Nat Neurosci︱ Jingjing Liu et al reveal the regulatory effect of hypothalamic melanin-clustering hormone on the activity of the hippocampal-dorsolateral septal circuit

Written by ︱Lu Yizang Editor ︱Wang Sizhen In our brain, information transmission is mainly carried out through various neurotransmitters
.

The classic neurotransmitters are fast-release neurotransmitters, mainly excitatory glutamate (Glu) and inhibitory γ-aminobutyric acid (GABA)
.

At the same time, there are also slow-release neurotransmitters in the brain, such as neuropeptides, which play an important role in the regulation of synaptic information transmission and behavior [1]
.

The previous research on the function of neuropeptides is relatively small, especially the role in the circuit and behavior, is not very clear
.

 Melanin-concentrating hormone (MCH) is a very important neuropeptide
.

In the brain, MCH-expressing neurons are mainly located in the lateral hypothalamic area (LHA) and zona incerta (zona incerta) [2], and project to a wide range of brain regions [3-5]
.

Studies have shown that MCH plays an important role in the mammalian brain, including the regulation of energy homeostasis, metabolism, reward, sleep, learning and memory, social interaction and other processes [6-13]
.

MCH neurons also express other neuropeptides, such as visfatin, cocaine and amphetamine-regulated transcription peptides, and also release glutamate or γ-aminobutyric acid [14-16]
.

Little is known about the role of MCH in neuronal activity, circuits, or behavior, as the role of MCH in MCH neurons is not well understood
.

 At the neural circuit level, MCH neurons of the hypothalamic LHA have mutual projections to the dorsolateral septum (dLS), which receives projections from hippocampal neurons
.

The CA3 (dCA3) pyramidal neurons of the dorsal hippocampus (dHP) contain place cells that encode spatial information about the environment [17-20]
.

A study of in vivo electrophysiological recordings in freely moving animals revealed that a subset of MCH neurons had specific action potential firing during exploratory behavior [21], indicating the location of MCH neurons in the hypothalamus and dorsal hippocampus Cells can co-activate in spatial exploration behaviors
.

However, the mechanism is unclear, and it is possible that MCH neurons, as well as MCH itself, alter information transmission in the hippocampal-septal loop
.

 On January 3, 2022, Zhiping Pang's team at Rutgers University's Robert Wood Johnson School of Medicine's Children's Health Institute and Richard W.
Tsien's team at NYU School of Medicine's Neuroscience Institute collaborated to publish a paper titled in Nature Neuroscience.
The research paper "Hypothalamic melanin-concentrating hormone regulates hippocampus-dorsolateral septum activity" describes the regulation of hypothalamic melanin-concentrating hormone (MCH) on the activity of the hippocampus-dorsolateral septum circuit
.

Dr.
Liu Jingjing is the first author of the paper, and Prof.
Zhiping P.
Pang is the corresponding author of the paper
.

In this study, the authors found that MCH release can affect dLS information transfer, thereby enhancing dorsal hippocampus-dependent spatial navigation
.

MCH neuropeptides have multiple effects on dLS signaling, and these unique and synergistic effects enable dLS neurons to fire faster to high-frequency dCA3 inputs, resulting in MCH-mediated enhancement of spatial learning and memory
.

From the synapse to the behavioral level, this paper explains how the neuropeptidergic input of the dLS modulates the neural output of the dHP, thereby affecting the behavioral state, further enriching our understanding of synaptic plasticity and memory, and revealing neuropeptide A regulatory role in the transmission of brain information to eventual behavioral presentation
.

1.
MCH can reduce the spontaneous firing of neurons in the dLS-LHA loop.
Studies have shown that dLS has a projection relationship to LHA ​​[22]
.

Therefore, MCH secreted by MCH neurons of LHA may have a regulatory effect on the related circuits of dLS
.

To confirm this conjecture, the authors directly optogenetically manipulated MCH neurons in the LHA
.

Additionally, to confirm the projection relationship of dLS to LHA, the authors injected microfluorescent RetroBeads into the LHA of the same MCH-Cre mice (Fig.
1a)
.

After 2-3 weeks of virus injection, electrophysiological experiments on isolated brain slices were performed.
It was found that LHA 470nm blue light stimulation could induce action potentials of virus-infected neurons (MCH neurons), and the light frequency could reach 20Hz (Fig.
1 b, c), the frequency is consistent with the physiological firing frequency of MCH neurons recorded during exploratory behavior
.

MCH neurons have extensive projections to the whole brain and dense, varicose-like projections to the dLS (Fig.
1d,e)
.

Consistent with previous studies, light stimulation of the axon terminals of MCH neurons elicited glutamate release-mediated excitatory postsynaptic currents (EPSCs) in dLS neurons (Fig.
Mesoglutamatergic synaptic vesicles have a high probability of release
.

Figure 1 Exogenous MCH can inhibit the spontaneous firing of dLS neurons (Source: JJ Liu, et al.
, Nat Neurosci, 2022) dLS contains a large number of GABAergic projection neurons, which forms an extensive, confined The axonal plexus, which is the anatomical basis of local lateral inhibition or feedforward inhibition
.

Indeed, light-evoked excitatory postsynaptic currents (oEPSCs) in MCH neurons are accompanied by a secondary inhibitory postsynaptic currents (oFFI-IPSCs) with a delay of several milliseconds (Fig.
2a,b)
.

Therefore, during short-duration stimulation (10 s, 10 Hz or 20 Hz), the effect of MCH neuronal activation on dLS neuron firing was variable and dependent on excitatory (oEPSC) and inhibitory (oFFI) -IPSCs) (Fig.
2 cf)
.

 To induce MCH release and detect the slow effects of neuropeptides, the authors applied long-term photostimulation
.

They first examined the spontaneous action potential firing of dLS neurons after long-term light stimulation of MCH neuronal fibers
.

During 80s, 10 Hz or 20 Hz stimulation, low glutamate levels could be detected as glutamate release decayed very quickly (Fig.
1fg)
.

However, after prolonged light stimulation, spontaneous firing (sAP) of dLS neurons was significantly inhibited, and this inhibition continued for several minutes after the light was stopped (Fig.
1hk, Fig.
2gh)
.

This inhibitory effect is MCH-mediated as it can be reversed by the MCH-type 1 receptor-specific antagonist Tc-MCH7c
.

At the same time, exogenous administration of MCH can also simulate the effect of light stimulation of MCH neuronal fibers on dLS neurons
.

These results suggest that MCH release into the dLS induces a delayed and sustained inhibitory effect on the firing and excitability of dLS projection neurons to the LHA
.

Figure 2.
Immediate effect of light stimulation of MCH neurons projecting to dLS in LHA on spontaneous firing of dLS neurons (Source: JJ Liu, et al.
, Nat Neurosci, 2022) 2.
MCH can promote excitatory salient input in dLS Although MCH neurons have a large number of projections in the dLS, studies have shown that the dLS in mice rarely expresses the MCH receptor [23, 24]
.

To further confirm this, the authors used RNAscope in situ hybridization to examine the expression of MCH-type receptor (MCHR1) in mouse dLS
.

The results showed that there was almost no expression of MCHR1 mRNA in dLS (Fig.
3a, Fig.
4a)
.

Therefore, the inhibitory effect of MCH on the excitability of dLS neurons found in the previous experiments is unlikely to be achieved by MCH directly acting on MCHR1 on postsynaptic dLS neurons
.

Figure 3 MCH can enhance excitatory synaptic transmission in dLS neurons (Source: JJ Liu, et al.
, Nat Neurosci, 2022) Next, the authors investigated whether MCH inhibited excitatory synaptic transmission in dLS, thereby reducing the Excitability of dLS neurons
.

They first recorded spontaneous excitatory postsynaptic potentials (sEPSCs) and electrical stimulation-evoked excitatory postsynaptic potentials (eEPSCs) of dLS neurons, and then examined the effects of photostimulation on these two potentials before and after light stimulation of MCH neuron fibers
.

The results showed that 20Hz light stimulation enhanced sEPSC and eEPSC (Fig.
3b,c, Fig.
4b)
.

It is possible that this enhancement is due to the MCH-mediated increase in presynaptic glutamate release
.

The main evidence is: (1) increased frequency of sEPSCs (Fig.
4b); (2) increased amplitude of AMPA receptor (AMPAR) and NMDA receptor (NMDAR)-mediated eEPSCs, but no change in AMPAR to NMDAR ratio ( Fig.
3 b, c); (3) The paired-pulse ratio (PPR) of eEPSCs decreased (Fig.
3 d, e)
.

And this effect of light stimulation of MCH neuronal fibers on dLS neuronal EPSCs could be reversed by MCHR1 antagonists TC-MCH7c 35 or SNAP94847 (Fig.
4ce)
.

In addition, exogenous MCH (or the MCH analog [Ala17]-MCH) also consistently enhanced sEPSCs in dLS-projecting neurons to the LHA (Fig.
5ac)
.

Therefore, it is very likely that photostimulation leads to the release of endogenous MCH, which then activates presynaptic MCHR1 to enhance glutamate release in dLS
.

And these effects were not found in eYFP-expressing control mice (Fig.
5df), suggesting that light stimulation had no effect on neuronal activity
.

Figure 4 MCH enhances the excitatory synaptic transmission of dLS-LHA projection neurons through a presynaptic mechanism (Source: JJ Liu, et al.
, Nat Neurosci, 2022) In order to further prove that MCH mediates the enhancement of EPSCs in dLS neurons , the authors injected a virus with shRNA-Pmch into the hypothalamus, which suppressed MCH expression (Fig.
3f, Fig.
5gi)
.

To test the effect of shRNA-Pmch inhibition of MCH expression on function, the authors injected a mixture (3:1) of shRNA-Pmch-GFP virus and cre-dependent AAV-C1V1-mCherry virus into the LHA of MCH-cre mice
.

The results showed that the expression of MCH in shRNA-Pmch-GFP cells was significantly decreased compared with the control group (GFP-) (Fig.
3g); at the same time, the expression of MCH was also significantly decreased in MCH neurons expressing C1V1 (Fig.
5j).
)
.

And electrophysiological experiments showed that, compared with the control group, light stimulation of MCH neuronal fibers did not enhance excitatory synapses and reduce PPR in shRNA-Pmch group animals (Fig.
3hk, Fig.
5k,l)
.

This further proves that MCH indeed mediates the enhancement of EPSCs in dLS neurons
.

Figure 5.
The regulatory effect of MCH on excitatory synaptic transmission in dLS neurons (Source: JJ Liu, et al.
, Nat Neurosci, 2022) The above experiments show that MCH mainly affects dLS neurons by acting on the dLS presynaptic mechanism MCHR1 expression may exist in presynaptic neurons of dLS
.

Meanwhile, it has been shown that a major dLS excitatory input comes from the CA3 pyramidal neurons of the dHP
.

Therefore, the authors performed RNAscope in situ hybridization and found a large amount of MCHR1 mRNA expression in CA3 pyramidal neurons (Fig.
6a, Fig.
7ac)
.

Therefore, MCH is likely to regulate the activity of the CA3-dLS loop
.

Sure enough, when Chris ChR2-eYFP was expressed in CA3 pyramidal neurons, exogenous MCH doubled the excitatory synaptic strength of photostimulation-induced HP-dLS loops (Fig.
6be, Fig.
7de), while PPRs drop (Fig.
7f)
.

To demonstrate whether endogenous MCH has a similar effect, the authors employed a dichromatic optogenetic approach
.

They injected cre-dependent AAV-C1V1-mCherry and AAV-CamkII-ChR2-eYFP into LHA ​​and CA3 of MCH-cre mice, respectively, so that MCH neurons express C1V1 and CA3 pyramidal neurons express ChR2 (Figure 6f, g, Fig.
7g, h)
.

Then, electrophysiological experiments of brain slices were carried out
.

Similar to the exogenous MCH effect (Fig.
6d,e), the amplitude of dLS neuron oEPSCs in the HP-dLS circuit evoked by 470 nm blue light stimulation doubled after 590 nm red light stimulation (Fig.
6h, i), and PPR also decreased significantly (Fig.
6 j, k)
.

At the same time this effect was reversed by antagonists of MCHR1 (Fig.
7i,j)
.

These experiments further suggest that the effect of MCH is a presynaptic mechanism, possibly through MCHR1 expressed on axons of CA3 pyramidal neurons projecting to the dLS
.

So endogenous MCH can enhance the excitatory input of the HP-dLS loop
.

Figure 6 MCH can enhance the excitatory synaptic transmission of the dHP-dLS loop (source: JJ Liu, et al.
, Nat Neurosci, 2022) Figure 7 MCH can promote the excitatory input to dLS neurons in the hippocampus (source: JJ Liu, et al.
, Nat Neurosci, 2022) 3.
The regulatory effect of MCH on inhibitory synaptic transmission in dLS Although MCH can enhance presynaptic excitatory input in dLS, MCH inhibits the spontaneous firing of dLS neurons
.

Therefore, it is possible that MCH can enhance inhibitory synaptic transmission in dLS, thereby regulating the balance of excitatory-inhibitory synaptic transmission in dLS, and finally inhibiting the firing of dLS neurons
.

To confirm this, the authors performed electrophysiological experiments on brain slices
.

They found that long-term light activation of MCH fibers in dLS significantly increased the frequency of spontaneous IPSCs (sIPSCs) and the amplitude of electrical stimulation-evoked IPSCs (eIPSCs) (Fig.
Figure 8c, d)
.

These effects were blocked by MCHR1 antagonists (Fig.
8c,d, Fig.
9b,c), and exogenous MCH also had these effects (Fig.
9d,e)
.

Because the previous results showed that there is almost no expression of MCHR1 in dLS
.

Therefore, the above results suggest that MCH is likely to inhibit the excitability of dLS neurons by enhancing long-range inhibitory projections from other brain regions
.

 As shown in the previous results, there are a large number of GABA neurons in the dLS
.

Therefore, it is very likely that there is a feedforward inhibition (FFI) phenomenon
.

To confirm this, the authors went on to perform electrophysiological experiments to verify the FFI (Fig.
8e)
.

They first expressed ChR2 in HPCA3 pyramidal neurons and then photoactivated dHP-dLS excitatory inputs to induce a polysynaptic inhibitory effect
.

The results showed that CNQX inhibited both light-induced FFI (oFFI) and HP-dLS loop excitatory input (Fig.
8f), demonstrating the polysynaptic nature of oFF
.

Moreover, even though ChR2-induced HP-dLS EPSCs were enhanced in the presence of MCH, HP-dLS oFFI was suppressed (Fig.
8g, h)
.

These results further demonstrate that MCH inhibits the excitability of dLS neurons
.

Figure 8 MCH-mediated inhibitory synaptic transmission reduces dLS neuron activity and excitability (Source: JJ Liu, et al.
, Nat Neurosci, 2022) Next, the authors investigated the role of GABA signaling in the inhibitory effect of MCH role
.

First, they used the GABAAR antagonist picrotoxin (PTX), and the inhibitory effect of MCH was not completely eliminated
.

However, the GABABR antagonist CGP55845 could completely reverse the inhibitory effect of MCH (Fig.
8i,j), suggesting that postsynaptic GABABR in dLS is a key factor in the inhibitory effect of MCH
.

Further experiments revealed that GABABR is involved in synaptic transmission within dLS as well as oFFI of HP dCA3 afferents (Fig.
8kn), suggesting that GABABR-mediated inhibitory effects play a critical role in the regulation of MCH on dLS neuron firing.
role
.

 The above experimental results as a whole show that MCH mediates the enhancement of inhibitory synaptic strength of dLS neurons through GABABR, thereby reducing the excitability of dLS neurons
.

Figure 9.
Endogenous MCH can enhance the activity of GABA in dLS (Image source: JJ Liu, et al.
, Nat Neurosci, 2022) 4.
The effect of MCH on the high-frequency firing of dLS neurons The previous experiments explained the slow effect The effect of MCH on GABA- and glutamate-mediated rapid synaptic transmission, the authors next investigated the role of this regulatory effect on information transmission at the overall circuit level
.

The dorsal hippocampus (dHP) uses high-frequency discharges to encode spatial information [17-20]
.

The resting-state firing frequency of most dHP pyramidal cells is less than 2 Hz, but the firing frequency is as high as 50 Hz when encoding positional information [25, 26]
.

In addition, this high-frequency discharge encoded with location information is also associated with dLS [19, 26]
.

Therefore, the authors investigated how changes in the frequency of hippocampal (HP) inputs affect dLS neuron firing
.

It was found that in the absence of MCH, the firing of dLS neurons induced by HP activation was limited and could not form high-frequency firing, and this inhibitory effect was caused by GABABR-mediated FFI
.

In the presence of MCH, dLS neurons induced by HP activation can maintain high-frequency firing, and this effect can also be seen in the presence of GABABR antagonists (Fig.
10), indicating that GABABR is indeed mediated.
inhibitory effect
.

These results indicate that in the body, dLS acts as an information transfer station, which can transmit high-frequency information of hippocampal CA3 downstream, and GABABR-mediated FFI is an inhibitory factor.
MCH can remove this inhibitory factor, thereby ensuring the high level of upstream nuclei.
transmission of frequency information (Fig.
10c)
.

Figure 10 MCH can maintain the information flow of high-frequency encoding of HP-dLS-LHA loop (Source: JJ Liu, et al.
, Nat Neurosci, 2022) 5.
MCH in dLS can enhance hippocampal-dependent spatial memory Since MCH can Regulating the information transmission of the HP-dLS-LHA loop enables the transmission of high-frequency information in the coding space of the hippocampus.
So, can this regulation affect behavior? The authors then conducted a series of behavioral experiments to test this hypothesis
.

 The author first used the Morris water maze test to test spatial memory
.

By the method of cannula, in bilateral dLS, they gave mice dLS MCH receptor agonist [Ala17]-MCH, antagonist TC-MCH7c and blank control, respectively, and divided the mice into agonist group, antagonist group and control group
.

The results showed that compared with the control group, the agonist group performed better, but the antagonist group performed worse, regardless of the proportion of time in the platform area, the number and time of entering the platform area (Figure 11 af ).
), suggesting that MCH can enhance the occurrence of spatial memory
.

To further verify this result, the authors used chemogenetic methods to inject cre-dependent retro-AAV virus with DREADD (excitatory hM3Dq or inhibitory hM4Di) into the dLS of MCH-cre mice, respectively, so that the projected dLS MCH neurons in the LHA express hM3Dq or hM4Di, and intraperitoneal injection of CNO promoted (hM3Dq mice) or inhibited (hM4Di mice) MCH release
.

The Morris water maze test was performed after CNO injection, and the results showed that compared with the control group, the mice in the hM3Dq group had better performance, and the mice in the hM4Di group performed worse, but there was no difference in the exercise capacity of the three groups (Figure 11 gl ), which is consistent with the previous pharmacological experimental results
.

Therefore, both pharmacological and chemogenetic experiments suggest that MCH signaling in dLS can enhance hippocampal-dependent spatial memory
.

Figure 11 MCH can enhance the maintenance of hippocampus-dependent spatial memory (Source: JJ Liu, et al.
, Nat Neurosci, 2022) Figure 12 Summary of the mechanism by which MCH regulates the hippocampal-septal neural circuit (Source: JJ Liu, et al.
al.
, Nat Neurosci, 2022) article conclusion and discussion, inspiration and prospect In summary, the study combined electrophysiology, pharmacology, optogenetics, chemical genetics, behavioral and other methods, mainly expounded the dorsal hippocampus ( dHP) The synapse and circuit regulation mechanism of the neuropeptide melanin-clustering hormone (MCH) signaling in the CA3-dorsolateral septal (dLS) neural circuit (Fig.
12)
.

 Here we show that both exogenous and endogenous MCH can enhance both excitatory (including CA3 input) and inhibitory synaptic strength in dLS through presynaptic mechanisms
.

Furthermore, GABABR-mediated inhibitory effect plays an important role in the excitability of dLS neurons, while feedforward inhibition mediated by intralateral inhibition of dLS inhibits the firing frequency of dLS neurons induced by excitatory input from the hippocampus
.

MCH regulates this process, increases the fidelity of dLS neuron firing, and can more accurately respond to the signal input from the upstream dorsal hippocampus, thereby promoting the occurrence of spatial learning and memory
.

 In conclusion, the present study clearly illustrates the regulatory effect of the slow-acting neuropeptide MCH on the classical fast-acting neurotransmitters GABA and glutamate-mediated synaptic transmission, and the effect of this effect on circuit information transmission, which ultimately affects the occurrence of behavior
.

It further enriches our understanding of the mechanism of action of neuropeptides in the brain and contributes to our better understanding of how the brain works at the synaptic and circuit level
.

Link to the original text: https://doi.
org/10.
1038/s41593-021-00984-5 Corresponding author Prof.
Zhiping P.
Pang (left), first author Dr.
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