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 0.3times 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 receptormediated 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 zprojection 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), controlCNiFERs 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 M1CNiFERs. 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.
CNiFER FRET
ratio, ΔR/R
(a)
30 nM ACh
Ca2+
1.0
0
0
200
CNiFER FRET
ratio, ΔR/R
(b)
800
1000
1200
800
1000
1200
800
1000
1200
30 nM ACh
Ca2+
Ca2+
Ca2+ free
1.0
0
0
200
(c)
CNiFER FRET
ratio, ΔR/R
400
600
Time (s)
400
600
Time (s)
30 nM ACh
Ca
Ca2+ free
2+
1.0
0
0
200
400
600
Time (s)
Supplemental figure 1. Q.-T. Nguyen*, L F. Schroeder*, M. Mank, A. Muller, P. W. Taylor, O. Griesbeck and D. Kleinfeld
M1-CNiFER ΔR/R (%)
100
80
60
40
20
0
ACh (pulses, 30s isi)
0
50
100
150
200
250
Time (seconds)
300
350
400
Supplemental figure 2. Q.-T. Nguyen*, L F. Schroeder*, M. Mank, A. Muller, P. W. Taylor, O. Griesbeck and D. Kleinfeld
M1-CNiFER FRET Signal,
ΔR/R
[Acetylcholine]
(µM)
1.6
1.4
1.2
1.0
10
5
0
0
500
1000
1500
2000
2500
3000
Time after initial bolus (s)
Supplemental figure 3. Q.-T. Nguyen*, L F. Schroeder*, M. Mank, A. Muller, P. W. Taylor, O. Griesbeck and D. Kleinfeld
p < 0.001
M1-CNiFER FRET ratio, ΔR/R
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
100 nM
ACh
100 nM
ACh
+
100 nM
atropine
100 nM
ACh
+
3 μM
clozapine
100 nM
ACh
+
3 μM
olanzapine
Supplemental figure 4. Q.-T. Nguyen*, L F. Schroeder*, M. Mank, A. Muller, P. W. Taylor, O. Griesbeck and D. Kleinfeld
ΔR/R
CNiFER FRET ratio, ΔR/R
ACh
Vehicle
1.4
1.3
1.2
1.1
1.0
1 2 3
Sample
1.25
20 s
puff
Time
Supplemental figure 5. Q.-T. Nguyen*, L F. Schroeder*, M. Mank, A. Muller, P. W. Taylor, O. Griesbeck and D. Kleinfeld
1
2
3
200 μm
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
control-CNiFERs
50 μm
Supplemental figure 7. Q.-T. Nguyen*, L F. Schroeder*, M. Mank, A. Muller, P. W. Taylor, O. Griesbeck and D. Kleinfeld
b
Spectral power
pre-NBM (n=10)
post-NBM (n=10)
pre-NBM, after clozapine (n=8)
post-NBM, after clozapine (n=8)
1.5
Z-score of -log[ECoG δ-band]
a
1.0
0.5
p = 0.04
0
p = 0.005
−0.5
0
1
2
3
4
Frequency [Hz]
5
6
−1.0
p = 0.008
Pre-NBM
n = 30
Post-NBM
n = 30
Pre-NBM
n = 30
Post-NBM
n = 30
Clozapine
Supplemental figure 8. Q.-T. Nguyen*, L F. Schroeder*, M. Mank, A. Muller, P. W. Taylor, O. Griesbeck and D. Kleinfeld
CNiFER FRET ratio, ΔR/R
NBM stimulus
600 μA
Olanzapine
3 mg/kg
Vehicle
NBM stimulus
600 μA
0.05
2 min
NBM stimulus
600 μA
NBM stimulus
600 μA
10 s
10 s
Time
Supplemental figure 9. Q.-T. Nguyen*, L F. Schroeder*, M. Mank, A. Muller, P. W. Taylor, O. Griesbeck and D. Kleinfeld