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org/cgi/content/full/4/161/161ra152/DC1



Supplementary Materials for

Optogenetic and Potassium Channel Gene Therapy in a Rodent Model
of Focal Neocortical Epilepsy

Robert C. Wykes, Joost H. Heeroma, Laura Mantoan, Kaiyu Zheng, Douglas C.
MacDonald, Karl Deisseroth, Kevan S. Hashemi, Matthew C. Walker,* Stephanie
Schorge,* Dimitri M. Kullmann*

*To whom correspondence should be addressed. E-mail: m.walker@ucl.ac.uk (M.C.W.);
s.schorge@ucl.ac.uk (S.S.); d.kullmann@ucl.ac.uk (D.M.K.)

Published 21 November 2012, Sci. Transl. Med. 4, 161ra152 (2012)
DOI: 10.1126/scitranslmed.3004190

The PDF file includes:

Fig. S1. Correlation of EEG coastline length with increases in high-frequency
power.
Fig. S2. Correlation of burst counting by a blinded observer with high-frequency
power and coastline length.
Fig. S3. Electroclinical features of the tetanus toxin model.
Fig. S4. Automated event classification.
Fig. S5. Criteria used to identify adapting layer 5 pyramidal neurons.
Fig. S6. Biophysical properties of type 2 pyramidal neurons in animals injected
tetanus toxin.
Fig. S7. Estimation of volume of tissue transduced with Kv1.1-GFP lentivirus.
Fig. S8. Preferential transduction of excitatory neurons with Kv1.1 lentivirus.
Fig. S9. Identification of layer 5 neurons.
Fig. S10. Behavioral assessment in animals injected with Kv1.1-GFP lentivirus.
Fig. S11. Immunohistochemical analysis of neuronal and glial markers after
Kv1.1-GFP lentivirus injection.
Fig. S12. Stable neuronal transduction with Kv1.1 lentivirus.
Figure S1. Correlation of EEG coastline length with increases in high-frequency power.
(A) Scatter plots from 58 TT-injected animals showing power in 4 8 Hz (left) or 120 160 Hz
(right) frequency bands, plotted against coastline length (red lines, linear regression). (B)
Coefficients of determination (r
2
) obtained from plots as in (A) for each frequency band. ***, P
<0.001.


Figure S2. Correlation of burst counting by a blinded observer with high-frequency power
and coastline length. (A) Representative EEG segments identified as seizure-like bursts. (B)
Scatter plots showing absence of correlation between beta (12 30 Hz) power and burst count
during one hour of EEG recording 7 days after TT (n =19; red line, linear regression). (C) as in
(B) for 120 160 Hz band. (D) Correlation of coastline and burst count. (E) Coefficients of
determination obtained from plots as in (B D) (filled bar, coastline). **, P <0.01; ***, P <
0.001.


Figure S3. Electroclinical features of the tetanus toxin model. Contralateral dystonia, >15 %
weight loss, or death following a severe seizure, were seen in a subset of animals which
exhibited especially high HF power (Fig. 1F). (A) Dystonic contralateral forelimb posture. (B)
EEG recording from an animal showing a buildup of electrographic seizure activity prior to
death.
High Frequency Power
I
n
t
e
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m
i
t
t
e
n
c
y
Library Events Plot Eating
Grooming
High Frequency High Amp
High Frequency Low Amp
High Frequency Spike
High Frequency Short
500 ms
1

m
V
Left to right: File name, Second, Channel, Classification status, Baseline value, Metrics
M1310061660.ndf 3072.0 14 U 3.1 0.684 0.213 0.874 0.634 0.619 0.932
a
b

Figure S4. Automated event classification. The program stepped through consecutive 1 s EEG
epochs, and updated a running estimate of the baseline power as the lowest power between 4 and
160 Hz in any 1 s epoch during the preceding 20 minutes. Epochs whose power exceeded 5 x
baseline were defined as putative events. For each such event, 6 parameters were estimated:
Power (in the 4 160 Hz band), Transient Power (power in the 1 4 Hz band), High Frequency
Power (60 160 Hz), Spikiness (voltage range/standard deviation), Voltage Asymmetry (balance
of points exceeding 2 standard deviations either side of the mean), and Intermittency (low-
frequency power of the rectified high-frequency signal). We applied a sigmoidal function to
these 6 characteristics so as to obtain metrics bounded between zero and one. (A) Example of a
metrics processor output line. The last 6 numbers are the metrics. The event is thus represented
as a point in a 6-dimensional hypercube. (B) Two-dimensional projection of the metric space,
showing High Frequency Power and Intermittency selected as the X and Y axes, with colors
corresponding to different classes of normal behavior-related and epileptiform events. The event
library was constructed by an operator who, with reference to synchronized video recordings,
classified events as no event (no obvious electrographic or behavioral event), short high
frequency bursts (<250 ms), long high frequency bursts (>250 ms, event power >6 x
baseline), long high frequency bursts of low amplitude (>250 ms, event power 5 6 x
baseline), high frequency spikes, eating-related or grooming-related. The archetypical
epileptiform EEG event exemplars are shown on the right. Other classes of events, which bore
no relation to seizures in either behavior or EEG signature, such as head-shake and other/non-
specified, are not illustrated (such events occurred at similar frequencies in TT-injected and
control animals). As the algorithm stepped through subsequently identified events these were
provisionally identified as belonging to one or other category according to its Euclidean distance
to previously classified neighbors. The identity of each new event proposed by the algorithm was
overruled by the observer if necessary until it reached a false allocation rate <1 %. Once this
criterion was satisfied, all EEG data were classified without further operator interference.

Figure S5. Criteria used to identify adapting layer 5 pyramidal neurons. Type 1 non-
adapting neurons have larger somata and thicker-tufted dendrites than type 2 neurons and exhibit
non-adapting trains of action potentials (APs) (30). (A) Sample traces showing APs at threshold
in both types of neurons. (B) Schematic showing measurement of AP waveform parameters. (C)
AP waveform parameters in type 1 and type 2 pyramidal neurons. (D) Biophysical properties and
location of type 1 and type 2 pyramidal neurons (left to right: resting membrane potential (RMP),
resting input resistance (R
N
), current threshold (I
Thresh
), membrane time constant, capacitance,
distance from pia). (E) Firing patterns evoked by constant current injection (left, black trace,
type 1; right, gray trace, type 2). (F) Interspike interval normalized by the interval between the
second and third spikes. (G) Voltage threshold and waveform during trains of APs (left to right:
threshold plotted against AP number, peak AP voltage, halfwidth). (H) Expanded traces
showing representative AP trains from type 1 and type 2 neurons showing distinct
afterhyperpolarization shapes. (I) At sub-physiological temperatures type 1 neurons can exhibit
AP bursting behavior, consistent with previous reports in the rodent motor cortex (57). At 34
37 C neither type 1 nor type 2 neurons exhibited burst firing. Therefore, all recordings were
conducted at 36 C.



Figure S6. Biophysical properties of type 2 pyramidal neurons in animals injected
tetanus toxin. (A) Sample traces showing action potentials at threshold from two control (black)
and two tetanus-injected animals (red). The proportion of neurons firing doublets was not
significantly different between conditions. (B) Parameters measured as explained in figure S5.
Control (Ct) data are replotted for comparison. (C) Trains of action potentials in neurons from
both control animals and animals injected with tetanus toxin showed adaptation of firing
frequency. (D) Summary of inter-spike intervals. * P <0.05 (not significant when corrected for
multiple comparisons).


Figure S7. Estimation of volume of tissue transduced with Kv1.1-GFP lentivirus. (A) Bright
field and GFP fluorescence showing an area of transduced neurons. (B) Sequential 50-m slices
from one animal, used to estimate the area of transduced tissue in each slice (outlined in slice
number 7). (C) Estimated transduced volume: 0.14 0.01 mm
3
(n =5).


Figure S8. Preferential transduction of excitatory neurons with Kv1.1 lentivirus.
Immunofluorescence images obtained 3 weeks after injection of lentivirus encoding Kv1.1 and
GFP showing colocalization of GFP with the glutamatergic neuron marker CaMKII, and little
or no colocalization with either the GABAergic marker GAD67 or the glial marker GFAP. Kv1.1
lentivirus injection resulted in GFP expression in 678 121 neurons (avg s.e.m., n =5 animals
tested). Of these, 72 8% (n =3) were CaMKII-positive, corresponding to 53 19 % of the
local CaMKII-positive population; 1.5 1.5 % (n =4) were GAD67-positive (6% of the local
population) and 0 cells (n =4) were GFAP-positive.

16
Action potential number Action potential number
Action potential number

Figure S9. Identification of layer 5 neurons. Cells included in Fig. 3 were adapting type 2
pyramidal cells (UI, uninjected animals; G, GFP-positive neurons from animals injected with
GFP-only lentivirus; UT, Kv, untransduced and transduced neurons from animals injected with
Kv1.1 lentivirus respectively; T1, type 1 neurons shown for comparison). Cell capacitance,
normalized inter-spike interval, and AP halfwidth were all clearly different from type 1 neurons.


Figure S10. Behavioral assessment in animals injected with Kv1.1-GFP lentivirus. (A)
Number of ipsilateral (right) and contralateral (left) foot faults during 2 minutes of free
exploration on an elevated wire grid in animals injected with Kv1.1-GFP lentivirus (n =5
animals, mean SEM). (B) Data from animals injected with GFP-only lentivirus (n =5). The
performance improved with time non-significantly in both groups, consistent with motor
learning, but neither group showed a difference between left and right forelimb function.
Grooming and nesting behavior were also unaffected.


Figure S11. Immunohistochemical analysis of neuronal and glial markers after Kv1.1-GFP
lentivirus injection. (A) GFP fluorescence (left) and NeuN immunofluorescence in a section
obtained 4 weeks after injection of lentivirus encoding Kv1.1 and GFP. (B) GFP and GFAP
immunofluorescence at 4 weeks.


Figure S12. Stable neuronal transduction with Kv1.1 lentivirus. (A) GFP
immunofluorescence 6 months after injection, showing similar size and location of transduced
area. (B) High magnification of transduced neurons at 6 months, showing pyramidal cell
morphology. (C) Serial sections illustrating transduced volume, which was similar to data
sacrificed at 4 weeks.