Supplementary Methods
Whole-cell Voltage-clamp analysis
To study voltage-dependant potassium (K+) channels in dLS neurons without
the influence of either sodium- or calcium-dependant currents, a modified
CoCl2 artificial cerebral spinal fluid (ACSF) was used for whole-cell voltageclamp recordings consisting of (in mM): 135 NaCl, 25 NaHCO3, 3 KCl, 0.5
CaCl2, 1 MgCl2, 25 glucose, 2 CoCl2, pH 7.3 with KOH, containing 1M
tetrodotoxin (TTX, Sigma). Experiments were carried out following protocols
adapted from (Jagger and Housley, 2002). Transient A-type K+-currents (IA)
rapidly activate and inactivate with a time constants 1-10 msec and 23-30
msec respectively, at potentials positive to -60 mV (Connor and Stevens,
1971; Rogawski et al, 1985). Because of these rapid changes, IA is likely to
prevent neurons from responding to fast depolarizations, by delaying the
onset of discharge in response to a depolarizing stimulus (Connor et al, 1971).
In addition, IA plays part in the repolarization of the AP and may regulate the
firing pattern. IA persists in the presence of Ca2+ channel blocker, such as Co
(Gustafsson et al, 1982; Numann et al, 1987; Storm, 1990). However, IA is
sensitive to the K+-channel blocker 4-aminopyridine (4-AP) at concentrations
between 1 and 5 mM (Nakajima et al, 1986; Numann et al, 1987; Segal and
Barker, 1984a; Segal et al, 1984b).
Isolation of fast transient K+-currents (IA and ID) and 4-AP sensitivity
In order to establish the characteristics of fast activating K+-currents
(putatively IA and ID) in septal neurons, cells were held at -60 mV prior to a
500ms pre-conditioning voltage step to either -70 mV or -30 mV. This pre-
1
conditioning, which enables A-type channel activation, was followed by a
depolarizing test-step to +40mV for 200ms. The fast activating currents were
then isolated by subtracting residual outward currents resulting from the preconditioning to -30 mV from the currents measured at pre-conditioning to -70
mV. To elucidate the sensitivity to 4-AP, neurons were pre-conditioned to -50
mV prior to a depolarizing step to +40 mV, first in control, and after
presentation of 4-AP-containing CoCl2 ACSF. To fully block the fast outward
potassium currents, CoCl2 ACSF with 4-AP was superfused for at least 5 min
prior to recordings.
A-type current activation
In order to maximize A-type current activation, cells were pre-conditioned
at -100 mV for 1s prior to 200 ms test-pulses ranging from -90 mV to +50 mV
(10 mV increments) (Connor et al, 1971). Leak voltage was subtracted off-line
using Axograph by accumulation of the average of sweeps at the holding
potential, and subtracting this average offset current from each sweep to be
analysed. For activation curves, the current-voltage relationship (I/V) of the
fast 4-AP sensitive peak was plotted as a function of the step potential.
A-type current inactivation
A-type current inactivation was studied by applying, from a holding potential of
-60 mV, 500 ms pre-pulses to potentials of -100 mV to 0 mV (10 mV
increments) followed by a 200 ms voltage-step to +40 mV. For inactivation
curves, peak current amplitude (I) for the test-pulse to +40 mV was
2
normalized after leak subtraction to maximum current (I/Imax) and plotted
against pre-pulse conditioning potential.
LS punches and RT-PCR analysis of Glp-1r expression
Mice (n = 3 per group) were deeply anaesthetized with isoflurane (5%) and
decapitated. Brains were then rapidly removed and snap frozen in liquid
nitrogen, before they were placed in a cryostat and 250 m thick sections
containing the LS were cut. dLS punches were obtained using an unbevelled
18G syringe and were immediately submerged in cold RLT buffer (Qiagen)
containing
-mercaptoethanol.
RNA
was
extracted
following
the
manufacturer’s instructions (RNeasy MiniKit, Qiagen Pty Ltd). DNase
treatment was performed using on-column DNase digestion and RNA was
eluted in 30 l RNAse free water. Following determination of RNA content,
integrity and purity [NanoDrop ND-1000 (NanoDrop Technologies, Inc.,
Wilmington, DE, USA) and agarose gel electrophoresis], cDNA was obtained
from 20 ng total RNA according to the manufacturer’s instructions (Applied
Biosystems Mulgrave, VIC, Australia).
Quantitative reverse transcription PCR utilizing SYBR green (Qiagen)
was performed using the ABI7700 real time PCR cycler (Applied Biosystems
Mulgrave, VIC, Australia) with primers designed to Glp-1r (mGLP-1R fwd,
5’-ATCCACCTGAACCTGTTTGC–‘3
5’-AGATACACGCCTTCCACCAG-3’)
5’-GATGTATGAAGGCTTTGGTC-3’;
mGLP-1R
and
-actin
-actin
rev,
(-actin
fwd,
rev,
5’-TGTGCACTTTTATTGGTCTC-3’). The comparative CT method (CT) for
relative quantitation of GLP-1R expression was used. Data obtained were
3
normalized using -actin as a control transcript. The normalized expression
value (CT) for tissue samples treated with AAV-GLP-1R-GFP is compared to
the equivalent CT for the control AAV-GFP treated tissue samples (Ctr). The
CT-Ctr is calculated as follows: CT-Ctr = (CT-Ctr with -actin primers in
AAV-GFP probe) – (CT-Ctr with GLP-1R primers in AAV-GFP probe). The
normalized
expression
values
of
GLP-1R
were
compared
in
the
AAV-GLP-1R-GFP samples and controls by calculating CT: CT = CT
AAV-GLP-1R-GFP - CT AAV-GFP. CT is used to calculate the relative
expression of GLP-1R.
Data analysis and statistics. For whole-cell electrophysiology, the resting
membrane potential and membrane capacitance were recorded shortly after
breakthrough. The voltage deflection evoked by small hyperpolarizing (-20 pA,
-10 pA) and depolarizing (10 pA, 20 pA) current injections were used to
calculate the membrane time constant and the input resistance. The
measures were obtained for each voltage trace and the values average
responses were used to obtain a single measure for each neuron. The
membrane time constant was calculated by fitting the first 100 ms of the
voltage deflection to a single exponential. The input resistance was calculated
from the plateau voltage deflection (last 100 ms).
The action potential waveform parameters, action potential latency and
the fast after-hyperpolarization (AHP) were calculated from the first action
potential evoked at the threshold current injection. The fast AHP was defined
as difference between action potential threshold and the most negative
potential within a 6 ms window following the action potential peak. Cell
4
capacitance was cancelled and series resistance was compensated by ~80%.
Access resistance (15 M) was monitored and data were discarded if the
access resistance changed by 25%.
All data are expressed as mean  SEM. Statistical analyses were
performed using GraphPad Prism 6 (GraphPad Software, La Jolla, CA, USA).
Normally distributed variables were analyzed using unpaired, two-tailed
student’s t-test, ordinary one-way ANOVA or two-way repeated-measures
ANOVA followed by Bonferroni’s posthoc test to evaluate statistical
significance (P < 0.05).
5
References
Connor JA, Stevens CF (1971). Voltage clamp studies of a transient outward
membrane current in gastropod neural somata. J Physiol 213(1): 21-30.
Gustafsson B, Galvan M, Grafe P, Wigstrom H (1982). A transient outward
current in a mammalian central neurone blocked by 4-aminopyridine. Nature
299(5880): 252-254.
Jagger DJ, Housley GD (2002). A-type potassium currents dominate
repolarisation of neonatal rat primary auditory neurones in situ. Neuroscience
109(1): 169-182.
Nakajima Y, Nakajima S, Leonard RJ, Yamaguchi K (1986). Acetylcholine
raises excitability by inhibiting the fast transient potassium current in cultured
hippocampal neurons. Proceedings of the National Academy of Sciences of
the United States of America 83(9): 3022-3026.
Numann RE, Wadman WJ, Wong RK (1987). Outward currents of single
hippocampal cells obtained from the adult guinea-pig. J Physiol 393: 331-353.
Rogawski MA, Beinfeld MC, Hays SE, Hokfelt T, Skirboll LR (1985).
Cholecystokinin and cultured spinal neurons. Immunohistochemistry, receptor
binding, and neurophysiology. Annals of the New York Academy of Sciences
448: 403-412.
6
Segal M, Barker JL (1984a). Rat hippocampal neurons in culture: potassium
conductances. J Neurophysiol 51(6): 1409-1433.
Segal M, Rogawski MA, Barker JL (1984b). A transient potassium
conductance regulates the excitability of cultured hippocampal and spinal
neurons. J Neurosci 4(2): 604-609.
Storm JF (1990). Potassium currents in hippocampal pyramidal cells. Prog
Brain Res 83: 161-187.
7
Supplementary Figure legends
Supplementary Figure S1. Whole-cell Voltage-clamp analysis of septal
neurons. (A) Protocol employed to evoke fast activating K +-currents.
Pre-conditioning to -70mV but not to -30mV prior to test-step at +40mV
evoked fast outward currents as shown in representative traces for a septal
neuron in (B). Grey box is indicating region of interest shown in (B1) after leak
subtraction. Isolation of IA and ID was obtained by subtraction of residual
currents evoked by pre-conditioning to -30 mV from evoked currents of preconditioning to -70mV and representative trace is shown in (B2). (C) Glp-1r-/neurons (n = 5) exhibit a slightly larger current, which did not reach
significance compared to Glp-r+/+ cells (n = 8; P = 0.17). (D) Protocol
employed to activate A-type current in Glp-r+/+ and Glp-1r-/- septal neurons.
Representative response of a septal neuron in control (E) and 4-AP (1 mM)
containing CoCl2 ACSF (E1). (E2) Difference between (E) and (E1) (grey
boxes) after leak subtraction. (F) Peak was measured as indicated by grey
box and plotted as a function of the current - voltage relationship of activation
for A-type current (Glp-r+/+, n =5; Glp-r-/-, n = 4). (G) Protocol employed to
evoke inactivation of the A-type current. (H) Representative response of a
septal neuron in CoCl2ACSF containing 4-AP. Grey box indicates region of
interest shown in (H1) after leak subtraction. (I) Data obtained from shaded
region in (H1) was used to generate the I/V plot. Inactivation curve for the Atype current of septal neurons is not altered by loss of GLP-1R signalling (Glpr+/+, n = 8; Glp-r-/-, n = 5).
8
Supplementary Figure S2. AAV spread covers the dLS in the rostro-caudal
dimension and is confined to target area. Representative pictures showing
GFP expression after vector mediated gene delivery in the anterior (A) and
posterior part of the dLS (B).
Supplementary Figure S3. Locomotor activity after AAV-meditated gene
delivery. (A) Exploratory behaviour of Glp-1r-/- + AAV-GLP-1R-GFP mice (n =
9) compared with Glp-1r-/- mice (n = 15) and Glp-1r+/+ controls (n = 17) was
unaltered in (A) the EPM (one-way ANOVA F = 2.69, P = 0.08) or (B) during
baseline locomotion assessment preceding the cocaine sensitization (oneway ANOVA F = 1.43, P = 0.25).
Supplementary Figure S4. Additional measures to interpret cocaine-induced
locomotor sensitization. (A) Magnitude of change of increase in locomotor
behaviour was not statistically different between groups (two-way repeated
measures ANOVA genotype effect F(2,41) = 0.2887, P = 0.7508). Data
represent the mean increase in percentage in activity in respect to acute
cocaine (dotted line) over 30 min recording period. (B) Stereotypic time over
the course of the experiment was not statistically different between groups
(two-way repeated measures ANOVA genotype effect F(2,36) = 1.417, P =
0.2557).
Supplementary Figure S5. Glp-1r-/- mice exhibit altered response in the CPP
paradigm using cocaine (15 mg/kg). * P < 0.05
9
Supplementary Figure S6. Locomotor activity during CPP expression.
Locomotion was comparable between groups during the CPP expression test
(one-way ANOVA F = 0.7677, P = 0.4727).
Supplementary Figure S7. Characterization of Glp-1r mRNA expression
following AAV-mediated gene delivery. Left panel, illustration of a coronal
mouse brain section; the dashed line indicates the region where tissue
punches were taken. AAV-mediated gene delivery results in a 4-fold increase
of Glp-1r transcript in Glp-1r-/- injected with AAV-GLP-1R-GFP mice compared
with Glp-1r+/+ controls. As expected, no Glp-1r transcript was detected in
Glp-1r-/- mice.
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