Download Supplemental Material Results A B Supplemental Figure 1: Bath

Supplemental Material
Results
A
B
Supplemental Figure 1: Bath application of tianeptine does not affect the reversal potential of
NMDA-EPSCs in hippocampal CA1 pyramidal neurons. A, Current-Voltage (IV) plots for
NMDA-EPSCs, normalized to the peak amplitude at +40 mV, did not differ between ACSF and
tianeptine-treated cells when measured at the end of washout. B, Reversal potential, computed
from a linear-fit of values close to the x-axis, was also similar between the two groups (Control:
15±5 mV, n = 7; Tianeptine: 12±5 mV, n = 9, n = 9, p > 0.9).
A
B
Supplemental Figure 2: Bath application of tianeptine does not affect the reversal potential of
NMDA-EPSCs in LA principal neurons. A, Current-Voltage (IV) plots for NMDA-EPSCs,
normalized to the peak amplitude at +40 mV, did not differ between ACSF and tianeptine-treated
cells when measured at the end of washout. B, Reversal potential, computed from a linear-fit of
values close to the x-axis, was also similar between the two groups (Control: 11±5 mV,
n=7; Tianeptine: 16±3 mV, n = 13, p > 0.4).
Supplemental Figure 3: Bath application of tianeptine has no effect on voltage sag in response
to a hyperpolarizing current step. The voltage sag during prolonged hyperpolarizing direct
current steps and the rebound overshoot after cessation of the current step are hallmarks of the
presence of Ih. A, To obtain independent, nonpharmacological evidence for a role of h-channels,
the voltage sag ratio in response to a prolonged (600 ms) hyperpolarizing direct current step and
the normalized voltage rebound were examined. Representative traces of voltage responses to
hyperpolarizing current steps injected into the same neuron before (pre) and after (post)
tianeptine treatment. B, The voltage sag that can be observed after hyperpolarization of the
membrane with current steps was not affected 30 min after tianeptine application, measured as
the ratio between the voltage deflection at steady-state and peak voltage did not change
significantly. C, In control cells, the voltage sag ratio measured 30 min after ACSF application
did not show any significant difference compared to baseline (p > 0.05).
Supplemental Figure 4: Changes in peak amplitude of the after-hyperpolarizing potential
(AHP) in LA neurons after in vitro treatment with tianeptine and ACSF. A, Representative AHP
traces before (pre) and after (post) bath application of tianeptine (TIA; 50 M, 15 min) on
amygdalar slices. B, The amplitude of AHP decreased after tianeptine treatment when measured
at the end of washout, i.e. 30 min after treatment (Pre-tianeptine: -4.95±0.68 mV, posttianeptine: -1.74±0.45 mV, n = 8, p < 0.01, paired t-test). C, A similar decrease in AHP
amplitude was also evident in ACSF applied control neurons. (Pre-ACSF: -5.23±0.31 mV, postACSF: -1.82±0.68 mV, n = 7, p < 0.01, paired t-test) indicating that the decrease in AHP in
tianeptine treated cells is most likely caused by rundown, as has been reported in previous
studies (Kaczorowski, J Physiol. 2007).
Supplemental Figure 5: The frequency of mIPSCs in LA principal neurons, measured as interevent intervals, was not affected by bath application of tianeptine. A, Representative traces of
mIPSCs during baseline, tianeptine application and washout. B, Mean values of inter-event
interval did not differ between the epochs of baseline, treatment and washout in tianeptinetreated neurons (Baseline: 665.2±38 ms, tianeptine: 703.4±39.6 ms, washout: 677.8±38.8 ms, n =
5, p > 0.05, one-way ANOVA). C, Cumulative probability distribution of mIPSC inter-event
intervals indistinguishable between synaptic events recorded during baseline, tianeptine
application and washout (p > 0.05, K-S test).
Supplemental Figure 6: The amplitude of mIPSCs in LA principal neurons was not affected by
bath application of tianeptine. A, Mean values of mIPSC amplitudes were not affected by
tianeptine treatment (Baseline: -37.9±1.43 pA, tianeptine: -34.49± 1.3 pA, washout: -36.3± 1.4
pA, n = 5, p > 0.05, one-way ANOVA). B, Cumulative probability distributions of mIPSC
amplitude values were indistinguishable between recordings during baseline, tianeptine
application and washout.
MATERIALS AND METHODS
Slice electrophysiology
After anesthesia (with halothane) the animals were decapitated and the brain was rapidly
dissected into a beaker containing oxygenated (95% O2, 5% CO2) ice-cold artificial
cerebrospinal fluid (ACSF). The ACSF had the following composition (in mM): 126 NaCl, 2.5
KCl, 26 NaHCO3, 1.25 Na2H2PO4, 10 D-(+)-glucose, 2 CaCl2 and 1 MgSO4, pH 7.4 (290-300
using a Vibrotome 1000 Plus (Vibratome, St. Louis, MO, USA) and were transferred to a
holding chamber containing oxygenated ACSF at room temperature, and allowed to recover until
ready for recording. The holding and cutting ACSF additionally contained 3.3 mM of HEPES to
improve the slice health. All solutions were oxygenated before perfusion (2.5 ml/min) and were
maintained at room temperature (23–25°C) during recording. Healthy looking neurons (with
membranes which are smooth and not too contrasting with background) were identified using an
infrared differential contrast video microscopy attached to an Olympus BX-50WI microscope
(Olympus, Melville, New York). Cells that did not seal (> 2 GΩ
discarded. Patch electrodes (3-5 µM, ~ 2 µm tip dia.) were pulled from thick walled borosilicate
glass pipettes (O.D.: 1.5 mm; I.D.: 0.86 mm; Warner Instruments, Hamden, CT, USA) on a P-97
Flaming-Brown Micropipette Puller (Sutter Instruments, Novato, CA, USA). Low-intensity
stimulations (100 μs duration; 15–80 μA intensity) were delivered from a bipolar electrode (25
Iso-Flex stimulus isolator
(A.M.P. Instruments Ltd., Jerusalem, Israel). Monosynaptic excitatory post-synaptic currents
(EPSCs) were evoked by stimulating either the Schaffer collateral (SC) inputs to CA1 or
thalamic afferents to LA projection neurons at a constant rate of 0.05 Hz. All recordings were
made using an EPC-9 amplifier (HEKA Elektronik, Lambrecht, Germany) filtered at 2.9 kHz,
and digitized at 20 kHz. Stimulus delivery and data acquisition were performed through PULSE
software (HEKA Elektronik, Lambrecht, Germany).
For all EPSC experiments, the internal solution contained (in mM): 100 gluconic acid,
100 cesium hydroxide, 17.5 CsCl2, 2 MgCl2, 8 NaCl, 10 HEPES, 1 EGTA, 3 MgATP, 0.3
NaGTP and 10 phosphocreatine (pH 7.3, CsOH, ~ 290 mOsm, sucrose/water). Picrotoxin (PTX:
bicuculline (10 μM) was included in the ACSF to block GABAA receptor-mediated
inhibitory postsynaptic potentials. AMPAR mediated EPSCs were recorded at -70 mV while
NMDAR currents were isolated by depolarizing the cell to -30 mV in the presence of CNQX (10
µM). The holding potential (- 30 mV) for NMDA EPSCs was chosen on the basis of maximum
inward current in the current-voltage relationship for neurons in both the LA and CA1 regions.
Further, for recording NMDA-mediated EPSCs, the cells were voltage-clamped at -30 mV for 2
s prior the stimulus delivery. This was done to partially relieve the Mg2+ block of NMDA
channels and also to allow the return of voltage-activated currents to steady state. EPSC
amplitudes were obtained from mean values in a time window (AMPA-EPSC: 2 ms, NMDAEPSC: 4 ms) centered at the peak and were holding current subtracted (obtained by averaging an
equivalent window 20 ms before the stimulus artifact). For each cell, recording began by
collecting steady baseline EPSCs, NMDA or AMPA receptor mediated, for at least 5 min before
bath application of tianeptine or ACSF alone for 15 min and was followed by another 30 min of
washout with ACSF alone. Statistical comparisons were made from averaged values at the last 4
min of three epochs – baseline (5 min), application of drug or ACSF alone (15 min) and washout
(30 min).
Neuronal excitability was measured with a current clamp internal solution having the
following composition (in mM): 115 K-gluconate, 20 KCl, 10 HEPES, 0.5 EGTA, 3 MgATP
and 0.3 NaGTP (pH 7.3, KOH, ~ 290 mOsm). For excitability experiments, we injected
suprathreshold current (500 ms duration) whose intensity was fixed before baseline recording to
elicit a short-train of action potentials (~ 5 in number). The current injection protocol was
repeated at 0.05 Hz. In order to reflect physiological conditions, excitatory and inhibitory
synaptic currents were not blocked in the excitability experiments.
The depolarizing voltage sag, indicative of the hyperpolarization-activated cation current
(Ih), was induced by injecting hyperpolarizing currents (600 ms duration) of varying amplitudes
(-600 to -50 pA in steps of 50 pA). The sag voltage ratio was calculated by dividing the
maximum negative voltage response after current injection to the steady-state amplitude just
before the end of the current step. After-hyperpolarizing potential (AHP) was measured from the
peak negative voltage response after a single super-threshold current step (400 pA, 600 ms).
All mIPSCs were recorded in the presence of 20 µM CNQX to block AMPAR-mediated
excitatory currents and 0.5 µM TTX for blocking sodium channels. mIPSC recording were
carried out at -70 mV using a chloride loaded internal solution (Cl- reversal ~ 0 mV) consisting
of the following composition (in mM): 120 CsCl, 2 MgCl2, 10 HEPES, 1 EGTA, 3 MgATP, 0.3
NaGTP and 10 phosphocreatine (pH 7.3, CsOH, ~ 300 mOsm, sucrose/water). The mIPSC
protocol consisted of 10 min baseline which was immediately followed by 10 min of either
tianeptine or ACSF application and a washout (10 min). Statistical comparisons were performed
on 300 events/epoch each for baseline, tianpetine and washout and were sampled from the first
30 events of every minute for each cell. This method ensured us to avoid any biasing of the
results due to the unusually high number of events that was observed in some cells.
The following parameters were measured by offline analysis (IGOR Pro) from each cell:
(1) Average spike frequency was computed from the number of spikes observed within the 500
ms depolarizing pulse. (2) Input resistance was determined from a hyperpolarizing step (200 ms)
delivered 300 ms before the depolarizing pulse. (3) First-spike latency: the time between start of
the depolarizing current pulse and the peak of the first action potential. (4) Half-width of the
action potential was measured as the duration of the first action potential at half-amplitude. (5)
Action potential threshold was taken as the voltage at which the slope of the rising phase of the
first action potential crossed 20 V/s. (6) Amplitude of the action potential was measured from the
spike threshold to the peak of the first spike.
For every cell, the resting membrane potential was measured as soon as the whole cell
configuration was established and cells with values more positive than -60 mV were discarded.
Series resistance and input resistance were continuously monitored by hyperpolarizing current or
voltage pulse. Cells were eliminated if series resistance changed by more than 12.5% in either
direction from the average value. All analysis of the electrophysiological data was carried offline
using IGOR PRO software (Wavemetrics, Lake Oswego, OR, USA).
Chemicals
Most of the chemical and toxins were obtained from Sigma (St. Louis, MO, USA) unless
mentioned otherwise. Bicuculline methiodide was from Tocris (Tocris Bioscience, Bristol, UK).
All ACSF chemicals were purchased from Mallinckrodt (Mallinckrodt Chemicals, Hazelwood,
MO, USA). The chemicals used in histology except silver nitrate (Merck Specialities Pvt. Ltd.,
Mumbai, India) were from Qualigens (Qualigens Fine Chemicals, Mumbai, India). Tianeptine
was obtained from Servier, France and Serdia, India.
Histology
On the last day of the experimental protocol, after completion of the EPM test, animals
from all the four groups were anesthetized with Halothane and killed by decapitation. The brain
was removed quickly and the tissue containing amygdala was processed for rapid Golgi staining
(14). The staining procedure consists of 7 days and is briefly explained below. On day 1, the
brains were transferred to semi-opaque amber bottles filled with rapid Golgi fixative containing
(per 100 ml of sol. in water): 5g Potassium dichromate, 5g Chloral hydrate, 8 ml Glutaraldehyde,
6 ml Formaldehyde and 1 ml of Dimethyl sulfoxide. For the next 4 days, the solutions in the
bottles were replaced with fresh prepared fixative. On the 6th day, the fixative was drained out
from all the bottles and, after thorough rinsing of all the tissues with water, was immersed in
silver nitrate solution (0.75% in H2O) for two days. The tissues were paraffin embedded for
sectioning on the 8th day after cleaning (in water) and dehydration (in absolute alcohol). The
paraffin blocks were sectioned on a rotary microtome (Jung RM 2055, Leica, Nussloch,
Germany) at 120 µm and were subsequently dehydrated (absolute alcohol) and cleared (xylene)
before mounting them on DPX coated glass slides. The slides were kept undisturbed roughly a
week for drying and were coded before morphological analysis.
Morphological analysis
The Golgi-impregnated neurons that had untruncated dendrites with consistent and dark
impregnation along the entire extent of all of its dendrites were selected for tracing. All
morphological quantifications were carried out on the two major classes of BLA projection
neurons (pyramidal and stellate). The dendrites were traced in 3D using the 40X objective on an
Olympus microscope (Olympus, Melville, New York) equipped with motorized stage (Ludl
Electronic, Products, Hawthorne, NY). Tracing and quantification (Sholl analysis) was done with
Neurolucida software (Microbrightfield Inc. Williston, VT, USA).