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The paper

The paper we will discuss today is titled - REM sleep–active MCH neurons are involved in forgetting hippocampus-dependent memories. It was published by Izawa et al., from Thomas S. Kilduff's and Akihiro Yamanaka's labs. It was published on science on 20 sep 2019.

So what's the fuss about - Background.

It appears that when we sleep our memories undergo maintenance. Some memories are strengthened, while others are deleted. We don't know yet how and which memories are targeted to be strengthened and which are targeted to be forgotten. But a big part of this can be attributed to we don't know the cellular and molecular mechanisms of exactly what goes on in our dreams, during our sleep.

In a very old post of mine, I talked about how memories are replayed in a brain region called hippocampus. In fact, you can kind of even engineer dreams. That is if you associate a spatial task with a cue like a tone or smell, you can play the tone when the animal is asleep. And, the same pattern of neurons, which got activated when the animal was exploring some space, will be activated again. In fact, it has been shown that a novel task learnt by an animal is replayed in its sleep for hours (Giri et al., 2019). And, this replay is not only limited to animal models but has also been shown to occur in resting humans (Schuck and Niv, 2019).

It has been shown in humans and animals that replay during sleep helps in memory enhancement. That is to say that memories that are replayed get stronger (Rasch, 2018). It is believed that during sleep when the replay happens in the absence of sensory cues, the hippocampus replay help forms the memory engrams in other cortical parts of the brain. This changes the weak short term memory to strong long term memory (Jordan, 2016).

But, as I said before, not all memories are enhanced. When we sleep some are deleted as well. Francis Crick in 1983 suggested that we dream to forget. And, now evidence has accumulated that forgetting during sleep is perhaps also an important part of memory consolidation. Perhaps it works to reduce information overload (Feld and Born, 2017). Then Li et al., 2017 showed that there is indeed pruning of synapses (connections between neurons) in the hippocampus. They showed that while some synapses are selectively pruned others are selectively enhanced.

I know one of the burning questions this leads us to is - how does brain make a decision for which memories to keep and which to forget. There can be multiple parameters dictating this - for instance how well the memory was formed, to begin with, or how much positive or negative reward was associated with that memory (Schapiro et al., 2018, Michon et al., 2019). But, this still doesn't tell us how that decision is made in the brain at cellular levels. If we want to know how that decision is made, we need to know what neurons fire, how and when to make that decision.

Think of it this way - there is an archivist sitting in your brain that presses one button when it wants to store the memory and another when it wants it to be deleted. But understanding the mind of archivist can only be achieved if we know what buttons the archivist is pressing and when. In today's journal club I talk about the paper which has figured out one such button for forgetting. We will see what that button is and how they found it.

How did they find the button?

Mapping the input connections.

![IMG_20191002_205123.png]()

The researchers found that MCH neurons from hypothalamus in mouse brain project into hippocampus
Drawn by @scienceblocks
Inspiration used from image by Smittawiki | CC BY 3.0

Now if you wanted to know which buttons are being pressed to regulate memory, you need to figure out which buttons are connected to the place where memories are being formed, recalled and replayed. One way to do this is to label the neurons which fire to this region. You can inject the brain of the mice with something called - retrogradely transported beads or retrobeads. The thing with these beads it that they will be transported back to neurons that make connections to the region they are injected in. Hence, by injecting these beads in the hippocampus Izawa et al., were able to find MCH neurons in the hypothalamus, among others projecting to the hippocampus.

Melanin-concentrating hormone (MCH) neurons are known for regulating exploring behaviour, feeding, mood and sleep (Blanco-centurion et al., 2019, Verret et al., 2003, Torterolo et al., 2015, Dilsiz et al., 2019). However, there isn't much to say about there role in the regulation of memory formation or retention.

Activating MCH neurons makes the mice forget.

Hence, to explore the possibility of their involvement in memory, the researcher used chemical and optogenetic tools to activate and inhibit MCH neurons. First, they created a MCH-Cre mouse. Then they injected that mice brain with an adeno associated virus (AAV) containing a FLEX-hM3Dq gene. This way hM3Dq protein is specifically expressed in MCH neurons. The thing about hM3Dq is that it a receptor that can be activated by a chemical called clozapine-N-
oxide. On activation, the receptor causes a release of calcium ions in the neurons and hence activates it.

Now all they needed to do was to teach the mice something and activate MCH neurons after the learning to see if the memory of the mice improves or worsens. To do so they put the mice in novel object recognition (NOR) test, and then gave it CNO during the reception period. During NOR test the mice are exposed to novel objects. Now, mice love novelty and spend most of its time exploring the novel objects. Then after some hours to days of retention period you expose the mice to the same object and even another novel object. If mice remember the previous object, it would spend less time with it. If it doesn't it would re-explore the old object as it did before (Lueptow, 2017).

Izawa et al., found that if they activated MCH neurons by giving CNO to mice after exploring the novel object, the mice failed to remember it during the test. This also held true for context-dependent fear memories. The context-dependent fear memory is created by exposing the mice to a new cage and giving it a foot shock in the new cage. That way mouse learns to fear this new context. But if you activate the MCH neurons then it appeared that mice did not remember the new context so well.

Inhibiting or ablating MCH neurons enhances memory

Now, I remember this!

Ideally, if activation of MCH neurons is required for forgetting then inhibiting them would enhance the memory. To test this hypothesis. The authors this time injected the MCH-cre mice with a virus contacting gene for FLEX-hM3Di. hM3Di protein now expressed in MCH neurons acts in a direction opposite to hM3Dq. Hence, on getting CNO injection MCH neurons will be inhibited. So what happened when after learning in NOR test the mice received CNO? Turns out it remembered things better. It even feared the context when it received shock more than the mice that got saline instead of CNO.

No MCH neurons - longer retention of memory.

To see for how long will the mice retain the memory if MCH neurons did not fire, the authors genetically ablated the MCH neurons after a learning session. To do so they took a MCH-tTA mice and expressed diphtheria toxin A fragment (DTA) gene them under the control of TetO sequence. What happens here is that MCH neurons do not express DTA gene unless there is doxycycline in the diet of mice (because tTA protein can't bind to TetO sequence in presence of doxycycline). However, once doxycycline is removed from the diet, tTA binds to TetO, DTA gets expressed and kills the cells it is expressed in.

When Izawa et al., ablated the MCH neurons, the mice remembered the novel object it was exposed to for more than 48 hours. It was also more scared of the context where it received foot shock in. However, to see if forgetting MCH neurons only affected hippocampus based contextual memories the authors also conditioned the mice to be scared of a tone. In this scenario, the mice would hear a tone and right after the tone, it will receive a foot shock. This kind of fear memory comes majorly from the amygdala (another brain region). Hence, it wasn't surprising to see that ablation of MCH neurons did not improve tone dependent fear memory.

MCH neurons make mice forget only during retention phase.

All this is fine, but you might want to ask, at what point after learning, is the activation of MCH neurons important for forgetting something? It would be nice if you can control exactly when the MCH neurons get activated. One way to do this by using optogenetics. That is to express the Channelrhodopsin in just MCH neurons and then inserting blue LED lights in the mice brain. Now you can shine the blue light, which will turn on the channelrhodopsin causing the MCH neurons to fire. And this is what researchers did. They activated the MCH neurons with blue light either during training or during retention period or during the final test. Only when the MCH neurons were activated during the retention period it led to forgetting memories. That is to say, MCH neurons did not play a role in learning and recall but it does something to memories probably when they are being replayed. So when do MCH neurons really fire during retention period?

Different MCH neurons fires during REM sleep and awake state.

To figure out when certain kind of neurons fire you need to label them in a way that they shine only when they are firing. One way to do this is by expressing a protein called GCaMP6 in cells of your interest. This protein is a calcium sensor. When a neuron fires the calcium concentration in its cytoplasm increases. This causes this protein to give out green light in response. To do this the authors injected virus contains FLEX-GCaMP6 gene in the brain of MCH-Cre mice. Now whenever the MCH neuron will fire it will shine green and fibre optics inserted in mice brain will record the cell that fired.

There was some activity of MCH neurons when mice were awake. But this activity increased further when the mice were sleeping. Especially during their REM sleep (the dream sleep). The activity was mainly quite high when mice went from NREM to REM sleep and NREM to wakefulness transitions. When they resolved the MCH neuron activity to a single cell state, the authors discovered that there are 3 sub-populations of MCH neurons. One that is active during wakefulness, another cluster active during sleep and a third small cluster active both during sleep and awake state. This indicates that MCH neurons might have a dedicated role during sleep - like deleting memories.

The MCH neurons thar are active during REM sleep are required for forgetting.

To check if the sleep active MCH neurons are indeed those that are required for forgetting, they had to inhibit these neurons when the mice were sleeping or awake. Then see which inhibition actually improves memory. To do so the authors expressed a protein called ArchT in MCH neurons of the mice. ArchT acts on direction opposite to that of channelrhodopsin. That is when turned on by yellow-green light, the ArchT inhibits the firing in neuron it is expressed in. The NOR test revealed that memories were improved only when MCH neurons were activated during REM sleep. Activating them during NREM sleep or during wakeful state had little to no effect.

Summary

Hence, the paper clearly demonstrates that the firing of MCH neurons during sleep is involved in forgetting memories. However, this makes me further curious to know what makes the MCH neurons fire. Like do they fire randomly or do certain kind of feedback from memory and reward systems control them? What I am saying is that if this is indeed one of the buttons of archivist now it boils down to knowing how and when this button is pressed.

There is one point to note in the study though. The MCH neuron activity is quite high during NREM to REM transition states. It is possible that by inhibiting these neurons we may be affecting the state itself. Authors do show that REM sleep time is affected by inhibiting these neurons. Hence, the study doesn't completely rule our other indirect mechanism for forgetting related to sleep state.

Nevertheless, this is still pretty cool. I mean if this is really how it works imagine what it means for therapy of those suffering from memory-related anxiety disorders like PTSD. Maybe, someday in future, it would be possible to give a drug to activate these neurons while someone is sleeping and make them forget specific memories by cues. Let's see.

If you have any questions or insights that I might have missed, do let me know in comments. Let me know if you enjoyed this journal club or if you would like something to be improved.

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References

1. Izawa et al., 2019. REM sleep–active MCH neurons are involved in forgetting hippocampus-dependent memories

2. Dream within a dream - Editing and inception of memories (Research Highlights)

3. Giri et al., 2019. Hippocampal Reactivation Extends for Several Hours Following Novel Experience

4. Schuck and Niv, 2019. Sequential replay of nonspatial task states in the human hippocampus.

5. Rasch, 2018. Memory Formation: Let’s replay

6. Jordan, 2016. Dendritic Spikes Provide a Mechanism for Hippocampal Replay and Sharp-Wave/Ripple Generation

7. Francis Crick & Graeme Mitchison, 1983. The function of dream sleep

8. Feld et al., 2017. Sculpting memory during sleep: concurrent consolidation and forgetting

9. Li et al., 2017. REM sleep selectively prunes and maintains new synapses in development and learning.

10. Schapiro et al., 2018. Human hippocampal replay during rest prioritizes weakly learned information and predicts memory performance

11. Michon et al., 2019. Post-learning Hippocampal Replay Selectively Reinforces Spatial Memory for Highly Rewarded Locations

12. Cornwall et al., 1988. Quantitative analysis of axonal branching using the retrograde transport of fluorescent latex microspheres

13. Blanco-Centurion et al., 2019. Dynamic Network Activation of Hypothalamic MCH Neurons in REM Sleep and Exploratory Behavior

14. Verret et al., 2003. A role of melanin-concentrating hormone producing neurons in the central regulation of paradoxical sleep.

15. Torterolo P, Scorza C, Lagos P, et al. Melanin-Concentrating Hormone (MCH): Role in REM Sleep and Depression. Front Neurosci. 2015;9:475. Published 2015 Dec 17. doi:10.3389/fnins.2015.00475

16. Dilsiz et al., 2019. MCH neuron dependent reward and feeding.

17. Leuptow, 2017. Novel Object Recognition Test for the Investigation of Learning and Memory in Mice.