Técnicas Neurofuncionais e Vetores Virais

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Neurofunctional 
Techniques
Lessons 9/10/12
16 October 2023
17 October 2023
23 October 2023
Viral delivery methods
Optogenetics
X-genetics
Calendar • M 25 Sept: Course introduction
• T 26 Sept: Functional imaging
• W 27 Sept: Functional imaging
• M 2 Oct: Biophysics of diffusion
• T 3 Oct: Functional imaging
• W 4 Oct: Practical exercise: paper presentations 
• M 9 Oct: Modeling in neuroscience + Exercises on the first part of the course
• T 10 Oct: Exercises on the first part of the course
• W 11 Oct: Statistics (Cesca)
• M 16 Oct: Molecular approaches in modern neuroscience
• T 17 Oct: Optogenetics
• W 18 Oct: Practical exercise: paper discussion
• M 23 Oct: X-genetics + Exercises on the second part of the course
• T 24 Oct: Practical exercise: paper discussion 
• W 25 Oct: Statistics (Cesca)
• M 30 Oct: Practical exercise: paper discussion
• T 7 Nov: Genome editing in neuroscience (Dr. Jaudon)
• M 20 Nov: In vivo Ca2+ imaging (Dr. Riccardi)
• W 15 Nov: Statistics (Cesca)
• T 28 Nov: Seminar Francesco Papaleo (IIT, Genova) 
• W 29 Nov: Statistics (Cesca)
• M 4 Dic: Questionnaire (date to be confirmed)
• 11, 12, 13, 14, 18, 19 Dic: Presentation paper
Objectives
1. Introduce most commonly used viral delivery systems
2. Being able to chose the most appropriate one for a 
specific experimental purpose
Lentiviruses vs.
recombinant adeno-associated viruses (rAAVs)
Infection localized with lenti
Broader with rAAVs
Lenti better for primary cultures
rAAV (vs. lentivirus) properties
Inverted Terminal Repeat
• Size: 1 hr/animal)
2) Intracerebroventricular injections in pups
Technically similar to in utero electroporation
Intracerebroventricular injections in pups can be 
combined with Intersectional expression
Intra-
cerebral
injection
P0
Dual AAV system
Increased 
expression in 
cortical neurons
Rescue of 
behavioural 
phenotype 
EGFP
dCas9-VP64
gRNA gRNA
tdTomatoF/+; CaMKII-Cre/+
3) Systemic delivery
3) Systemic delivery
Chan et al., 2017
3) Systemic delivery
Chan et al., 2017
Tremendous potential for gene therapy
Anterograde - retrograde labeling
rAAV2-retro
Retrograde labeling of projection 
neurons with rAAV2-retro
Retrograde labeling of 
projection neurons 
with rAAV2-retro
Tervo et al., 2016
rAAV2-retro to distinguish cortical pyramidal 
neurons based on their projection sites
Intratelencephalic (IT) neurons  contralateral cortex
Pyramidal tract (PT) neurons  outside of the cortex
Layer V
Bsn
rAAV2-retro to distinguish cortical pyramidal 
neurons based on their projection sites
Injection in the Pons
PT neurons
Injection in the contro-lateral cortex
IT neurons
Retrograde AAV-mediated EGFP labeling
Intratelencephalic (IT) neurons
Pyramidal tract (PT) neurons
Layer V
Bsn
rAAV2-retro identifies neuron type- and genotype-
specific differences in dendritic spine morphology
Celora et al., 2023
PT IT
β
3
 in
te
gr
in
Tervo et al., 2016
rAAV2-retro to distinguish cortical 
projection neurons: activity
Tervo et al., 2016
rAAV2-retro to distinguish cortical 
projection neurons: rescue experiments
Anterograde - retrograde labeling
rAAV2-retro
Monosynaptic retrograde labelling 
• It requires recombinant rabies viruses (RABVs)
• In contrast to non-viral tracers, trans-synaptic viral labeling is amplified rather than
diluted. This occurs thanks to the replicative nature of viruses
• The CVS derived strains of RABVs spread exclusively in a synapse-specific manner;
(other polysynaptic viral tracers do not spread exclusively at synaptic sites and
label neurons that are not necessarily connected by synapses)
• RABVs spreads retrogradely (from the post- to pre-synaptic neuron)
• Other advantages of RABVs: reduced cytotoxicity and the possibility to use them in
primates
• Native RABVs are polysynaptic tracers, causing ambiguity in the interpretation of
how many synaptic steps have been crossed at any given time
• RABVs are negative-sense single-stranded RNA viruses, making impossible the use
of useful genetic tools, such as lox P sites, tet-regulatory sequences or cell-specific
promoters
Retrograde labeling
rAAV2-retro
Monosynaptic retrograde labelling 
Production of recombinant 
RABVs with either:
• native glycoprotein or
• a different glycoprotein (EnvA) 
Native glycoprotein: monosynaptic but not neuron-type specific
Native glycoprotein
Monosynaptic retrograde labelling 
Production of recombinant 
RABVs with either:
• native glycoprotein or
• a different glycoprotein (EnvA) 
EnvA
EnvA: monosynaptic and neuron-type specific
Monosynaptic retrograde labelling CA1  subiculum
Cre-dependent helper rAAV expressing the 
TVA receptor + EGFP + the glycoprotein EnvA(G) 
are injected in the subiculum
Use of transgenic mouse 
expressing Cre in the subiculum 
thanks to the fibronectin promoter
One week later, recombinant RABVs coated 
with EnvA(G) and expressing mCherry are 
injected in the subiculum
Conclusions - viruses
1. From an experimental point of view consider:
a. Promoter type
b. Serotype type
c. Delivery method
2. Tremendous potential for therapeutic applications in brain 
disorders (e.g. BBB-permeable injectable rAAVs)
3. Retrograde labelling for neuronal connectivity
a. rAAV2-retro
b. RABVs
Objectives
1. Appreciate the power of X-genetics
2. Introduce the most commonly X-genetic systems
X-genetics
• Optogenetics: introduction
• Cellular precision
• Opsin types
• Light delivery systems
• Examples of optical interrogation of neural circuits
• Other uses of optogenetics
• Non-opotogentics
- Chemogenetics
- Magnetogenetics
X-genetics
• Optogenetics: introduction
• Cellular precision
• Opsin types
• Light delivery systems
• Examples of optical interrogation of neural circuits
• Other uses of optogenetics
• Non-opotogentics
- Chemogenetics
- Magnetogenetics
Why we need optogenetics
In describing unrealized prerequisites for assembling a
general theory of the mind, Francis Crick underlined
the need of ‘a method by which all neurons of just
one type could be inactivated, leaving the others
more or less unaltered’ (Crick 1979).
Optogenetics: Combining genetics and optics to achieve loss- or gain-of-
function of well-defined neuronal circuits with high temporal precision.
Why we need optogenetics
Conceptually similar to knock out/down and over-expression experiments but 
the aim is to silence or activate neuronal populations rather than genes
Optogenetics : the basic priniples
Channelrhodopsin:
An ion channel 
activated by light
Optogenetics to study neural circuits of behavior
Advantages of optogenetics
1) Advantages over electrophysiology:
 High cellular precision, mainly because of the ‘genetics’ part
2) Advantages over pharmacology:
 High temporal precision, mainly because of the ‘optics’ part
Traditionally, we could study the neural bases of behavior by combining
electrophysiological methods and pharmacology
X-genetics
• Optogenetics: introduction
• Cellular precision
• Opsin types
• Light delivery systems
• Examples of optical interrogation of neural circuits
• Other uses of optogenetics
• Non-opotogentics
- Chemogenetics
- Magnetogenetics
Targeting strategies for optogenetics
Site-directed 
intracranial 
injections
Site-directed 
intracranial 
injections
+ light delivery
Site-directed intracranial 
injections
+ cell-type specific 
promoter or Cre-
dependent expression
Targeting strategies for optogenetics
Site-directed 
intracranial 
injections + Cre
in rAAV2-retro
Site-directed intracranial 
injections
+ cell-type specific 
promoter or Cre-
dependent expression + 
wavelength
Site-directed intracranial 
injections
+ cell-type specific 
promoter or Cre-
dependent expression + 
wavelength
Advantages of optogenetics
1) Advantages over electrophysiology:
 High cellular precision, mainly because of the ‘genetics’ part
2) Advantages over pharmacology:
 High temporal precision, mainly because of the ‘optics’ part
Traditionally, we could study the neural bases of behavior by combining
electrophysiological methods and pharmacology
X-genetics
• Optogenetics: introduction
• Cellular precision
• Opsin types
• Light delivery systems
• Examples of optical interrogation of neural circuits
• Other uses of optogenetics
• Non-opotogentics
- Chemogenetics
- Magnetogenetics
Opsins
Opsins: seven-transmembrane proteins that are light-sensitive thanks 
to the chromophore retinal, a vitamin A-related organic cofactor. 
Retinal + opsin = rhodopsin
Upon absorption of a photon, retinal isomerizes and triggers a
sequence of conformational changes within the opsin partner, which
mediate phototransduction.
There are two distant families of opsins
• Type II: G-protein coupled receptors (GPCRs) found in higher eukaryotes,
responsible for vision (but also circadian rhythm and pigment regulation).
In the dark, type II opsins bind retinal in the
11-cis configuration. Upon illumination,
retinal isomerizes to the all-trans
configuration and initiates the second
messenger cascade of phototransduction.
After photoisomerization, the retinal-protein
linkage is hydrolyzed; all-trans retinal
diffuses out of the protein and is replaced by
a fresh 11-cis retinal molecule for another
round of signaling.
• Type I: broad family of seven-transmembrane proteins found
in prokaryotes, algae and fungi. They are not G-protein
coupled receptors (GPCRs), rather they combine the two tasks
of light sensation and ion flux into a single protein (with
bound retinal), encoded by a single gene.
There are two distant families of opsins
They use retinal in the all-trans configuration, which
photoisomerizes upon photon absorption to the 13-cis
configuration.
Unlike the situation with type II rhodopsins, the activated
retinal molecule in type I rhodopsins does not dissociate from
its opsin protein but thermally reverts to the all-trans state
while maintaining a covalent bond to its protein partner.
Major opsin types
Cation-permeable channels
for membrane 
depolarization (such as 
channelrhodopsins (ChRs))
ChR2
Chloride pumps (e.g, 
halorhodopsin (NpHR)) and
proton pumps (such as 
bacteriorhodopsin or 
proteorhodopsin (BR/PR)) for 
membrane hyperpolarization
Light-activated 
membrane-bound G 
protein-coupled 
(OptoXR) receptors 
for activation of 
signaling pathways
Opsin diversification / optimization
• Large & non-inactivating currents:
 To elicit effectively and consistently APs with minimal light stimulation
• Optimization of kinetic properties:
 Either fast deactivation, to elicit APs that are time-locked to the light 
pulses
 Or very slow deactivation (bi-stable opsins), to change spiking 
propensity over defined time periods
• Diversification/optimization of spectral prosperities
 For multiple independent optical stimulations
 For concomitant imaging readout
Opsin diversification / optimization
Point mutation for larger 
photocurrent
Chimera ChR1/2 + point 
mutations for reduced 
inactivation
Genomic identification 
of red-shifted opsins
Chimera VChR1/ChR1 for 
Larger photocurrent
Point mutations for 
faster deactivation
Point mutations for 
faster deactivation
First opsin
Spectral diversification of opsins
Klapoetke al., 2014
Far red
Green
Blue
Spectral diversification
Improved Kinetics for eliciting APs at high frequency
Klapoetke al., 2014
Bistable opsins
Step-function opsins (SFOs)/bistable opsins have mutations that stabilize the active
retinal isomer, thereby prolonging the active state of the channel even after light is off.
Some SFOs can also be deactivated by a pulse of yellow light; the yellow pulse drives
isomerization of retinal back to the non-conducting state.
Bistable opsins
Berndt al., 2009
Opsin diversification / optimization
X-genetics
• Optogenetics: introduction
• Cellular precision
• Opsin types
• Light delivery systems
• Examples of optical interrogation of neural circuits
• Other uses of optogenetics
• Non-opotogentics
- Chemogenetics
- Magnetogenetics
Light sources
• Intensity of light (few mW/mm2)
• Frequency of stimulation (few ms)
• Hologen/xenon arc lamps
• Light emitting diodes (LEDs)
• Lasers
Wide-field vs. cellular resolution optogenetics
Light sources in vitro
(b) LED- or a laser-coupled fiber placed near the brain slice.
(c) Arc lamp, LED, or laser as a light
source through the microscope objective
(d) Digital mirror device (dMd) to illuminate specific pixels within the field of view
simultaneously. Cellular-resolution optogenetics.
(e) Laser scanning photostimulation (LSPS) where the laser beam is focused to a small point.
LSPS illuminates with higher intensity. Unlike dMds, LSPS cannot simultaneously illuminate
multiple regions of interest, but with sufficiently fast galvanometric mirrors, multiple regions can
be illuminated with submillisecond delays. Cellular-resolution optogenetics.
Wide-field 
optogenetics
Laser scanning photostimulation
vs Digital mirror device 
Laser scanning photostimulation to map the subcellular 
organization of neocortical excitatory connections
Petreanu al., 2009
How do you get light deep into the brain tissue 
for in vivo experiments ?  Optical fibers
Only neurons that are both in the cone of illumination and express ChR2 will be activated to 
fire action potentials.
Optogentics for Parkinson
Dopamine depletion in the basal ganglia leads to altered activity of the subthalamic nucleus
(STN), which has been linked to clinical deficits in movement. Electrical high-frequency (>90
Hz) stimulation (HFS) of the STN (deep brain stimulation or DBS) is a highly effective
treatment for medically refractory Parkinson. But why?
This highlights the importance of (i) cell-type specificity and (ii) temporal precision
Gradinaru al., 2009
X-genetics
• Optogenetics: introduction
• Cellular precision
• Opsin types
• Light delivery systems
• Examples of optical interrogation of neural circuits
• Other uses of optogenetics
• Non-opotogentics
- Chemogenetics
- Magnetogenetics
Major opsin types
Cation-permeable channels
for membrane 
depolarization (such as 
channelrhodopsins (ChRs))
ChR2
Chloride pumps (e.g, 
halorhodopsin (NpHR)) and
proton pumps (such as 
bacteriorhodopsin or 
proteorhodopsin (BR/PR)) for 
membrane hyperpolarization
Light-activated 
membrane-bound G 
protein-coupled 
(OptoXR) receptors 
for activation of 
signaling pathways
They change the propensity for AP firing
They regulate 
signaling pathways
Major opsin types
Light-activated 
membrane-bound G 
protein-coupled 
(OptoXR) receptors 
for activation of 
signaling pathways
They regulate 
signaling pathways
LOV protein-based tools
LOV domains are photosensory modules found in photoreceptors from plants, bacteria,
fungi and algae. Upon blue light absorption, the flavin mononucleotide (FMN) cofactor
forms a transient covalent bond with a cysteine residue, resulting in a conformational
change.
This process reverts in the dark over the course of seconds to minutes via hydrolysis of the
FMN–cysteine bond.
In optogenetics, LOV domains coupled to non-
endogenous effector proteins enable blue light-
mediated modulation of protein accessibility
and activity.
 Gene expression
 Enzyme activity
 Membrane recruitment
LOV protein-based tools
LOV2
LOV2
Conclusions - optogenetics
1. Optogenetics allows for loss- or gain-of-function of well-defined 
neuronal circuits with high temporal precision
2. Types:
a. for membrane depolarization (ChR2)
b. for membrane hyperpolarization (NpHR)
c. for activation of signaling pathways (OptoXR) receptors
3. Optimizations/diversifications:
a. Large non-inactivating currents (ChR2; H134R)
b. Fast kinetic properties (Chronos)
c. Spectral properities (Chrimson)
d. Step-function opsins (SFOs)/bistable opsins
4. Light delivery systems:
a. Wide-field
b. cellular resolution optogenetics
5. LOV protein-based tools
X-genetics
• Optogenetics: introduction
• Cellular precision
• Opsin types
• Light delivery systems
• Examples of optical interrogation of neural circuits
• Other uses of optogenetics
• Non-opotogentics
- Chemogenetics
- Magnetogenetics
Chemogenetics
Designer Receptors 
Exclusively Activated by 
Designer Drugs (DREADDs)
Gq
Gs
Gi
Gi
Gi
Not as invasive as optogenetics
Slower than optogentics
Metabotropic receptorsMetabotropic receptors
Metabotropic
receptor
Gq/11 protein
Metabotropic receptorsMetabotropic receptors
Gs protein
Metabotropic
receptor
Metabotropic
receptorAdenylyl
cyclase
PKA / EPAC /direct targets
Gi protein
Metabotropic receptorsMetabotropic receptors
Chemogenetics
hM4D as a Synaptic 
Silencer
In the presence of the hM4D agonist 
CNO, the presynaptic AP is still elicited, 
but postsynaptic currents are inhibited
Chemogenetics therapies for epilepsy
Focal epilepsy is commonly pharmacoresistant, and resective surgery is often contraindicated by 
proximity to eloquent cortex. Many patients have no effective treatment options.
Tetanus toxin model of chronic epilepsy, which responds poorly 
to antiepileptic drugs
X-genetics
• Optogenetics: introduction
• Cellular precision
• Opsin types
• Light delivery systems
• Examples of optical interrogation of neural circuits
• Other uses of optogenetics
• Non-opotogentics
- Chemogenetics
- Magnetogenetics
Magnetogenetics
It promises:
- Not as invasive as optogenetics
- As fast as optogenetics
Why magnetic stimulation?
Unlike light, magnetic fields penetrate tissue unimpeded, and indeed, magnetic therapies
have been used for over 3 decades to remotely stimulate the brain without the need for
surgery. Transcranial magnetic stimulation (TMS) is for example used as a treatment for
multiple brain disorders, including major depression. rTMS suffers however from technical
limitations, including poor spatial resolution, which makes it impossible to target specific
neuronal circuits.
Molecular targets
of magnetogenetics
Mechano-sensitive ion channels Mechano-sensitive receptors
Integrins
Cadherins
 Fast opening/closing of ion channels
 Neuron-wide membrane hyper/de-polarization 
 Neuron-wide change in AP firing propensity
 Prolonged stimulation of mechano-receptors
 Localized subcellular (synaptic) effects on cytoskeleton
 Long-term rearrangement neural connectivity
How to stimulate mechano-sensitive proteins
Ferritin is paramagnetic!
All ‘genetic’ approach: ferritin + receptor
How to stimulate mechano-sensitive proteins
All ‘genetic’ approach: ferritin + receptor ‘Non-genetic’:
Magnetic particle
+
‘Genetic’: receptor
Ferritin is paramagnetic!
How to stimulate mechano-sensitive proteins
All ‘genetic’ approach: ferritin + receptor ‘Non-genetic’:
Magnetic particle (MNP)
+
‘Genetic’: receptor
Ferritin is paramagnetic!
How to stimulate mechano-sensitive proteins
All ‘genetic’ approach: ferritin + receptor ‘Non-genetic’:
Magnetic nanoparticle (MNP)
+
‘Genetic’: receptor
Ferritin is paramagnetic!
How to stimulate mechano-sensitive proteins
How to stimulate mechano-sensitive proteins
Magneto-mechano-stimulation in vitro:
multi-pole magnetic tweezers
Magneto-mechano-stimulation in vivo:
repetitive transcranial magnetic stimulation (rTMS)
rTMS w/o the ‘genetic’ counterpart is not magneto-
genetics
rTMS directly evokes APs via electromagnetic 
induction
rTMS is used as a treatment for major depression
rTMS has poor spatial resolution (cm3)
The rTMS setup could be repurposed to mechanically 
stimulate MNPs for magnetogenetics
Possible molecular targets
for magnetogenetics
Mechano-sensitive ion channels
Integrins
Cadherins
 Fast opening/closing of ion channels
 Neuron-wide membrane hyper/de-polarization 
 Neuron-wide change in AP firing propensity
 Prolonged stimulation of mechano-receptors
 Localized subcellular (synaptic) effects on cytoskeleton
 Long-term rearrangement neural connectivity
Neuron-wide resolution 
As for optogenetics
Synaptic resolution 
Not possible with optogenetics
Mechano-sensitive receptors
Synaptic mechanogenetics
Synaptic mechanogenetics
www.synmech.eu
http://www.synmech.eu/
Conclusions - chemogenetics/magnetogenetics
1. Chemogenetics:
 is less invasive than optogenetics
 but is slower than optogenetics
 relies on engineered
G-protein coupled receptors (GPCRs):
Designer Receptors Exclusively Activated by Designer Drugs (DREADDs)
2. Magneto-genetics:
 is less invasive than optogenetics
 is as fast as optogenetics
 can achieve a subcellular (synaptic) resolution
 generally needs a ‘genetic’ component (the mechano-sensitive ion channel or 
receptor) and a ‘non genetic’ component (the magnetic nanoparticle; MNP)
 can be used in vitro (with magnetic tweezers) and in vivo (with rTMS)