ACETYLCHOLINE ( ACh) modulation of circuit excitability

Bio 337 Neurotransmission and Neuromodulation

Learning objective: If someone asks you How does Acetylcholine effect excitability ? Your answer will be: That depends on ..

Cholinergic Circuits in the CNS: under-rated & problematic! Acetylcholine ( ACh) is important in the Brain:

ACh modulates the activity of circuits that underlie attention, memory & motivated behaviors
The Challenges:

BASAL FOREBRAIN CHOLINERGIC NUCELI PTG CHOLINERGIC NUCELI

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 in circuits of motivation & emotional memory

Acetylcholine: (ACh) what you may already know: 1. ACh basics 2. muscle-type nicotinic AChRs and the NMJ 3. Peripheral muscarinic AChRs

Acetylcholine Basics

From lecture by Professor Wollmuth (fonts changed)

Synthesis and Packaging of Acetylcholine (ACh)

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

Na+ choline [choline] in serum ~ 10 M

choline + acetyl-CoA

Axon

Choline Acyltransferase ACh

Pre-synaptic Terminal

The NMJ : the archetypal synapse

motoneuron

Muscle fiber
myelin

The NMJ

axon
Active zone Syn cleft nAChRs NMJ fold

Presynaptic terminals

NMJ/ MUSCLE-TYPE NICOTINIC AChRs: The archetypal ligand-gated ion channel (= ionotropic receptor)

Studies of the NMJ nAChR have guided the structural & biophysical dissections of other ionotropic channels
g subunit
ACh binding sites

a subunit

Acetylcholine
d subunit

Muscarinic ACh receptors in cardiac & smooth muscle

--- 7TMs, 2nd messenger cascades ..indirect mechanisms of channel gating..

ACh binds to M2 muscarinic acetylcholine receptor activates Gi protein bg subunits bind to IK,ACh channel activate channel hyperpolarize membrane potential HR

ACh in the brain :

Key concepts:
1. There are &*(^% few cholinergic neurons in the brain. 2. The projections/ terminal fields are REALLY extensive 3. There is reciprocal innervation of cholinergic nuclei with numerous brain regions ( many related to emotional memory)

ACh in the Brain

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

BASAL FOREBRAIN CHOLINERGIC NUCELI

PTG CHOLINERGIC NUCELI

Cholinergic Circuits in the CNS (AChE)

VAChT Ab

Cholinergic Circuits in the CNS (VACh Transporter Ab)

B = Nucleus Basalis MAJOR cholinergic nucleus

VAChT Ab
BLA Lat CeA

BLA BLA VAChT Ab

JCN 519: 790805 (2011)

Cholinergic projections to the BLA (highest density terminal field)

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

PFC

Ac

Basal Forebrain ACh nuclei

VTA

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

Role of AChR signaling in neuropsychiatric disorders Alzheimers Disease

Impaired short term memory, dysnomia, progressive aphasia, disorientation, dementia Auguste Deter, 51 yo , admitted: 1901 d.1906

Nicotine

Alois Alzheimer 1864-1915

psychosis (hallucinations, delusions) Affective/mood dysregulation (depression, mania) Thought disorder Altered information processing (cognitive impairment, sensory gating deficits)

Schizophrenia

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 depends of type and location *

: Two types of ACh receptors in brain

Ligand-gated ion channels = nicotinic AChRs

G-protein coupled receptors = muscarinic AChRs

neuronal nicotinic AChRs (nAChRs)

Heteropentameric nAChRs

Homopentameric nAChRs

neuronal nicotinic AChRs

Agonists: nicotine, acetylcholine carbachol Antagonists: mecamylamine, atropine! (aBgTx a7 specific)

neuronal nicotinic AChRs

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

: Two types of ACh receptors in brain

Ligand-gated ion channels = nicotinic AChRs

G-protein coupled receptors = muscarinic AChRs

From lecture by Professor Wollmuth

Muscarinic (M2) acetylcholine receptor

bg-Subunits of G proteins may have regulatory activity, too

Kir

AC inactive
g b K+ ai

GTP

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
BUT M1 receptors: are unusual because they gK & increase excitability
+5 mV
-80 mV -80 mV

1 nA 5 ms

mAChR

control

M2 and M4 receptors can also be coupled to inhibition of N type Ca channels to decrease release

(so much more than you wanted to know and NO you do not have to)
Gene Function Effectors Agonists acetylcholine oxotremorine muscarine carbachol McNA343 77-LH-28-1 Antagonists atropine scopolamine dicycloverine Thorazine tolterodine ]pirenzepine EPSP in autonomic ganglia secretion from salivary glands and stomach
In CNS (memory?) cortex, hippo, striatum

neuronal muscarinic AChRs

(Gi), (Gs )

Gq

M1

CHRM1

K+ conductance
(aka M current)

induces slow EPSP

M2

*slow heart rate; reduce contraction atrium reduce conduction velocity of AV node
CHRM2 In CNS: homotropic inhibition, basal forebrain, thalamus

Gi
K+ conductance
(aka GIRK)

Ca2+ conductance

acetylcholine methacholine carbachol Oxotremorine muscarine

atropine dicycloverine Thorazine Diphenhydramine tripitramine Gallamine

smooth muscle contraction Increase intra Ca vascular endothelium M3 CHRM3 increased endocrine and exocrine secretions, In CNS coretex hippocampus ,thalamus

Gq

acetylcholine bethanechol carbachol oxotremorine pilocarpine

atropine Diphenhydramine dicycloverine Tolterodine oxybutynin ipratropium darifenacin tiotropium atropine Dicycloverine oxybutynin mamba toxin
atropine Diphenhydramine dicycloverine ipratropium

M4

CHRM4

decreased locomotion In CNS; cortex , striatum, hippocampus

Gi
K+ conductance Ca2+ conductance

acetylcholine carbachol oxotremorine acetylcholine carbachol oxotremorine

M5

CHRM5

In CNS substantia nigra, VTA?

Gq

Acetylcholine (ACh) release

Q: Is ACh released via classic point to point transmission at axo-dendritic synapses or by volume transmission ? A: Yes (& no). phasic release of ACh and a role for overflow ** Axo-axonic synapses ***

other

BASICS OF CNS SYNAPTIC STRUCTURE

Type I

Type I

Type I Type II Type II

Type II

ACh release near soma & dendritic shaft (as in PNS & some CNS)

ACh release at spines (?)

spine

soma

By far the most common situation in the CNS: ACh is released at Axo-axonic synapses to modulate release of other neurotranmitters

ACh input Glutamate or GABA-ergic synapse Ca2+

Na

Ca2+

CICR

[Ca2+]int

G protein Ligand gated coupled AChRs AChRs (nicotinic) (muscarinic)

ACh in the brain:

SO: How does the activation of pre &/or

post synaptic nAChRs &/or mAChRs add up to modulate excitability? That depends on.. which TYPES of AChRs

& LOCATION

LOCATION

LOCATION

Studies of cholinergic modulation in vitro

Hipp or cortical input from YFP mice

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

A B
WT or +/-

C
D
Electrophysiological recording of synaptic interactions

nAcc or Amyg (WT or +/-)

Synaptic facilitation by nicotine at inputs from Hipp nAcc

NICOTINE APPLICATION PIPETTE

+NIC

In vitro analysis vs in vivo reality

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

& what about the effects of ACh?

Hipp
mAChRs
a7, a4 b2

mAChRs

mAChRs mAChRs

mAChRs

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 ACh per se influences circuit activity ?? so we could have a way to FIX it when it goes wrong?

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

: major obstacles to studying effects of endogenous

cholinergic circuits..
Cholinergic neurons are few in number & at low density within disperse nuclear groups
NBM

NBM

ChAT Tau GFP line from SJ. Vijayaraghavan

Cholinergic Neurons in the basal forebrain 2011 (Chat GFP transgenic)

ChAT Tau GFP line from SJ. Vijayaraghavan

Dorsal and ventral striatum (emotion motion)

Cholinergic projections in the CNS (Chat GFP transgenic)

Amygdala (BLA) emotional memories

ventral hippocampus ChAT Tau GFP line from SJ. Vijayaraghavan

OPTOGENETIC LABELING OF CHOLINERGIC NEURONS ChAT-Tau GFP; ChAT- ChR2 AAV- DiO- floxed ChR2- RED into NBM
ChAT Tau GFP x ChAT-CRE double transgenic
Cholinergic neurons in NBM OPTO probe labeled Cholinergic neruons NBM

2 weeks
Cholinergic axons in terminal fields PTO probe labeled Cholinergic axons

Direct, electrophysiological stimulation of cortical projections + recording in BLA -/+ optogenetic stimulation of cholinergic inputs (L.Jiang) Light Stim Cortical Input Stimulating
Recording electrode Light electrode Record BLA

ELEC STIM

OPTO +

CORTICAL INPUT

OPTO TAGGED CHOLINERGIC INPUT Target neuron

OPTO stimulation of NBM inputs to BLA, like nicotine, elicits LTP (Li Jiang)
Stim Cortical Recording Input, 1 Hz electrode

Record BLA

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 in circuits of motivation & emotional memory