SUPPLEMENTARY MATERIAL “An in vivo biosensor for neurotransmitter release and in situ receptor activity”
FIGURE CAPTIONS
Supplementary Figure 1. M1-CNiFER Dynamics in calcium-free media. These data
demonstrate that the tonic M1-CNiFER response is dependent upon calcium in the
media. (a) M1-CNiFERs respond with typical phasic and tonic components to 30 nM
acetylcholine in the constant presence of calcium. (b) M1-CNiFERs respond with only
the phasic component in calcium free media. Calcium returned to the media in the
continued presence of 30 nM acetylcholine results in a response with phasic and tonic
components, though possibly a slower phasic onset. (c) Calcium withdrawal from media
during the M1-CNiFER tonic response abolishes the response.
Supplementary Figure 2. Repetitive stimulation of M1-CNiFERs with pulses of acetylcholine. Two second pulses of 300 nM acetylcholine pulses, with a 30 second
interstimulus interval, were applied. Stabilization occurs at to an asymptotic value of 0.3-
times the initial change with a time-constant or 70 s.
Supplementary Figure 3. M1-CNiFERs respond to slowly-increasing concentrations of acetylcholine. A linear gradient of acetylcholine from 0 to 10 nM
was presented to M1-CNiFERs on a period of 1200 s; the gradient was calibrated by
concurrent measurements of co-dissolved alexa-594. The M1-CNiFER response (black
trace) to the slowly-increasing acetylcholine lacks a prominent phasic component as is
characteristic of the response to a step of acetylcholine (Fig. 1b). The M1-CNiFER
response to a bolus of 10 nM acetylcholine applied at the end of the experiment is
largely unchanged from that applied prior to start of the gradient. Note that there is a
small offset at the end of the experiment of possible technical origin; a similar level of
drift is seen without the addition of acetylcholine (gray trace).
Supplementary Figure 4. Atropine, clozapine and olanzapine suppress receptor- mediated responses in vitro. In Flexstation™ 3 assay, the M1-CNiFER FRET
response to 100 nM acetylcholine chloride is abolished by 100 nM atropine sulfate,
3 µM clozapine, and 3 µM olanzapine. All three effects are significant by t-test,
p < 0.001, n = 6 for each group. Bars represent standard error.
Supplementary Figure 5. M1-CNiFER response to acetylcholine is maintained
after brain implantation. M1-CNiFERs respond to intracortical 27 nl puffs of 1 mM acetylcholine chloride (cyan) but not PBS (grey). Time trace shows average response and standard error for 3 injections, same animal. Inset shows fractional change of 1/3
the area under the ∆R/R curve for 30 s after the stimulus as compared to that for 10 s
before the stimulus, for either acetylcholine or PBS (3 animals, 3 trials each). Bars
represent standard error.
Supplementary Figure 6. Intact vasculature surrounding M1- and control-CNiFER sites in the frontal cortex of a live rat implanted two days earlier. Image is a z-
projection spanning 140 µm from the cortical surface. Cerebrovasculature is visualized
by 500 µl intravenous injection of 5 % (w/w) fluorescein dextran (yellow). Visibility of the
fluorophore shows the vasculature is intact. M1-CNiFERs labeled in cyan (1,3), control-
CNiFERs labeled in red (2).
Supplementary Figure 7. Immunostain against GFAP reveals no astrocytic scar
around chronically implanted M1- and control-CNiFERs. In top image, mCherry and
TN-XXL fluorescence are overlayed on a brightfield 3,3'-diaminobenzidine α-GFAP stain
for astrocytes. Scale bar represents 200 µm. In bottom image, only TN-XXL
fluorescence is overlayed to better visualize the morphology of CNiFER cells. Scale bar
represents 50 µm. α-GFAP stain reveals cell bodies and long complex processes
apparent at the surface of the brain and in deeper layers at ~ 1 mm (see white arrows).
There is no astrocytic concentration (glial scar) along the implantation site. Functional
data with in vivo two photon microscopy is typically acquired at depths of 50 to 200 µm
below the pia, where few astrocytes in this section are present. Preparation was 2 d in
vivo; M1-CNiFER column extends to the cortical surface in a neighboring section (data
not shown).
Supplementary Figure 8. Clozapine suppresses NBM-evoked activation of ECoG, consistent with suppression of M1-CNiFER response. (a) Spectral analysis of
electrocorticogram reveals the hallmark of NBM-evoked cortical activation: a
suppression of power in the δ-band. Intraperitoneal clozapine (5 mg/kg) abolishes this
effect, consistent with its antimuscarinic properties and consistent with its effect on M1-
CNiFERs. Pre-NBM spectra in blue, post-NBM spectra in green. (b) Summary graph
showing statistical significance of NBM-evoked cortical activation as measured by
minus one times the logarithm of the inverse power of delta band,
-log[power in ECoG δ-band], and its modulation by clozapine. As expected, NBM
stimulation significantly decreases power in the δ-band (p = 0.005, t-test). In the
presence of clozapine, NBM stimulation mildly increases the power in the δ−band
(p = 0.04, t-test). Furthermore, clozapine slows the baseline spontaneous
electrocorticogram (p = 0.008, t-test). Bars represent standard error.
Supplementary Figure 9. Olanzapine suppression of NBM-evoked M1-CNiFER
response is not activity dependent. Intraperitoneal olanzapine (3 mg/kg) suppresses
the M1-CNiFER response to a single NBM stimulation delivered 20 minutes after
injection of the antipsychotic. Black vertical dashed lines represent 600 µA NBM
stimulations.
0 200 400 600 800 1000 1200
0 200 400 600 800 1000 1200
30 nM AChCa2+Ca2+ Ca2+ free
30 nM AChCa2+ Ca2+ free
0 200 400 600 800 1000 12000
1.0
0
1.0
0
1.0
Time (s)
Time (s)
Time (s)
(a)
(b)
(c)
30 nM AChCa2+
Supplemental figure 1. Q.-T. Nguyen*, L F. Schroeder*, M. Mank, A. Muller, P. W. Taylor, O. Griesbeck and D. Kleinfeld
CN
iFE
R F
RE
Tra
tio, Δ
R/R
CN
iFE
R F
RE
Tra
tio, Δ
R/R
CN
iFE
R F
RE
Tra
tio, Δ
R/R
0 50 100 150 200 250 300 350 400
0
20
40
60
80
100
Time (seconds)
M1-
CN
iFE
R Δ
R/R
(%)
ACh (pulses, 30s isi)
Supplemental figure 2. Q.-T. Nguyen*, L F. Schroeder*, M. Mank, A. Muller, P. W. Taylor, O. Griesbeck and D. Kleinfeld
[Ace
tylc
holin
e](µ
M)
M1-
CN
iFE
R F
RE
T S
igna
l, Δ
R/R
1.0
1.2
1.4
1.6
0
10
5
Time after initial bolus (s)
0 500 1000 1500 2000 2500 3000
Supplemental figure 3. Q.-T. Nguyen*, L F. Schroeder*, M. Mank, A. Muller, P. W. Taylor, O. Griesbeck and D. Kleinfeld
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
100 nMACh
100 nMACh
+100 nMatropine
100 nMACh
+3 μM
olanzapine
100 nMACh
+3 μM
clozapine
p < 0.001
M1-
CN
iFE
R F
RE
T ra
tio, Δ
R/R
Supplemental figure 4. Q.-T. Nguyen*, L F. Schroeder*, M. Mank, A. Muller, P. W. Taylor, O. Griesbeck and D. Kleinfeld
20 s
AChVehicle
CN
iFE
R F
RE
T ra
tio, Δ
R/R
1 2 31.01.11.21.31.4
ΔR
/R
puff
1.25
Time
Sample
Supplemental figure 5. Q.-T. Nguyen*, L F. Schroeder*, M. Mank, A. Muller, P. W. Taylor, O. Griesbeck and D. Kleinfeld
200 μm
1 2 3
Supplemental figure 6 Q.-T. Nguyen*, L F. Schroeder*, M. Mank, A. Muller, P. W. Taylor, O. Griesbeck and D. Kleinfeld
control-CNiFERs
M1-CNiFERs
200 μm
50 μm
control-CNiFERs
Supplemental figure 7. Q.-T. Nguyen*, L F. Schroeder*, M. Mank, A. Muller, P. W. Taylor, O. Griesbeck and D. Kleinfeld
0 1 2 3 4 5 6
post-NBM (n=10)pre-NBM (n=10)
post-NBM, after clozapine (n=8)pre-NBM, after clozapine (n=8)
Frequency [Hz]
Spe
ctra
l pow
er
−1.0
−0.5
0
0.5
1.0
1.5
Pre-NBMn = 30
Post-NBMn = 30
Pre-NBMn = 30
Post-NBMn = 30
Clozapine
p = 0.04
p = 0.008
p = 0.005
a b
Z-sc
ore
of -l
og[E
CoG
δ-b
and]
Supplemental figure 8. Q.-T. Nguyen*, L F. Schroeder*, M. Mank, A. Muller, P. W. Taylor, O. Griesbeck and D. Kleinfeld
2 min
Vehicle
NBM stimulus600 μA
Olanzapine3 mg/kg
NBM stimulus600 μA
10 s10 s
NBM stimulus600 μA
NBM stimulus600 μA
0.05
CN
iFE
R F
RE
T ra
tio, Δ
R/R
Time
Supplemental figure 9. Q.-T. Nguyen*, L F. Schroeder*, M. Mank, A. Muller, P. W. Taylor, O. Griesbeck and D. Kleinfeld