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Learning objective: If someone asks you How does

Acetylcholine effect excitability ?

Your answer will be: That depends on ..

ACETYLCHOLINE ( ACh)

modulation of circuitexcitability

Bio 337Neurotransmission and Neuromodulation

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Cholinergic Circuits in the CNS:under-rated & problematic!

Acetylcholine ( ACh) is important in the Brain:

ACh modulates the activity of circuits that underlieattention, memory & motivated behaviors

The Challenges:

BASAL FOREBRAINCHOLINERGIC NUCELI

PTG CHOLINERGIC NUCELI

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1. Gentle introduction to Acetylcholine: (ACh)the ACh synapses and receptors you probably already know

2. ACh neurons and their projections in the brain Distribution & why should you care?

3. Brain ACh receptor types focus on neuronal type nAChRs( but dont forget mAChRs)

4. Release of ACh in the brain

5. ACh as a modulator : Location, Location, Location

6. Data based examples : ACh as modulatory transmitter incircuits of motivation & emotional memory

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Acetylcholine: (ACh)

what you may already know:

1. ACh basics

2. muscle-type nicotinic AChRsand the NMJ

3. Peripheral muscarinic AChRs

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Acetylcholine

B

asics

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Synthesis and Packaging of

Acetylcholine (ACh)

Axon

Na+

choline

[choline] in

serum ~ 10 Mmitochondria choline+

acetyl-CoA

Choline Acyltransferase

ACh

Pre-synaptic Terminal

(CH3)3-N-CH2CH2-O-C-CH3+ =

O

From lecture by ProfessorWollmuth (fonts changed)

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eNMJ:

thearch

etypalsy

napse

Muscle fiber

motoneuron

myelin

The NMJ axon

Presynaptic terminals

Activezone

Syn cleft

nAChRs

NMJ fold

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NMJ/ MUSCLE-TYPE NICOTINIC AChRs:The archetypal ligand-gated

ion channel (= ionotropic receptor)

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Studies of the

NMJ nAChR

have guided thestructural &

biophysical

dissections of

other ionotropicchannels

ACh bindingsites

Acetylcholine

subunit

subunit

subunit

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Muscarinic ACh receptors in cardiac & smooth muscle--- 7TMs, 2nd messenger cascades

..indirect mechanisms of channel gating..

ACh binds to M2 muscarinicacetylcholine receptor

activates Gi protein

subunits bind to IK,ACh

channel

activate channel

hyperpolarize membranepotential

HR

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ACh in the brain :

Key concepts:

1. There are &*(^% few cholinergic neurons in

the brain.

2. The projections/ terminal fields are REALLYextensive

3. There is reciprocal innervation of cholinergicnuclei with numerous brain regions ( many relatedto emotional memory)

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ACh in the Brain

Ok.. As brain transmitters/ modulators go .. ACh IS a bit of asports challenge..

BASAL FOREBRAIN

CHOLINERGIC NUCELI

PTG CHOLINERGIC NUCELI

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Cholinergic Circuits

in the CNS

(AChE)

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BLA

VAChT Ab

JCN 519:790805(2011)

Lat

CeA

BLABLA

VAChT Ab

VAChT Ab

Cholinergic projections to the BLA (highest densityterminal field)

B = Nucleus Basalis MAJORcholinergic nucleus

Cholinergic Circuits in the CNS(VACh Transporter Ab)

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RECIPROCAL REGULATION of CHOLINERGIC, GABA-ergic,GLUTAMATergic & DOPAMIN-ergic CIRCUITS

PFC

AcVTABasal

ForebrainACh

nuclei

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ACh in the brain -- why should you care?

ACh and AChRs as therapeutic targets

a. neurological ALS, My Gravis, AD, PD

b. neuropsychiatric SZ, ADHD, depression, addiction

c. inflammation

R l f AChR i li i hi i di d

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Alzheimers Disease Impaired short termmemory, dysnomia,progressive aphasia,

disorientation,dementia

Auguste Deter, 51 yo ,admitted: 1901 d.1906 Alois Alzheimer

1864-1915

psychosis (hallucinations, delusions)Affective/mood dysregulation (depression, mania)Thought disorderAltered information processing (cognitive

impairment, sensory gating deficits)

Schizophrenia

Nicotine

Role of AChR signaling in neuropsychiatric disorders

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Acetylcholine Receptors in the CNS:

(AChRs) Key concepts:

1. There are multiple classes of neuronal nicotinic

AChRs ( muscle-type nicotinic AChRs )*altho all cationic, net effect depends on location *

2. There are multiple classes of neuronal muscarinic (7TM)

AChRs with different effector channels

* Some hyperpolarize, some depolarize, net effect dependsof type and location *

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: Two types of ACh receptors in brain

G-protein coupled

receptors =

muscarinic AChRs

Ligand-gated ion

channels = nicotinic

AChRs

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uronaln

icotinicAChRs(n

AChRs)

Homopentameric

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neuro

na

lnico

tinicAC

hRs

Heteropentameric nAChRsHomopentameric

nAChRs

Agonists: nicotine, acetylcholine carbacholAntagonists: mecamylamine, atropine! ( BgTx 7 specific)

l i i i h

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neuronal nicotinic AChRs

(so much more than you wanted to know and NO you do not have to)

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: Two types of ACh receptors in brain

Ligand-gated ion

channels = nicotinic

AChRs

G-protein coupled

receptors =

muscarinic AChRs

From lecture by Professor

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bg-Subunits of G proteins may have regulatory

activity, too

K+

Muscarinic (M2

)

acetylcholine receptor

Kir

bg

AC

inactive

ai

GTP

From lecture by ProfessorWollmuth

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Muscarinic Acetylcholine receptors (mAChRs)ACh activation of many 4/5 mAChRs leads to a decrease in excitability

&/or release due to increased K (gK) conductance

M2 and M4 receptors can also becoupled to inhibition of N type Cachannels to decrease release

BUT M1 receptors: areunusual because they gK &

increase excitability

-80 mV -80 mV

+5 mV

1 nA

5 ms

mAChR

control

l i i AChR

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Gene Function Effectors Agonists Antagonists

M1 CHRM1

EPSP in autono mic ganglia

secretion from salivary glands and stomach

In CNS (memory?) cortex, hippo, striatum

Gq(G

i

), (Gs

)

induces slow EPSP

K+ conductance(aka M current)

acetylcholine

oxotremorine

muscarine

carbachol

McNA343

77-LH-28-1

atropine

scopolamine

dicycloverine

Thorazine

tolterodine

]pirenzepine

M2 CHRM2

*slow heart rate; reduce contraction atrium

reduce conduction velocity o f AV node

In CNS: homotropic inhibition, basal

forebrain, thalamus

Gi

K+ conductance(aka GIRK)

Ca2+ conductance

acetylcholine

methacholine

carbachol

Oxotremorine

muscarine

atropine

dicycloverine

Thorazine

Diphenhydramine

tripitramineGallamine

M3 CHRM3

smooth muscle contraction

Increase intra Ca vascular endothelium

increased endocrine and exocrine secretions,

In CNS coretex hippocampus ,thalamus

Gq

acetylcholine

bethanechol

carbachol

oxotremorine

pilocarpine

atropine

Diphenhydramine

dicycloverine

Tolterodine

oxybutynin

ipratropium

darifenacin

tiotropium

M4 CHRM4decreased loco motion

In CNS; cortex , striatum, hippocampus

Gi

K+ conductance Ca2+ conductance

acetylcholine

carbachol

oxotremorine

atropine

Dicycloverine

oxybutynin

mamba toxin

M5 CHRM5 In CNS substantia nigra, VTA? Gqacetylcholine

carbachol

oxotremorine

atropine

Diphenhydramine

dicycloverine

ipratropium

neuronal muscarinic AChRs(so much more than you wanted to know and NO you do not have to)

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Acetylcholine (ACh) release

Q: Is ACh released via classic point to pointtransmission at axo-dendritic synapses

or by volume transmission ?

A: Yes (& no).phasic release of ACh and a role for

overflow

** Axo-axonic synapses ***

other

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Type I

Type II

Type

I

Type II

Type I

Type II

BASICS OF CNS SYNAPTIC STRUCTURE

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soma

ACh release near soma &dendritic shaft

(as in PNS & some CNS)

spine

ACh release at spines (?)

By far the most common situation in the CNS:

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By far the most common situation in the CNS:ACh is released at Axo-axonic synapses to

modulate release of other neurotranmitters

Glutamate or

GABA-ergicsynapse

ACh input

Ligand gatedAChRs (nicotinic)

G proteincoupled AChRs

(muscarinic)

Ca2+

NaCa2+

CICR [Ca2+]int

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ACh in the brain:

SO: How does the activation of pre &/orpost synaptic nAChRs &/or mAChRs add upto modulate excitability?

That depends on..

which TYPES of AChRs

&LOCATION LOCATION LOCATION

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Studies of cholinergic modulation in vitro

B

Hipp or corticalinput from YFPmice

Optical (SDCM) recording of Ca++signaling along individual axons

nAcc or Amyg(WT or +/-)

CA

DElectrophysiological recording ofsynaptic interactions

WT or +/-

S naptic facilitati n b nic tine at

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Synaptic facilitation by nicotine atinputs from Hipp nAcc

NICOTINEAPPLICATIONPIPETTE

+NIC

In vitro analysis vs in vivo reality

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In vitro analysis vs in vivo reality

Sotell me, what would be the effect of (just) nicotine onthis motivational circuit?

& what about the effects of ACh?

mAChRsmAChRs

mAChRs

mAChRs

mAChRs

Hipp

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Understanding the effects of nicotine

(tho very relevant to the physiology, morbidity and mortality of

zillions of people world wide)

is still only part of the picture..

How do we figure out how AChper se influencescircuit activity ??

so we could have a way to FIX it when it goes

wrong?

(NB! the next 6 slides are for enjoyment andpersonal edification only)

: j b l d i ff f d

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: major obstacles to studying effects of endogenouscholinergic circuits..

NBM

Cholinergic neurons are few in number & at lowdensity within disperse nuclear groups

ChAT Tau GFP line from SJ. Vijayaraghavan

NBM

Ch li i N i th b l f b i 2011

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Cholinergic Neurons in the basal forebrain 2011

(Chat GFP transgenic)

ChAT Tau GFP line from SJ. Vijayaraghavan

Ch li i j ti i th

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Cholinergic projections in the

CNS (Chat GFP transgenic)

ChAT Tau GFP line from SJ. Vijayaraghavan

Dorsal and ventral striatum(emotion motion)

ventral hippocampus

Amygdala (BLA)emotional memories

OPTOGENETIC LABELING OF CHOLINERGIC NEURONS

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ChAT Tau GFP x ChAT-CRE

double transgenic

AAV- DiO- floxed

ChR2- RED intoNBM

2 weeks

ChAT-Tau GFP;

ChAT- ChR2

Ch

olinergicaxonsin

termina

lfie

lds

OPTOpro

be

labe

ledC

ho

linergicaxons

Cho

linergicne

uronsinNBM

OPTOpro

be

lab

eledC

ho

linergic

neruon

sNBM

OPTOGENETIC LABELING OF CHOLINERGIC NEURONS

Direct, electrophysiological stimulation of cortical projections +

http://localhost/var/www/apps/conversion/tmp/scratch_9/NBM_60x_proj_over_z_red_green_max_speed_Chat_GFP_cre_1600_1_slice5_06092011003_zstk.avihttp://localhost/var/www/apps/conversion/tmp/scratch_9/BLA_20x_proj_over_z_red_3_frames_per_sec_Chat_GFP_Cre_1600_3_slice1_05262011004_zstk_3_avi.avi

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OPTO +

OPTO TAGGEDCHOLINERGICINPUT

Target neuron

ELEC STIMCORTICAL

INPUT

Stimulatingelectrode

Light

, p y g p jrecording in BLA -/+ optogenetic stimulation of cholinergic inputs

(L.Jiang)

Recordingelectrode

Stim Cortical

Input

Record BLA

Light

OPTO stimulation of NBM inputs to BLA, like nicotine, elicits LTP

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Recording

electrode

Stim Cortical

Input, 1 Hz

Record

BLA

OPTO stimulation of NBM inputs to BLA, like nicotine, elicits LTP(Li Jiang)

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1. Gentle introduction to Acetylcholine: (ACh)the ACh synapses and receptors you probably already know

2. ACh neurons and their projections in the brain Distribution & why should you care?

3. Brain ACh receptor types

focus on neuronal type nAChRs( but dont forget mAChRs)

4. Release of ACh in the brain

5. ACh as a modulator : Location, Location, Location

6. Data based examples : ACh as modulatory transmitter incircuits of motivation & emotional memory