payant--mikayla-ann--cellular-and-functional-role-of-melaninconcentrating-hormone-in-the-lateral-septum-html.html

Abstract

Melanin-concentrating hormone (MCH) is a neuropeptide exclusively produced in the lateral

hypothalamus and zona incerta with diverse functions including prominent effects on energy

balance and anxiety that can impact the development of obesity and mood disorders. MCH

neurons project to many brain regions, and MCH can have site-specific effects on neuronal

activity and behavioural outcomes. Thus, in addition to global manipulations of MCH function, it

is important to characterize the role of MCH at specific target sites. The lateral septum (LS)

possesses MCH receptors and receives the strongest projection from MCH neurons. However,

the function of MCH in the LS is not known. The LS has known roles in the regulation of

feeding and anxiety-related behaviours, and such overlapping functions with those of MCH

suggest the LS is a key target site of MCH in the brain. This thesis focused on understanding the

role of MCH in the LS by 1) determining the mechanisms of MCH action on LS cells; 2)

defining the behavioural significance of MCH on feeding and anxiety-like behaviour in the LS;

and 3) elucidating the properties of MCH release and action within the LS. We found that MCH

directly inhibited cells in the lateral LS through a novel chloride-mediated mechanism (Chapter

2), and this inhibition promoted a context-dependent increase in feeding behaviour (Chapter 3).

Furthermore, MCH was released and inhibited nearby cells predominantly separate from those

that were directly innervated by MCH neurons, revealing three distinct populations of LS cells

within this circuit. The activation of these parallel glutamate and MCH-mediated circuits

increased feeding and decreased anxiety-like behaviour, respectively (Chapter 4). Together, we

have shown that the LS is an important target of MCH neurons and that MCH and glutamate

interactions are important for mediating behavioural outcomes. This work will improve our

Acknowledgements

I would like to thank my thesis committee, Dr. Alfonso Abizaid and Dr. Michael Hildebrand.

Your continued guidance, feedback, and encouragement over the years at committee meetings,

conferences, and through conversations in the hallway is truly appreciated. Dr. Jenny Bruin,

thank you for serving on my final thesis and comprehensive examination committee. I have

learned a lot from you throughout the years and have enjoyed time spent with you and your lab

through journal club and collaborations. Thank you to Dr. Christian Burgess for serving as my

external examiner and taking the time to read my thesis and attend my defense.

I would also like to thank Dr. Christian Burgess and Dr. Sylvain Williams for their help

with setting up and troubleshooting my

in vivo

optogenetic experiments.

A tremendous thank you to my supervisor Dr. Melissa Chee for taking a chance on me as

a student with very little experience and teaching me the skills necessary to be a successful

scientist and thoughtful person. I sincerely appreciate the care and effort you put in to create as

many opportunities as possible for the growth and success of your students. You have always

known when to encourage me to step out of my comfort zone and pursue opportunities that I

would not have had the confidence to purse on my own. I will always be grateful for the

opportunity to learn from you.

To the Cheetos, past and present, thank you for being the best group of lab mates. It has

been a pleasure working alongside so many wonderful people. Everyone has always been kind

and generous, and this environment has made my time in the Chee lab very special. A special

thank you to Duncan, Persephone, Anjali, Jesukhogie, Haneen, and Marina for their help with

this project. Thank you to Bianca, Aditi, Persephone, Nikita, Yasmina, Duncan, and Alex for

many great memories in the lab and for making every conference experience so much fun. I will

always be proud to be a Cheeto.

To my support system outside of the lab: I would not have made it to this point without

your unwavering support and encouragement. Thank you to my parents, you have always been

my biggest cheerleaders and have gone above and beyond to support me in any venture. Your

passion for healthcare, nutrition, and electrical work have serendipitously manifested into this

research project that combines all three. To my brother Chris, thank you for your consistent

support and for always having new ideas to think about and discuss at every family gathering.

Your curiosity and desire to learn is inspiring. To Megan and Lisa, thank you for being the best

and most supportive friends possible. I am lucky to have friends that are always a text or phone

call away, ready with kind words and great advice. To my dog Sadie, thank you for keeping me

company while writing and always being available for a hug.

Finally, to Damian, I am so grateful for you. Thank you for being supportive and

understanding of many long days spent at the lab and late nights at my computer. Thank you for

always having food ready when I got home. You have helped me become a better person and

scientist by encouraging me to also take care of myself. I will always appreciate your support

throughout this chapter of our lives.

Table of contents

Abstract

Acknowledgements

Table of contents

List of Abbreviations

List of Publications

Chapter 1: Melanin-concentrating hormone actions in the brain

1.1 Overview

1.2 MCH neurons

1.2.1 Distribution of MCH projections

1.3 Widespread distribution of MCH receptors

1.3.1 MCH receptor activation

1.4 MCH functions

1.4.1 MCH and energy balance

1.4.1.1 MCH transgenic models

1.4.1.2. MCH administration promotes feeding

1.4.1.3 Hypothalamic regions underlying MCH-mediated homeostatic feeding

1.4.1.4 Brain regions underlying MCH-mediated effects on hedonic feeding

1.4.1.5 The LS as a possible mediator of MCH-induced feeding

1.4.2 MCH and anxiety

1.4.2.1 Brain regions underlying MCH-mediated effects on anxiety

1.4.2.2 LS as an integrative site for the expression of MCH-mediated anxiolysis

1.5 Specific aims and hypotheses

1.6 References

Chapter 2: Inhibitory actions of melanin-concentrating hormone in the lateral septum

2.1 Key Points

2.2 Abstract

2.3 Introduction

2.4 Materials and Methods

2.4.1 Neuroanatomy

2.4.2 Microscopy

2.4.3 Image analysis

2.4.4 Electrophysiology

2.4.5 Experimental design and statistical analysis

2.5 Results

2.5.1 Distribution of MCH-ir fibers throughout the LS

2.5.2 Distribution of

Mchr1

-expressing LS cells

2.5.3 Distribution of MCHR1-expressing LS cells

2.5.4 Proximity of MCH-ir fibers at MCHR1-expressing LS cells

2.5.5 MCH inhibited LS cells

vii

2.5.6 MCH-mediated hyperpolarization is MCHR1-dependent

2.5.7 MCH activated a chloride channel to hyperpolarize LS cells

2.5.8 MCH did not inhibit GABAergic or glutamatergic input on LS cells

2.6 Discussion

2.7 References

2.8 Supporting figures

Chapter 3: Melanin-concentrating hormone promotes feeding through the lateral septum

3.1 Abstract

3.2 Introduction

3.3 Materials and Methods

3.3.1 Cannula Implantation

3.3.2 Intra-LS infusion

3.3.3 Food intake

3.3.4 Locomotor activity

3.3.5 Open field movement

3.3.6 Implant site validation

3.3.7 Experimental design and statistical analyses

3.4 Results

3.4.1 MCH infusion into the LS increased chow intake in male and female mice

3.4.2 MCH infusion into the LS increased intake of a palatable diet in male and
female mice

3.4.3 MCH in the lateral LS increased locomotor activity

3.4.4 LS MCH infusion did not alter food seeking in an anxiogenic environment

3.5 Discussion

101

3.6 References

105

Chapter 4: Melanin-concentrating hormone and glutamate release in the lateral septum
act on different cells to regulate feeding and anxiety-like behaviour

110

4.1 Abstract

111

4.2 Introduction

112

4.3 Materials and Methods

114

4.3.1 Stereotaxic surgery

114

4.3.2 Neuroanatomy

116

4.3.3 Construction of implants and patch cords

118

4.3.4 Behavioural testing

120

4.3.5 Confirmation of injection and implant sites

121

4.3.6 Electrophysiology

121

4.3.7 Data analysis

124

4.4 Results

125

4.4.1 Medial MCH neurons projected to the LS

125

4.4.2 MCH terminals did not overlap with MCH-immunoreactive fibers

126

4.4.3 MCH and glutamate innervated separate LS cells

128

viii

4.4.4 Channelrhodopsin-mediated MCH release and transmission extracellularly in
the LS

131

4.4.5 Glutamate-mediated feeding by MCH nerve fibers in the LS

134

4.4.6 MCH-mediated anxiolysis in the LS

136

4.5 Discussion

138

4.6 References

143

4.7 Supporting figures

147

Chapter 5: Integrated Discussion

148

5.1 Summary

148

5.2 Local MCH release inhibited LS cells

148

5.3 Differential MCH and glutamate targets and function in the LS

150

5.3.1 Orexigenic effects of MCH and glutamate in the LS

150

5.3.2 Anxiolytic effect of MCH in the LS

151

5.4 Proposed model of MCH action in the LS

151

5.5 Conclusion

153

5.6 References

154

List of Abbreviations

Abbreviation

Full Form

ACB

Nucleus Accumbens

ACSF

Artificial cerebrospinal fluid

AgRP

Agouti-Related peptide

AMP

Adenosine monophosphate

ANOVA

Analysis of variance

ARA

Allen Reference Atlas

Anteroposterior

AAV

Adeno-associated virus

BLA

Basolateral amygdala

CaCl2

Calcium chloride

CART

Cocaine and amphetamine-regulated transcript

ccg

Corpus callosum, genu

ChR2

Channelrhodopsin-2

Caudoputamen

Crfr2

Corticotropin-releasing factor receptor 2

DAPI

4',6-diamidino-2-phenylindole

DAT

Dopamine transporter

Dorsoventral

EYFP

Enhanced yellow fluorescent protein

EGTA

Ethylene glycol-

bis(β

-aminoethyl ether)-N,N,N',N'-tetraacetic acid

FC/PC

Ferrule connector/physical contact

Fornix

Genome copies

GABA

Gamma-aminobutyric acid

G protein, G

subtype

G protein, G

subtype

G protein, G

subtype

GFP

Green fluorescent protein

HEPES

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

HGHpA

Human growth hormone polyadenylation signal

HDx

High-dextrose diet

i.p.

Intraperitoneal

icv

Intracerebroventricular

–

Current

–

Voltage

Immunoreactive

K-gluconate

Potassium gluconate

Knockout

LHA

Lateral hypothalamus

Lateral septum

LSr

Lateral septal nucleus, rostral part

LSc

Lateral septal nucleus, caudal part

LSv

Lateral septal nucleus, ventral part

MCH

Melanin-concentrating hormone

MCHR1

Melanin-concentrating hormone receptor 1

MCHR2

Melanin-concentrating hormone receptor 2

mEPSC

Miniature excitatory postsynaptic current

mIPSC

Miniature inhibitory postsynaptic current

MSH

Melanocyte-stimulating hormone

Medial septal nucleus

mTOR

Mammalian target of papamycin

mRNA

Messenger ribonucleic acid

mOsm/L

Milliosmoles per liter

NeuN

Neuronal nuclei

NDS

Normal donkey serum

NPY

Neuropeptide Y

ob/ob

Leptin knockout mouse

opt

Optic tract

PBS

Phosphate buffered saline

PBT

PBS with Triton-X

PKC

Protein kinase C

POMC

Proopiomelanocortin

RMP

Resting membrane potential

RRID

Research resource identifier

Room temperature

sEPSC

Spontaneous excitatory postsynaptic current

sIPSC

Spontaneous inhibitory postsynaptic current

TTX

Tetrodotoxin

TC-MCH 7c

MCHR1 antagonist

Third ventricle

Vglut2

Vesicular glutamate transporter 2

Vglut2-flox

Vesicular glutamate transporter 2 floxed

Holding potential

WPRE

Woodchuck hepatitis virus posttranscriptional regulatory element

Wild type

Zeitgeber time

List of publications

Payant MA

Miller PA, Shankhatheertha A, Williams-Ikhenoba, El Khalili HB, Guirguis M,

Chee MJ (2023). Melanin-concentrating hormone and glutamate release in the lateral septum act
on different cells to regulate feeding and anxiety-like behaviour.

In preparation for submission.

Payant MA

, Shankhatheertha A, Chee MJ (2023). Melanin-concentrating hormone promotes

feeding through the lateral septum.

In preparation for submission.

Payant MA

, Spencer CD, Chee MJ (2023). Inhibitory actions of melanin-concentrating hormone

in the lateral septum. Revisions requested. Preprint available at bioRxiv:

https://doi.org/10.1101/2023.10.21.562777

Payant MA

, Sankhe AS, Dumiaty Y, Levy Z, Campbell J, Chee MJ. Dietary fructose induces

synaptic plasticity at neuropeptide Y neurons (2023).

In preparation for submission.

Payant MA

, Chee MJ (2021). Neural mechanisms underlying the role of fructose in

overfeeding. Neurosci Biobehav Rev 128: 346-357.

Hoyek MP, Merhi RC, Blair HL, Spencer CD,

Payant MA

, Martin Alfonso DI, Zhang M,

Matteo G, Chee MJ, Bruin JE (2020). Female mice exposed to low doses of dioxin during

pregnancy and lactation have increased susceptibility to diet-induced obesity and diabetes. Mol

Metab 42:101104.

Negishi K,

Payant MA

Schumacker KS, Wittman G, Butler RM, Lechan

RM, Steinbusch HWM, Khan AM, Chee MJ (2020). Distributions of hypothalamic neuron

populations co-expressing tyrosine hydroxylase and the vesicular GABA transporter in the

mouse. J Comp Neurol 528(11): 1833-1855.

denotes equal authorship

Chapter 1. General introduction: Melanin-concentrating hormone actions in the brain

1.1

Overview

Feeding is a complex behaviour that is influenced by many factors which may include energy

status, emotional state, palatability, and nutritional content of food. The hypothalamus has long

been known as a master regulator of homeostatic feeding as it possesses many neuropeptide-

expressing cell populations that can increase or decrease food intake. Information from the

hypothalamus may be communicated to higher order brain regions through the release of

neuropeptide and neurotransmitter messengers to allow for the integration of physiological state

with other sensory and environmental information. This integration can result in the modification

of behaviour based on context and may, for example, decrease feeding in a dangerous or anxiety-

provoking environment. Investigating how neuropeptides and their co-expressed

neurotransmitters act within target brain regions is critical to understanding the mechanisms

underlying context-dependent feeding behaviour and may assist in developing treatments for

feeding-related disorders such as obesity or anorexia. This thesis focuses on one population of

hypothalamic neurons that express the neuropeptide melanin-concentrating hormone (MCH) and

the function of MCH neuron projections to the lateral septum (LS) to advance our understanding

of brain circuitry regulating feeding behaviour.

MCH is a neuropeptide produced in the lateral hypothalamus (LHA) and has been

implicated in widespread physiological functions such as feeding, mood disorders, memory, and

sleep. These functional insights have been gained largely from transgenic mouse models of

global MCH or MCH receptor (MCHR1) deletion, thus the specific target sites and neural

circuits that underlie these MCH functions are not well-defined. Recent developments using viral

tracing approaches revealed that the LS receives the most synaptic terminals from MCH neurons.

The coincident distribution of MCH fibers in the LS also overlap with known functions of the LS

that regulate feeding and anxiety. In this thesis, we investigated the role of MCH within the LS

by elucidating the neuronal mechanism underlying MCH action in the LS (Aim 1) and

characterizing the contributions of MCH projections in the LS on food-related and anxiety-like

behaviours (Aim 2 and Aim 3).

1.2

MCH neurons

MCH production is largely restricted to neurons in the LHA and zona incerta (Bittencourt et al.,

1992; Broberger et al., 1998), though

Mch

mRNA has also been reported in the medial preoptic

nucleus, paraventricular nucleus, and ventral LS (LSv) during lactation (Beekly et al., 2020).

Within the LHA, medial MCH neurons appear first in development and their distribution spreads

laterally, anteriorly, and posteriorly to peak between embryonic day 12 and 13 (Brischoux et al.,

2001). In adult mice, MCH neurons are interspersed around hypocretin/orexin neurons but are

spread more dorsoventrally and rostrocaudally to cover a larger area (Skofitsch et al., 1985;

Broberger et al., 1998).

MCH neurons are heterogeneous and can also express additional chemical messengers.

MCH is synthesized from the prepro-MCH gene

Pmch

, thus nearly every MCH neuron co-

expresses with Neuropeptide EI and Neuropeptide GE that are also made from

Pmch

(Bittencourt et al., 1992). Moreover, there are subpopulations of MCH neurons that produce

cocaine and amphetamine related transcript (CART) and dynorphin (Broberger, 1999; Harthoorn

et al., 2005; Croizier et al., 2010). MCH neurons that co-express CART are found medially

within the LHA (Broberger, 1999; Croizier et al., 2010) and thus may represent an anatomically

and functionally distinct subpopulation of MCH neurons.

In addition to neuropeptides, MCH neurons may also colocalize with classical

neurotransmitters. MCH neurons reliably colocalize with the vesicular glutamate transporter,

vGLUT2, and can release glutamate at downstream projection sites (Chee et al., 2015; Mickelsen

et al., 2017; Beekly et al., 2020). MCH neurons also express GABA synthesizing enzymes and

have been shown to release glutamate in the tuberomammillary nucleus (Jego et al., 2013) but

interestingly, MCH neurons do not express traditional vesicular GABA transporters, such as the

vesicular GABA transporter

Vgat

, thus they may rely on non-canonical mechanisms or alternate

vesicular transporters for the packaging and release of GABA (Beekly et al., 2020; Chee et al.,

2015; Mickelsen et al., 2017).

1.2.1

Distribution of MCH projections

MCH-immunoreactive fibers can be detected in most of the central nervous system with the

medial MCH cells co-expressing CART projecting rostrally, and lateral MCH cells that do not

co-express CART project caudally toward the spinal cord (Brischoux et al., 2002; Croizier et al.,

2010). MCH-immunoreactive fibers have been found in the LS (Skofitsch et al., 1985;

Bittencourt et al., 1992), with a higher density in the ventral LS (Bittencourt et al., 1992) and

likely originating from medial MCH neurons. Expression in the LSv may overlap with Substance

P-immunoreactive fibers and cells expressing the estrogen receptor (Risold and Swanson,

1997b). Tracing studies from MCH neurons have shown that there is strong innervation from

virally-labeled projections of MCH neurons that were concentrated more dorsally in the LS and

reflect sites for glutamate release, though it is not clear if these projections also carry MCH

(Chee et al., 2015; Liu et al., 2022).

1.3

Widespread distribution of MCH receptors

MCH has two endogenous receptors in the human brain, MCHR1 and MCHR2 (Hill et al.,

2001), however only MCHR1 is present in the rodent brain (Tan 2002). MCHR1 distribution

parallels the extensive expression of MCH-immunoreactive fibers with many regions expressing

Mchr1

mRNA in the rat (Lembo et al., 1999; Saito et al., 2001) and mouse brain (Chee et al.,

2013). Moderate expression of

Mchr1

mRNA and ciliary MCHR1 immunoreactivity in the LS

(Lembo et al., 1999; Hervieu et al., 2000; Saito et al., 2001; Chee et al., 2013) is more prominent

in the LSv (Diniz et al., 2020). However, injections of rhodamine-conjugated MCH led to a high

expression of rhodamine-labeled cells in the LS and suggests the presence of functional MCHR1

at levels higher than what may be detected with traditional staining methods (Ruiz-Viroga et al.,

2021).

1.3.1

MCH receptor activation

MCHR1 is a G-protein coupled receptor that can couple to G

-, G

-, and G

-protein-mediated

pathways

in vitro

to both increase intracellular calcium levels and inhibit cyclic AMP production

(Bachner et al., 1999; Hawes et al., 2000; Pissios et al., 2003). Furthermore, MCHR1 signaling

can lead to the inhibition of voltage-dependent calcium channels (Gao et al., 2003), activation of

calcium-induced chloride currents (Bachner et al., 1999), and activation of G-protein-coupled

inwardly-rectifying potassium channels (Bachner et al., 1999) in cultured cells.

In brain slices, MCH application can lead to neuronal inhibition in several brain regions

(Zheng et al., 2005; Wu et al., 2009; Sears et al., 2010; Devera et al., 2015; Al-Massadi et al.,

2019; Liu et al., 2022), however the mechanism of MCHR1 activation is site-specific. MCH-

mediated inhibition occurs postsynaptically through the activation of potassium channels in the

nucleus accumbens (Sears et al., 2010) and medial septum (Wu et al., 2009). By contrast, MCH

has presynaptic inhibitory effects on glutamatergic input to neurons in the nucleus of the solitary

tract (Zheng et al., 2005) but presynaptically enhances glutamatergic and GABAergic input in

the LS (Liu et al., 2022).

1.4

MCH functions

MCH was first isolated from the pituitary of salmon where it aggregates melanin granules to

lighten the skin color of fish (Kawauchi et al., 1983). MCH is a highly conserved neuropeptide

found in many species. The first report of MCH action in mammals described its orexigenic

actions that stimulated feeding upon intracerebroventricular (ICV) administration, thus

implicating its contribution to energy balance (Vaughan et al., 1989; Qu et al., 1996). Ensuing

studies have since determined that MCH can modulate many other functions, including sleep,

memory, motivation, olfaction, maternal behaviour, mood disorders, and immune function

(Lakaye et al., 2009; Takase et al., 2014). This thesis focuses on two major roles of MCH

—

energy balance and anxiety

—

as these pertain to overlapping functions of the LS.

1.4.1

MCH and energy balance

1.4.1.1

MCH transgenic models

Genetic manipulations have established that MCH is necessary for the maintenance of body

weight and energy expenditure. Knocking out

Pmch

in mice leads to decreased body weight,

reduced fat mass, hypophagia, and increased oxygen consumption (Shimada et al., 1998). MCH-

knockout mice are also resistant to diet-induced obesity and adverse metabolic effects of aging,

like insulin resistance, due to their increase in energy expenditure (Kokkotou et al., 2005; Jeon et

al., 2006). Unsurprisingly, a similar phenotype can also be produced by ablating MCH neurons

(Alon and Friedman, 2006; Whiddon and Palmiter, 2013) or knocking out the MCH receptor,

MCHR1 (Chen et al., 2002). MCH deletion, MCHR1 deletion, or MCH neuron ablation in leptin

deficient

mice can also increase energy expenditure and lessen the obese phenotype

(Segal-Lieberman et al., 2003; Alon and Friedman, 2006; Bjursell et al., 2006). On the other

hand, overexpressing the MCH gene leads to mice that are hyperphagic and develop symptoms

of metabolic disorder, such as elevated insulin, leptin, and glucose levels compared to wildtype

controls (Ludwig et al., 2001).

MCH decreases energy expenditure through downregulation of the mesolimbic dopamine

system. MCH knockout mice and mice with ablated MCH neurons have increased expression of

markers of dopamine transmission such as DAT, have increased dopamine release in the nucleus

accumbens, and are more sensitive to blocking dopamine reuptake (Pissios et al., 2008; Whiddon

and Palmiter, 2013). Specifically, MCH can inhibit dopamine release in the nucleus accumbens,

and deletion of MCHR1 on GABAergic neurons in the nucleus accumbens increases locomotor

activity (Chee et al., 2019).

1.4.1.2

MCH administration promotes feeding

Acute and chronic MCH administration can promote feeding leading to diet-induced obesity.

Acute MCH infusion into the lateral ventricle or third ventricle of rats dose-dependently

increases food intake primarily within the first 1

–

2 hours after infusion and decreases toward

baseline levels within 6 hours (Qu et al., 1996; Rossi et al., 1997; Kela et al., 2003). Consistent

with this idea, endogenous MCH levels are highest in the cerebrospinal fluid at the beginning of

the dark phase, prior to animals eating their first meal (Noble et al., 2018). Specifically, MCH

administration can increase the rate of licking a sweet solution during the first minute of a meal,

suggesting that MCH is involved in post-oral appetition to increase food consumption (Baird et

al., 2006; Lord et al., 2021).

Chronic intracerebroventricular (ICV) infusion of MCH, lasting between 6 and 14 days,

produces an obesogenic phenotype, increasing food intake (Rossi et al., 1997; Marsh et al., 2002;

Kawata et al., 2017), body weight (Marsh et al., 2002; Kawata et al., 2017), body fat (Marsh et

al., 2002), circulating glucose and insulin, and increased liver adiposity (Kawata et al., 2017).

The hallmarks of metabolic disorder are enhanced when MCH-treated mice were given a high fat

diet (Gomori et al., 2003). Infusions of an MCH receptor agonist replicates this phenotype

(Shearman et al., 2003) and correspondingly, ICV infusions or oral administration of an MCH

receptor antagonist can suppress the development of diet-induced obesity (Shearman et al., 2003;

Ito et al., 2010; Kawata et al., 2017). Although the orexigenic effects of MCH are robust in

males, the increased estradiol in females reduces the effects of MCH (Messina et al., 2006;

Santollo and Eckel, 2008, 2013).

1.4.1.3

Hypothalamic regions underlying MCH-mediated homeostatic feeding

The neural circuitry that underlies the orexigenic effect of MCH is largely unknown but may be a

result of MCH release into the cerebrospinal fluid to reach target sites within the forebrain (Baird

et al., 2008; Noble et al., 2018).

MCH-induced hyperphagia may be mediated through several hypothalamic nuclei,

including the arcuate nucleus, and lateral hypothalamic nucleus (Rossi et al., 1999; Abbott et al.,

2003; Romero-Pico et al., 2018). In the arcuate nucleus, neurons co-expressing agouti-related

peptide (AgRP) and neuropeptide Y (NPY), and neurons co-expressing proopiomelanocortin

(POMC) and CART are well known to have orexigenic and anorexigenic functions, respectively

(Andermann and Lowell, 2017). Although MCH function is not required for the orexigenic

effects of NPY or AgRP (Marsh et al., 2002), MCH application to hypothalamic explants leads

to increased release of NPY and AgRP while decreasing CART and alpha-MSH release (Abbott

et al., 2003). Moreover, MCH inhibits POMC neurons to increase feeding, body weight,

adiposity, and glucose intolerance (Al-Massadi et al., 2019). In the LHA, antagonism or

knockdown of opioid receptors blocks the ability of MCH to stimulate feeding (Romero-Pico et

al., 2018) which may be a result of acting on neurons that co-express MCHR1 (Imbernon et al.,

2016).

1.4.1.4

Brain regions underlying MCH-mediated effects on hedonic feeding

In addition to driving homeostatic feeding to promote a positive energy balance, MCH also

interacts with the reward system to enhance the value and intake of sweet foods. The preference

for the non-nutritive sweetener, sucralose, surpasses the preference for sucrose when paired with

optogenetic activation of MCH neurons (Domingos et al., 2013). This response is associated with

activation of ventral tegmental area dopamine neurons and higher dopamine levels within the

nucleus accumbens (Domingos et al., 2013). Additionally, MCH infused directly into the nucleus

accumbens or activation of neurons that project to the nucleus accumbens drives consumption of

chow and sucrose in males (Georgescu et al., 2005; Lopez et al., 2011; Terrill et al., 2020) and

may be mediated through activation of opioid receptors (Lopez et al., 2011).

MCH may also be involved in learned aspects of feeding such as recognizing food cues

that can lead to overeating. MCHR1-KO mice can still learn a conditioned stimulus but no longer

overeat when the cue was present, suggesting MCH signaling is necessary for the expression of

cue-induced overeating (Sherwood et al., 2015).

1.4.1.5

The LS as a possible mediator of MCH-induced feeding

Like MCH, the LS is also known to regulate homeostatic and hedonic feeding. However, while

MCH actions are orexigenic, the LS mediates anorexigenic actions, as loss of LS function via

lesions to the entire septal area or inhibition by GABA

or GABA

receptor activation increases

food intake and body weight (Singh and Meyer, 1968; Stoller, 1972; King and Nance, 1986;

Calderwood et al., 2020; Gabriella et al., 2022). In particular, the ventral LS (LSv) is most

prominent for the anorexigenic effects of the LS (Pankey et al., 2008), as optogenetic activation

of LSv neurons decreases feeding and LSv neurons decrease their activity during a feeding bout

(Xu et al., 2019). Thus, the inhibitory effects of MCHR1 activation within the LS may contribute

to driving homeostatic feeding.

Opioid signaling within the LS plays an important role in LS modulation of food intake.

There are enkephalinergic neurons within the LS along the lateral border as well as enkephalin-

immunoreactive fibers (Risold and Swanson, 1997b). Leu-enkephalin is downregulated in fasted

male animals (Kovacs et al., 2005) but injections of a µ-opioid receptor agonist and morphine

into the LS increased feeding (Calderwood et al., 2020; Calderwood et al., 2022). In fact, it was

found that the LS is one of the most effective site of morphine-stimulated feeding (Stanley et al.,

1988). Since the orexigenic effects of MCH are in part dependent on the activation of µ-opioid

receptors, the LS represents a possible region mediating this effect.

The inhibitory effect of the LS on hedonic feeding is related to GABAergic transmission

in the LSv, which can paradoxically also increase food seeking. LSv GABAergic neurons are

important for inhibiting palatable foods but do not affect homeostatic-driven feeding following a

fast (Chen et al., 2022). LS lesions and intra-LS infusions of GABA

and GABA

agonists

increase motivation (Pubols, 1966; Lorens and Kondo, 1971) and consumption of a palatable diet

independent of their nutritional status (Beatty and Schwartzbaum, 1968; Mitra et al., 2014). In

the LSv, strong activation of neurotensin expressing neurons allowing for neuropeptide release

can suppress all feeding, however stimulation that would only release GABA inhibits hedonic

feeding. There may be additional complexity to this circuit as there are different subtypes of

neurotensin-expressing LSv neurons that are activated during approach and those that are

inhibited during consumption of palatable food. The activation of these neurons can both

decrease latency to food approach but suppress food intake when stimulated at different times

(Chen et al., 2022). Food approach may depend on the ability to remember the location of food

and consistent with this, activation of inputs from the ventral hippocampus engages LS

GABAergic neurons to enhance food-related spatial memory (Decarie-Spain et al., 2022). MCH

can potentiate hippocampal input to the LS (Liu et al., 2022), thus this circuit may also be

involved in recognizing and approaching food cues in the environment that can lead to

overeating.

Summary

. The LS is involved in both homeostatic and hedonic feeding. The orexigenic

effects of MCH may be mediated through the LS by directly inhibiting LS neurons, acting

through opioid signaling, or modulating hippocampal input to the LS to alter food-seeking

behaviour.

1.4.2

MCH and anxiety

Studies from genetic or chemogenetic manipulations of the MCH system have most consistently

supported an anxiogenic role for MCH. Chemogenetic activation of MCH neurons increases

anxiety-like behaviour in the elevated plus maze, open field test, marble burying test, and

sucrose preference test (He et al., 2022). By contrast, MCH-KO mice are more resistant to stress-

induced hyperthermia (Smith et al., 2006). Likewise, anxiety-like behaviours are minimized in

MCHR1-KO animals, as they spend more time in the center of the open field or open arms of the

elevated plus maze, have no increase in temperature in the stress-induced hyperthermia

paradigm, and spend more time interacting with an intruder in the social interaction test (Roy et

al., 2006). Furthermore, MCHR1 antagonists on their own can decrease behavioural measures of

stress and anxiety in a social interaction test, pup separation test, stress induced hyperthermia,

and elevated plus maze making them a potential therapeutic treatment (Borowsky et al., 2002;

Chaki et al., 2005; Chaki et al., 2015), but importantly, MCHR1 antagonism can reverse anxiety-

like behaviours caused by MCH infusion (Smith et al., 2006) or by stress (Chaki et al., 2015).

While global MCH manipulations through genetic deletion of MCH receptors or

activation of all MCH neurons suggest an anxiogenic function of MCH, it is not clear if this

effect is due to compensatory mechanisms from developmental models or contribution of other

chemical messengers. The role of MCH on anxiety can be heterogenous because despite reports

that central administration of MCH can be anxiogenic (Smith et al., 2006), some report

anxiolytic outcomes (Kela et al., 2003) or no effects (Duncan et al., 2005). This distinction may

be clarified by investigating the effects of MCH in specific brain regions, which may reveal

anxiogenic and anxiolytic effects.

1.4.2.1

Brain regions underlying MCH-mediated effects on anxiety.

Anxiety is regulated by many different brain regions, and differential MCH action in the

prefrontal cortex and dorsal raphe or basolateral amygdala (BLA) may mediate the anxiolytic

and anxiogenic effects of MCH, respectively. MCH injection into the dorsal raphe is anxiogenic

(Oh et al., 2020) and may be a result of decreased activity of serotonin neurons (Devera et al.,

2015). In addition, injections of MCH into the BLA increases anxiety-like behaviour and

interferes with the beneficial effects of exercise on anxiety (Kim et al., 2015; He et al., 2022).

Furthermore, administration of an MCHR1 antagonist into the BLA blocks increases in anxiety-

like behaviour observed with chemogenetic activation of MCH neurons (He et al., 2022).

In contrast to the anxiogenic effects of MCH in the dorsal raphe and BLA, global

MCHR1 deletion decreases serotonin in the prefrontal cortex and eliminates the surge of

serotonin caused by a forced swim stressor (Roy et al., 2006). Moreover, intranasal MCH

improves stress-induced decreased phosphorylation of transcription factors, including mTOR,

and synaptic proteins in the prefrontal cortex. The anxiolytic effects of MCH were blocked by

co-administration with the mTOR inhibitor, rapamycin, suggesting an important role of this

pathway in the prefrontal cortex in MCH-mediated decreases in anxiety (Oh et al., 2020).

1.4.2.2

LS as an integrative site for the expression of MCH-mediated anxiolysis

The LS has long been known to play a role in stress and anxiety but may have a modulatory role

depending on the context and chemical messengers involved. LS cells are activated by several

stressful paradigms including the foot shock avoidance test, elevated plus maze, and air puff-

induced ultrasonic vocalization test (Duncan et al., 1996; Thomas et al., 2013), but their firing

rate may decrease following the stressor (Contreras et al., 2004). Specifically, a subpopulation of

LS cells that express neurotensin are activated only in response to a stressor that can be escaped

and may contribute to the context dependent regulation of behaviour (Azevedo et al., 2020).

Septal lesions reduce anxiety in the elevated plus maze and shock probe-burying test

(Treit and Pesold, 1990; Menard and Treit, 1996), and pharmacological inhibition of the LS via

intra-septal infusions of GABA agonists were anxiolytic in punished drinking, shock probe

burying task, and elevated plus maze (Drugan et al., 1986; Pesold and Treit, 1996; Lamontagne

et al., 2016). In conjunction with this, blocking glutamatergic transmission in the LS also has an

anxiolytic effect in females (Molina-Hernandez et al., 2006). The release of glutamate from

MCH neurons may thus have anxiogenic actions, but as the net effect of MCH neuron activation

inhibits LS activity and that MCHR1 activation is known to inhibit postsynaptic cells (Chee et

al., 2015; Liu et al., 2022), MCH projections may have net anxiolytic actions in the LS.

Furthermore, the expression of stress and anxiety in the LS may be attributed to

dysfunctions in the brain serotonin system. The stress hormone, corticotropin releasing hormone,

can depress serotonin levels in the LS (Price and Lucki, 2001). While MCH can depress neuronal

firing in the dorsal raphe (Devera et al., 2015), it may also act presynaptically at serotonergic

projections to suppress transmitter release (Sears et al., 2010) and reduce anxiety through the LS

(Viana Mde et al., 2008).

1.5

Specific aims and hypotheses

Rationale

The LS is one of the strongest projection sites of MCH neurons and possesses both

MCH-immunoreactive fibers and MCH receptors. However, the effects of MCH in the LS have

not been well characterized. Given the anatomical evidence for MCH action in the LS and the

functional similarities between known roles of MCH and those supported by the LS, I

hypothesize that the LS is a brain region that mediates MCH action in the regulation of food-

related and anxiety-like behaviours. My thesis aims to define and characterize the mechanisms of

MCH action (

Aim 1

;

Chapter 2

), behavioural contributions of MCH projections (

Aim 2

;

Chapter 3 and 4

), and conditions underlying MCH release (

Aim 3

;

Chapter 4

) in the LS. These

objectives will elucidate whether the LS is a prominent brain region underlying MCH-mediated

behaviours.

Aim 1. Determine the neuroanatomical and cellular basis of MCH action in the LS

(Chapter 2).

The LS is a diverse and heterogenous structure, and while the expression of MCH fibers and

receptors have previously been shown, the precise LS region that supports MCH action has not

been defined. Therefore, we generated neuroanatomical maps to chart the distribution of MCH

fibers and receptors within the entire LS (

Aim 1a

) and used these maps to triangulate hotspots

for MCH action. We then performed electrophysiological recordings to define the neuronal

mechanisms underlying MCH action (

Aim 1b

Aim 1a. Map the distribution of MCH fibers,

Mchr1

mRNA, and MCHR1 protein

expression within the LS.

We will use a combination of immunohistochemistry and

in situ

hybridization to generate

standardized neuroanatomical maps of MCH-immunoreactive fibers,

Mchr1

mRNA, and

MCHR1 protein expression throughout the whole LS in male and female mice. I hypothesize that

LS regions where MCH fiber and receptor distribution overlap constitute hotspots of MCH

action.

Aim 1b. Determine the cellular mechanisms of MCH action at LS cells.

Whole-cell patch-clamp recordings from LS cells will assess the effect of bath-applied MCH on

afferent input and excitability of LS cells. I hypothesize that MCH application can produce long-

lasting inhibition of LS cells in a MCHR1-dependent manner.

Aim 2. To determine the effects of MCH on feeding and anxiety-like behaviour in the LS

(Chapter 3 and 4).

The LS comprises the densest projections from MCH neurons and has known functions

mediating feeding and anxiety-like behaviour that overlap with known functions of the MCH

system. As MCH neurons may release both MCH and/or glutamate in the LS, we will directly

compare the behavioural effect following MCH infusion (

Aim 2a

) or optogenetic stimulation of

MCH projections, which may include the release of both MCH and glutamate (

Aim 2b

). We will

isolate the effect of MCH using pharmacological or transgenic approaches, which includes

generating the

Pmch-cre;Vglut2-flox

mouse where glutamate release was disabled.

Aim 2a. Define the behavioural response of intra-LS MCH administration on food

intake (Chapter 3).

We will determine the effect of intra-LS infusion of MCH and MCHR1 activation on context-

dependent feeding. I hypothesize that intra-LS infusion will promote food intake and that this

orexigenic action will be blocked when pretreated with a MCHR1 antagonist.

Aim 2b. Define the behavioural response following optogenetic stimulation of fiber

projections from MCH neurons (Chapter 4).

We will (

) optimize optogenetic protocols, including the conditions for viral expression, optic

fiber implants, and photostimulation intensity or duration, and (

) compare behavioural

outcomes following the optogenetic stimulation of channelrhodopsin-expressing fiber projections

from

Pmch-cre

and

Pmch-cre;Vglut2-flox

mice during

ad libitum

feeding and the open field test.

The specificity of MCH actions will also be assessed following treatment with a MCHR1

antagonist. I hypothesize that photostimulating MCH terminals in the LS of

Pmch-cre

mice will

produce similar behavioural results as MCH infusion thereby increasing food intake and anxiety-

like behaviour but to a lesser extent than in

Pmch-cre;Vglut2-flox

because glutamate release

from MCH neurons may oppose the actions of MCH.

1.6

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2. 1 Key points

•

RESEARCH QUESTION.

Melanin-concentrating hormone (MCH) neurons have dense

nerve terminals within the lateral septum (LS), a key region underlying stress- and

anxiety-like behaviours that are emerging roles of the MCH system, but it is not known if

the LS is a MCH target site.

•

NEUROANATOMY.

We found spatial overlap between MCH-immunoreactive fibers,

Mchr1

mRNA, and MCHR1 protein expression especially along the lateral border of the

LS.

•

ELECTROPHYSIOLOGY.

Within MCHR1-rich regions, MCH directly inhibited LS

cells by increasing a chloride conductance in a protein kinase C-dependent manner.

•

SIGNIFICANCE.

Electrophysiological MCH effects in brain slices have been elusive

and even fewer have described the mechanisms of MCH action. Our findings

demonstrated, to our knowledge, the first description of MCHR1 Gq-coupling in brain

slices, which was previously predicted in cell or primary culture models only. Together,

these findings defined hotspots and mechanistic underpinnings for MCH effects such as

in stress- and anxiety-related behaviours.

2.2

Abstract

Melanin-concentrating hormone (MCH) neurons can co-express several neuropeptides or

neurotransmitters and send widespread projections throughout the brain. Notably, there is a

dense cluster of nerve terminals from MCH neurons in the lateral septum (LS) that innervate LS

cells by glutamate release. The LS is also a key region integrating stress- and anxiety-like

behaviours that are also emerging roles of MCH neurons. However, it is not known if the MCH

peptide acts within the LS or whether MCH target sites are localized. We analysed the

projections from MCH neurons in male and female mice anteroposteriorly throughout the LS and

found spatial overlap between the distribution pattern of MCH-immunoreactive (MCH-ir) fibers

with MCH receptor

Mchr1

mRNA hybridization or MCHR1-ir cells. This overlap was most

prominent along the ventral and lateral border of the rostral part of the LS (LSr). Most MCHR1-

labeled LS neurons laid adjacent to passing MCH-ir fibers, but some MCH-ir varicosities

directly contacted the soma or cilium of MCHR1-labeled LS neurons. We thus performed whole-

cell patch-clamp recordings from MCHR1-rich LSr regions to determine if and how LS cells

respond to MCH. Bath application of MCH to acute brain slices activated a bicuculline-sensitive

chloride current that directly hyperpolarized LS cells. This MCH-mediated hyperpolarization

was blocked by calphostin C and suggested that the inhibitory actions of MCH were mediated by

protein kinase C-dependent activation of GABA

receptors. Taken together, these findings

defined potential hotspots within the LS that may elucidate the contributions of MCH to stress-

or anxiety-related feeding behaviours.

2.3

Introduction

Neurons that produce melanin-concentrating hormone (MCH) are found primarily within the

lateral hypothalamic area (LHA) (Broberger

et al.

, 1998; Broberger, 1999; Croizier

et al.

, 2010;

Beekly

et al.

, 2020), but they can send widespread projections throughout the brain (Skofitsch

al.

, 1985; Bittencourt

et al.

, 1992). MCH neurons can express additional neuropeptides

(Harthoorn

et al.

, 2005; Mickelsen

et al.

, 2017) and neurotransmitters like GABA (Jego

et al.

2013) and glutamate (Chee

et al.

, 2015). MCH has well-established functions in energy balance

(Qu

et al.

, 1996; Shimada

et al.

, 1998; Ludwig

et al.

, 2001; Kokkotou

et al.

, 2005; Pissios

et al.

2006) and sleep (Verret

et al.

, 2003; Ferreira

et al.

, 2017), but recent findings have also

elaborated on the roles of MCH for regulating stress (Kim & Han, 2016), motivation (Mul

et al.

2011), and memory (Monzon et al., 1999; Adamantidis et al., 2005; Adamantidis and Lecea,

2009). The diverse functions of MCH thus implicate distinctive target sites for MCH.

MCH neurons strongly innervate the lateral septum (LS) via direct glutamatergic

projections (Chee

et al.

, 2015), but it is not known whether MCH plays a role in the LS. MCH

immunoreactivity has been detected in the LS of the rat brain (Skofitsch

et al.

, 1985; Bittencourt

et al.

, 1992), but this has not been examined in detail for the mouse brain. In rats,

immunohistochemical staining showed that the LS comprises moderate levels of MCH-

immunoreactive (MCH-ir) fibers, with the highest level of immunoreactivity in the ventral part

of the LS (Bittencourt

et al.

, 1992). In addition to the presence of MCH-ir fibers, the expression

of MCH receptors (MCHR) also aid in identifying the LS as a potential target site of MCH

action. There are two known MCH receptors in the human brain, MCHR1 and MCHR2 (Hill

al.

, 2001), but only MCHR1 is present in the rodent brain (Tan

et al.

, 2002). Similar to the

widespread distribution of MCH-ir fibers, many brain regions can express

Mchr1

mRNA within

the rat (Lembo

et al.

, 1999; Saito

et al.

, 2001) and mouse brain (Chee

et al.

, 2013). Indeed, there

is a moderate level of

Mchr1

mRNA in both the rat (Lembo

et al.

, 1999; Saito

et al.

, 2001) and

mouse LS (Chee

et al.

, 2013).

MCHR1 is a G-protein coupled receptor that can couple to G

- (Hawes

et al.

, 2000), G

(Hawes

et al.

, 2000), or G

-protein-mediated pathways (Pissios

et al.

, 2003). However, MCH

action in the brain is largely inhibitory by hyperpolarizing the membrane and suppressing action

potential firing (Gao, 2009), for example at the lateral hypothalamus (Rao

et al.

, 2008), nucleus

accumbens (Georgescu

et al.

, 2005; Sears

et al.

, 2010), or medial septal nucleus (Wu

et al.

2009). In this study, we assessed the neuroanatomical and electrophysiological premise for MCH

action in the LS and determined whether MCH could inhibit the activity of LS cells.

We described the distribution of MCH-ir fibers,

Mchr1

mRNA, and MCHR1 protein in

the mouse LS, and we used these fiber and cell maps to guide patch-clamp recordings to identify

putative sites and mechanisms of MCH action. As the MCH system (Mystkowski

et al.

, 2000;

Mogi

et al.

, 2005; Rondini

et al.

, 2007; Takase

et al.

, 2014; Terrill

et al.

, 2020; Teixeira

et al.

2020) as well as the LS has been shown to be sexually dimorphic, we completed our analyses in

the male and female brain but determined that there were no sex differences in the

neuroanatomical and electrophysiological effects of MCH. We observed similar distribution

patterns between MCH-ir,

Mchr1

mRNA, and MCHR1 protein throughout the entire

rostrocaudal extent of the LS and found that MCH directly inhibited LS cells by recruiting

protein kinase C (PKC) and activating a GABA

receptor-mediated chloride conductance. These

findings indicate that MCH can act in the LS to regulate neuron activity and suggest that the LS

is an important projection site for MCH functions.

2.4

Materials and Methods

The use of all animals has been approved by the Carleton University Animal Care Committee on

Animal Use Protocol 110940 in accordance with guidelines provided by the Canadian Council

on Animal Care. All C57BL/6J

wild type mice (stock 000664; Jackson Laboratory, Bar Harbor,

ME) were bred in house and maintained on a 12-hour light-dark cycle (22

–

24°C; 40

–

60%

humidity). All mice were given

ad libitum

access to food (Teklad Global Diets 2014, Envigo,

Mississauga, Canada) and water.

2.4.1 Neuroanatomy

Tissue processing.

Mice

were anesthetized with an intraperitoneal injection (i.p.) of chloral

hydrate (700 mg/kg; MilliporeSigma, Burlington, MA) prepared in sterile saline, transcardially

perfused with cold (4



C) saline (0.9% NaCl), then followed by fixation with 10% formalin

(VWR, Radnor, PA). The brain was extracted from the skull, post-fixed overnight in 10%

formalin (24 hr, 4



C), and cryoprotected in phosphate buffered saline (PBS) containing 20%

sucrose and 0.05% sodium azide (24 hr, 4



C). Mice whose brains were processed for MCHR1

immunohistochemistry were perfused with saline followed by 250 mL of 10% formalin. Brains

were post-fixed in 20% sucrose dissolved in 10% formalin (4 hr, 4



C) then cryoprotected as

above.

All brains were sliced into five series of 30



m coronal sections using a freezing

microtome (Spencer Lens Co., Buffalo, NY). Two tissue series remained free-floating in PBS-

diluted formalin (comprising PBS-azide and formalin in a 9:1 ratio) prior to

immunohistochemical staining for MCH or MCHR1. Three tissue series were mounted onto

Fisherbrand Superfrost Plus Microscope Slides (Fischer Scientific, Waltham, MA) to use for

situ

hybridization. One series, designated as probe tissue, was used for

Mchr1

hybridization.

Two adjacent series served as a positive control and a negative control to the probe tissue. The

negative control tissue was later used for Nissl staining to parcellate and define the

neuroanatomical boundaries of each slice. After tissues were mounted, the glass slides were air

dried at room temperature (RT, 20

–



C; 1 hr), and then at −20



C (30 min) before being stored

at −80



Single-label immunohistochemistry.

To detect MCH immunoreactivity, the tissue was

washed in six 5-min exchanges of PBS and pretreated with 10 mM sodium citrate for 5 min

(75°C) followed by 0.3% hydrogen peroxide in PBS for 20 min (RT). Following three 10-min

PBS exchanges, the tissue was then blocked with 3% normal donkey serum (Jackson

ImmunoResearch Laboratories, Inc., West Grove, PA) dissolved in PBS with 0.25% Triton-X

(PBT) and 0.05% sodium azide for 2 hr (NDS; RT). After blocking, the tissue was incubated

with an anti-rabbit MCH antibody (1:2,000; kindly provided by Dr. E. Maratos-Flier, Beth Israel

Deaconess Medical Center; RRID: AB_2314774; (Elias

et al.

, 1998; Chee

et al.

, 2013) overnight

in NDS (RT). The following day, the tissue was washed six times in PBS (5 min each) then

incubated with a biotinylated goat anti-rabbit antibody (1:500; Jackson ImmunoResearch

Laboratories; RRID: AB_2337965) prepared in NDS for 1 hr (RT). The tissue was washed three

times in PBS for 10 min each and treated with avidin biotin horseradish peroxidase (PK-6100,

Vector Laboratories, Newark, CA) in PBT for 30 min (RT). Tissue was washed in three 10-min

PBS exchanges and underwent tyramine signal amplification by treating with PBT comprising

0.005% hydrogen peroxide and 0.5% borate-buffered biotinylated (Sulfo-NHS-LC biotin; 21335,

Thermo Fisher Scientific, Waltham, MA) tyramine (T90344, MilliporeSigma) for 20 min (RT).

Following three 10-min washes in PBS, the tissue was incubated with an Alexa Fluor 647-

conjugated streptavidin antibody (1:500; Jackson ImmunoResearch Laboratories; RRID: AB_

2341101) and NeuroTrace 435/455 (1:50; N21479, Thermo Fisher Scientific) in NDS without

sodium azide for 2 hr (RT). Slices were then mounted on SuperFrost Plus microscope slides and

coverslipped with ProLong Diamond Antifade Mountant (Thermo Fisher Scientific).

Dual-label immunohistochemistry

To detect MCHR1 immunoreactivity, tissue was first

washed in six 5-min PBS exchanges and pretreated with 0.3% hydrogen peroxide in PBS for 20

min (RT). Following a set of three 10-min washes, the tissue was blocked in NDS for 2 hr (RT)

and then incubated in anti-rabbit MCHR1 antibody (1:3,000; Thermo Fisher Scientific; RRID:

AB_2541682) prepared in NDS for 48 hr (4°C). The tissue was rinsed with six 5-min PBS

exchanges and incubated with a biotinylated goat anti-rabbit antibody (1:5,000) in NDS without

azide for 1 hr (RT). Following three 10-minute washes, the tissue was incubated in avidin biotin

horseradish peroxidase in PBT for 30 min (RT). The tissue was washed three times (10 min each,

RT) and underwent tyramine signal amplification. After washing the tissue three times with PBS

(10 min each, RT), it was incubated with a Cy3-conjugated streptavidin antibody (1:200, RT;

Jackson ImmunoResearch Laboratories; RRID: AB_2337244).

The tissue was then washed three times with PBS (10 min each) and incubated with an

anti-rabbit NeuN antibody (1:2,000; MilliporeSigma; RRID: AB_2571567) in NDS overnight

(RT). The following day, the tissue was washed in six 5-min PBS exchanges and incubated with

a donkey anti-rabbit Alexa Fluor 488-conjugate (1:500; Thermo Fisher Scientific; RRID:

AB_2535792) and NeuroTrace 435/455 (1:50) in NDS without azide. Finally, the tissue was

washed for 2 hr in PBS (RT) prior to mounting on SuperFrost Plus slides and coverslipped with

ProLong Diamond Antifade Mountant. This MCHR1 antibody has been previously validated for

ciliary expression (Diniz et al., 2020) and we have determined that there is no MCHR1 staining

in the LS of male or female MCHR1-knockout mice (data not shown).

Triple-label immunohistochemistry

To determine the proximity of MCHR1- and MCH-

immunolabeling at NeuN-labeled cells, brain tissues were prepared using procedures for

optimized MCHR1 labeling. Tissues were treated to label MCHR1 immunoreactivity, as

described above, followed by tyramine signal amplification and treatment with an Alexa Fluor

647-conjugated streptavidin antibody (1:200; Jackson ImmunoResearch Laboratories; RRID:

AB_ 2341101). After rinsing with three PBS exchanges (10 min each), they were immediately

incubated anti-rabbit MCH (1:2,000; RRID: AB_2314774) and anti-mouse NeuN (1:1,000;

HB6429, Hello Bio, Princeton, NJ) in NDS overnight (RT). After the tissues were washed in six

5-min PBS exchanges, they were incubated with an NDS cocktail comprising donkey anti-rabbit

Alexa Fluor 568-conjugate (1:1,000; Thermo Fisher Scientific; RRID: AB_2534017) and donkey

anti-mouse Alexa Fluor 488-conjugate (1:500; Thermo Fisher Scientific; RRID: AB_141607) for

2 hr at RT, rinsed with PBS, then mounted onto SuperFrost Plus slides and coverslipped with

ProLong Diamond Antifade Mountant.

In situ hybridization.

We optimized

in situ

hybridization procedures using a RNAscope

Multiplex Fluorescent Reagent Kit v2 (Advanced Cell Diagnostics (ACD), Newark, CA) and

manufacturer instructions for fixed-frozen mouse brain tissue (Document 323100-USM, ACD).

To promote tissue adherence, slides w

ere removed from storage at −80°C, baked at 37°C for 45

min, dehydrated in an ethanol gradient (50%, 70%, 100%; 5 min each), and then air-dried for 15

min (RT) immediately prior to the start of tissue treatments.

Tissue was rehydrated in PBS for 5 min (RT), pretreated with 5

–

8 drops of hydrogen

peroxide (323110, ACD) for 10 min (RT), washed twice in distilled water for 1 min each, and

submerged in 100% ethanol for 15 min (RT) to promote tissue adherence. The slides were then

placed inside a steamer (Oster, Boca Raton, FL) using a coplin jar filled with preheated distilled

water for 10 s (99°C) before transferring into the Target Retrieval Reagent (322000, ACD) for 5

min (99°C). Following two 15 s washes in distilled water (RT), the slides were dehydrated in

100% ethanol for 3 min, and then washed in three PBS exchanges (1 min each). A hydrophobic

barrier was then drawn around each slide with an ImmEdge pen (Vector Laboratories), and the

slides were dried overnight (RT). The following day, the slides were washed twice in PBS for 2

min and then placed in 10% formalin for 30 min (RT). Slides were then washed twice in PBS for

2 min and the tissue was treated with 5

–

8 drops of Protease Plus (322331, ACD) and incubated

in a HybEZ oven (310010, ACD) at 40°C for 30 min. After protease treatment, the slides were

washed with two exchanges of distilled water for 1 min each.

RNAscope probes for

Mm-Ppib

(313911, ACD),

Bacillus

dapB

(320871, ACD), and

Mm-Mchr1

(317491, ACD) were designated for positive control, negative control, or

experimental targeting, respectively, and were applied directly to the slides to cover the tissue.

The tissue was hybridized for 2 hr at 40°C in the HybEZ oven, then washed with three fresh

exchanges (2 min each; RT) of 1



Wash Buffer (310091, ACD).

The hybridization signal was

amplified by alternating incubations in AMP-1 (40°C, 30 min; 323110, ACD), AMP-2 (40°C, 30

min; 323110, ACD), and AMP-3 (40°C, 15 min; 323110, ACD) with two Wash Buffer washes

(2 min each).

Mchr1

hybridization was then labeled with Cyanine 3 (Cy3) by treating tissue with HRP-

C1 (40°C, 15 min; 323110, ACD), washing the tissue twice in Wash Buffer for 2 min (RT), and

incubating the tissue with TSA plus Cy3 (1:750; NEL44E001KT, PerkinElmer, Waltham, MA)

in TSA Buffer (322809, ACD) for 30 min in the 40°C oven. Slides were then washed twice in

Wash Buffer (2 min each, RT) and incubated with HRP Blocker (323110, ACD) in the oven at

40°C for 15 min.

Where applicable, the tissue underwent immunohistochemical staining to label MCH-

immunoreactive fibers, as adapted from Mickelsen and colleagues (2019). The tissue was

blocked with NDS without sodium azide and applied to each slide for 30 min (RT). After

blocking, the tissue was incubated with an anti-rabbit MCH antibody (1:2,000; RRID:

AB_2314774) for 1 hr (RT). The tissue was thoroughly rinsed with two exchanges in PBS (2

min each) then incubated with a donkey anti-rabbit Alexa Fluor 647 conjugate (1:500;

ThermoFisher Scientific; RRID: AB_2536183) for 30 min (RT).

After washing the slides twice in Wash Buffer for 2 min (RT), 4

–6 drops of 4′,6

-diamidino-2-

phenylindole (DAPI; 323110, ACD) were applied for 30 s, and the slides were coverslipped

using ProLong Diamond Antifade Mountant. Slides were dried in the dark overnight at RT and

then stored at −20°C.

2.4.2 Microscopy

All images were acquired using a Nikon Ti2-E inverted microscope (Nikon Instruments Inc.,

Mississauga, Canada) and processed using NIS-Elements Imaging Software (Nikon).

Confocal imaging.

Tiled confocal images were acquired with a Nikon C2 confocal

system using 405-nm, 488-nm, 561-nm, and 640-nm excitation lasers to visualize DAPI or

NeuroTrace, Alexa Fluor 488, Cy3, and Alexa Fluor 647 fluorophores, respectively. Full brain

overview images of DAPI-labeled nuclei from

Mchr1

stained slices were acquired using a 4



objective (0.20 numerical aperture). Higher magnification images of the LS used for analysis

were imaged for DAPI/NeuroTrace, Alexa Fluor 488, Cy3, and/or Alexa Fluor 647 signals with

a Plan Apochromat 10



objective (0.45 numerical aperture) or 20



objective (0.75 numerical

aperture) at a single image plane and stitched with NIS-Elements Imaging Software. Where

applicable, Z-

stacks of 1 μm optical slices were acquired with a Plan Apochromat 40



objective

(0.95 numerical aperture) or 60



objective (1.40 numerical aperture) and displayed as orthogonal

, and

projections or projected by their maximum intensity values (NIS-Elements

Imaging Software).

In situ hybridization signals.

The negative control slices were imaged at a single image

plane using the Plan Apochromat 10



objective and the 561-nm laser. The positive control

Ppib

hybridization signals were imaged to assess tissue and RNA quality. Images of all the sections

containing the LS for both negative control and experimental probe series were acquired using

the same settings to ensure that any differences observed between sections were not due to a

difference in magnification, scan area, laser power, or gain. Tiled images of the LS from each

probe section were then acquired at 10



magnification using the 405-nm, and 561-nm lasers to

image DAPI- and

Mchr1

-labeling. Images were saved and exported such that the different

channels could be toggled on and off to allow visualization of individual channels.

Brightfield imaging.

Large field-of-view images of Nissl-stained tissue were viewed and

imaged using a CF160 Plan Apochromat 10



objective lens and acquired with a DS-Ri2 colour

camera (Nikon). Shading correction was applied during image acquisition to adjust for

illumination inconsistences at the edge of each image tile. The tiled images were stitched with

NIS-Elements Imaging Software.

2.4.3 Image analysis

Plane-of-section analysis.

To assess the neuroanatomical distribution of MCH-ir fibers and

MCHR1 protein, we used unique cytoarchitectural features seen in tiled, confocal

photomicrographs of NeuroTrace-staining to parcellate and draw boundaries corresponding to

brain regions defined in the

Allen Reference Atlas

(

ARA

; Dong, 2008;

Supporting figure 1A

We used tiled, brightfield photomicrographs of Nissl-staining to parcellate tissue used to

analyse

Mchr1

hybridization signals (

Supporting figure 1B

). Following confocal imaging,

coverslipped tissue that served as the negative control was soaked in PBS overnight (RT) until

the coverslip slid off. The exposed brain tissue was then treated for Nissl staining, as previously

described (Negishi

et al.

, 2020; Bono

et al.

, 2022). Where necessary, DAPI-labeled overview

images were aligned with parcellated images of the Nissl-stained tissue. Confocal images of

Mchr1

mRNA hybridization signal in the LS were imported into Adobe Illustrator 2021 (Adobe

Inc., San Jose, CA) and aligned to DAPI-labeled overview images. White matter, ventricles,

blood vessels, and other easily identifiable landmarks were used to ensure slices were properly

aligned.

All parcellations were drawn in Illustrator using an Intuos graphic tablet (Wacom, Kazo,

Japan) with reference to nomenclature and atlas levels provided by the

ARA

Fiber density.

Confocal images of MCH immunoreactivity in the LS were visualized by

Alexa Fluor 647 emission. MCH-ir axon fibers and varicosities were traced in a new layer within

Illustrator using an Intuos graphic tablet (

Supporting figure 1A

). Fiber tracing was restricted

to the LS only and then mapped to

ARA

brain templates (see

Mapping

description below). For

each atlas level, another layer was added to the Illustrator file so that a filled shape can be drawn

to encompass each LS subregion and the entire LS area. A clipping mask of this filled shape was

then applied to isolate, as separate image files, the filled shape of the total LS area, filled shape

of each subregion, mapped fiber tracing in the full LS, and mapped fiber tracing in each

subregion. The images were then analysed in MATLAB (MathWorks, Natick, MA) to determine

the total number of pixels encompassing the subregions or entire LS area (

pixels

total

) and the

number of pixels occupied by the fiber tracings (

pixels

fibers

). As the LS is a heterogenous three-

dimensional structure, we analysed fiber density at all LS levels (Risold and Swanson, 1997a;

Risold and Swanson, 1997b). The density of MCH-ir fibers at each

ARA

level (

) was expressed

on a ratio scale as:

D = 100

(

pixels

fibers

/pixels

total

) to capture nuanced changes in fiber density

throughout the rostrocaudal axis of each LS subregion.

Quantification of Mchr1 mRNA expression.

Representative images from negative

control tissue, corresponding to each probe slice, were adjusted using lookup table values (LUTs;

NIS Elements) until the image appeared black to eliminate background fluorescence from any

dapB

hybridization. This set of LUTs were averaged and applied to images of

Mchr1

hybridization signals to subtract background fluorescence resulting from non-specific binding.

Mchr1

hybridization was visualized by Cy3 fluorescence and appeared as punctate red

dots, which were far fewer after background correction. Only dots colocalizing to a DAPI-

stained nuclei were included in our analyses (

Supporting figure 1B

). A DAPI-stained nucleus

colocalizing with clusters of 3+ red dots were labeled as a

Mchr1

-expressing neuron and marked

by a red-filled circle (

Supporting figure 1B

). In the event that mRNA dots appeared between

two DAPI-labeled nuclei, only one cell would be reported, thus it is possible that we are

underestimating the number of

Mchr1

cells available in the LS. We counted the number of red-

filled circles within the LS of each available brain slice.

Quantification of MCHR1 protein expression.

Neuronal MCHR1 expression was

counted from confocal images of NeuroTrace-labelled cells, ciliary MCHR1, and NeuN

immunoreactivity visualized by NeuroTrace 435/455, Cy3, and Alexa Fluor 488 fluorescence,

respectively. An orange-filled circle (Illustrator) was placed over NeuroTrace and NeuN-ir

neurons marked by an MCHR1-ir primary cilium (

Supporting figure 1C

). The number of

circles were quantified within the LS of each available brain slice.

Mapping.

The fiber tracings and filled circle labels were kept in individual layers of the

Adobe Illustrator file so that each layer could be easily separated and mapped onto the

corresponding level of the

ARA

template (Dong, 2008). The collection of fiber and circle labels

was copied, resized, and adjusted so that the representation of the experimental LS fit the shape

of the LS shown in the atlas reference template. In this way, neurons were mapped to their

correct position relative to the unique neuroanatomical boundaries specific to the animal, despite

physical differences unique to the animal (such as size and shape of brain regions). Individual

subregions of the LS were mapped one-by-one to maintain accuracy in relative position and

distribution of fibers and neurons (

Supporting figure 1A

iii

Appositions

Direct physical contact between fiber and soma or cilia was assessed using

consecutive confocal Z-stack slices. Fiber contacts were referred to as appositions to the

membrane where no visible space appeared between the fiber and cell membrane along the

orthogonal

and

projections (Krimer

et al.

, 1997; Lambe

et al.

, 2000; Bouyer & Simerly,

2013). Contacts were determined at a physical zoom magnification of 2400



or greater, which

permitted the detection of at least 0.4 μm gaps.

2.4.4 Electrophysiology

Slice preparation

Mice were anesthetized with an injection of chloral hydrate (700 mg/kg, i.p.)

and transcardially perfused with a carbogenated (95% O

, 5% CO

), ice cold artificial

cerebrospinal fluid (ACSF) solution containing (in mM) 118 NaCl, 3 KCl, 1.3 MgSO

, 1.4

NaH

, 5 MgCl

, 10 glucose, 26 NaHCO

, 0.5 CaCl

(300 mOsm/L). The brain was removed

from the skull and sliced at 250



m using a vibrating microtome (VT1000s, Leica Biosystems,

Buffalo Grove, IL) in cold, carbogenated ACSF. Slices containing the LS were transferred to

glucose-based ASCF containing (in mM) 124 NaCl, 3 KCl, 1.3 MgSO

, 1.4 NaH

, 10

glucose, 26 NaHCO

, 2.5 CaCl

(300 mOsm/L) for 10 min (37



C) and then allowed to recover at

RT for at least one hour prior to slice recording.

Slice recording.

Slices containing the LS were bisected and transferred to the recording

chamber where they were continuously perfused with carbogenated, glucose-based ACSF

(31



C). Slice recordings were performed on three separate electrophysiology rigs. Cells were

visualized with infrared differential interference contrast microscopy at 40



magnification on

either an Examiner.A1 microscope (Zeiss, Oberkochen, Germany) equipped with an AxioCam

camera (Zeiss) and Axiovision software (Zeiss), or with an Eclipse FN1 microscope (Nikon)

equipped with a pco.panda 4.2 camera (Excelitas PCO GmbH, Kelheim, Germany) and NIS-

Elements Imaging software (Nikon).

Whole-cell patch-clamp recordings were performed using borosilicate glass pipettes (7

–



) backfilled with a potassium-based internal pipette solution containing (in mM) 120 K-

gluconate, 10 KCl, 10 HEPES, 1 MgCl

, 1 EGTA, 4 MgATP, 0.5 NaGTP, 10 phosphocreatine

(290 mOsm/L, pH 7.24) to assess membrane properties, ionic conductances, and glutamatergic

events. Internal pipette solution with an increased chloride concentration contained (in mM) 109

K-gluconate, 22 KCl, 10 HEPES, 1 MgCl

, 1 EGTA, 0.03 CaCl

, 4 MgATP, 0.5 NaGTP, 9

phosphocreatine (290 mOsm/L, pH 7.24). A cesium-based internal pipette solution used to

record GABAergic events contained (in mM) 128 CsMS, 11 KCl, 10 HEPES, 0.1 CaCl

, 1

EGTA, 4 MgATP, 0.5 NaGTP (290 mOsm/L, pH 7.24). For recordings measuring membrane

properties, 0.4% biocytin (Cayman Chemical, Ann Arbor, MI) was added to the internal pipette

solution to allow for post-hoc immunohistochemical labeling and visualization of recorded cells.

Recordings of electrical activity were generated using a MultiClamp 700B amplifier (Molecular

Devices, San Jose, CA) and digitized by a Digidata 1440A (Molecular Devices) or using an

Axopatch 200B amplifier (Molecular Devices) and digitized by a Digidata 1322A (Molecular

Devices). All traces were acquired using pClamp 10.3 software (Molecular Devices) and filtered

at 1 kHz.

Drug treatment.

Following a baseline period of at least 5 min, MCH (3



M; H-1482;

Bachem, Torrance, CA) was bath applied into the recording chamber for approximately 5 min

followed by a washout period in ACSF. Where applicable, tetrodotoxin (TTX; 500 nM; T-550,

Alomone labs, Jerusalem, Israel), TC-MCH 7c (10



M; 4365, Tocris, Toronto, Ontario, Canada),

and bicuculline (30



M; 14343, MilliporeSigma) were applied to the slice during the baseline

period approximately 10 min prior to MCH application and maintained over the washout period.

Calphostin C (100 nM; HB0160, Hello Bio Inc., Princeton, NJ) prepared and maintained in the

dark until it was illuminated by a bright light within the slice recording chamber was applied to

the slice for 20

–

30 min prior to MCH application. Antagonists were only added to cells that were

hyperpolarized by a puff of MCH. All drugs were prepared from stock solution then dissolved

into ACSF immediately prior to application.

Puff application.

In experiments elucidating the membrane or intracellular mechanisms

underlying the effects of MCH, we first delivered a short puff of MCH to a patched cell to

identify those cells that responded with a reversible membrane hyperpolarization. To deliver the

MCH puff, a second borosilicate glass “puff” pipette was filled with 3



M MCH solution and

lowered into the slice within 30

–



m from the patched cell. A gradual positive pressure was

manually applied to the puff pipette for 5

–

10 seconds until the MCH solution reached the

patched cell.

Biocytin immunohistochemistry.

Some brain slices used for electrophysiology

recordings were post-fixed with 10% formalin to use for post-hoc immunohistochemical

staining. The slices containing the biocytin-filled cells were rinsed in PBS (six 5-min washes),

blocked in NDS (2 hours; RT), incubated with a streptavidin-conjugated Cy3 antibody (1:500)

prepared in NDS (2 hours; RT), and then washed in PBS for 10 min. The slices were then

washed with two more exchanges of PBS containing DAPI (1:2,000; Thermo Fisher Scientific)

for 10 min. Brain slices were then mounted to Superfrost Plus microscope slides and

coverslipped with ProLong Diamond Antifade Mountant.

2.4.5 Experimental design and statistical analyses

Anatomical studies.

Male and female mice wildtype mice (8

–

10 weeks) were used in a between-

subject design to assess the distribution of MCH-ir fibers,

Mchr1

mRNA, and MCHR1

immunoreactivity. Comparisons between LS subregions or across LS levels were determined by

two-way mixed model ANOVA with Tukey post-hoc testing, as not all LS levels can be captured

in every brain sample.

Slice recording

. Acute brain slices were prepared from male and female wildtype mice

(38 male, 30 female) aged 5

–

23 weeks. Cells were recorded from two to three slices containing

the LS and corresponding to Bregma 1.145

–

0.345 mm. Data sets included 1

–

4 cells per mouse.

Resting membrane potential (RMP).

Only neurons that exhibited a stable membrane

potential (varied <5 mV) for 5 minutes prior to drug application were included in our data

analyses. All voltages were corrected for a +15 mV or +14 mV liquid junction potential when

using the internal pipette solution containing 12 mM or 24 mM chloride, respectively. For bath

applications, RMP was sampled every 1 s using Clampfit 10.7 (Molecular Devices) and binned

into 30 s increments. Control value was the mean RMP averaged over 1 min immediately prior

to MCH application. The change in RMP (



RMP) was determined at the peak effect of MCH,

which was within 4

–

8 min of MCH application and following washout 5

–

10 min later. In puff

experiments, RMP was sampled every 500 ms and binned into 2-second increments, the



RMP

elicited by MCH was sampled 10

–

25 s after the puff. Within-group designs comparing control,

MCH, and washout conditions were analysed using a repeated measure one-way ANOVA with

Tukey post-hoc testing. Comparisons of



RMP over time between two drug treatment

conditions were analysed using a repeated measure two-way ANOVA. Comparisons of



RMP

after or at peak effect of drug treatment were compared using a one-way ANOVA with Tukey

post-hoc testing.

–

V curve.

Ionic conductance was measured in voltage clamp from a holding potential

) of −75 mV. Descending 10 mV voltage steps (250 ms) were applied from −55 mV to −125

mV. The mean reversal potential (V

rev

) was averaged based on the V

rev

for each cell, which was

determined as the

-intercept calculated from a line equation where the slope is calculated from

the −55 mV and −65 mV steps or from current values at two adjacent voltage steps where the

current changes from a negative to a positive value, where applicable. The V

rev

was compared to

the theoretical equilibrium potential of the chloride ion (E

) using a one-sample

test. Between-

group differences in net currents evoked at each voltage step following MCH application in the

absence or presence of bicuculline were compared using repeated measures two-way ANOVA

with Bonferroni post-hoc comparison. The net current evoked at the

–

105 mV voltage step was

compared using a one-way ANOVA with Tukey post-hoc comparison.

Synaptic activity.

Spontaneous (sIPSC) or miniature (mIPSC) inhibitory postsynaptic

current events were recorded at V

–

15 mV while excitatory post synaptic currents (sEPSC,

mEPSC) were recorded at V

–

75 mV. The IPSC and EPSC frequency were analysed using

MiniAnalysis (Synaptosoft) and binned into 30-second increments. The control value was taken

as the mean of a 1 min sample between 0 and 4.5 min prior to MCH application. The percent

change in frequency and amplitude were determined at the peak effect of MCH between 2 and

9.5 min after the onset of MCH application. The washout was taken between 10 and 19 min after

MCH application. Statistical significance was determined using a repeated measure one-way

ANOVA with Tukey post-hoc testing.

We generated cumulative probability plots by pooling the amplitude and interevent

intervals from 200 IPSC events or 50 EPSC events from each cell from baseline, MCH, and

washout recording periods. Differences in the distribution of IPSC or EPSC amplitudes or

interevent intervals in cumulative probability plots were analysed using the Kolmogorov-

Smirnov

test.

Graphs and illustrations

. All data graphs were generated using Prism 9 (GraphPad

Software, San Diego, CA). Results were considered statistically significant at

< 0.05.

Representative sample traces data were exported from Clampfit and plotted in Origin 2018

(OriginLab Corporation, Northampton, MA). Manuscript figures were assembled in Illustrator.

2.5

Results

In order to identify potential sites of MCH action within the LS, we quantified the relative

expression of MCH-ir fibers (

Figure 1A

Mchr1

mRNA (

Figure 1B

), and MCHR1 receptors

(

Figure 1C

) in each subregion and level of the LS and then mapped their distribution throughout

the rostrocaudal axis of the LS that spans 2.125 mm between

ARA

level (L) 36 and L57.

2.5.1 Distribution of MCH-ir fibers throughout the LS

To maximize the detection of MCH-ir fibers, we performed our immunohistochemical stains

using tyramide signal amplification. We then traced these fiber projections so that they can be

mapped onto

ARA

templates with reference to Nissl-based parcellations and systematically

examine the distribution of MCH-ir fibers throughout the LS. The density and pattern of MCH-ir

fiber expression was comparable between males and females (

Supporting figure 2

), so their

datasets were combined to assess the overall MCH-ir fiber density across the LS.

The LS includes the rostral LS (LSr), caudal LS (LSc), and ventral LS (LSv). The LSr

comprised the largest cytoarchitectural subdivision of the LS, and majority of MCH-ir fibers in

the LS were found in the LSr (F(2, 12) = 10.33,

= 0.0025). Notably, MCH-ir fiber density was

more abundant in the ventral than dorsal aspects of the LSr (

Figure 1A

–

iii

). Near the peak

ARA

level of MCH-ir expression, there was a distinctive pattern of MCH-ir fibers that were more

concentrated at the midline or along the medial LSr border adjacent to the medial septal nucleus,

along the lateral LSr border adjacent to the lateral ventricle, and along the ventral LSr border

abutting the nucleus accumbens, lateral preoptic area, or bed nuclei of the stria terminalis

(

Figure 1A

; see

Supporting figure 2

for MCH-ir fiber maps at all

ARA

levels of the LS).

MCH-ir fiber density differed throughout the anteroposterior axis of the LS (F(16, 99) =

4.46, p < 0.0001), and MCH-ir fiber density in the LSr gradually increased by nearly two-fold at

its peak between L45

–

L49 and then diminished posteriorly (

Figure 1A

). The cytoarchitectural

boundary of the LSc is dorsal to the LSr, begins around L44, and then persists throughout the LS.

The LSc is a small LS subregion and comprised relatively few dispersed MCH-ir fibers within

the overall LS (

Figure 1A

). The LSv emerged posteriorly in the LS starting at L52, and the LSv

contained a moderate and evenly distributed MCH-ir fiber density (

Figure 1A

2.5.2 Distribution of

Mchr1

-expressing LS cells

To determine if the LS expressed receptors for MCH, we used RNAscope to label

Mchr1

mRNA

hybridization in the LS; positive staining for

Mchr1

mRNA appeared as punctate dots. We

observed cells that contained 1

–

2 dots, and while even low amounts of mRNA may be translated

to protein (Greer et al., 2016; Lipo et al., 2022), we only considered cells with 3+ dots to provide

a conservative estimation of

Mchr1

-positive cells (

Figure 1B

). Similar to the distribution of

MCH-ir fibers,

Mchr1

hybridization was more prominent in the LSr (F(2, 72) = 116.1, p <

0.0001), where

Mchr1

cells were most prevalent in the ventral than dorsal LSr (

Figure 1B

–

iii

where they tend to be along the lateral LSr borders (

Figure 1B

; see

Supporting figure 3

for

representative maps of

Mchr1

hybridization at all

ARA

levels of the LS).

The distribution of

Mchr1

-expressing cells was similar between males and females

(

Supporting figure 3

). The number of

Mchr1

cells differed rostrocaudally within the LS (F(16,

72) = 3.47,

= 0.0001) and peaked at L49 (

Figure 1B

). Of the identified

Mchr1

-expressing

cells, 89% were found in the LSr and only 10% and 1% were located in the LSc and LSv,

respectively. In the posterior levels at L52 and L53, there were few

Mchr1

cells in the LSr but

there was an increasing proportion of

Mchr1

cells dorsally in the LSc.

2.5.3 Distribution of MCHR1-expressing LS cells

To determine if

Mchr1

transcripts were translated to protein, we performed an

immunohistochemical stain to label MCHR1 immunoreactivity. As MCHR1 is concentrated on

the primary cilium of neurons (Diniz et al., 2020), we determined its colocalization to a

NeuroTrace and/or NeuN-ir soma (

Figure 1C

). The vast majority (93%) of MCHR1 cells were

in the LSr (F(2, 69) = 84.95, p < 0.001), and the pattern of MCHR1 immunoreactivity was

similar to

Mchr1

mRNA hybridization. MCHR1-expressing cells clustered toward the lateral

border and ventral half of the LSr, while the medial LSr bordering the medial septum had few

MCHR1 cells at all levels of the LS (

Figure 1C

–

; see

Supporting figure 4

for representative

maps of MCHR1-expressing cells at all

ARA

levels of the LS).

MCHR1-expressing cells were differentially distributed throughout the anteroposterior

axis of the LS (F(16, 69) = 3.65, p < 0.0001) and peaked between L48 and L49, where there

were 5-fold more MCHR1 cells than in the anterior or posterior LS (

Figure 1C

). About 5% of

MCHR1 cells were found in the LSc and were distributed across several

ARA

levels. The LSv

comprised ~2% of MCHR1 cells, which emerged posteriorly and were clustered at L55 (

Figure

). Interestingly, while 99.9% MCHR1-ir cilia in LSr and LSc colocalized to a NeuN-labeled

soma, about 56% of MCHR1-ir cilia in the LSv did not colocalize to NeuN, though they were

associated with a NeuroTrace-labeled cell.

Figure 1. Relative expression of MCH-immunoreactive fibers,

Mchr1

mRNA, and MCHR1

protein throughout the LS.

Representative confocal photomicrographs of MCH-

immunoreactive (MCH-ir) fibers (arrowheads) amid NeuroTrace-labeled soma (

) in the dorsal

(

Aii

) and ventral regions of the LS (

Aiii

). Coronal map of traced MCH-ir fibers in the LS (

Aiv

) at

a representative

Allen Reference Atlas

level (

ARA

; Dong, 2008). Fiber density was expressed as

the percent area covered by MCH-ir fibers at each

ARA

level in the rostral LS (LSr), caudal LS

(LSc), and ventral LS (LSv;

). Representative confocal photomicrographs of

Mchr1

mRNA

hybridization amid DAPI-labeled nuclei (

) showing 1

–2 “dots” (open arrowhead; not included

in subsequent analyses) or 3+ dots (white arrowhead) in the dorsal (

Bii

) and ventral regions of

the LS (

Biii

). Only dots that surrounded a DAPI-labeled nucleus were included in our analyses.

Coronal map of

Mchr1

hybridization distributed within the LS at a representative

ARA

level

(

Biv

). Percent of

Mchr1

cells (comprising 3+ dots) at the LSr, LSc, and LSv of each

ARA

level

was relative to the total number of

Mchr1

cells per brain (

). Representative confocal

photomicrographs of MCHR1 immunoreactivity on the primary cilium (arrowhead) of NeuN-
immunoreactive neurons (

) in the dorsal (

Cii

) and ventral regions of the LS (

Ciii

). Coronal

map of MCHR1-expressing cells at a representative

ARA

level (

Civ

). Percent of total MCHR1

cells in the LSr, LSc, and LSv at each

ARA

level (

). Only

ARA

levels captured by our dataset

were included. Scale bar: 200

μm (

), 25

μm (

Aii

Aiii

), 10 μm (

Bii

Biii

), 20

μm (

iii

). Significance (

< 0.05) was determined with a two-way mixed-effect model ANOVA with

Tukey post-hoc testing: **

< 0.01, ****

<0.0001

ACB, nucleus accumbens; cc, corpus

callosum; CP, caudoputamen; LSc, lateral septal nucleus, caudal part; LSr, lateral septal nucleus,
rostral part; MS, medial septal nucleus; SH, septohippocampal nucleus; VL, lateral ventricle.

2.5.4 Proximity of MCH-ir fibers at MCHR1-expressing LS cells

MCH is known to reach its receptor and target site by volume transmission (Noble

et al.

, 2018).

However, as MCH fibers and MCHR1 are featured in similar LS regions dorsoventrally and

rostrocaudally (

Figure 1

), we also assessed the proximity between MCH-ir fibers and

Mchr1

mRNA or MCHR1 protein expression. MCH-ir fibers were present around

Mchr1

-expressing

cells in the ventral LSr (

Figure 2A

) and in some cases appeared to be in close contact with an

Mchr1

-expressing cell (

Figure 2B

). However, since

Mchr1

hybridization was localized to DAPI-

labeled nuclei, it was not possible to assess if MCH-ir fibers formed direct appositions with a

Mchr1

cell. To determine if MCH fibers may come in direct contact with MCHR1-expressing LS

cells, we performed a stain for MCHR1 protein, MCH-ir fibers, and the neuronal marker NeuN

to mark the cell body and examined MCH fiber appositions on the soma or cilia of MCHR1-

expressing cells. The majority of MCHR1-expressing LS cells (111 of 123) were not contacted

by MCH-ir fibers either at their cell bodies or cilia (

Figure 2C

). Most MCHR1 cells were not

immediately adjacent to visible MCH-ir fibers, and some MCH-ir varicosities came within

0.4

μm of MCHR1

-labeled cilia or their affiliated cell body without making direct physical

contact (

Figure 2D

). Interestingly, MCH-ir fibers directly contacted about 10% of MCHR1 LS

cells examined, and these appositions may occur at the NeuN-labeled cell body (

Figure 2E

) or

MCHR1-ir cilium (

Figure 2F

). These findings suggested that MCH is preferentially transmitted

through diffusion from local MCH fibers in the LS, but MCH fibers may also be in direct contact

with LS cells for localized actions.

Figure 2. Proximity of MCH-immunoreactive fibers to MCHR1-expressing LS cells.

Representative merged-channel confocal photomicrographs from the lateral and ventral LSr
border (

inset

dashed outlined area) of

Mchr1

mRNA on DAPI stained nuclei (white arrow) in

relation to MCH-ir fibers (

) in close proximity to

Mchr1

-expressing cells (

). Representative

merged-channel confocal photomicrographs from the lateral and ventral LSr border (

inset

dashed outlined area) of MCHR1-ir cilia on NeuN-ir neurons in relation to MCH-ir fibers (

which may be adjacent but relatively distant (asterisk) from MCHR1-ir LS cells. High
magnification confocal photomicrographs with orthogonal projections in the

-plane of MCH-

ir varicosities in

that are closely associated but do not make physical contact (

, open

arrowhead) or that form appositions (filled arrowhead) at the NeuN-ir soma (

) or associated

MCHR1-ir cilia (

). Appositions were observed when no visible space was discerned between

the MCH-ir varicosity and NeuN-ir soma or MCHR1-ir cilium in both the

- and

-plane at

the same optical section (yellow line). Scale bars: 10 μm (

–

); 100 μm (

inset

);

X, Y

axis 5 μm each (

–

). ACB, nucleus accumbens; cc, corpus callosum; CP, caudoputamen; LSc,

lateral septal nucleus, caudal part; LSr, lateral septal nucleus, rostral part; LSv, lateral septal
nucleus, ventral part; MS, medial septal nucleus; VL, lateral ventricle.

2.5.5 MCH inhibited LS cells

As local diffusion may be the primary mode of MCH transmission within the LS, we

hypothesized that substantive spatial overlap between the distribution of MCH-ir fibers and

MCHR1 would define putative hotspots for MCH action. We found that the spatial overlap

between the expression of MCH-ir fibers,

Mchr1

mRNA, and MCHR1 protein in the LS was

most prominent toward the lateral and ventral borders of the LSr (

Figure 3A

), which formed our

focus region for identifying MCH-responsive LS cells and defining the mechanisms of MCH

action.

We prepared acute brain slices containing the LS and performed whole-cell patch-clamp

recordings from cells along the ventrolateral border of the LSr (

Figure 3B

). Bath application of

MCH (3 µM) significantly hyperpolarized a subset of MCH-sensitive LS cells, which were

distinguished from MCH-insensitive cells where MCH application did not affect the RMP (F(1,

19) = 11.91;

= 0.003;

Figure 3C

). This MCH-mediated hyperpolarization was observed

in about half of LSr neurons recorded from both male (7/12 cells) and female (5/9 cells) mice

and reversibly hyperpolarized the RMP

by −6.1 ± 1.1 mV (n = 12; F(2, 22) = 13.38,

= 0.002;

Figure 3C

iii

). We did not detect any differences in the magnitude of hyperpolarization between

cells from male (−6.3 ± 1.5 mV, n = 7) or female mice (−5.6 ± 2.0, n = 5;

(10) = 0.29,

= 0.78),

thus data from both male and female mice were combined.

To determine if MCH acts directly on LS cells, we pretreated the slice with TTX (500

nM) to block action potential-dependent activity. Subsequent co-application of MCH in the

Figure 3. MCH directly hyperpolarized LS cells.

Overlaid maps of MCH-immunoreactive

(MCH-ir) fibers (blue) with low-

Mchr1

(pink circles;

), high-

Mchr1

(red circles;

), and

MCHR1-ir cells (purple circles;

Aii

) at

Allen Reference Atlas

Level 47 (Dong, 2008). Shape of

the LS in acute brain slices guided whole-cell patch-clamp recordings (

inset

) from cells near

the ventrolateral border of the LSr (

). The position of the biocytin-filled recorded cell (

inset

Bii

) was verified by post hoc staining (

Bii

). Representative sample trace of MCH-mediated

hyperpolarization following bath application of 3 μM MCH (

). Time course of the MCH-

mediated change in RMP (



RMP) at MCH-sensitive (filled circles) and MCH-insensitive cells

(open circles;

Cii

). Mean



RMP from each MCH-sensitive cell before MCH application (con),

at the peak effect of MCH, and after MCH washout (wash) (

Ciii

). Representative sample trace of

MCH-induced hyperpolarization in the presence of 500 nM TTX (

). Time course of



RMP

(

Dii

) was summarized as the mean



RMP from each cell before MCH application (con), at the

peak effect of MCH, and after MCH washout (wash) (

Diii

). Scale bar: 200 µm (

Bii

); 20 µm

(

inset

);

50 μm (

Bii

inset

); 25 mV, 2 min (

); 4 mV, 2 min (

). Significance (

< 0.05) was

determined with a repeated measures two-way mixed-effect model ANOVA (

) or repeated

measures one-way ANOVA with Tukey post-hoc testing (

iii

): *

< 0.05, **

< 0.01, ***

<0.001. ACB, nucleus accumbens; ccg, corpus callosum, genu; CP, caudoputamen; LSc, lateral

septal nucleus, caudal part; LSr, lateral septal nucleus, rostral part; MS, medial septal nucleus;
VL, lateral ventricle.

2.5.6 MCH-mediated hyperpolarization is MCHR1-dependent

We screened for MCH-sensitive LS neurons by applying a short puff of MCH (

Figure 4A

). We

found that a single MCH puff (MCH

puff

) produced a small but reversible RMP hyperpolarization

(−2.0 ± 0.4 mV, n = 8) that was sufficient to identify an MCH

-sensitive neuron (

Figure 4A

). A

second MCH

puff

applied at least three minutes later also produced a similar (−1.6 ± 0.3 mV, n =

8) hyperpolarization (

Figure 4A

iii

). By contrast, a puff application of bath ACSF did not

alter the RMP (0.5 ± 0.3 mV, n = 9; F(2, 22) = 18.17,

< 0.0001;

Figure 4A

iii

To confirm that MCH-mediated hyperpolarization was occurring via the MCHR1

receptor, we applied MCH in the presence of the MCHR1 antagonist, TC-MCH 7c. After

identifying an MCH-sensitive cell (MCH

puff

: −1.6 ± 0.3 mV, n = 7), we pretreated the slice with

10 µM TC-MCH 7c for 10

–

15 min. There was a main effect of the MCHR1 antagonist (F(2, 18)

= 21.24;

< 0.0001), as a second MCH

puff

applied in the presence of TC-MCH 7c no longer

produced a hyperpolarization (−0.2 ± 0.2 mV, n = 7;

= 0.013;

Figure 4B

iii

). Similarly,

MCH application via the slice chamber over a longer duration also did not elicit a membrane

hyperpolarization in the presence of TC-MCH 7c (MCH

bath

: 1.2 ± 0.4 mV, n = 7;

< 0.0001;

Figure 4B

iii

). These findings indicated that MCHR1 activation mediated the inhibitory

effects of MCH.

Figure 4. MCHR1-mediated hyperpolarization at LS cells.

Brightfield photomicrograph

showing the placement of a pipette for puff application during patch-clamp recording (

top

Representative sample trace of the change in resting membrane potential (



RMP) following a

10-

second puff of ACSF or 3 μM MCH (MCH 1) followed by a subsequent MCH puff 3 minutes

later (MCH 2) (

bottom

). Comparison of



RMP following a puff of ACSF or 3 μM MCH

over time (

Aii

) or immediately after the puff application (

Aiii

). Comparison of



RMP elicited in

the presence or absence of the MCHR1 antagonist TC-

MCH 7c (10 μM) by a 10

-second puff of

MCH (

), 5-minute bath application of MCH over time (

Bii

), or immediately after MCH

application (

Biii

). Scale bar: 20 µm (

top

) 2 mV, 10 s (

bottom

). Significance (

< 0.05)

was determined with a Two-way ANOVA (

, B

) or one-way ANOVA with Tukey post-hoc

testing (

iii

, B

iii

): *

< 0.05, ***

< 0.001, ****

<0.0001.

2.5.7 MCH activated a chloride channel to hyperpolarize LS cells

We next sought to determine the ionic basis of the MCH-mediated hyperpolarization. We

compared the current

–

voltage (I

–

V) relationship of MCH-sensitive cells before and after a full

bath application of MCH. We determined the I

–

V relationship of the cell by recording the

steady-state current change elicited by each voltage step (

Figure 5A

). MCH application elicited

a membrane current with a V

rev

of −63.7 ± 8.1 mV (n = 8), which corresponded to the predicted

(−63.1 mV) under our conditions (

(7) = 0.17,

= 0.87;

Figure 5A

). We then elevated the

internal chloride concentration to determine if MCH-mediated inhibition was chloride-

dependent. With an elevated chloride internal, MCH activated a current that corresponded with

the new predicted E

of −44.8 mV

(

(6) = 0.64,

= 0.55;

Figure 5A

iii

The net current elicited by MCH revealed an outward rectification at depolarizing

potentials. These properties are consistent with that of ionotropic GABA

receptor currents

(Valeyev

et al.

, 1999) expressed in the LS (Heldt & Ressler, 2007; Hörtnagl

et al.

, 2013) and that

contribute a tonic chloride conductance (Lee & Maguire, 2014). To determine if the MCH-

mediated chloride current is related to the activation of GABA

receptors, we next pretreated the

slice with the GABA

receptor antagonist bicuculline (30 μM), and subsequent MCH co

application did not evoke a change in membrane current (

(11) = 2.57,

= 0.026;

Figure 5A

iii

There was a significant interaction in the effect of bicuculline on the MCH-mediated current

elicited across the voltage range (F(7, 77) = 7.38,

< 0.0001;

Figure 5A

) thus supporting a

MCH-mediated activation of a GABA

receptor.

To determine if this GABA

receptor chloride conductance underlies the MCH-mediated

hyperpolarization, we applied bicuculline to an MCH

puff

sensitive cell (−1.3 ± 0.2 mV, n = 9).

Bicuculline pretreatment abolished the inhibitory effects of MCH (F(2, 24) = 31.78,

< 0.0001),

as neither a short MCH

puff

(−0.1 ± 0.2 mV, n = 9;

= 0.027) nor prolonged MCH

bath

application

(2.0 ± 0.4 mV, n = 9;

< 0.0001) elicited a membrane hyperpolarization (

Figure 5B

). These

findings indicated that a bicuculline-sensitive current underlies the inhibitory effect of MCH at

LS cells and suggested that MCH may regulate the activation of a chloride conductance.

MCHR1 activation can couple to multiple intracellular G proteins, but most commonly to

proteins or G

proteins (Saito

et al.

, 1999; Hawes

et al.

, 2000). As PKC can be activated

following MCHR1 stimulation (Pissios

et al.

, 2003) and is linked to GABA

receptor activation

(Poisbeau

et al.

, 1999) and increased chloride conductance via GABA

receptors (Lin et al.,

1996), we determined if MCH-mediated inhibition was linked to PKC activity. We first

identified MCH-responsive cells (MCH

puff

: −2.1 ± 0.4 mV, n = 5) and pretreated the LS brain

slice with 100 nM calphostin C, a PKC inhibitor. Interestingly, MCH application in the presence

of calphostin C did not hyperpolarize the RMP (MCH

puff

: −0.2 ± 0.2, n = 5;

= 0.038) even

when MCH was applied for an extended duration (MCH

bath

2.3 ± 1.1 mV, n = 5;

= 0.030).

These findings indicated a main effect of calphostin C treatment (F(2, 8) = 11.92;

= 0.004;

Figure 5C

). Taken together, these results suggested that MCH activated a GABA

receptor

chloride current in a PKC-dependent manner.

Figure 5. MCH-mediated activation of protein kinase C-dependent GABA

receptors.

Representative current output from MCH-sensitive cells immediately before (control), during a 3

μM MCH application, and following MCH washout (wash) in response to voltage steps (250 ms)
applied at −10 mV increments from −55 mV to −125 mV (bottom right panel) (

). Comparison

of the current

–voltage relationship of net current elicited by 3 μM MCH in the absence (red filled

circles) or presence of the GABA

receptor antagonist bicuculline (BIC, 30 μM; blue filled

circles) or with a high internal chloride concentration (green open squares) (

Aii

). Comparison of

the change in resting membrane potential (



RMP) in the absence or presence of 30 μM BIC by

a 10-second puff application of MCH over time (

), 5-minute bath application of MCH over

time (

Bii

), or immediately after MCH application (

Biii

). Comparison of



RMP in the absence or

presence of the protein kinase C inhibitor Calphostin C (CalC, 30 μM) by a 10

-second puff

application of MCH over time (

), 5-minute bath application of MCH over time (

Cii

), or

immediately after MCH application (

Ciii

). Scale bar: 100 pA, 100 ms (

). Significance (

0.05) was determined using a one-way ANOVA with Tukey post-hoc testing (A

iii

), repeated

measures two-way ANOVA (

, C

), or repeated measures one-way ANOVA with Tukey post-

hoc testing (

iii

, C

iii

): *

< 0.05, ****

<0.0001.

2.5.8 MCH did not inhibit GABAergic or glutamatergic input on LS cells

Previous MCH research showed that MCH can act via a presynaptic mechanism (Zheng

et al.

2005), therefore we next determined if MCH also acts presynaptically in the LS. The LS

comprises primarily GABAergic neurons with reciprocal connections to other LS cells

(Gallagher & Hasuo, 1989; Sheehan

et al.

, 2004; Zhao

et al.

, 2013). We observed a high baseline

frequency of sIPSC events (7.2 ± 3.1 Hz, n = 8) that was consistent with high GABAergic tone at

LS cells (Carette et al., 2001;

Figure 6A

). MCH application produced a reversible rightward

shift in the distribution of sIPSC interevent intervals (control vs. MCH: n = 8; D(8) = 0.27,

0.0001; MCH vs. wash: n = 8; D(8) = 0.30,

< 0.0001;

Figure 6B

) that reflected a 43%

decrease in sIPSC frequency (baseline: 7.2 ± 3.1 Hz; MCH: 5.6 ± 2.7 Hz; wash: 6.4 ± 3.0 Hz; n

= 8; F(2, 21) = 17.39,

< 0.0001;

Figure 6C

inset

). In addition to decreasing sIPSC frequency,

MCH produced a reversible leftward shift in the distribution of sIPSC event amplitudes (control

vs. MCH: n = 8, D(8) = 0.23,

< 0.0001; MCH vs. wash: n = 8, D(8) = 0.30,

< 0.0001;

Figure

) but did not decrease the mean amplitude of sIPSC events (control: 32.2 ± 5.0 pA; MCH:

29.8 ± 5.5 pA; wash: 31.9 ± 5.9 pA; n = 8; F(2, 21) = 0.67,

= 0.52;

Figure 6D

inset

We pretreated the slice with 500 nM TTX to abolish activity-dependent synaptic

transmission and recorded mIPSC events to determine if MCH would act on GABAergic

presynaptic terminals (

Figure 6E

–

). Interestingly, co-application of MCH in TTX did not

affect the distribution of interevent intervals (control vs MCH: n = 8; D(8) = 0.10;

= 0.27;

Figure 6G

) and, accordingly, did not change mIPSC frequency (control: 1.3 ± 0.4 Hz; MCH: 1.2

(

) or mIPSC frequency over time (

). The cumulative distribution of sIPSC (

) and mIPSC

interevent intervals (

) and amplitudes (

)

were sampled immediately before (con), after

MCH application, or following MCH washout (wash). Percent change in event frequency or
amplitude was summarized in the

inset

(

). Scale: 50 pA, 150 s (

). Significance (

< 0.05) was determined with a Kolmogorov-Smirnov test (

) or repeated measures one-way

ANOVA with Tukey post-hoc testing (

inset

): *

< 0.05, ***

< 0.001, ****

<0.0001.

The LS also receives glutamatergic input from different regions such as the hippocampus

(Swanson & Cowan, 1977; Risold & Swanson, 1997

), which has also been shown to express

MCHR1 (Saito

et al.

, 1999). Therefore, we next examined if MCH can also change excitatory

input to the LS. Spontaneous glutamatergic inputs to LS cells occur at a low frequency (1.3 ± 0.5

Hz, n = 9), and there was a slow rundown of sEPSC frequency over time (

Figure 7A

) that

shifted the distribution of interevent intervals throughout the recording (control vs MCH: n = 9,

D(9) = 0.54,

< 0.0001; control vs wash: n = 9, D(9) = 0.42,

= 0.0003;

Figure 7C

Nonetheless, MCH did not alter sEPSC frequency (control: 1.3 ± 0.5 Hz; MCH: 1.3 ± 0.5 Hz;

wash: 1.4 ± 0.6 Hz; n = 9; F(2, 16) = 0.60,

= 0.55;

Figure 7C

inset

). MCH application also did

not change the amplitude of sEPSC events (control: 15.5 ± 2.6 pA; MCH: 15.3 ± 2.3 pA; wash:

14.8 ± 2.0 pA; n = 9; F(2, 16) = 0.07,

= 0.91), which also reflected a gradual rundown over

time (control vs MCH: n = 9, D(9) = 0.20,

= 0.21; control vs wash: n = 9, D(9) = 0.26,

0.05;

Figure 7D

In the presence of TTX, MCH had no effect on the frequency (control: 2.7 ± 1.0 Hz;

MCH: 2.6 ± 1.0 Hz; wash: 2.6 ± 1.0 Hz; n = 6; F(2, 10) = 1.35;

= 0.32;

Figure 7E

–

) or the

amplitude (control: 10.4 ± 1.1 pA; MCH: 10.1 ± 1.0 pA; wash: 10.2 ± 1.0 pA; n = 6; F(2, 10) =

0.06;

= 0.85;

Figure 7H

) of mEPSC events. Taken together, these findings suggested that

MCH did not affect excitatory synaptic input.

presence of 500 nM TTX (

) were summarized as the change in sEPSC (

) or mEPSC

frequency over time (

). The cumulative distribution of sEPSC (

) and mEPSC interevent

intervals (

) and amplitudes (

) were sampled immediately before (con), after MCH

application, or following MCH washout (wash). Percent change in frequency or amplitude was
summarized in the

inset

(

). Scale: 10 pA, 150 s (

); 50 pA, 150 s (

). Significance (

< 0.05) was determined with a Kolmogorov-Smirnov test (

): *

< 0.05, ***

< 0.001, ****

<0.0001.

2.6

Discussion

MCHR1-expressing cells concentrated along the ventral and lateral boundaries of the LS and

overlapped with the distribution pattern of MCH-ir fibers. We performed patch-clamp recordings

from these MCHR1-rich hotspots and revealed a novel mechanism of MCH action in the LS.

Pharmacological application of MCH directly hyperpolarized LS cells, but MCH did not alter

synaptic input to LS cells. The inhibitory effects of MCH were MCHR1-dependent and mediated

by an increased chloride current.

The coincident distribution pattern of

Mchr1

mRNA and MCHR1-ir cells in the LS

implicated that these were MCH target sites.

Mchr1

hybridization signals can be seen throughout

the LS but was most prominent in the LSr. This corresponded with

Mchr1

mRNA expression in

the rat LS that was higher in the intermediate (Saito et al., 2001; i.e., mouse LSr) and ventral part

of the LS (Bittencourt et al., 1992; i.e., mouse LSv) and lower in the dorsal part of the LS

(Hervieu et al., 2000; i.e., mouse LSc).

Mchr1

hybridization signals reflected the presence of

MCHR1 cells along the ventrolateral edge of the LSr anteriorly, as well as within the small LSv

cluster posteriorly.

MCHR1-positive LS cells were distributed within or near MCH-ir fiber fields, which

were also more abundant in the LSr than the LSc or LSv. Nerve terminals from MCH neurons

are known to terminate in the LSr and form glutamatergic synapses to innervate LS cells (Chee

et al.

, 2015). Interestingly, glutamatergic nerve endings from MCH neurons terminated in the

dorsal zones of the LSr (Chee

et al.

, 2015; Liu

et al.

, 2022), where MCH-ir fibers were relatively

sparse. Rather, MCH-ir fibers were prominent within the ventrolateral zones of the LSr where

they spatially overlapped with MCHR1-expressing cells. MCH-ir fibers also extend into the

medial zones of the LSr from the medial septum, but medially distributed MCH-ir fibers did not

overlap with MCHR1-rich zones.

The proximity between MCH fibers and MCHR1-expressing LS cells implicated hotspots

for MCHR1 activation. Most MCHR1 cells lie adjacent to but were not in contact with passing

fibers and suggested that MCH is preferentially released extrasynaptically in the LS. MCH

availability within the LS may also be a result of uptake from the lateral ventricle (Ruiz-Viroga

et al.

, 2021) to then reach MCHR1 LS cells by volume transmission (Noble

et al.

, 2018). This

could be facilitated by MCHR1 expression along the primary cilium of neurons (Berbari

et al.

2008; Diniz

et al.

, 2020) to detect MCH in the extracellular space (Diniz

et al.

, 2020).

Furthermore, like in the nucleus accumbens (Sears

et al.

, 2010) and nucleus of the solitary tract

(Zheng

et al.

, 2005) MCH-ir fibers can also be closely associated with LS cells, including

MCHR1-expressing cells in the ventrolateral LS that were in direct physical contact with MCH-

ir fibers. However, ultrastructural analyses would be required to determine if these contacts

reflect active release sites. The functional contributions of local MCH release or MCH uptake

from the ventricular space are not known (Ruiz-Viroga

et al.

, 2021) but may influence

behaviours that transpire over a longer timeframe. Meanwhile, local sources of MCH like those

in direct contact with MCHR1 LS cells may mediate rapid, acute responses to environmental

stressors.

Functional MCHR1 activation directly hyperpolarized and suppressed action potential

firing of LS cells. While MCH can act presynaptically by inhibiting glutamatergic input (Zheng

et al.

, 2005), it did not regulate either glutamatergic or GABAergic input to the LS. The

inhibitory effect of MCH was mediated by a bicuculline-sensitive chloride current, which

suggested the activation of ionotropic GABA

receptors. While MCHR1 activation is known to

couple to G

-proteins (Gao, 2009), it can also couple to G

-proteins (Bächner

et al.

, 1999;

Pissios

et al.

, 2003). MCHR1 coupling to the G

pathway may activate chloride currents

(Bächner

et al.

, 1999) in a PKC-dependent manner (Pissios

et al.

, 2003), as PKC activation can

increase chloride influx via GABA

receptors (Lin

et al.

, 1994, 1996

). The postsynaptic

mechanisms linked to MCH-mediated inhibition, including at the medial septal nucleus (Wu

al.

, 2009) and nucleus accumbens (Sears

et al.

, 2010), have involved the activation of potassium

channels, thus our findings implicate a novel chloride-mediated mechanism underlying the

inhibitory actions of MCH.

The LS may integrate MCH roles that increase feeding (Qu

et al.

, 1996; Rossi

et al.

1997; Dilsiz

et al.

, 2020) and anxiety-related behaviours (Smith et al., 2006; Dilsiz et al., 2020)

by suppressing LS activity. Photostimulating GABAergic LS cells reduces feeding (Xu

et al.

2019), and GABA

and GABA

-mediated inhibition of LS cells can increase feeding (Gabriella

et al., 2022; Calderwood et al., 2020). Since MCH can inhibit LS cells, MCH may thus increase

feeding by downregulating GABAergic output from the LS. The LS is a notable region that

regulates anxiety (Sheehan

et al.

, 2004), and this may be ascribed to its afferent or efferent

connections. MCH dampens LS activity, which can elicit anxiogenesis. For example, inhibiting

hippocampal projections to the LS increased anxiety-like behaviours (Parfitt

et al.

, 2017).

The LSc, LSr, and LSv designations by the

Allen Reference Atlas

are based on

cytoarchitectonic parcellation (Dong, 2008), but LS divisions can also be informed by afferent

and efferent connections that overlap with the distribution of MCHR1 cells (Risold and

Swanson, 1997a; Risold and Swanson, 1997b). The ventrolateral LSr receives strong

glutamatergic input from the ventral hippocampal CA1 (Risold and Swanson, 1997a) to suppress

feeding and anxiety (Parfitt

et al.

, 2017). Additionally, there is a concentration of cells

expressing the type 2 corticotropin-releasing factor receptor (

Crfr2

) in the lateral LSr that

innervate the anterior hypothalamic nucleus (Risold & Swanson, 1997

; Bang

et al.

, 2022).

Activating LS

Crfr2

terminals in the anterior hypothalamus promotes anxiety (Anthony

et al.

2014).

The LS can also be divided into band-shaped domains informed by its chemoarchitecture

(Risold and Swanson, 1997b). The distribution pattern of MCHR1-expressing LS cells

corresponded to dorsolateral and ventrolateral LSr bands that overlap with enkephalin and

urocortin immunoreactive fibers (Risold & Swanson, 1997

; Chen

et al.

, 2011) anteriorly. These

fibers have been implicated in feeding and anxiety-regulated behaviours at the LS. Enkephalin

immunoreactivity is lower when food is less abundant and may reflect food availability (Kovacs

et al.

, 2005). Enkephalin acts via the µ-opioid receptor in the LS (Mansour

et al.

, 1994), and

administration of a µ-opioid receptor agonist into the LSr increases feeding (Calderwood

et al.

2020) and anxiety-like behaviour in mice (le Merrer

et al.

, 2006). Urocortin activates CRFR2

receptors in the ventrolateral LS (Van Pett

et al.

, 2000; Anthony

et al.

, 2014) and can inhibit

feeding during stress (Stengel & Taché, 2014). Urocortin administration into the LS inhibits

feeding and increases anxiety-like behaviour (Wang & Kotz, 2002; Bakshi

et al.

, 2007; Noguchi

et al.

, 2013) and activation of

Crfr2

LS cells also promotes anxiety (Anthony

et al.

, 2014).

Interestingly, urocortin infusion into the LS can reduce orexin-mediated feeding (Wang & Kotz,

2002) and given that orexin and MCH have complimentary roles on feeding (Barson

et al.

2013), interactions between urocortin and MCH may modulate feeding behaviour in response to

stress.

MCH may also act via the LSv to mediate anxiety behaviours. MCHR1-expressing cells

in the LSv may overlap with Substance P-immunoreactive fibers (Risold & Swanson, 1997

and Substance P can have an anxiogenic effects in the LS (Gavioli

et al.

, 1999, 2002; Ebner

al.

, 2008). The LSv receives glutamatergic input from the ventral hippocampus to promote

coping mechanisms such as stress-induced grooming, which could help relieve feelings of stress

and anxiety (Mu

et al.

, 2020). Given the inhibitory actions of MCH on the LS, it may thus act via

the LSv to dampen such neuronal coping mechanisms.

In conclusion, our findings showed that MCH inhibits the LS and suggests that MCH

might converge with enkephalin and urocortin at the LSr, or with Substance P in the LSv, to

fine-tune feeding and anxiety-related behaviours. We anticipated that the convergence of MCH

fibers and MCHR1-expressing cells would assist in identifying hotspots underlying the action of

MCH in the LS, and our electrophysiological recordings in these regions revealed a novel

mechanism of MCH-mediated action that inhibited LS cells by increasing chloride conductance.

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Supporting figure 1. Schematic for mapping MCH-immunoreactive fibers,

Mchr1

mRNA

hybridization, and MCHR1 immunoreactivity in the LS.

NeuroTrace- (

) or Nissl-

stained tissue (

) were parcellated to define the neuroanatomical boundaries of the LS and

surrounding regions with reference to the

Allen Reference Atlas

(ARA; Dong, 2008). The

parcellations were aligned to confocal photomicrographs of MCH-immunoreactive fibers (

Aii

Mchr1

mRNA hybridization signals (

Bii

), and MCHR1-immunoreactive cells (

Cii

). The traced

fibers (

Aiii

), labeled

Mchr1

cells (

Biii

), or labeled MCHR1 cells (

Ciii

) were mapped onto

ARA

brain templates. ACB, nucleus accumbens; ccg, corpus callosum, genu; CP, caudoputamen; LSc,
lateral septal nucleus, caudal part; LSr, lateral septal nucleus, rostral part; MS, medial septal
nucleus; VL, lateral ventricle.

Supporting figure 2. Distribution pattern of MCH-immunoreactive fibers in the LS.

Representative

coronal maps of MCH-

immunoreactive (MCH-ir) fibers throughout the anteroposterior extent of the male (

) and female LS (

), as defined by nomenclature

and cytoarchitectural boundaries designated by the

Allen Reference Atlas

(ARA; Dong, 2008). MCH-ir fibers were traced then

mapped to corresponding

ARA

templates. Each panel includes the atlas level and stereotaxic coordinate inferred from Bregma (



)

assigned to that tissue. Only MCH-ir fibers within the LS were traced. ACB, nucleus accumbens; aco, anterior commissure; BST, bed
nuclei of the stria terminalis; cc, corpus callosum; ccg, corpus callosum, genu; CP, caudoputamen; df, dorsal fornix; fa, anterior
forceps; fi, fimbria; fx, columns of the fornix; isl, islands of Calleja; islm, major island of Calleja; LPO, lateral preoptic area; LSc,
lateral septal nucleus, caudal part; LSr, lateral septal nucleus, rostral part; LSv, lateral septal nucleus, ventral part; MEPO, median
preoptic nucleus; MS, medial septal nucleus; PVT, paraventricular thalamic nucleus; PT, paratenial nucleus; SF, septofimbrial
nucleus; SH, septohippocampal nucleus; TRS, triangular nucleus of septum; TTd, taenia tecta, dorsal part; V3, third ventricle; VL,
lateral ventricle.

Supporting figure 3. Maps of cells expressing

Mchr1

mRNA hybridization throughout the LS.

Representative coronal maps of

cells expressing

Mchr1

hybridization throughout the anteroposterior extent of the male (

) and female LS (

), as defined by

nomenclature and cytoarchitectural boundaries designated by the

Allen Reference Atlas

(ARA; Dong, 2008).

Mchr1

-expressing cells

were mapped to corresponding

ARA

templates. Each panel includes the atlas level and stereotaxic coordinate inferred from Bregma (



)

assigned to that tissue. Only

Mchr1

hybridization signals within the LS were analysed. ACB, nucleus accumbens; aco, anterior

commissure; BAC, bed nucleus of the anterior commissure; BST, bed nuclei of the stria terminalis; cc, corpus callosum; ccg, corpus
callosum, genu; CP, caudoputamen; fa, anterior forceps; fx, columns of the fornix; isl, islands of Calleja; islm, major island of Calleja;
LPO, lateral preoptic area; LSc, lateral septal nucleus, caudal part; LSr, lateral septal nucleus, rostral part; LSv, lateral septal nucleus,
ventral part; MEPO, median preoptic nucleus; MS, medial septal nucleus; NDB: diagonal band nucleus; SF, septofimbrial nucleus;
SH, septohippocampal nucleus; TRS, triangular nucleus of septum; TTd, taenia tecta, dorsal part; V3, third ventricle; VL, lateral
ventricle.

Supporting figure 4. Maps of MCHR1-immunoreactive cells throughout the LS.

Representative coronal maps of MCHR1-

immunoreactive NeuN-positive (filled circle) or NeuN-negative cells (open circle) throughout the anteroposterior extent of the male
(

) and female LS (

) were mapped to

Allen Reference Atlas

(Dong, 2008)

templates according to the corresponding cytoarchitectural

boundaries. Each panel includes the atlas level and stereotaxic coordinate inferred from Bregma (



) assigned to that tissue. Only

MCHR1 cells within the LS were included. ACB, nucleus accumbens; aco, anterior commissure; BAC, bed nucleus of the anterior
commissure; BST, bed nuclei of the stria terminalis; cc, corpus callosum; ccg, corpus callosum, genu; CP, caudoputamen; fa, anterior
forceps; fx, columns of the fornix; isl, islands of Calleja; islm, major island of Calleja; LPO, lateral preoptic area; LSc, lateral septal
nucleus, caudal part; LSr, lateral septal nucleus, rostral part; LSv, lateral septal nucleus, ventral part; MEPO, median preoptic nucleus;
MS, medial septal nucleus; NDB: diagonal band nucleus; SF, septofimbrial nucleus; SH, septohippocampal nucleus; TRS, triangular
nucleus of septum; TTd, taenia tecta, dorsal part; V3, third ventricle; VL, lateral ventricle.

3.1

Abstract

Melanin-concentrating hormone (MCH) is a neuropeptide produced in the lateral hypothalamus

and is well-established to regulate homeostatic and hedonic feeding behaviour.

Intracerebroventricular administration of MCH promotes feeding in male, but not female

rodents, but the specific brain regions underlying the orexigenic effects of MCH are not well-

defined. While some regions such as the nucleus accumbens and arcuate nucleus contribute to

MCH-mediated feeding, the lateral septum (LS), a major target site of MCH neuron projections

has not been examined. MCH can inhibit LS cells through the activation of the MCH receptor,

MCHR1. Interestingly, LS inhibition stimulates feeding, thus it may mediate the orexigenic

actions of MCH. To determine if MCH stimulates feeding via the LS, we bilaterally infused

MCH into the LS of male and female mice and found that MCH elicited a rapid and long-lasting

increase in consumption of standard chow and a palatable, high sugar, diet in both male and

female mice. Importantly, infusing the MCHR1 antagonist TC-MCH 7c into the LS blocked the

orexigenic actions of MCH. Although MCH and the LS can regulate food seeking and anxiety-

like behaviour, the orexigenic effects of MCH in the homecage did not translate to more time

spent investigating a food reward in an anxiogenic environment. Taken together, these findings

indicated that MCH can stimulate feeding in both male and female mice via the LS in a context-

dependent manner.

3.2

Introduction

Melanin-concentrating hormone (MCH) is a hypothalamic neuropeptide known to regulate

several behaviours including feeding, the first ascribed role of MCH (Qu et al., 1996).

Mch

mRNA expression is elevated in obesity, and this increase may be linked with hunger, as fasting

also promotes

Mch

expression (Qu et al., 1996). These transcriptional changes suggested that

MCH promotes feeding, and indeed, MCH administration into the lateral (Qu et al., 1996; Kela

et al., 2003) or third ventricles (Rossi et al., 1997; Duncan et al., 2005) acutely increased chow

intake and sustained this orexigenic effect for up to six hours (Qu et al., 1996; Rossi et al., 1997;

Kela et al., 2003). Moreover, the chronic intake of palatable high-fat diets also elevated the gene

expression for

Mch

and the MCH receptor,

Mchr1

(Elliott et al., 2004). By contrast,

Mch

deletion (Pissios et al., 2008; Mul et al., 2011) or MCHR1 antagonism (Karlsson et al., 2012)

attenuated the intrinsic reward properties of palatable food.

MCH has been associated with multiple aspects of feeding behaviour but is most

consistently involved in prolonging food bouts to promote overeating (Lee et al., 2021). MCH

neurons are active during, but not preceding, food consumption (Subramanian et al., 2023), and

while activating MCH neurons does not consistently stimulate feeding, activating MCH neurons

during a food bout increases the amount of food consumed (Dilsiz et al., 2020). Consistently,

MCHR1 deletion prevents cue induced overeating (Sherwood et al., 2015). This may be related

to improved taste evaluation and the taste-independent reward associated with nutrient-dense

foods. MCH infusion increased initial rate of licking a sweet, but not a bitter, solution suggesting

that increased intake was based on positive taste (Baird et al., 2006). MCHR1 antagonism also

reduced effort for a sucrose reward but not a saccharin reward (Karlsson et al., 2012) suggesting

that MCH preferentially reinforces calorically-dense substances. Consistent with the role of

MCH to prolong food bouts or extend ongoing feeding, the orexigenic effects of MCH were

more robust in the dark than light cycle when rodents have already initiated feeding (Terrill et

al., 2020). In addition, MCH may regulate context dependent, reward motivated food seeking

(Subramanian et al., 2023) and given the involvement of MCH neurons in memory (Izawa et al.,

2019; Liu et al., 2022) and anxiety-like behaviour (He et al., 2022), it is plausible that MCH

action at downstream sites could help integrate contextual information to determine when it is

appropriate to prolong eating.

The distributions of MCH projections and MCHR1 are widespread throughout the brain,

but the brain regions underlying MCH-mediated feeding are not fully characterized. The

orexigenic effects of MCH have largely been described in rats by infusing MCH into discrete

brain regions, which included the arcuate nucleus (Abbott et al., 2003), paraventricular nucleus

(Rossi et al., 1999; Abbott et al., 2003), the lateral hypothalamic area (Romero-Pico et al., 2018).

Notably, the orexigenic effects of MCH are most prominently featured in the nucleus accumbens

(Georgescu et al., 2005; Guesdon et al., 2009; Terrill et al., 2020), where MCH infusion

promoted both homeostatic and hedonic feeding (Terrill et al., 2020). However, the orexigenic

effects of MCH have only been reported in male subjects (Terrill et al., 2020), as MCH-mediated

feeding is inhibited by estradiol in female subjects (Messina et al., 2006; Santollo and Eckel,

2008). While MCH infusion in the nucleus accumbens consistently promoted feeding, activating

accumbens-projecting MCH neurons has yielded mixed results (Noble et al., 2018; Terrill et al.,

2020). Chemogenetic activation of MCH neurons projecting to the nucleus accumbens increased

chow intake in both the light and dark cycle (Terrill et al., 2020), however, in a different study,

the same procedure had no effect on chow intake in the light cycle (Noble et al., 2018).

Given the relative paucity of targets supporting the orexigenic actions of MCH, we

determined if the lateral septum (LS) is a target site of MCH action. The LS receives dense

projections from MCH neurons (Chee et al., 2015) and expresses MCHR1 (Lembo et al., 1999;

Saito et al., 2001; Chee et al., 2013; Diniz et al., 2020), and MCH can directly inhibit LS cells

(Payant et al., 2023). LS inhibition stimulates feeding (Mitra et al., 2014; Gabriella et al., 2022),

thus MCH action in the LS may promote feeding behaviour. We infused MCH bilaterally into

the LS of male and female wildtype mice and determined home-cage feeding of regular chow or

a highly palatable sugar pellet. Since the LS can regulate both feeding and anxiety-like behaviour

(Bakshi et al., 2007; Xu et al., 2019), we also assessed whether food availability promoted entry

into anxiogenic environments. We found that intra-LS MCH infusion significantly increased the

consumption of chow or a palatable diet in both males and females, especially when MCH was

infused directly above MCHR1-rich LS regions. However, when a food reward like a familiar

palatable food pellet was placed in the center of an open field, which engenders avoidant

anxiety-like behaviours, MCH did not facilitate food exploration in sated or fasted mice. Taken

together, these findings highlight the LS as an important structure underlying MCH-induced

feeding, but this orexigenic effect was present only in the habituated home-cage and not apparent

in novel, anxiogenic environments.

3.3

Materials and Methods

All procedures described herein were approved by the Carleton University Animal Care

Committee in accordance with guidelines provided by the Canadian Council on Animal Care. All

C57BL/6J

wildtype mice (stock 000664; Jackson Laboratory, Bar Harbor, ME) were bred in-

house, maintained on a 12-hour light-dark cycle (22

–

24 °C; 40

–

60% humidity), and given

libitum

access to regular chow (2.9 kcal/g; Teklad Global Diets 2014, Envigo, Mississauga,

Canada) or 60% high dextrose diet (3.6 kcal/g; Teklad Custom Diet, TD.05256, Envigo,

Mississauga, Canada) and water.

3.3.1

Cannula implantation.

Male and female mice (8

–

14 weeks old) received a

subcutaneous (sc) injection of carprofen (20 mg/kg; Zoetis, Kirkland, QC, Canada) analgesia 30

minutes prior to the start of surgery. They were then anesthetized with isoflurane, secured in a

stereotaxic apparatus (David Kopf Instruments, Tujunga, CA), and their incision sites were

sterilized before making a rostrocaudal incision to expose the skull. The skull surface was

cleaned and air-dried, and the head position was leveled within the stereotaxic frame using

bregma and lambda coordinates read from the skull.

A bilateral 26 gauge stainless steel guide cannula (C235DCS-5/SPC, Protech

International, Boerne, TX) was lowered into the central LS (in mm; anteroposterior (AP) +0.7,

mediolateral (ML)



0.4, dorsoventral (DV) −3.3;

Paxinos and Franklin, 2001; Dong, 2008) or

lateral LS (AP +0.7, ML



0.6, DV −3.3

). Dental cement, formed by mixing Jet denture repair

powder (1230P1, Lang Dental Manufacturing, Wheeling, IL) with Jet self-curing acrylic resin

liquid (1404CLR, Lang Dental Manufacturing), was applied to secure the cannula pedestal to the

top of the skull. The skin at the incision sites was pulled around the implant and sutured around

the implant to minimize skull exposure. A mating dummy cannula (C235GS-5/SPC, Protech

International) was inserted in the guide cannula and secured with a dust cap (303DC/1, Protech

International) while the animal recovered in its home-cage for at least two weeks.

3.3.2

Intra-LS infusion.

Animals were habituated to handling for 10 consecutive days (5

min per day). An internal cannula with a 0.25 mm projection at the tip of the guide cannula

(C235IS-5/SPC, Protech International) was inserted into the guide cannula. Thin-walled PE50

tubing (C232CT, Protech International) was fitted onto each injector, and the infusate (1 μl total

volume per side) was delivered over 4 min and then allowed to penetrate the target region for 2

min before the tubing was removed and attached to the other side of the bilateral cannula. MCH

(1 μg;

4025037

, Bachem, Torrence, CA), or the MCHR1 antagonist TC-

MCH 7c (1 μg;

4365

Tocris, Toronto, Canada) was delivered via a sterile vehicle of artificial cerebrospinal fluid

(aCSF) comprising (in mM) 148 NaCl, 3 KCl, 1.4 CaCl

, 0.8 MgCl

, 0.8 Na

HPO

, 0.2 NaH

prepared as described (ALZET Osmotic Pumps). In experiments where MCH was applied

following MCHR1 antagonist pretreatment, TC-

MCH 7c (0.5 μg) was infused first and a

solution comprising 1 μg MCH and 0.5 µg TC

-MCH 7c was infused 5 min later.

3.3.3

Food intake.

A chow or 60% high dextrose food pellet was presented on the floor

of the home-cage. The weight of the food remaining was determined every hour for four hours.

Infusions began at ZT 1

–

3 immediately prior to the start of the 4-h feeding period. Ramekins

with regular chow and 60% high dextrose diet were placed on the floor of the home cage

overnight and intake of each diet was measured to determine food preference.

3.3.4

Locomotor activity.

Micromax infrared beam-break system (Omnitech, Columbus,

OH) was used to determine homecage ambulatory activity by recording the total number of beam

breaks over four hours following infusion (ZT1

–

3).

3.3.5

Open field movement.

Mice were habituated to an opaque open field arena (45 x 45

x 45 cm) at least 3 times prior to testing. On the day of testing, sated or fasted mice where food

was removed from their homecage for 6 or 16 h, respectively were

habituated to

the testing room

for

approximately one hour. Infusions began at ZT6

–

11 in the sated condition and ZT1

–

4 in the

fasted condition, 30 min prior to testing. Mice were then placed into an opaque open field box

with a piece of 60% high dextrose diet affixed to the middle of the arena and allowed to explore

the arena for 20 min. Movement in the open field arena was recorded with an overhead camera

(Hue HD camera, Hue, London, UK) and tracked using ANY-maze (Stoelting Co, Wood Dale,

IL). The open field arena was divided into the outside perimeter and center area (22.5 x 22.5

cm). The amount of time the mouse spent in each zone was based on tracking the center-point of

the mouse. Plots of animal tracking were generated in ANY-maze and exported as PNG files.

3.3.6

Implant site validation.

At the end of the study, mice received an infusion of 0.5%

Evans Blue dye in saline (1 µl; MilliporeSigma, Burlington, MA) and were euthanized 30 min

later. Mice were anesthetized with chloral hydrate (700 mg/kg; MilliporeSigma), decapitated,

and their brains were rapidly extracted from the skull and then post-fixed in 10% formalin

(VWR, Radnor, PA) for at least 24 h. The brains were cryoprotected in phosphate buffered saline

(PBS) containing 20% sucrose and 0.05% sodium azide (24 hr, 4



C) and sliced into five series of

30 µm coronal sections using a freezing microtome (Spencer Lens Co., Buffalo, NY). Brightfield

images of LS-containing brain slices were acquired using a Nikon Ti2-E inverted microscope

(Nikon Instruments Inc., Mississauga, Canada) and processed using NIS-Elements Imaging

Software (Nikon). Cases where the bilateral cannula landed outside the boundaries of the LS, as

defined by the Allen Reference Atlas (Dong, 2008), were excluded from analysis.

3.3.7

Experimental design and statistical analyses.

Experiments were conducted using a

within-group design where mice were tested in the paradigm two to four times, once for each

drug condition, in a counterbalanced design. The first hour of food intake was analyzed using a

two-tailed, paired

-test. Cumulative food intake, locomotor activity, and open field movement

were compared by two-way repeated measures ANOVA with Bonferroni post-hoc testing. A

three-way ANOVA was used to compare responses between male and female mice. In some

cases, a mixed-effect model was used to account for missing datapoints. Results were considered

statistically significant at

< 0.05. All data graphs were generated using Prism 9 (GraphPad

Software, San Diego, CA). Manuscript figures were assembled in Adobe Illustrator 2024 (Adobe

Inc., San Jose, CA).

3.4

Results

3.4.1 MCH infusion into the LS increased chow intake in male and female mice

To determine if the LS mediated the orexigenic actions of MCH, we bilaterally infused MCH (1

µg) into the center of the rostral LS (LSr;

Figure 1A

) of sated male and female mice and

measured chow intake over a 4-h period. There was an increase in chow intake within the first

hour of MCH infusion (vehicle: 0.06



0.01 g; MCH: 0.11



0.01 g;

(10) = 2.93,

= 0.015) in

male mice leading to a main effect of MCH that more than doubled cumulative intake over 4 h

(F(1, 10) = 13.74;

= 0.004) and reflected a significant interaction of MCH infusion on chow

intake over time (F(4, 40) = 11.46; p < 0.0001;

Figure 1B

). In female mice, MCH infusion into

the LS did not significantly increase chow intake in the first hour (vehicle: 0.09



0.03 g; MCH:

0.19



0.07 g;

(7) = 1.46,

= 0.189), and while we did not detect a significant main effect of

MCH (F(1, 7) = 4.75;

= 0.066), there was a significant interaction of MCH over time, as MCH

increased chow feeding (F(4, 28) = 5.49;

= 0.002;

Figure 1C

). The orexigenic effect on MCH

was comparable between male and female mice, as there was no main effect of sex (F(1, 85) =

2.27;

= 0.136) and no interaction between sex and MCH infusion (F(1, 85) = 0.31;

= 0.581).

To determine if this orexigenic response was mediated by the activation of MCHR1

receptors in the LS, we co-infused MCH with the MCHR1 antagonist TC-MCH 7c (1 µg).

MCHR1 antagonism has been reported to suppress feeding (Ito et al., 2010), but TC-MCH 7c

infusion into the LS alone had no main effect on feeding in male (F(1, 10) = 0.70;

= 0.424) or

female mice (F(1, 7) = 0.36;

= 0.0567) nor a significant interaction of TC-MCH 7c over time in

male (F(4, 40) = 1.62;

= 0.187) or female mice (F(4, 28) = 0.62;

= 0.653) when compared to

vehicle infusion. TC-MCH 7c infusion suppressed MCH-mediated feeding, as MCH infusion in

the presence of TC-MCH 7c resulted in a similar amount of cumulative feeding in male (F(1, 10)

= 3,15;

= 0.11) and female mice (F(1, 7) = 2.54;

= 0.155). TC-MCH 7c-mediated suppression

was sustained over time, as chow intake remained at a similar level over time in both male (F(4,

40) = 2.56;

= 0.053;

Figure 1D

) and female mice (F(4, 28) = 1.63;

= 0.196;

Figure 1E

) as

when the LS was treated with TC-MCH 7c only.

We recently determined that MCHR1 expression concentrated along the lateral LSr

border (Payant et al., 2023), thus we performed targeted infusions toward the laterally-distributed

MCHR1-expressing LS cells (

Figure 1F

). MCH infusion into the lateral LSr significantly

increased feeding within the first hour in both male (vehicle: 0.15



0.03 g; MCH: 0.28



0.03 g;

(5) = 3.24,

= 0.023) and female mice (vehicle: 0.11



0.02 g; MCH: 0.22



0.06 g;

(7) = 2.48,

= 0.042). There was a main effect of MCH infusion on cumulative chow intake over four hours

in both male (F(1, 5) = 10.38;

= 0.023;

Figure 1G

) and female mice (F(1, 7) = 9.19;

= 0.019;

Figure 1H

). MCH produced a similar 2-fold increase of chow intake, which was apparent within

2 h of MCH infusion into the lateral LS of male mice (F(4, 20) = 8.13;

= 0.0005;

Figure 1G

)

and just 1 h in female mice (F(4, 28) = 3.99;

= 0.011;

Figure 1H

). Although MCH infusion

doubled chow intake relative to vehicle in both male and female mice, there was a main effect of

sex (F(1, 60 = 7.18;

= 0.010) and an interaction between sex and MCH infusion which

produced a larger increase in chow consumption in male mice (F(1, 60) = 6.72;

= 0.012). This

Schematic of lateral LS infusions with bilateral cannulas implanted 1.2 mm apart (

top

Representative infusion spread following dye infusion (

bottom

;

). Cumulative chow intake of

aCSF vehicle and MCH (

), or TC-MCH 7c (1 µg/µl) and MCH with TC-MCH 7c (1 µg/µl

each;

) into the lateral LS of male and female mice. Scale bar: 200 µm (

). Significance (

< 0.05) was determined using a two-way ANOVA with Bonferroni post- comparison of drug
conditions at each timepoint: **,

< 0.01; ***,

< 0.001; ****,

< 0.0001. ACB, nucleus

accumbens; cc, corpus callosum; LS, lateral septum; VL, lateral ventricle.

3.4.2 MCH infusion into the LS increased intake of a palatable diet in male and female

mice

To determine if MCH also promoted hedonic feeding through the LS, we measured the intake of

a palatable, high dextrose diet (HDx) over four hours in sated mice following MCH infusion into

the central or lateral LS. Mice preferentially consumed HDx (11.3 ± 1.7 kcal/day) over chow

(5.7 ± 1.2 kcal/day) thus confirming the palatability of the HDx. Both male and female mice

displayed robust hyperphagia when given access to HDx relative to chow, but when delivered in

the central LS, MCH further increased HDx intake in male mice (F(1, 10) = 5.80;

= 0.037)

over time (F(4, 40) = 3.05;

= 0.028;

Figure 2A

). Male mice consumed more of the HDx

overall (F(1. 85) = 16.88;

< 0.0001), but there was no significant interaction between sex and

MCH infusion (F(1, 85) = 3.44;

= 0.067). The orexigenic effect of MCH was blocked by co-

infusion of TC-MCH 7c (F(1, 10) = 3.08;

= 0.110) and there was a small decrease in feeding

over time compared to TC-MCH 7c alone (F(4, 40) = 2.72;

= 0.043;

Figure 2B

). By contrast,

MCH infusion into the central LS did not further increase HDx intake in female mice (F(1, 7) =

0.024;

= 0.880) nor have an effect over time (F(4, 28) = 0.38;

= 0.819;

Figure 2C

), and there

were also no differences in HDx intake following the co-infusion of MCH with TC-MCH 7c

(F(1, 7) = 0.35;

= 0.571; MCH x time: F(4, 28) = 0.43;

= 0.785).

When MCH was infused into the lateral LSr, it increased HDx intake in both sexes, and

this orexigenic effect was abolished following TC-MCH 7c co-infusion. In males, there was no

main effect of MCH infusion (F(1, 6) = 3.59;

= 0.107), but MCH increased HDx intake over

time and within 4 h (F(4, 23) = 4.54;

= 0.008;

Figure 2E

). This MCH-mediated feeding in the

lateral LSr was blocked by TC-MCH 7c co-infusion (F(1, 5) = 5,17;

= 0.072; MCH x time:

F(4, 20) = 2.83;

= 0.052;

Figure 2F

). Interestingly, there was a robust increase in HDx intake

following MCH infusion into the lateral LSr of female mice (F(1, 7) = 8.00;

= 0.025) that was

apparent within an hour of MCH infusion (F(4, 28) = 4.54;

= 0.006;

Figure 2G

). This

orexigenic MCH effect was blocked by co-infusion of TC-MCH 7c (F(1, 7) = 1.77;

= 0.225;

MCH x time: F(4, 28) = 1.69;

= 0.180;

Figure 2H

). While male mice consumed more of the

HDx (F(1, 129) = 18.25;

< 0.0001), there was no interaction of sex with MCH infusion (F(1,

129) = 0.11;

= 0.741). These findings indicated that MCH further increased feeding even when

baseline feeding is elevated, but this effect was stronger in female mice when directed towards

the lateral LSr.

(

< 0.05) was determined using a two-way ANOVA with Bonferroni post- comparison of drug

conditions at each timepoint: **,

< 0.01; ***,

< 0.001; ****,

< 0.0001.

3.4.3 MCH in the lateral LS of male mice increased locomotor activity

While MCH increased food intake when infused throughout the LS, we did not detect differences

in ambulatory or locomotor activity during the feeding period in male mice when MCH was

administered alone (F(1, 10) = 0.06;

= 0.806; MCH x time: F(3, 30) = 0.29;

= 0.830;

Figure

) or in the presence of TC-MCH 7c (F(1, 10) = 0.07;

= 0.802; MCH x time: F(3, 30) = 1.35;

= 0.278;

Figure 3B

). Similarly, there were no locomotor activity differences following MCH

infusion alone (F(1, 7) = 0.07;

= 0.801; MCH x time: F(3, 21) = 1.27;

= 0.310;

Figure 3C

) or

when co-infused with TC-MCH 7c (F(1, 7) = 1.16;

= 0.317; MCH x time: F(3, 21) = 2.89;

0.060;

Figure 3D

However, when MCH was infused over the lateral LSr, there was an overall increase in

ambulatory activity of male mice (F(1, 5) = 9.71;

= 0.026). This MCH-mediated hyperactivity

in male mice was most prominent within the first hour following MCH infusion and decreased

over time (F(5, 25) = 4.08;

= 0.008;

Figure 3E

). The increased ambulatory activity was

abolished by TC-MCH 7c treatment (F(1, 5) = 0.24;

= 0.643; MCH x time: F(5, 25) = 1.34;

0.279;

Figure 3F

). No differences in ambulatory activity were seen in female mice with MCH

treatment (F(1, 7) = 0.86;

= 0.383; MCH x time: (F(5, 35) = 0.60;

= 0.701;

Figure 3G

) nor

TC-MCH 7c treatment (F(1, 7) = 3.97;

= 0.087; MCH x time: F(5, 35) = 2.11;

= 0.088;

Figure 3H

). Although the locomotor effect was only present in males, there was no significant

interaction between sex and MCH infusion when compared directly (F(1, 72) = 0.255;

0.615).

(

< 0.05) was determined using a two-way ANOVA with Bonferroni post- comparison of drug

conditions at each timepoint: **,

< 0.01; ***,

< 0.001; ****,

< 0.0001.

3.4.4 LS MCH infusion did not alter food seeking in an anxiogenic environment

As MCH stimulated feeding in the absence of increasing ambulatory activity, it is unlikely that

the hyperactivity of male mice treated with MCH in the lateral LSr was directly related to

feeding. We hypothesized that the observed hyperactivity was related to an anxiogenic and/or

stress response, which are LS- associated functions (Bakshi et al., 2007; Xu et al., 2019)

Furthermore, as MCH-mediated feeding may be context-dependent (Subramanian et al., 2023),

and MCH may increase feeding and locomotor activity in the home-cage environment, we

determined whether MCH would alter food-seeking behaviour when placed in an anxiogenic

environment. We infused MCH into sated mice and placed them into an open field with a HDx

food reward placed in the center of the open field to determine if the orexigenic effect of MCH in

the LS could overcome their native fear of travelling into the center.

While MCH infusion can drive feeding in sated animals in a non-stressful environment, it

was not sufficient to overcome anxiety in order to increase time spent in the center of the open

field when infused into the central (F(1, 10) = 0.27;

= 0.613;

Figure 4A

) or lateral LS of male

and female mice (F(1, 7) = 2.2;

= 0.182

Figure 4B

Being in a state of negative energy balance is an endogenous motivator to overcome

anxiogenesis and promote food approach behaviours, thus fasted mice will spend more time

exploring food placed in the center of an open field (Lockie et al., 2017). We thus determined if

MCH infusion in fasted mice would promote food approach in the open field. As anticipated,

fasted animals spent more overall time in the center of the open field investigating the food

101

aCSF vehicle (1 μl) or MCH (1 μg/μl) infusion into the central LS of male and female mice that

were sated (

) or fasted (

), and into the lateral LS of mice that were sated (

) or fasted (

Significance (

< 0.05) was determined using a two-way ANOVA with Bonferroni post-hoc

comparisons between drug conditions.

3.5

Discussion

Intracerebroventricular administration of MCH elicited robust feeding effects (Qu et al., 1996;

Rossi et al., 1997; Kela et al., 2003), but few brain regions have been identified to support the

orexigenic effects of MCH. We showed here that the LS is an important region underlying

MCH-mediated feeding. MCH infusion into the LS, especially when directed over the lateral LSr

where MCHR1-expressing cells were concentrated (Payant et al., 2023), increased feeding in

both male and female mice. However, the orexigenic effects of MCH when the mouse is in its

habituated homecage was abolished when placed in an anxiogenic environment, even when the

mouse was hungry.

The LS is a novel brain region underlying the orexigenic effect of MCH. Acute MCH

infusion into the LS increased consumption of a standard chow or palatable, high-dextrose, diet.

MCH induced feeding within the first hour and persisted over four hours which corresponded

with the timeline of intracerebroventricular administration of MCH (Qu et al., 1996; Rossi et al.,

1997; Kela et al., 2003). MCH infusion into both the central and lateral LS promoted feeding, but

consistent with our recent findings showing the concentration of MCHR1 expression towards the

lateral LS (Payant et al., 2023), we found that directing MCH towards the lateral LS elicited a

more robust orexigenic effect of MCH. The properties of MCH-mediated feeding in the LS are

unique from that in the hypothalamus (Rossi et al., 1999; Abbott et al., 2003; Al-Massadi et al.,

2019) or nucleus accumbens (Georgescu et al., 2005; Terrill et al., 2020), and it is noteworthy

that MCH initiated feeding even during the light cycle when animals were sated. This is in

102

contrast to orexigenic MCH actions in the nucleus accumbens that enhanced chow and sucrose

intake in the dark cycle when mice are naturally-feeding but increased only sucrose intake within

a 4 hour period in the light cycle (Georgescu et al., 2005; Terrill et al., 2020).

The orexigenic effect of MCH in the LS was uniquely prominent in both male and female

mice. Prior studies reported MCH-mediated feeding primarily in males (Terrill et al., 2020), as

elevated levels of estrogen in females blunted the orexigenic effect of MCH, and they are

restored in ovariectomized females (Messina et al., 2006; Santollo and Eckel, 2008). Estradiol

treatment decreases the efficacy of MCH-induced feeding (Messina et al., 2006; Santollo and

Eckel, 2008) by reducing the number of MCH cells and downregulating MCHR1 expression in

the hypothalamus (Santollo and Eckel, 2013). Although the LS expresses estrogen receptors

(Hasunuma et al., 2023), estrogen does not appear to strongly regulate MCH effects in this

region. There was a subtle sex difference in MCH-mediated chow consumption whereby MCH

infused into the lateral LS of females produced less hyperphagia than in males. However, MCH

doubled chow intake in both males and females, therefore the difference in consumption may be

related to the higher baseline feeding in males. The distribution and amount of

Mchr1

mRNA or

MCHR1 expression is comparable in the LS of male and female mice, and there were no

discernable differences in the magnitude of MCH-mediated inhibition in the LS (Payant et al.,

2023). The comparable effects of MCH-mediated feeding in the LS of male and female mice

were thus consistent with the distribution and functional activation of MCHR1 in the LS.

The orexigenic effect of MCH in LS was context-dependent. MCH-mediated feeding was

prominent in the homecage but did not extend to food-seeking in an anxiogenic environment. In

the wild, feeding and anxiety are intricately related, as anxiety may suppress feeding but a

hungry animal may take risks to feed even in anxiogenic, suboptimal environments. We

103

observed a mild increase in locomotor activity in male mice when MCH was infused into the

lateral LS. Since this effect was absent in the other groups exhibiting feeding effects, it appeared

to be unrelated to feeding. As such, we hypothesized that the hyperactivity could be related to an

anxiolytic or exploratory behavioural outcome. LS inhibition often suppresses anxiety-like

behaviours (Pesold and Treit, 1996; Wang et al., 2023) and a switch to exploratory behaviour in

the face of risk (Zhong et al., 2022). Therefore, we determined if MCH infusion into the LS may

increase time spent investigating a food reward placed in the center of an open field. However, in

sated or fasted mice, MCH did not increase the amount of time mice spent in the center of the

open field. While MCH in the LS did not mediate the interaction between feeding and anxiety, it

may still extend beyond homecage feeding to other facets of feeding-related behaviour. The LS

receives strong input from the hippocampus (Risold and Swanson, 1997), which promotes spatial

food memory (Decarie-Spain et al., 2022), and LS activation shortens the latency to food

approach (Chen et al., 2022). As the infusion of MCH or activation of MCH terminals in the LS

enhanced spatial memory by potentiating hippocampal inputs (Liu et al., 2022), future studies

may evaluate whether MCH acts in the LS to regulate spatial memory-dependent food-seeking.

The magnitude of the orexigenic effect with intra-LS MCH infusion was larger with

chow feeding than palatable diet feeding. This result was unexpected because MCH is thought to

reinforce the consumption of calorically dense solutions (Duncan et al., 2005; Karlsson et al.,

2012) by increasing their hedonic value (Baird et al., 2006; Lopez et al., 2011) and by recruiting

post-oral processes that detect and reinforce the consumption of nutrient-dense substances

(Domingos et al., 2013). However, this is often shown through the comparison of sweetened

solutions in the presence and absence of calories, for example via sucrose and sucralose,

respectively, and therefore differential outcomes could be expected when comparing two

104

nutrient-containing solid diets. Furthermore, as baseline feeding was greatly elevated when given

access to HDx, ceiling levels of baseline palatable HDx feeding would mask any orexigenic

effects of MCH, such as that seen with standard chow.

Chronic MCHR1 antagonism can suppress feeding (Shearman et al., 2003; Ito et al.,

2010; Kawata et al., 2017), but acute MCHR1 antagonism has no effect (Shearman et al., 2003)

or may decrease feeding in the dark cycle on feeding (Georgescu et al., 2005). Unsurprisingly,

we did not see any reductions in feeding within four hours of LS infusion during the light cycle.

We noted that administration of TC-MCH 7c with MCH did not fully block the effects of MCH

in males. It is not clear if this is due to the timing or insufficiency of MCHR1 antagonism. We

administered TC-MCH 7c a few minutes prior to MCH infusion and this may have been

insufficient time for the antagonist to block MCHR1 prior to the co-infusion of TC-MCH 7c with

MCH. Additionally, TC-MCH 7c may not fully block the cellular effects of MCH, as TC-MCH

7c pretreatment at LS cells only partly blocked the inhibitory effects of MCH (Liu et al., 2022).

In conclusion, MCH infusion into the LS promoted hyperphagia in male and female mice

and these results implicated the LS as an important structure underlying MCH-mediated

behaviour. As prior studies were conducted in rat, cross species differences hindered our ability

to assess the relative contribution of the LS to MCH-mediated feeding compared to other brain

targets. However, MCH uniquely initiated feeding via the LS, and as LS inhibition can stimulate

feeding (Mitra et al., 2014; Gabriella et al., 2022), and MCH inhibits LS cells (Payant et al.,

2023), the MCH system is an emerging regulator of LS-mediated feeding.

105

3.6

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