Förster resonance energy transfer (FRET) is the process of nonradiative energy transfer from an

excited donor fluorophore to an acceptor fluorophore through dipole–dipole interactions. The

effi- ciency of FRET strongly depends on the distance between the donor and the acceptor
(Förster 1993; Lakowicz 2006), making this phenomenon a useful tool for monitoring
interactions between proteins that are fused to fluorophores (Miyawaki 2003). Because FRET
increases acceptor fluorescence and decreases donor fluorescence, the ratio between the
fluorescence intensities in the donor and the acceptor emission wavelengths is often used to
measure FRET (Wallrabe and Periasamy 2005). Alternatively, the fluorescence lifetime of the
donor, which is the time between the excitation of the fluorophore and emission of a photon, can
be used as a readout of FRET because the fluorescence lifetime shortens as FRET efficiency
increases (Wallrabe and Periasamy 2005; Lakowicz 2006; Yasuda 2006). There are several
advantages to using fluorescence lifetime imaging microscopy (FLIM) compared with
ratiometric imaging. First, the signal is independent of the relative concentration of the donor and
the acceptor. Second, the signal is relatively independent of light scattering by the tissue
compared with ratiometric imaging, which is affected by the wavelength dependency of light
scattering. Third, when multiple populations with different FRET efficiency coexist, the
fluorescence decay curve becomes multiexponential, and one can deconvolve each component
by fitting the curve (Lakowicz 2006). FLIM has been combined with two-photon laser-scanning
microscopy (2pLSM) to image samples in light-scattering tissue. This technique has enabled the
measurement of signaling events in small neuronal compartments in brain slices (Yasuda 2006).

The fluorescence from a fluorophore following a short laser pulse typically decays exponentially
(Fig. 1B). Thus, the fluorescence lifetime of the free donor tD can be measured by fitting the
fluorescence decay curve with an exponential function convolved with the Gaussian pulse
response function of the system (Fig. 1B):

Since the first FRET-based calcium sensors were developed (Miyawaki et al. 1997), a number of
genetically encoded FRET sensors of signaling have been created (most of them use FRET
between eCFP and eYFP). For example, a class of kinase activity reporters has been produced,
which consist of four linked components: a donor, a specific kinase target polypeptide, a
phosphoamino-acidbinding domain, and an acceptor (Ni et al. 2006). Phosphorylation of the
kinase target polypeptide causes binding between the kinase target polypeptide and the
phosphoamino-acid-binding domain, thereby altering the FRET efficiency between the donor
and the acceptor. Another class of kinase activity reporter uses fluorescent proteins tagged to
both ends of the kinase to measure the conformational change of the kinase that occurs when the
protein is activated (Takao et al. 2005; Ni et al. 2006). Most of these sensors are optimized for
ratiometric imaging. Because the design criteria for ratiometric measurements and FLIM are
different, the sensors must be reoptimized for FLIM (Yasuda 2006; Yasuda et al. 2006).

One way to evaluate the dynamic range of a sensor is to coexpress the sensor with an activator or
an inactivator protein within cells and to measure the sensor signals (Mochizuki et al. 2001). For
some proteins, constitutively active or inactive mutants of the target protein could be used to test
the dynamic range (Mochizuki et al. 2001; Yasuda et al. 2006). The dynamic range of FLIM
sensors can be up to 50% in terms of the binding fraction or 25% in the mean fluorescence
lifetime (Yasuda et al. 2006; Lee et al. 2009).
The fluorescence lifetime (τ) is defined as the average time that a molecule remains in
an excited state prior to returning to the ground state. For a single exponential decay,
the fluorescence intensity as a function of time after a brief pulse of excitation light is
described as

I (t) = I0 exp (-t/τ)

where I0 is the initial intensity immediately after the excitation pulse.

Many currently available fluorescence microscopic techniques, such as confocal or

multi-photon excitation, cannot provide detailed information about the organization and
dynamics of complex cellular structures. In contrast, time-resolved fluorescence
microscopy allows the measurement of dynamic events at very high temporal resolution
and can monitor interactions between cellular components with very high spatial
resolution as well. Fluorescence lifetime imaging microscopy (FLIM) was developed to
obtain a spatial and temporal distribution of molecular lifetime in a single living cell
instead of decay curves only. Both time and frequency domain FLIM imaging systems
are available on the market.

The combination of lifetime and FRET (FLIM-FRET) provides high spatial (nanometer)
and temporal (nanoseconds) resolution (Bacskai et al., 2003; Elnagovan et al.,
2002; Krishnan et al., 2003). The presence of acceptor molecules within the local
environment of the donor that permits energy transfer will influence the fluorescence
lifetime of the donor. By measuring the donor lifetime in the presence and the absence
of acceptor one can estimate the distance between the donor- and acceptor-labeled
proteins. While 1p-FRET produces 'apparent' E%, i.e. efficiency calculated on the basis
of all donors (FRET and non-FRET), the double-label lifetime data in 2p-FLIM-FRET
usually exhibits two donor lifetimes (FRET and non-FRET), allowing to estimate the
distance.

Alexis (1)

We’ve seen both of these images in class → we know BDNF and TrkB are involved with LTP in
the hippocampus and but we mostly discussed the presynaptic mechanisms that can lead to
LTP (may or may not bring up the controversial paper here)

Alexis (2)

Tropomyosin receptor kinase B / Brain derived neurotrophic factor

Alexis (3)

Temporal and spatial analysis paired with pharmacological tools to see how BDNF induces LTP

Alexis (5)
Depleted Mg++ and TTX added

Alexis (6)

Excite fluorophore of donor which transfers energy upon close enough proximity and acceptor
fluorophore will fluoresce (emission)
GFP tagged TrkB and RFP tagged domain that interacts with TrkB on PLCgamma (downstream
in TrkB signaling pathway)
Therefore activation of TrkB should result in FRET signal

Alexis (7)

Traditionally measure ratio of intensity → increased intensity of acceptor = increased

association
2pFLIM measures time it takes for acceptor to fluoresce after excitation → called lifetime
Shorter lifetime = increased association

Advantage is that it is independent of intensity and concentration of donor/acceptor

Jeff (1)

2pFLIM of dendritic spine upon Glu uncaging

Heat map of lifetimes (red = shorter lifetime aka more TrkB activity) → can see its isolated to
dendritic spine of uncaging (done w laser for precision)
Volume of spine also changes upon Glu uncaging / TrkB activity

Jeff (2)

Why are they measuring spine volume?

Two types of LTP → we’ve been introduced to fLTP
dependent on NMDA current inducing increased AMPA receptor expression → ^ Na+
current through ^ AMPA = ^ excitability of spine
Hebbian Theory depicted in schematic
sLTP however...
Size of spine increases → makes sense bc increased AMPA receptor expression
Not only AMPA receptor expression but also involves structural remodeling of
cytoskeleton
They are using change in volume as measurement of sLTP
Most likely just different measurements (electrical vs. structural) for the same idea of synaptic
potentiation

Jeff (3)

Shows that TrkB activity, although fairly localized, does spread to adjacent spines and dendrite
over time

Jeff (5)
Not only is BDNF released at the post-synaptic neuron, it is necessary for sLTP

BDNF floxed mice w/cre recombinase (cre+ = KO)

Jeff (6)

Important because not only is NMDA/CaMKII needed for BDNF release, they are also needed
for sLTP

Jeff (7)

Mention segway into visualising the BDNF release itself rather than just TrkB activity post Glu
uncaging

Jeff (8)

pH inside vesicle is different than extracellular space

When fusion occurs, SEP fluoresces

Alexis (8)

Image depicting fluorescence of BDNF-SEP (green) and dendritic spines (mCherry) after Glu
uncaging (each black bar = Glu uncaging pulse)
BDNF is released specifically at the spine, not so much dendrite (^ green fluorescence)
Volume increases (^ response from mCherry)

(these just prevent all NT release / BDNF release so are kinda like controls)
TeTx = tetanus toxin → prevents vesicle fusion
POMC → prevents activity dependent BDNF release

(This implicates these as upstream of the BDNF release)

AP5 → NMDA antagonist
NBQX → AMPA antagonist
CN21 → CaMKII antagonist

Important: we know this is post-synaptic BDNF release because tetrodotoxin (prevents action
potentials) is in solution. Because pre-synaptic release of most neurotransmitters/signaling
molecules are dependent on action potentials, inhibiting action potentials should isolate post-
synaptic release.

Alexis (9)

When BDNF knocked out, sLTP does not occur but can be rescued by BDNF-SEP → validates
mutant BDNF can induce sLTP like WT
Show that fluorescent signal from BDNF-SEP release is not dependent on endogenous BDNF

Genetic Info:
transgenic BDNF fl/fl mouse line injected with GFP for all cells, diluted Cre to knock out BDNF in
some cells, and tdTomato that is Cre dependent so that Cre negative = green, Cre positive =
red (AAV1.CAG.GFP, AAV1.hSyn.Cre, AAV1.CAG.Flex.TdTomato injected into embryos in
utero surgery)

Alexis (13)

eEPSC by extracellular schaffer collateral stim at 0.03Hz, LTP induced by 2Hz stim & 0mV
depol.
Held at -70 mV

Alexis (14)

Cre- = eGFP and Cre+ = tdTomato

Jeff (10)

Dieni et al. showed that BDNF (stained with alpha myc) is localized to soma and axon terminals
(arrows pointing to cell bodies and axon terminals) (neuron morphology stained with MAP2) and
is not found in dendrites
However the paper we presented immunostained for BDNF w HA tag and visualized BDNF via
EM in post synaptic structures
Quantification of HA-stained observations in BDNF-HA and WT BDNF

Bdnf-HA mice were generated as previously described (another Nature paper from 2009)