4.

Optical tools for measurement and

manipulation of neuronal activity and for
functional analysis of neural circuits

--Optical techniques for measurement of neuronal activity:

advantages and disadvantages in comparison with
electrophysiology

--Optogenetics (activation or inhibition of specific neurons

with light stimulation)

--Examples of applications of optogenetics in combination

with optical or electrophysiological measurements of neural
activity in modern neurobiology
 Electrophysiology
Advantages:

--high sensitivity (very high signal to noise ratio) and high temporal resolution
allowing the exact measure of the time course of electrical signals on very different time scales

--the only technique allowing voltage-clamping and hence measurements of ionic

currents
Patch-clamp on neurons and dendrites in brain slices

10 um
 Patch clamp in vivo:

---Blind whole cell

---TPTP (two-photon targeted patch):

2 photon microscopy to direct the pipette filled with a fluorescent dye on a
neuron (e.g. expressing GFP) in the intact brain
Whole-cell patch-clamp on a hippocampal neuron in a moving rat
Electrophysiology
Disadvantages:

--necessity of physical contact between electrode and tissue (invasive)

--it is not possible to record simultaneously from many cells with single cell resolution
(maximum around 10)
Optical measurements of electrical signals with molecules (probes) which convert the
variation of V (or its consequences) in an optical signal (typically, a change in fluorescence)

In vivo In vivo

Advantages:
--non invasive,
--high spatial resolution
--simultaneous measurements from several cells or brain areas

Scanziani and Hausser (2009) Nature Reviews Neuroscience

Optical measurements of electrical signals with molecules (probes) which convert the
variation of V (or its consequences) in an optical signal (typically, a change in fluorescence)

In vivo In vivo
In vivo

Advantages:
--non invasive,
--high spatial resolution
--simultaneous measurements from several cells or brain areas

Disadvantages:
--indirect measurements (necessity of calibration),
--limited temporal resolution (due to kinetics of probes and detection system)
--limited sensitivity (cf dependence of SNR from shot noise)
--limited depth in cerebral cortex in vivo (about 400 um, up to max 700 um)

Scanziani and Hausser (2009) Nature Reviews Neuroscience

Shot noise = F0 / √ N with F0= basal fluorescence and N=number of photons

Signal to noise ratio (SNR) is proportional to

ΔF/ shot noise = ΔF/F0 x √ N

To improve SNR:
try to optimize especially ΔF/F0 (which depends on the probe properties) and
the excitation and detection system, rather than increasing N, i.e. the probe
concentration (to avoid perturbation of the system, given the square root
dependence).
To be able to compete with electrophysiology (and be advantageous)
an optical V imaging method should:

1) record single subthreshold and suprathreshold electrical events

(without need to average over repeated trials)

2) be fast (submsec resolution)

3) to have sub-micron resolution deep into the tissue

4) be able to measure V from identified neurons

We are close to getting there

 Indirect measurement of neuronal activity
via [Ca2+]in measurements
Ca2+-sensitive organic fluorescent dyes (combination of Ca2+ chelator like
EGTA with a fluorescent chromophore: binding of Ca2+ changes the fluorescence
spectrum: e.g. Fura-2):
allow detection of single AP (Ca change produced by opening of CaVs during AP)
in neurons in brain slices (not easily in vivo)

Multicellular loading of membrane-permeable dyes:

simultaneous measurements in a population of neurons  network activity
(in brain slices and in vivo in anesthetized or awake animals)
If dye is injected or perfused in single neurons, one can measure subthreshold signals (e.g.
due to opening of NMDAR channels in spine activated by single vesicle release  optical
quantal analysis)
 Dye Loading

Measurement of Ca2+
signals in dendrites,
spines, terminals

Injection in brain
with a pulse of
pressure: loads
Simultaneous
neurons in area of measurement of activity
300-500 um in a population of neuron
diameter

Membrane-permeable
acetoxymethyl (AM) ester

Christine Grienberger , Arthur Konnerth (2012) Neuron

 Indirect measurement of neuronal activity
via [Ca2+]in measurements
Ca2+-sensitive organic fluorescent dyes (combination of Ca2+ chelator like
EGTA with a fluorescent chromophore: binding of Ca2+ changes the fluorescence
spectrum: e.g. Fura-2):
allow detection of single AP in neurons in brain slices (not easily in vivo)

Problems:
--Given the slow time course of the Ca signal, the single spikes in a train cannot be
resolved (unless sophisticated analysis is used).

--Little information on subthreshold signals (none on IPSPs).

--Difficult to obtain measurements from specific neurons (in some cases possible by
utilizing transgenic lines that express a fluorescent marker, eg GFP, in specific
cells).
Two-photon Ca2+ imaging in L2/3 pyramidal neurons in awake head-restrained rat

After anesthesia

Greenberg et al (2008) Nat Neurosci

 Indirect measurement of neuronal activity
via [Ca2+]in measurements

Genetically encoded Ca2+ indicators (GECI)

Are formed by a protein that changes conformation after binding Ca2+ (e.g.
calmodulin) fused with a fluorescent protein (e.g. GFP) or a couple of fluorescent
proteins suitable for FRET.

.
 Yellow Cameleon Ca2+ binding reduces the
distance between the two
Calmodulin- fluorescent proteins CFP
binding peptide
and YFP and produces
opposite changes in the
fluorescence of the donor
(blue decreases) and
acceptor (yellow increases);
the fluorescence ratio gives
Time of AP firing a measure of [Ca]in that is
independent of the dye
concentration.

In the presence of Ca2+

the interaction CaM-
M13 produces
conformational changes
around the
chromophore that lead
cpFP: circularly permuted to an increase in the
fluorescent protein emitted fluorescence.

Knopfel (2012) Nature Reviews Neuroscience

Genetically encoded optical indicators for the analysis of neuronal circuits
 Indirect measurement of neuronal activity
via [Ca2+]in measurements

Genetically encoded Ca2+ indicators (GECI)

Are formed by a protein that changes conformation after binding Ca2+ (e.g.
calmodulin) fused with a fluorescent protein (e.g. GFP) or a couple of fluorescent
proteins suitable for FRET.

Superior than organic Ca2+ sensitive dyes because:

--they allow Ca2+ measurements in specific neurons

--they allow chronic measurements

--they modify less the internal [Ca]in given their lower concentration

While sensitivity and speed of the first GECIs were Inferior to those of organic
dyes, the sensitivity and velocity of the most recently developed GECI are
comparable ( e.g. GCaMP6f: Chen et al, 2013 Nature)
 Insertion of indicator genes (GECI e GEVI) in specific neurons in vivo
The plasmid containing the indicator
gene under the control of a strong
promoter is injected in the lateral ventricle
of the embrio through the uterus wall.
Electroporation is applied to make the
plasmid entering into specific neurons,
whose identity depends on the age of the
embrio and the location of the
electropration

The viral construct containing the

indicator gene is adressed to specific
cortical areas via stereotaxic injection
and to specific neurons depending on
the specific promoter that is used

Transgenic mice express GECI in

specific neurons, based on the
specific promoter that is used in the
construct injected in the fertilized egg.

Problems:
--there are few specific promoters
--low expression
Specific expression of indicator genes using mouse that expresses the Cre-
ricombinase in specific neurons (Cre-driver mouse).

Crossing a Cre-driver mouse with

an indicator mouse carrying the
indicator gene under the control of
a strong promoter but inactivated
by lox-P flanked stop codon
cassettes, give rise to mice which
express specifically the indicator
in the neurons expressing the Cre
recombinase (that removes the
stop codon)

A viral construct dependent on

Cre-recombinase (containing the
indicator gene under the control of
a strong promoter but inactvated
by lox_P flanked stop codon
cassettes) is injected in Cre-driver
mouse in specific brain area.

Knopfel (2012) Nature Reviews Neuroscience

Genetically encoded optical indicators for the analysis of neuronal circuits
Problems

Viral vectors:
--variable concentration in different neurons and different types of neurons
--lthe expression continues to rise for months until it becomes toxic
--surgery is necessary

Transgenic mice:
Low level of expression for cellular imaging in vivo
Ziv and Ghosh, Curr Opin Neurobiol 2015
Miniaturized one photon microscope: no subcellular resolution, less depth
(but cf miniaturized endoscope), but allow measurements of thousands of neurons in freele behaving
mice with higher temporal resolution than with classical two photon microscope in head restrained
mice.

Since 2017 also good two-photon miniaturized microscopes: Zong et al (2017) Nature Methods
Imaging configurations: miniaturized
microscopes

Ghosh et al., Nature Methods 2011

 Optical measurement of V

Organic voltage-sensitive dyes: VSDs (voltage-sensitive dyes)

E.g. merocianines distribute in membrane in a V-dependent manner
generating fluorescence.

Problems:
--limited signal to noise ratio (SNR):
in single cell in slice one can measure single AP but not single EPSP
(it is necessary to average over many trials to measure EPSP)
Limited SNR is due:
1) small fluorescence change per unit V change
2) VSD distribute also in internal membranes thus increasing background fluorescence
3) Phototoxicity and photo bleaching

--lack of specificity
 Optical measurement of V
Genetically encoded voltage indicators (GEVI)
VSFPs (voltage-sensitive fluorescent proteins): formed by a protein with voltage-
sensor domain (S1-S4 of V-dependent channel or V-sensor of certain phophatases
VSP) fused with a fluorescent protein or couples of fluorescent proteins suitable
for FRET.
.
Superior than organic dyes (VSDs) because they allow measurements in specific
neurons

Inferior to VSDs in terms of velocity (on rates: tens of ms)

Similar to VSDs in terms of sensitivity (worse SNR compared to GECI),

Knopfel (2012) Nature Reviews Neuroscience

Genetically encoded optical indicators for the analysis of neuronal circuits
The efficacy of FRET
increases with
membrane
depolarization

VSFP3 Microbial

VSFP3: the
depolarization
determines fluorescence
quenching.
Kinetics are more rapid Changes in fluorescence are linked
to V-dependent protonation of
than those of VSFP2,
rhodopsin.
but amplitudes are lower.

Knopfel (2012) Nature Reviews Neuroscience

Genetically encoded optical indicators for the analysis of neuronal circuits
New GEVI with better SNR and kinetics continue to be developed

They allow measurement of single APs and subthreshold PSPs (including

IPSPs) in single neurons in slices and in vivo.
 eFRET GEVI: Ace2N-mNeon

Fig. 2 Ace sensors provide about threefold to tenfold better spike detection fidelity than
previous GEVIs.

Cultured neuron

In vivo

Yiyang Gong et al. Science 2015;350:1361-1366

Published by AAAS
 Fluorescent Indicators

Fluorescent proteins

GECIs Syntetic
GEVIs indicators
• Calcium
• Voltage

Others
• GABA.
Glut,
Dopamine,
ACh