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Slide 1
Module BIO00009I
Neuroscience
Lecture 5
Neuronal cell signalling
Sangeeta Chawla
sangeeta
...
ac
...
Neuronal cell signalling
Intracellular signalling
- GPCR and Ca2+ signalling
- Modulation of synaptic transmission

L6
...
Pharmacology of reward pathways
-The reward pathway
-Synaptic targets of dependence-producing drugs

L8
...
Understand how intracellular signalling contributes to
neurotransmission and its modulation
...
Relate major anatomical regions of the brain to their function
and neural circuits to behavioral responses
...
Understand how small molecules can interact with neuronal
proteins to modify connectivity within neural circuits and alter
behaviour
...
Appreciate the mechanisms by which behaviour is
synchronized to day-night cycles
...
Explain and evaluate how our understanding of behaviour and
neural circuits has been inferred from particular experiments
...
Intracellular signalling
Review of neurotransmission
presynaptic

Voltage-gated Ca2+ channels (1)
couple the AP to NT release (2)depolarises membrane

NT binds and opens ligand-gated
ion channels (3) to mediate
direct excitation or inhibition
of the postsynaptic cell
...
They open, Ca
comes in, binds to a protein which allows the
vesicles to fuse to the membrane and release their
contents
...


NT can also activate presynaptic “autoreceptors”,
which can be ligand-gated channels or GPCRs (5 & 6)

Post cell is mediated by opening on ligand gated ion
channels
...

Gives an excitatory post synaptic potential which if
depolarises the membrane potential enough to
reach the threshold will open voltage gated sodium
channels in the post membrane, causing an AP
...
They couple neurotransmitter to
initition of intracellular signalling, affecting

transmission
...
Synaptic transmission
Ligand= fast
...
Can
get synpatic inputs modulating pre
...


Slide 5

L5
...


Purves

This can:
1
...
Postsynaptic potential
b
...


2
...
Synaptic transmission- acting on
pre/post snaptic events
b
...
Intracellular signalling
Role in neurotransmission
GPCR-regulated ion channels

Not ion channels, but can affect excitability by
regulating volatge gated/ligand gated ion channels
...

Post synaptic cell GPCRs contribute to post synpatic
potential
...

All alpha and gamma subunits are associated with
the membrane
...
Can then
influence the activotry of effector proteins which
generate certain intracellular messengers that then
act on ion channels either directly or via protein
kinases

Pre synaptic targets of GPCRs
Voltage Gated Caclium Channels

Presynaptic targets
VGCCs that couple AP to NT
release (1)
Post-synaptic targets
Ligand-gated (3) and voltage-gated ion
channels
...
Intracellular signalling
GPCR signalling
Receptor activation causes the
 subunit to
exchange GDP for GTP leading
to dissociation of the -GTP
complex from the  subunits
and the receptor
...

This is terminated by
GTPase activity
of  subunits (enhanced by
GAPs)
GDP bound  subunit reunites with
 subunits
...
This happens because GDP is exchanged
for GTP
...
In process it hydrolyses
GTP
...
Intracellular signalling
GPCR signalling: Effectors

G effectors
Enzymes that generate second messengers

cholera toxin
activates

Pertussis toxin
blocks

CaM kinase II

Targets: voltage and ligand gated channels, vesicle proteins, transcription factors

Slide 9

L5
...
Intracellular signalling
G or G?

A test for second messenger vs G
mediated effects
Cell-attached patch
Giga-ohm seal prevents
movement of NT
between extracellular medium
and the solution in pipette

Control

+NT

Modulation of
Voltage-gated Ca2+
channels
Intracellular injection
of cAMP elicits same
effects as NT
Later shown these
effcst are
mediatedPKA
phosphorylates
channel to increase
open probability and
open time

G alpha effectors are various enzymes that generate
second messengers
...
The kinase phosphorylates
substrates such as ion ahnnels modulating their
activity
...

Gi- inhibit adenyl cyclase, so less cAMP so less
protein kinase A activated so less phosphorylation

G alpha at membrane can also directly ion cahnnels
(directly by interacting with volatge gated ion
channels)
...

Cannabinoids act on voltage gated channels on pre
synaptic membrane mediated by beta gamma
subunits associated with the cannabinoid receptor
...
Want to know if mediated by second
messenger or affecting ion channel by beta gamma
complex
...
Also helps
understand how channels are modulated
...
They applied a
Neurotransmitter to outside of patch pipette theres
a karge enhancement of current- channels have
higher open probability and open time
...

Activates a Gs coupled receptor generating

cAMP (messenger) shown by if you inject
cAMP into cell in the absence of
neurotransmitter, get same effect
...
Intracellular signalling
G activates a K+ channel in response to ACh

Muscarinic ACh
Receptors- GPCR

Detached membrane patch inside-out
for single channel recordings

If you put neurotransmitter in patch can make case
for direct signalling- they cant diffuse away from
membrane
...

Which allows us to show neurotransmitter is
quantitative
...

Recording potassium channels (reflections =
channel opening)
...
This is an inside out
patch, the inside the cell is facing out to the bathing
solution
...
We know theres no secondary
messenger involved!
Membrane delimited regulation- all the regulatory
protein are in membrane!
If you apply beta gamma subunits get same effect
...
Intracellular signalling
GPCR modulation of VGCC and K+ channels

Modulation of ion channel activity by GPCR signalling pathways
Neurotransmitter/Hormone

Gq-coupled receptor

Gs-coupled receptor

PLC
IP3

cAMP

+

DAG

PKA
Ca2+

Gi/o-coupled receptor

PKC

+

+
VSCC

-

cAMP

 subunits

+

-

GIRKs

VSCC voltage sensitive calcium channel
Increase cAMP increases PKA which phosphorylates
channel
...

So beta gamma subunits are activating calcium
channels

Reverse can also happen the beta gamma subunits
can activate K channels downstream of Gi/o

coupled receptors or can inhibit K channels
...
Decrease of cAMP via Gi/o can inhibit
these channels
...

GPCR can influnence the activity of voltage gated
channels and K channels
...


Slide 13

L5
...


Presynaptic facilitation- increased NT release
due to activation of VGCC by Gs-coupled
receptors

Slide 14

L5
...
Why? Repolarise the membrane
faster!
Inhibition/facilitation largely mediated by action of
GPCRs on calcium and Potassium channels on
presynaptic membrane

Pre synaptic neurone recieves inputs
...
If is receiving axyl axonic synapse at which
an inhibitory NT is released
...
Reverse can also occur, pre
synaptic neuron communicates to post synaptic
neuron has an excitatory input (increase in NT
release)
...

Glutamate binds to receptors, anchor receptors
allow Na influx and K effeluc (Post potential )
Gaba can bind GPCRs that are Gi (decrease cAMP)
the voltage gated ion channels in pre are reduced in
activity, get less NT released post synaptic potential
also reduced
...


Slide 15

Post synaptic modulation

L5
...
Also activates NMDA a ligand gated
glut
...
Why? The pore of the channel is blocked
by magneisum
...

The ca that comes in via nmda receptor triggers
intracellular signalling- activates ca calmodulin
kinase 2
...
This
means the next time the synpatic neuron fires an
AP, the post synaptic response will be greater
...

Phosphorylation of the receptors allows them to be
trafficked to the membrane
...

(influx of Ca)

Slide 16

The calcium can act as secondary messenger
...
Intracellular signalling
Ca2+ ions as crucial second messengers

Typucally Ca coming though ligand gated channel
like NMDA
...
This
ER stores intracellular ca store
...

IP3 interacts with another channel on ER called the
IP3 R (IP3 receptor) which also allows it to release
Calcium
...

As the ca is released from the ER it sends a signal to
the plasma membrane (store depletion signal) to
activate additional stores on plasma membrane
(called store operated channels)
...
All
the ca is integrated into the cellular response
...
Also have ca exchanger on membrane
exchanging 3 na ions for one calcium ion out
...


Slide 17

L5
...


Intracellular Ca2+ release channels on the ER
IP3 receptors- activated by increased IP3 levels via Gq-coupled metabotropic
glutamate receptors
Ryanodine receptors- activated by Ca2+ coming in through the NMDA receptor/VGCC
causing Ca2+-induced Ca2+ release
Removal of Ca2+
SERCA- Sarcoplasmic endoplasmic reticulum Ca2+ ATPase, pumps Ca2+ back into ER
PMCA- Pumps Ca2+ out of cell
NCX -Na+/Ca2+ exchanger

Slide 18

Cyclic-nucleotide gated channels

L5
...
can activate
directly channels on membrane called cGMP-gated
Na+ channels
...
Intracellular signalling
Mediating excitation of sensory cells

Transient receptor potential
cation channels

Some TRP channels in sensory neurons
are store-operated channels,
and modulated by second messengers

PKC

Slide 20

Learning outcomes
Lecture 5 broad summary

1
...

-

Modulation of presynaptic VGCCs and GIRKs by GPCR
signalling (in presynaptic facilitation and inhibition)

-

Modulation of postsynaptic receptors by CaM Kinase II and
PKC

-

Excitation of photoreceptors via CNG-gated Na+ channels

Slide 21 Overview5 to 8
Lectures
L5
...
Anatomy of neural circuits underlying behaviour
-Anatomical organisation of the brain
-Neural circuits underlying emotional responses and reward

L7
...
Synchronizing behaviour with the environment
-Sleep/wake cycles
-Circadian rhythms



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