MOLECULAR AND CELLULAR NEUROSCIENCE
Molecular toolbox for functional analysis of gene mutations
underlying cognitive disorders – Nael Nadif Kasri
Overview Course
Aims: comprehensive basic and advanced knowledge of the molecular and cellular molecular and
cellular processes in the CNS in normal conditions as well as in neural disorders
o Week 1&2:
Neuronal communication and synaptic plasticity
How to identify and manipulate genes and cells involved in L & M: memory engrams
Which cell types are involved in L&M?
o Week 3:
How to identify and manipulate engrams to understand: Memory / Forgetfulness -> Can
we trick the brain?
On Thursday a lecture and on Friday an article discussion
o Main goal article discussion: in a short time, being able to use the newly acquired knowledge
to critically evaluate and present resent literature reporting new molecular and cellular
mechanisms underlying learning and memory in health and disease
Written examen: 100%
o Critically evaluating a paper received 1 day before exam
o You can use everything you want
First question: Explaining in your own words where this article is about?
Design new experiments
Week 1: Learning goals/topics
Understand and explain current methodologies to identify and manipulate neural correlates of
learning, memory and behaviour
Apply such methodologies for dissecting molecular processes related to specific cognitive tasks in
a time and cell-specific way
Topics: Neuronal communication and synaptic plasticity, Genetics of neurodevelopmental
disorders, Cell types, Genetic manipulations to generate animal models, Viral delivery, Cre-Lox /
Brainbow, Tet-on/off, CRISPR/Cas9, Designer Receptors Exclusively Activated by Designer Drugs
(DREADD) and Optogenetics
Neuronal communication - Synaptic transmission
Each neuron may be connected to up to 10,000 other
neurons, passing signals to each other via as many as 1,000
trillion synapses
An action potential invades the presynaptic terminal ->
depolarization of presynaptic terminal causes opening of
voltage-gated Ca2+ channels -> influx of Ca2+ through
channels -> vesicle fuse with presynaptic membrane ->
transmitter is released intro synaptic cleft via exocytosis ->
transmitter binds to receptor in postsynaptic membrane ->
opening or closing of postsynaptic channels -> postsynaptic
current causes excitatory or inhibitory postsynaptic potential
that changes the excitability of the postsynaptic cell
, Excitatory synapse: Glutamate
binds to AMPA receptor ->
sodium / Na+ flues in the cell ->
depolarisation -> threshold
potential (if strong enough
depolarisation) -> voltage-gated
Na+ channels open -> action
potential -> voltage gated
potassium / K+ channels open ->
K+ flues out the cell ->
repolarization ->
hyperpolarization (sometimes
overshoots resting potential) ->
resting potential through Na+/K+ pomp
o Asymmetric, dendritic spines, post synaptic
density (PSD)
o Inside the cell normally high concentration of
K+ and low concentration of Na+ and outside
the cell normally high concentration of Na+
and low concentration of K+ -> Na+ ions out of
the cell and K+ ions into the cell
o NMDA receptors require both voltage change
(depolarization -> unblocking by deletering
magnesium) and glutamate binding to open,
allowing calcium ions in, contributing to
synaptic plasticity (LTP) -> coincidence detector
Inhibitory synapse: GABA binds to GABA-A receptor -> chloride / Cl- channels open -> Cl- flues in
the cell -> hyperpolarisation
o Symmetric, dendritic shaft, soma, axon initial segment, vesicles, mitochondria,
hyperpolarizing
Synaptic plasticity
Way synapses are connected can change, and this make learning and memory possible. Learning and
memory storage in neuronal circuit is achieved in two ways:
Particular synapses are strengthened or weakened, i.e. in terms of neuronal
network theory their weights are modified
o Strengthened: Long-term synaptic potentiation (LTP)
More neurotransmitters are released
More receptors (like AMPA receptors) can be inserted into
postsynaptic membranes
Associated with NMDA receptors, which allows Ca2+ to enter the
cell -> triggering signalling pathways that strengthen the synapse
o Weakened: Long-term depression (LTD)
Postsynaptic weakly or not depolarized -> NMDA receptor -> low
Ca influx -> phosphates -> internalization AMPA receptors -> fewer receptors
Formation of new and elimination of old synapses rewires and thereby reprograms the circuit;
structural changes at the synapse
, o Dendritic spines (small protrusions on neurons where synapses form) can grow in response
to activity, creating more connections between neurons
Excitatory (glutamatergic) synapses are situated on dendritic spines
80% of synapses in central nervous system are excitatory
Correlation between structure and function: You need more space for more receptors
‘Cells that fire together, wire together’
o LTP requires simultaneous a presynaptic action potential (release of neurotransmitters) and a
postsynaptic depolarization (removing Mg2+ block from NMDA receptors) for its induction
o The simultaneous firing leads to calcium influx -> synthesis of CaMKII -> strengthens synaptic
connection by increasing number AMPA receptor (function) or forming dendrites (actin / Rho
GTPase; structure)
Genetics of neurodevelopmental disorders
Disrupted synaptic processes and epigenetics are over-
represented causes of many NDDs
Synapse development occurs in distinct phases: before
and after birth, there's rapid synapse formation,
followed by synaptic pruning during adolescence, largely
driven by microglia. In adulthood, synapses are
maintained. Neurodevelopmental disorders often
involve disruptions in these phases, leading to atypical
synapse formation, pruning, or maintenance.
Cell types
Neurons are not isolated cells; they are surrounded by glial cells:
Ependymal cells
o Ependymal wall ventricles and choroid plexus
o Help produce and circulate CSF
Astrocytes
o Metabolic support and reaction to brain damage (astrogliosis)
o Tripartite synapse, regulate neurotransmission, synaptic plasticity (LTP)
o Gliotransmission, modulate synaptic and neural activity
Oligodendrocytes
o Axon myelination
o Experience and learning-dependent myelination in adults
o Alternations in white manner can occur with learning and in cognitive/psychiatric disorders
Microglia
o Immune surveillants, remove toxics and pathogens -> change morphology to activated state
o Synaptic maturation (pruning; elimination of synapses) during development
o Learning and memory in adults: synapse elimination, formation and potentiation
They affect neuronal function during brain development, health, disease
o Every cell type has a different time of abundancy in development
Genetic manipulations to generate animal models
Mouse models are popular to study disease for multiple reasons
Genetic manipulation of mice:
o Embryonic stem (ES) cells are cultured from mouse blastocyts
o Construction of a targeting vector (traditional or CRISP/Cas9 system)
o Transfection or transduction of targeting vector into ES cells or microinjecting zygote directly
(CRISPR/Cas9 only)
Molecular toolbox for functional analysis of gene mutations
underlying cognitive disorders – Nael Nadif Kasri
Overview Course
Aims: comprehensive basic and advanced knowledge of the molecular and cellular molecular and
cellular processes in the CNS in normal conditions as well as in neural disorders
o Week 1&2:
Neuronal communication and synaptic plasticity
How to identify and manipulate genes and cells involved in L & M: memory engrams
Which cell types are involved in L&M?
o Week 3:
How to identify and manipulate engrams to understand: Memory / Forgetfulness -> Can
we trick the brain?
On Thursday a lecture and on Friday an article discussion
o Main goal article discussion: in a short time, being able to use the newly acquired knowledge
to critically evaluate and present resent literature reporting new molecular and cellular
mechanisms underlying learning and memory in health and disease
Written examen: 100%
o Critically evaluating a paper received 1 day before exam
o You can use everything you want
First question: Explaining in your own words where this article is about?
Design new experiments
Week 1: Learning goals/topics
Understand and explain current methodologies to identify and manipulate neural correlates of
learning, memory and behaviour
Apply such methodologies for dissecting molecular processes related to specific cognitive tasks in
a time and cell-specific way
Topics: Neuronal communication and synaptic plasticity, Genetics of neurodevelopmental
disorders, Cell types, Genetic manipulations to generate animal models, Viral delivery, Cre-Lox /
Brainbow, Tet-on/off, CRISPR/Cas9, Designer Receptors Exclusively Activated by Designer Drugs
(DREADD) and Optogenetics
Neuronal communication - Synaptic transmission
Each neuron may be connected to up to 10,000 other
neurons, passing signals to each other via as many as 1,000
trillion synapses
An action potential invades the presynaptic terminal ->
depolarization of presynaptic terminal causes opening of
voltage-gated Ca2+ channels -> influx of Ca2+ through
channels -> vesicle fuse with presynaptic membrane ->
transmitter is released intro synaptic cleft via exocytosis ->
transmitter binds to receptor in postsynaptic membrane ->
opening or closing of postsynaptic channels -> postsynaptic
current causes excitatory or inhibitory postsynaptic potential
that changes the excitability of the postsynaptic cell
, Excitatory synapse: Glutamate
binds to AMPA receptor ->
sodium / Na+ flues in the cell ->
depolarisation -> threshold
potential (if strong enough
depolarisation) -> voltage-gated
Na+ channels open -> action
potential -> voltage gated
potassium / K+ channels open ->
K+ flues out the cell ->
repolarization ->
hyperpolarization (sometimes
overshoots resting potential) ->
resting potential through Na+/K+ pomp
o Asymmetric, dendritic spines, post synaptic
density (PSD)
o Inside the cell normally high concentration of
K+ and low concentration of Na+ and outside
the cell normally high concentration of Na+
and low concentration of K+ -> Na+ ions out of
the cell and K+ ions into the cell
o NMDA receptors require both voltage change
(depolarization -> unblocking by deletering
magnesium) and glutamate binding to open,
allowing calcium ions in, contributing to
synaptic plasticity (LTP) -> coincidence detector
Inhibitory synapse: GABA binds to GABA-A receptor -> chloride / Cl- channels open -> Cl- flues in
the cell -> hyperpolarisation
o Symmetric, dendritic shaft, soma, axon initial segment, vesicles, mitochondria,
hyperpolarizing
Synaptic plasticity
Way synapses are connected can change, and this make learning and memory possible. Learning and
memory storage in neuronal circuit is achieved in two ways:
Particular synapses are strengthened or weakened, i.e. in terms of neuronal
network theory their weights are modified
o Strengthened: Long-term synaptic potentiation (LTP)
More neurotransmitters are released
More receptors (like AMPA receptors) can be inserted into
postsynaptic membranes
Associated with NMDA receptors, which allows Ca2+ to enter the
cell -> triggering signalling pathways that strengthen the synapse
o Weakened: Long-term depression (LTD)
Postsynaptic weakly or not depolarized -> NMDA receptor -> low
Ca influx -> phosphates -> internalization AMPA receptors -> fewer receptors
Formation of new and elimination of old synapses rewires and thereby reprograms the circuit;
structural changes at the synapse
, o Dendritic spines (small protrusions on neurons where synapses form) can grow in response
to activity, creating more connections between neurons
Excitatory (glutamatergic) synapses are situated on dendritic spines
80% of synapses in central nervous system are excitatory
Correlation between structure and function: You need more space for more receptors
‘Cells that fire together, wire together’
o LTP requires simultaneous a presynaptic action potential (release of neurotransmitters) and a
postsynaptic depolarization (removing Mg2+ block from NMDA receptors) for its induction
o The simultaneous firing leads to calcium influx -> synthesis of CaMKII -> strengthens synaptic
connection by increasing number AMPA receptor (function) or forming dendrites (actin / Rho
GTPase; structure)
Genetics of neurodevelopmental disorders
Disrupted synaptic processes and epigenetics are over-
represented causes of many NDDs
Synapse development occurs in distinct phases: before
and after birth, there's rapid synapse formation,
followed by synaptic pruning during adolescence, largely
driven by microglia. In adulthood, synapses are
maintained. Neurodevelopmental disorders often
involve disruptions in these phases, leading to atypical
synapse formation, pruning, or maintenance.
Cell types
Neurons are not isolated cells; they are surrounded by glial cells:
Ependymal cells
o Ependymal wall ventricles and choroid plexus
o Help produce and circulate CSF
Astrocytes
o Metabolic support and reaction to brain damage (astrogliosis)
o Tripartite synapse, regulate neurotransmission, synaptic plasticity (LTP)
o Gliotransmission, modulate synaptic and neural activity
Oligodendrocytes
o Axon myelination
o Experience and learning-dependent myelination in adults
o Alternations in white manner can occur with learning and in cognitive/psychiatric disorders
Microglia
o Immune surveillants, remove toxics and pathogens -> change morphology to activated state
o Synaptic maturation (pruning; elimination of synapses) during development
o Learning and memory in adults: synapse elimination, formation and potentiation
They affect neuronal function during brain development, health, disease
o Every cell type has a different time of abundancy in development
Genetic manipulations to generate animal models
Mouse models are popular to study disease for multiple reasons
Genetic manipulation of mice:
o Embryonic stem (ES) cells are cultured from mouse blastocyts
o Construction of a targeting vector (traditional or CRISP/Cas9 system)
o Transfection or transduction of targeting vector into ES cells or microinjecting zygote directly
(CRISPR/Cas9 only)
