Dissecting the Functions and Connectivity of POMC Neurons

Minmin Luo

National Institute of Biological Sciences, Beijing

Tsinghua University School of Life Science

Sohn, Elmquist, & Williams Trends Neurosciences 2013

Circuit for Feeding Regulation

Adiposity signals (Leptin, Insulin)

Satiety signals (CCK)

Pro-opiomelanocortin (POMC) neurons are located in the arcuate nucleus (ARC) and

nucleus of the solitary tract (NTS)

The activity of POMC neurons reduces appetite, enhances metabolism, and increases energy expenditure

• Ablating POMC neurons in the entire brain or mutating the POMC gene causes obesity in rodents and humans (Yaswen et al., 1999; Coll et al., 2004; Gropp et al., 2005; Xu et al., 2005).

• Intraventricular administration of POMC-derived melanocortins such as -MSH suppresses food intake within hours (Fan et al., 1997).

• However, optogenetic stimulation of POMC neurons in the ARC for 2 h does not inhibit food intake (Aponte et al., 2011),

Questions:

• Do POMC neurons in the ARC and NTS contribute differently to feeding suppression?

• Do they receive different inputs and affect distinct brain centers?

*: Y149C and A239G in hM3

Chemogenetic control of POMC neurons using DREADD technology DREADD, Designer Receptors Exclusively Activated by Designer Drugs

CNO: clozapine-n-oxide

Targeting POMC neurons

POMC hM3Dq overlay

50 µm

ARChM3Dq NTShM3Dq

3V

100 µm ME cc 100 µm

D

mCherry

L-ITR CAG WPRE polyA R-ITR

+ Cre

mCherry L-ITR CAG WPRE polyA R-ITR

AAV DIO mCherry

POMC-Cre + AAV-DIO-mCherry

Zhan et al. J Neurosci 2013

Chemogenetic Activation of POMC neurons

c-Fos hM3Dq

ME

3V

100 µm

3V

ME

overlay

100 µm 100 µm

10 s

5 m

V

CNO

CNO ACSF

ΔV

m (

mV

)

0

2

4

6

**

CNO

10 s 100 p

A

20 40 60 80 100 0

2

4

6 CNO

# o

f sp

ikes

Time (s) CNO

10 m

V

5 s

cc

ARC

% o

f h

M3

Dq

+ c

ell

s

exp

ressin

g c

-Fo

s

%

of

c-F

os

+ c

ells

Lackin

g h

M3D

q

cc

100 µm

c-Fos hM3Dq overlay

0

20

40

60

NTS 0

20

60

ARC NTS

40

mCherry 2A hM3Dq

L-ITR CAG WPRE polyA R-ITR

+ Cre

mCherry 2A hM3Dq L-ITR CAG WPRE polyA R-ITR

AAV DIO hM3Dq-2A-mCherry

POMC-Cre + AAV-DIO-hM3Dq-2A-mCherry

Feeding suppression by chronic, but not acute, activation of ARC POMC neurons

Time after first injection (h)

0

1

2

3

4

5

2 5 8 24 Cu

mu

lati

ve

fo

od

in

tak

e (

g)

ARChM3Dq

Bo

dy w

eig

ht

(g)

0

5

10

15

20

25

Saline CNO CNO x2

ARChM3Dq

saline

CNO

CNO x2

Time after first injection (h)

ARCmCherry

Cu

mu

lati

ve

fo

od

in

tak

e (

g)

0

1

2

3

4

2 5 8 24

saline

CNO

CNO x2

1

2

3

4

5

1 2 3 4 5 6 7 8

Fo

od

in

tak

e (

g)

Days

CNO saline ARChM3Dq (n=5)

-10

-8

-6

-4

-2

0

Bo

dy w

eig

ht

gain

(%

)

Body weight Food intake

* *

**

* *

*

Time after first injection (h) Cu

mu

lati

ve

fo

od

in

tak

e (

g)

0

1

2

3

4

NTShM3Dq

2 5 8 24

** **

*** ***

***

saline CNO CNO x2

Cu

mu

lati

ve

fo

od

in

tak

e (

g)

2 5 8 24 Time after first injection (h)

NTSmCherry

0

1

2

3

4 saline CNO CNO x2

*

Me

al s

ize

(g

)

0

0.2

0.4

0.6

0.8

saline CNO

NTShM3Dq

Me

al n

um

ber

0

1

2

3

4

saline CNO

***

NTShM3Dq

NTShM3Dq

0

5

10

15

20

25

Bo

dy w

eig

ht

(g)

0.0

0.5

1.0

1.5

Fo

od

in

take (

g)

8-10 14-24

saline CNO CNO x2

Time after first injection (h)

0-2

** **

2-5

** **

5-8

**

10-12

** ***

12-14

***

Fo

od

in

take (

g)

Days

NTShM3Dq (n=5)

Bo

dy w

eig

ht

gain

(%

)

1

2

3

4

5

1 2 3 4 5 6 7 8

CNO saline

-10

-8

-6

-4

-2

0

Body weight

Food intake

**

*

0

100

200

300

400

500

600

700

2 5 8 12 24

Tra

vel d

ista

nce (

m)

NTShM3Dq

saline CNO CNO x2

Time after first injection (h)

Feeding suppression by acute activation of NTS POMC neurons

Genetic ablation of ARC POMC neurons increase food intake and causes obesity

ARCDTR & DT NTSDTR & DT

ME

3V

100 µm

CC

100 µm

DTR

L-ITR CAG WPRE polyA R-ITR

+ Cre

DTR L-ITR CAG WPRE polyA R-ITR

AAV DIO DTR

Bo

dy w

eig

ht

gain

(%

)

POMC cell number in ARC

ARCDTR & DT

-10 0

20

40

60

80

0 400 800 1200

ARCDTR (n = 18) ARCmCherry (n = 14) NTSDTR (n = 7) NTSmCherry (n = 16)

Bo

dy w

eig

ht

gain

(%

) 0 4 8 12 16 20 24

Days after DT injection

-10

0

10

20

30

40

50

DT

ARCDTR & DT (n = 12) ARCmCherry (n = 5) NTSDTR & DT (n = 6) NTSmCherry (n = 6)

Daily f

oo

d in

take (

g)

2

3

4

5

6

7

Days after DT injection 0 4 8 12 16 20 24

DT

ARCDTR & DT ARCmCherry & DT ARCDTR & DT ARCmCherry & DT

Ablation of ARC POMC neurons reduces energy expenditure

O2 c

on

su

mp

tio

n (m

l/g

/min

)

ARCDTR & DT

n = 6

control

n = 5

0

0.02

0.04

0.06

0.08 ***

ARCDTR & DT (n = 8)

ARCmCherry & DT (n = 8)

Tra

ve

l d

ista

nc

e (

m)

0

100

200

300

400

500

Dark phase Light phase

***

The effects of ablating POMC neurons on metabolism

Co

rtic

oste

ron

e (

ng

/ml)

NTSDTR & DT (n = 6) NTSmCherry & DT (n = 6)

ARCDTR & DT (n = 8) ARCmCherry & DT (n = 6)

0

1

2

3

4

Le

vels

(m

mo

l/L

)

CHO

*

TG HDL-C

*

LDL-C

*

0

20

40

60

80

100

% b

od

y w

eig

ht

fluid fat mass

***

lean mass

***

NTSDTR & DT (n = 6) NTSmCherry & DT (n = 6)

ARCDTR & DT (n = 8) ARCmCherry & DT (n = 6)

ARCDTR & DT (n = 8) ARCmCherry & DT (n = 6) NTSDTR & DT (n = 6) NTSmCherry & DT (n = 6)

5

10

15

20

25

30

0 15 30 60 90 120 Time (min)

Glu

co

se (

mm

ol/

L)

0

50

100

150

200 * 6 6

5

6

Summary 1

• Activation of NTS POMC neurons rapidly inhibits feeding. Chronic stimulation is required for ARC POMC neurons to suppress food intake.

• Ablation of POMC neurons in the ARC but not the NTS increases food intake, reduces energy expenditure, and ultimately causes obesity and metabolic and endocrine disorders.

• Different behavioral functions of POMC neurons in the ARC and NTS: POMC neurons regulate feeding and energy homeostasis by integrating long-term adiposity signals from the hypothalamus and short-term satiety signals from the brainstem.

Transsynaptic tracing of presynaptic inputs of POMC neurons

AAV-DIO-TVA-EGFP

CAG

EGFP-2A-TVA

WPRE

AAV-DIO-RG

CAG

RG

WPRE

loxP loxP2272

SADΔG-mCherry(EnvA)

(Rabies virus) mCherry

Day 1

AAV-DIO-TVA-EGFP

AAV-DIO-RG

Day 14

SADΔG-mCherry(EnVA) Day 21

histology

Virus

POMC-Cre or AgRP-Cre

Input

ARC Starter

Input

TVA-EGFP POMC Merge POMC-Cre

TVA-EGFP Rabies virus

100 µm 25 µm

1 mm

POMC-Cre AgRP-Cre wild-type

Wang et al. Frontier Neuroanatomy 2015

Input patterns of POMC and AgRP neurons

Ag

RP

VTg

AHi

LS

BST

LPO

MPA AH PVN

AHC

SO

DM

VMH

ARC

PH

VS

PAG

NI

RMg

1 mm

PO

MC

LS

VDB

HDB

BST

LPO

MPA AH PVN

AHC

SO

VTg

RMg AHi

ARC

PH

VS

PAG

MRN

DS

DM

VMH

NI RMg

1 mm

Bregma 0.70mm -0.10mm -0.60mm -0.90mm -1.60mm

-2.30mm -2.70mm -4.00mm -5.20mm

Bregma 0.70mm -0.10mm -0.60mm -0.90mm -1.60mm

-2.30mm -2.70mm -4.00mm -4.70mm -5.20mm

-0.90mm

-4.70mm

-0.90mm

-4.70mm

-0.10mm

-2.70mm

-0.10mm

Distance from

Bregma (mm)

0

500

1000

1500

2000

4 2 0 -2 -4 -6 -8

ARC

Ce

ll n

um

ber

POMC-Cre

AgRP-Cre

Input patterns of POMC and AgRP neurons infralimbic cortex (IL)

dorsal peduncular cortex (DP)

secondary motor cortex (M2)

medial orbital cortex (MO)

retrosplenial cortex (RS)

cingulate cortex (Cg)

retrosplenial granular cortex (RSG)

prelimbic cortex (PrL)

lateral septum (LS)

dorsal tenia tecta (DTT)

diagonal band of broca (DB)

medial septum (MS)

accumbens nucleus (Acb)

medial amygdaloid nucleus (MEA)

amygdalohippocampal area (AHi)

bed nucleus of the stria terminalis (BST)

ventral pallidum (VP)

sublenticular extended amygdala (SLEA)

anterodorsal preoptic nucleus (ADP)

medial preoptic area (MPA)

medial preoptic nucleus (MPO)

lateral preoptic area (LPO)

anterior hypothalamus (AH)

dorsomedial hypothalamus (DM)

lateroanterior hypothalamic nucleus (LA)

lateral hypothalamus (LH)

retrochiasmatic area (Rch)

tuber cinereum area (TC)

paraventricular hypothalamus (PVN)

supraoptic nucleus (SO)

posterior hypothalamus (PH)

ventromedial hypothalamus (VMH)

medial tuberal nucleus (MTu)

zona incerta (ZI)

premammillary nucleus (PM)

lateral habenular nucleus (LHb)

paraventricular thalamic nucleus (PVT)

subiculum (S)

periaqueductal gray (PAG)

supramammillary nucleus (SuM)

medial mammillary nucleus (MM)

Edinger-Westphal nucleus (EW)

ventral tegmental area (VTA)

dorsal raphe nucleus (DRN)

ventral tegmental nucleus (VTg)

raphe magnus nucleus (RMg)

nucleus incertus (NI)

raphe obscurus nucleus

(ROb) 0

2000

4000

6000

2000

4000

6000

POMC AgRP

Cell density (cells/mm2)

Cortex

0 5

10

15

5

10

15

POMC AgRP

Proportion of total inputs (%)

Septum

Striatum

Amygdala

Pallidum

Hypothalamus

Thalamus

Hippocampus

Midbrain

Pons

Medulla

median raphe nucleus (MnR)

anterior tegmental nucleu (ATg)

lateral parabigeminal nucleus (LPB)

ac

Olfactory

bulb

Cerebral cortex

cc LV

LS

Thalamus

Hypothalamus

Superior

Colliculus

Midbrain

Medulla Spinal

cord

Cerebellum

4V

BST

PVN

LH

Inferior

Colliculus

Pons

Cg

3V

VP HDB

LPO

MPO

MPA AHi

SO

PH

S

SuM

MM

MRN ATg VTg

CG

Inputs to the ARC POMC neurons

0.5-1%

1-2%

2-5%

5-15%

＞15%

Difference between ARC POMC and AgRP neurons

LS LV

MPO AH

VTg

NI

HDB

PVN SO

200 µm

200 µm

DRN

VS

200 µm

RMg IRt

POMC-Cre

Input Starter Input

Virus

NTS

CeM LH PSTh

PVN BST

Rn

LC

PnC IRt

Lat

Med

Su5

PnO

NTS

Gi

1 mm

100 µm

CC

TVA-EGFP

rabies virus

ARC

Bregma -0.10mm -0.90mm -1.50mm -2.00mm -3.20mm

-5.00mm -5.40mm -5.80mm -6.20mm -7.60mm

Input pattern for the NTS POMC neurons

Input pattern for the NTS POMC neurons

a

c

Olfactory

bulb

Cerebral cortex

cc LV

Septum Thalamus

Hypothalamus

Superior

Colliculus

Medulla

Spinal

cord

Cerebellum

4V

Preoptic

DCN

S1 M1

BST PSTh

PVN

CeM

LH

R

DPMe

PAG

Inferior

colliculus

0.5-1%

1-2%

2-5%

5-15%

＞15% PCRt

IRt

Gi

MdD

MdV

Pons

PnO

PnC

3V

200 µm

IRt CeM PVN PSTh Rn SU5

Distance from Bregma (mm)

ARC

0

500

1000

1500

2000

4 2 0 -2 -4 -6 -8

Ce

ll n

um

be

r

NTS

ARC POMC

NTS POMC

Projection patterns of axon fibers

AAV-FLEX-EmGFP

mGFP

L-ITR EF1a WPRE polyA R-ITR

+ Cre

mGFP L-ITR EF1a WPRE polyA R-ITR

mtdTomato

L-ITR EF1a WPRE polyA R-ITR

+ Cre

mtdTomato L-ITR EF1a WPRE polyA R-ITR

AAV-FLEX-mtdTomato

POMC-Cre AAV-FLEX-EmGFP

AAV-FLEX-mtdTomato

ARC

NTS

MdV

LPB

MVe

Gi

PnC BST

Acb +Acb

BST

HDB LH

PSTh

Rt

LPO

DM VMH

MPO

MPA

PVT

PH

DpMe

DpG

PAG + PAG

LDTg

PnO

GiA

Gi

NTS

LS

VDB ARC AHC

TC

PVN PVN

PH

DTg NTS

DPGi

PMn

NTS

PCRt

IRt

LPGi

MVe Su5

Mo5 PnO LH

PSTh

ZI

DpMe PAG

LPB

POMC-Cre; ARC vs. NTS

1 mm

AgRP-Cre; ARC

LPB

LH

PSTh

LPO

BST

Acb PVT

PH

DM VMH LH

LDTg

PAG

PVN

ARC

PAG

AHC TC

MPO ADP

DM

VMH

PAG

MPA

PVN

PVT

1 mm

Summary 2 • The presynaptic partners of ARC POMC neurons

largely overlap with those of ARC AgRP neurons, although POMC neurons receive broader and denser inputs.

• NTS POMC neurons receive direct inputs mainly from the brainstem and show very different innervation patterns for their counterparts in the ARC.

• Almost all of their major presynaptic partners are innervated by POMC neurons, suggesting strong reciprocal projections among the major POMC neural pathways.

Acknowledgements

• Cheng Zhan, Jingfeng Zhou, Daqing Wang, Qiru Feng, Zhe Zhao, Ju-en Zhang, Shuailiang Lin, Junhong Bao, Yue Sun, Ping Wu, Rui Lin (NIBS, Beijing)

• Xiaobing He, Ting Ding, Fuqiang Xu (Wuhan Institute of Physics and Mathematics, CAS)

Funding: China Ministry of Science & Technology; NNSF; Beijing Municipal Government