but found no connections to any of them.

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 PVH^MC4R neurons with ChR2, then put an optic fibre over the PVH to stimulate the AgRP fibre terminals simultaneously alongside PVH^MC4R cell bodies (panel g, below). When they did this, they found the food intake was lower than AgRP neuron stimulation alone (panel h).

g. Diagram of experiment. AgRP neurons express ChR2. In different experiments, MC4R or OXT neurons also express ChR2. The optic fibre is placed over the PVH to stimulate cell bodies and AgRP terminals. h. Stimulation of AgRP terminals increases feeding. This is reduced ("occluded") by simultaneous stimulation of MC4R neurons. It is NOT reduced by simultaneous stimulation of OXT neurons.

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 PKC-delta neurons in the CeA reduces feeding, and PKC-delta neurons are not connected to AgRP neurons. If you simultaneously stimulate AgRP and PKC-delta neurons, and one "occludes" the other, it won't mean they are directly connected. It only means one is stronger than the other!

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.

This raises question to me, how often do peptide neurons synapse onto peptide receptor neurons? (AgRP is certainly a strange case insofar as it doesn't have a natural receptor). There are hundreds of peptide-receptor pairs, and from the brief literature I've looked at, people don't always verify these cells are connected. For example, in the recent Dietrich paper about NPY5R (NPY is another neurotransmitter for AgRP neurons), they stimulated AgRP neurons and applied NPY5R antagonists, but never actually patched onto neurons to see if they were 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.

Collateral

Once they identified PVH^MC4R neurons as important for feeding, they wanted to know where they projected, and specifically if PVH^MC4R projected to multiple targets or single ones. They identified PBN as a major target of PVH^MC4R neurons, and used rabies virus to retrogradely label PVH^MC4R that project to the PBN. When they investigated other brain regions that PVH^MC4R projects to, they did not see axons, showing these neurons do not send collaterals.

(Top) Diagram of experiment. PVH^MC4R neurons are labeled in red. PVH^MC4R that project to the PBN are labeled in green. (Images) There are red and green cells in the PVH, and red and green terminals in the PBN. However, there are only red terminals in the vlPAG and NTS/DMV.

Previous research has shown that AgRP neurons do not have collaterals, and made me wonder whether this is a common feature of mid- or hind-brain neurons. However, a 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.