Neuroscience & Medicine, 2013, 4, 194-207
Published Online December 2013 (http://www.scirp.org/journal/nm)
http://dx.doi.org/10.4236/nm.2013.44031
Open Access NM
Kinetics of Norepinephrine- and Serotonin-Induced BDNF
Release in Cultured Embryonic Hippocampal Neurons*
Michael Chen, Amelia Russo-Neustadt
Department of Biological Sciences, California State University, Los Angeles, USA.
Received August 22nd, 2013; revised September 20th, 2013; accepted October 15th, 2013
Copyright © 2013 Michael Chen, Amelia Russo-Neustadt. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
ABSTRACT
The primary neurotransmitters targeted by currently used antidepressants, such as duloxetine, venlafaxine and fluoxet-
ine, are serotonin and norepinephrine, which also are released in significant amounts in the central nervous system in
response to sympathetic nervous system activation. In cultured hippocampal neurons, we have previously shown that
norepinephrine induces increased expression of brain-derived neurotrophic factor (BDNF), the PI-3 K/Akt, MAPK
pro-survival pathways, the BDNF receptor, TrkB, a transcription factor, and cyclic AMP response element binding pro-
tein (CREB). In the present study, we extend these findings of increased BDNF expression to its kinetics of release into
the surrounding media. We also evaluate these two cell survival pathways, TrkB and CREB, in response to application
of serotonin and/or norepinephrine. Serotonin elicits an earlier, but brief expression and release of BDNF, whereas
norepinephrine elicits a more delayed and sustained release of BDNF. In response to both norepinephrine and 5-HT, the
presence of BDNF in lysates and subsequent release into the media is significantly increased out to 4 h, as is PI-3 K/Akt
activation. Together, these two neurotransmitters increase BDNF expression and release covering the entire 8 h contin-
uum evaluated. The results of this study provide further evidence for a G protein-coupled receptor and a crosstalk-to-
TrkB receptor with transactivation signaling across pathways.
Keywords: BDNF; CREB; Serotonin; Norepinephrine; Hippocampus; Kinetics
1. Introduction
Neuronal activity that increases as a result of sympathetic
nervous system stimulation [1], such as physical activity
[2-4] or administration of antidepressant drugs [5], has
been shown to increase levels of peripheral [6] or CNS [4,
7-9] brain-derived neurotrophic factor (BDNF), and whe-
ther that activity is electrical stimulation [1,10], extracel-
lular application of K+ [1,11-17] or Ca2+ [13] or applica-
tion of excitatory neurotransmitter (e.g., kainic acid, [11,15,
18-21]), forskolin (cAMP activator [15]) or inhibitory
neurotransmitter antagonist (CGP 52432, GABAB recap-
tor antagonist [15]). Conversely, anything that blocks neu-
rotransmission, such as tetrodotoxin (Na+ channel block-
er [10,12,22]), nifedipine [11,22], BAPTA-AM (intra-
cellular Ca2+ chelator [12]) pertussis toxin (G-protein
inhibitor [23]), Y 25130 (5-HT3 antagonist [15]) or AMPA
or NMDA receptor blocker [21], has been shown to de-
crease BDNF secretion.
We have recently shown that BDNF levels in cultured
hippocampal neurons will increase in response to appli-
cation of norepinephrine [24-26]. Such cultured condi-
tions were initially designed to model any activity or
intervention in vivo [27] that results in greater release of
norepinephrine, which has been shown to be neuropro-
tective by increasing expression, secretion and release of
BDNF [7,28]. The putative purpose of antidepressants is
to inhibit the reuptake or metabolism of 5-HT or norepi-
nephrine; although it is not yet definitively known why
the therapeutic efficacy is such that these drugs take 2 - 4
weeks to work, the prevailing thinking suggests that
changes in gene expression require such a time scale
[29,30].
In our hippocampal tissue culture model, application
of norepinephrine elicits maximum BDNF expression at
2 hours [24]. In the present study, we extend these pre-
vious results obtained with norepinephrine and subse-
*Funding was provided by PHS grant MH 559776 to ARN.
The authors declare no conflict of interest.

Kinetics of Norepinephrine- and Serotonin-Induced BDNF Release in Cultured Embryonic Hippocampal Neurons 195
quent activation of the survival/neuroprotective PI-3 K/
Akt and MAPK pathways [24] to determine how long 5-
HT takes in eliciting maximum BDNF expression and
secretion and signaling of these same two pathways.
2. Materials and Methods
2.1. Drugs and Chemicals
Norepinephrine, serotonin (5-HT), PD 98059, LY
294002 and wortmannin were purchased from Sigma
Chemical Co. (St. Louis, MO). K252a was purchased
from Calbiochem (La Jolla, CA). Dulbecco’s modified
eagle medium (DMEM) was purchased from ATCC
(Manassas, VA). N 2 was purchased from Invitrogen
(Eugene, OR). The BDNF Emax Immunoassay System
was purchased from Promega (Madison, WI). PD 98059
and wortmannin were initially dissolved in DMSO; at no
time did the concentration of DMSO exceed 0.5% of the
total volume in each tissue culture well.
2.2. Antibodies
Anti-BDNF was purchased from Santa Cruz Biotech
(Santa Cruz, CA). Anti-phospho-CREB, anti-CREB,
anti-phospho-T 308-Akt, anti-Akt, anti-phospho TrkA (Y
490), which cross-reacts with anti-phospho-TrkB, and
anti-TrkB were purchased from Cell Signaling Technol-
ogy (Danvers, MA). Anti-phospho-MAPK, anti-MAPK,
and anti-tubulin were purchased from Millipore (Temec-
ula, CA). Secondary antibodies, anti-rabbit IgG and anti-
mouse IgG, were purchased from Amersham-Pharmacia
Biotech (Piscataway, NJ).
2.3. Animals: Ethics Statement
All efforts were made to minimize the number of animals
used. We have abided by the use of the ethical treatment
of laboratory animals as specified in the National Re-
search Council’s Guide for the Care and Use of Labora-
tory Animals (1996). The Institutional Animal Care and
Use Committee (IACUC) and the Institutional Review
Board-Human Subjects at California State University,
Los Angeles, approved this project. The IACUC Protocol
number is AW 10 - 1.
2.4. Hippocampal Dissection at Embryonic Day
18 (E 18)
Female rats (Sprague-Dawley, Charles River Corp., Wil-
mington, MA) were sacrificed with an overdose of iso-
fluorane on their 18th day of pregnancy. The embryos
were removed by cesarean section and immediately
placed on ice. The two hippocampi of each embryo were
excised and cultured in Ca-Mg-free medium according to
the method of Banker and Cowan [31]. The dissected
hippocampi were then rinsed twice with 5 - 10 ml/rinse
of Ca-Mg-free medium in 15-ml conical tubes. Trypsin
(0.125%) was added to the Ca-Mg-free medium (~4 - 5
ml) and then the conical tube was placed in a water bath
at 37˚C for 5 - 10 min with gentle inversion every 2 - 3
min. Cells were quenched in 10% fetal bovine serum/
DMEM (warmed to 37˚C, 1 - 2 volume ~8 ml) to stop
the reaction and centrifuged at 200 g for 5 min. The su-
pernatant was then removed and the pellet resuspended
in ~2 ml serum-free DMEM. Neurons were then tritu-
rated (pipetting up and down mechanically for separation)
with a silicote-coated constricted glass pipette and fil-
tered through a 40 μm cell strainer (Falcon) into a sterile
50 ml conical tube. The volume was then supplemented
with serum-free 1% N 2/DMEM to a total volume of 5
ml. Trypan blue (10 μl) was then added to 10 μl of cells,
which were counted using a hemacytometer. Neurons
were then plated in poly-l-lysine-coated plates at a den-
sity of 50,000 cells/cm2. Treatment of hippocampal
mixed neuronal/astrocytic cultures was conducted in trip-
licate where each experiment was performed at least
twice. Cells were then placed in an incubator at 37˚C and
5% CO2.
2.5. 5-HT Dose-/Time Response Evaluation
On the 3rd-4th day following plating, 5-HT was added to
the appropriate wells in triplicate at concentrations of
10−9, 10−8, 10−7, 10−6, 10−5, and 10−4 M and allowed to
incubate at 37˚C, 5% CO2, for 0, 5, 10, 30, 60, and 120
min, culminating in 72 combination treatments. These
concentrations and time points were partially guided by
experiments performed by Cowan et al. [32]. At each of
these time points, the media was aspirated off and the
cells in each respective well lysed with the addition of 75
μl lysis buffer (10 mM tris, pH 7.4, 1% SDS), scraped
from their wells, boiled for 5 min, triturated through a
26-gauge needle, centrifuged at 14,000 g for 5 min, and
an equal volume of gel-loading buffer [33] added to the
supernatant. Samples were stored at −80˚C until ready
for SDS-PAGE/Western Blotting.
2.6. Norepinephrine, 5-HT or the Combination
On the third day following plating, norepinephrine (10−7
M [24]) and/or 5-HT (10−5 M) was added to the appro-
priate treatment wells, and plates allowed to incubate at
37˚C, 5% CO2, for 10, 30, 60, 120, 240, or 480 min (with
norepinephrine only or norepinephrine + 5-HT) or for 0,
5, 10, 30, 60, 120, or 240 min (with 5-HT only). At each
of these time points, three 100 μl aliquots (triplicate) of
media was withdrawn from each well and stored at
−20˚C till assaying with BDNF ELISA kit. At the same
respective time point, the rest of the media was aspirated
off and the cells in each respective well processed for
SDS-PAGE just as described in the preceding section.
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Kinetics of Norepinephrine- and Serotonin-Induced BDNF Release in Cultured Embryonic Hippocampal Neurons 196
2.7. SDS-PAGE/Western Blotting
Equal amounts of cell lysates were applied to each lane
of an SDS-PAG, electrophoresed, and the separated pro-
teins subsequently electro-transferred to nitrocellulose
(Amersham-Pharmacia Biotech, Piscataway, NJ). West-
ern blotting on nitrocellulose membranes was performed
according to the specifications of the respective manu-
facturers. Protein immunoreactivity was visualized by
enhanced chemiluminescence (ECL), followed by appo-
sition of blots to hyperfilm (Amersham-Pharmacia Bio-
tech, Piscataway, NJ). To control for inadvertent differ-
ences in protein loading, all blots were then stripped in
100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM tris-HCl,
pH 6.7, at 55˚C for 10 min, with agitation and then re-
probed with the antibody against the respective total
form (anti-Akt, anti-MAPK, or anti-CREB), followed by
ECL and apposition to hyperfilm. Blots first probed with
anti-BDNF were stripped and then re-probed with anti-
tubulin. Blots first probed with the phospho-forms, fol-
lowed by the total forms, were then stripped once more
and re-probed with anti-tubulin. All anti-tubulin reactions
were then reacted once again with ECL and apposed to
hyperfilm.
Optical densities of lightly exposed phospho-proteins,
BDNF, total forms (e.g., total CREB), and tubulin bands
corresponding to the relative mobility of each respective
signaling protein were quantified using computer-as-
sisted densitometry (MCID, Interfocus Imaging Ltd,
London). Optical densities of the phospho-forms were
then arithmetically divided by those of their respective
total forms; this quotient was then arithmetically divided
by the respective tubulin band. Optical density of BDNF
bands was arithmetically divided by those of their re-
spective tubulin bands. All exposed bands quantified
within the linear range of a standard curve were used in
gathering densitometry data.
2.8. BDNF ELISA
The presence of BDNF in the media in response to nore-
pinephrine, 5-HT, or both at each time point was detected
using the BDNF Emax Immunoassay System (Promega,
Madison, WI).
2.9. Statistical Analyses
For the Western data generated from the initial 5-HT
dose-and time-response evaluation of BDNF immunore-
activity, a 2-way ANOVA (dose × time) was calculated,
using Fisher’s post hoc LSD to evaluate pair-wise com-
parisons. For both the Western blot-generated optical
densities and the BDNF Emax data, statistical analyses
were implemented using 1-way ANOVA, followed by
Fisher’s post hoc LSD to test for pair-wise statistically
significant differences between any two time points. For
analyzing how the activity of intracellular signaling
molecules changes with respect to each other and over
time, a 2-way ANOVA (molecule × time), followed by
Fisher’s post hoc LSD test was conducted. For analyzing
how the levels of BDNF change with respect to each
other and over time, a 2-way ANOVA (condition × time),
followed by Fisher’s post hoc LSD test was conducted,
where condition is either lysates or media.
3. Results
3.1. Dose- and Time-Response of BDNF
Immunoreactivity to 5-HT in Hippocampal
Cell Lysates
Application of 5-HT to E 18 hippocampal neurons sig-
nificantly induced BDNF immunorectivity in a dose-[F(6,
84) = 138.38, p < 0.0001] and time-[F(5, 84) = 736.65, p <
0.0001] dependent manner. In addition, there was sig-
nificant interaction between these two variables [F (30, 84)
= 35.45, p < 0.0001] (Figure 1). BDNF immunoreac-
tivity was increased significantly in response to only 10−9
M 5-HT in as little time as 5 min (p < 0.0001) and conti-
nuing through to 10 min (p < 0.0001), compared to that
of vehicle-treated controls at the corresponding zero
time-points of 5 and 10 min (Figure 1). This same trend
was also evident for all 5 of the higher doses evaluated
(10−8, 10−7, 10−6, 10−5, and 10−4 M 5-HT), compared to
vehicle-treated controls and compared to the zero time
point. However, at 10−7 M 5-HT, BDNF immunoreac-
tivity was significantly increased at 30 min, compared to
vehicletreated controls and compared to the zero time
point, but then decreased to levels comparable to those of
controls (vehicle-treated at zero min) at 60 through to
Figure 1. Serotonin elicits BDNF immunoreactivity in hip-
pocampal neurons in a dose- and time-dependent manner. *,
statistically significantly different from vehicle controls ([5-
HT] = 0 M at 5 min (p < 0.001); #, statistically significantly
different from vehicle controls ([5-HT] = 0 M at 10 min (p <
0.001); +, statistically significantly different from vehicle
controls ([5-HT] = 0 M at 30 min (p < 0.001). Data are the
mean ± SEM. Each experiment was conducted at least twice,
with each sample measured in triplicate.
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Kinetics of Norepinephrine- and Serotonin-Induced BDNF Release in Cultured Embryonic Hippocampal Neurons
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197
120 min (Figure 1).
3.2. Kinetics of BDNF Release into the Media in
Response to 5-HT, Norepinephrine, and Both
As per the results displayed in Figure 1, the rest of this
study was performed with 10−5 M 5-HT, as this concen-
tration was one of the two that yielded the most BDNF
immunoreactivity (the other being 10−4 M). Norepineph-
rine was applied at a final concentration of 10−7 M, as
this dose has been shown to elicit the highest BDNF
immunoreactivity in a similar dose-/time-dependent ex-
periment [24].
Application and incubation of the aforementioned con-
centrations of 5-HT +/− norepinephrine at 5, 10, 30, 60,
120, 240, and 480 min display the results in Figure 2.
There was a statistically significant interaction [F(13, 27) =
13.23, p < 0.0001] between time point and neurotrans-
mitter (5-HT, norepinephrine, both, or neither (vehicle
controls)) and a significant main effect of neurotransmit-
ter [F(3, 27) = 76.66, p < 0.0001], but not for time point.
For each time point up to and including 120 min, nore-
pinephrine-induced secretion of BDNF was no different
from that of vehicle; but at 240 min, norepinephrine lead
to significantly more BDNF secretion into the media than
that of vehicle (p = 0.002). In sharp contrast, however, 5-
HT led to significantly more BDNF secretion than that of
vehicle much earlier at 10 (p < 0.0001), 30 (p = 0.004)
and 60 (p = 0.015) min. At 120 and 240 min, however,
vehicle- and 5-HT-secreted BDNF values were compara-
ble (Figure 2). When both neurotransmitters were com-
bined, however, all time points revealed significantly
more BDNF secretion into the media than that of vehicle
alone [5 min (p < 0.0001), 10 min (p < 0.0001), 30 min
(p = 0.051), 60 min (p = 0.002), 120 min (p < 0.0001),
240 min (p < 0.0001)] (Figure 2).
Comparison of the amount of BDNF secretion be-
tween vehicle and either neurotransmitter or both: veh vs.
5-HT [10 min (p < 0.000), 30 min (p = 0.004), 60 min (p
= 0.015)]; veh vs. norepinephrine [240 min (p = 0.002),
480 min (p = 0.006)]; veh vs. norepinephrine + 5-HT (p
< 0.0001 - 0.05). And comparison of the amount of
BDNF secretion between either neurotransmitter alone
and that of the combination treatment resulted in either
statistically significant differences or a trend (Figure 2):
5-HT vs. norepinephrine + 5-HT [10 min (p = 0.057), 30
min (p = 0.031), 60 min (p = 0.056), 120 min (p <
0.0001), 240 min (p = 0.016)]; norepinephrine vs. nore-
pinephrine + 5-HT [10 min (p < 0.0001), 30 min (p =
0.03), 60 min (p = 0.002), 120 min (p = 0.001), 240 min
(p = 0.016), 480 min (p = 0.011)] (Figure 2).
Figure 2. Serotonin and/or norepinephrine elicit the secretion of BDNF into the media from hippocampal neurons in a dose-
and time-dependent manner. Serotonin caused significantly more BDNF secretion at 10, 30, and 60 min than at 5 min (p ≤
0.005); norepinephrine elicited significantly more BDNF secretion at 120 min than at 5, 10, 30, and 60 min (p ≤ 0.004); nore-
pinephrine elicited significantly more BDNF secretion at 240 min than at either 120 (p < 0.0001) or 480 (p < 0.001) min; and
both neurotransmitters combined elicited as much as 2 - 5 times the amount elicited by vehicle alone at all time points.
Among the combination treatment itself, there was a statistically significant decrease in BDNF secretion at 30 min, compared
to that at 5 min (p = 0.022) and a lower, but not statistically significantly lower, than that at 60 min (p = 0.077). *, statistically
significantly different from that of vehicle controls at the same corresponding time point at p < 0.05. #: significantly different
at p < 0.05. Data are the mean ± SEM. Each experiment was conducted at least twice, with each sample measured in tripli-
cate.

Kinetics of Norepinephrine- and Serotonin-Induced BDNF Release in Cultured Embryonic Hippocampal Neurons 198
3.3. Kinetics of Activation of Intracellular
Signaling in Hippocampal Cell Lysates in
Response to 5-HT
Both the PI-3 K and MAPK pathways were differentially
activated in response to application of 5-HT over time
(Figure 3). PI-3 K was significantly activated over time
[F(6, 17) = 7.2, p = 0.001]. Only at 30 (p = 0.007) and 60 (p
< 0.000) min was PI-3 K expression significantly higher
than that at the zero time point (Figure 3(a)); PI-3 K
expression at 30 min was significantly higher than that at
240 min (p = 0.015). Phosphorylation of Akt exhibited
overall statistical significance over time [F(6, 17) = 6.5, p =
0.015] with its activation being significantly higher at 30
(p = 0.026) and 60 (p = 0.041) min than at zero min
(Figure 3(a)).
BDNF expression was significantly activated in re-
sponse to application of 5-HT over time [F(5, 12) = 70.83,
p < 0.000], where the 5-, 10- and 30-min time points
were all significantly higher than that at the zero-min
time point (p < 0.000); BDNF expression at 5 min was
significantly higher than at all other time points, except
that at 10 min (p < 0.000); the 10-min time point was
significantly higher than at the 30-, 60-, and 120-min
time points (p < 0.000); finally, BDNF expression was
significantly higher at the 5-, 10-, and 30-min time points
than that at 60 min (p < 0.000) (Figure 3(b)).
Phosphorylation of the BDNF receptor, TrkB, was not
overall significant over time, although the 240-min time
point exhibited statistically significantly higher phos-
phorylation levels than that at zero (p = 0.012) and 60
min (p = 0.03) (Figure 3(b)).
Phosphorylation of the transcription factor, CREB, ex-
hibited no overall statistically significant difference
among any of the time points with each other (Figure
3(b)).
Phosphorylation of neither MAPK 1 nor MAPK 2 was
statistically significantly different over time (Figure
3(c)).
To verify that the signaling pathways evaluated were
indeed affected by our treatments of one or both neuro-
transmitters, the appropriate pathway inhibitor was incu-
bated with 5-HT +/− norepinephrine. LY 294002 or
wortmannin inhibits the PI-3 K/Akt pathway, PD 98059
inhibits the MAPK pathway and K252a inhibits TrkB
phosphorylation (Figure 3(d)).
3.4. Kinetics of Activation of Intracellular
Signaling in Hippocampal Cell Lysates in
Response to both 5-HT and Norepinephrine
Both the PI-3 K and MAPK pathways, as well as BDNF,
TrkB and CREB, were differentially activated in re-
sponse to application of 5-HT and norepinephrine over
time (Figure 4). PI-3 K was significantly activated over
time [F(6, 14) = 257.18, p < 0.000]. PI-3 K expression was
significantly higher at all time points than at zero min (p
< 0.000); compared to that at zero min, PI-3 K expres-
sion was significantly higher at the 5-(p = 0.016), 60-(p =
0.006), 120-(p = 0.003) and 240-(p < 0.000) min time
points. The 30-min time point expression of PI-3 K ex-
hibited significantly less than that at any other time point,
except for at zero-(p < 0.000) and at 240 min, compared
to which, it was statistically the same. Finally, at 60 min,
(a) (b)
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Kinetics of Norepinephrine- and Serotonin-Induced BDNF Release in Cultured Embryonic Hippocampal Neurons 199
(c) (d)
Figure 3. Serotonin differentially affects various signaling intermediates and pathways. For ease of comparison, all 7 signal-
ing molecules were divided into 3 graphs (a)-(c): (a) vehicle, PI-3 K and P-Akt, (b) vehicle, BDNF, P-TrkB and P-CREB, and
(c) vehicle, P-MAPK 1 and P-MAPK 2. These 3 graphs are displayed to provide maximum clarity with respect to the ordinate
and do not reflect the relative proportions of all 7 signaling molecules (P-Akt, PI-3 K, BDNF, P-TrkB, P-CREB, P-MAPK 1,
P-MAK 2). Vehicle represents the mean of the responses of all 7 signaling molecules in response to application of vehicle,
which serves as baseline; its small error bars reveal that there are no statistically significant differences among all 7 mole-
cules and are displayed as such to help minimize complexity of the graphs. For such direct comparisons among all 7 mole-
cules, a 2-way ANOVA was conducted (molecule x time, see Methods). Levels of both P-MAPK 1 and P-MAPK 2 are signifi-
cantly lower than levels of PI-3 K at 5 (p = 0.013 - 0.014), 10 (p < 0.000), 30 (p = 0.004 - 0.006), 60 (p < 0.000) and 120 (p <
0.000) min; levels of both P-MAPK 1 and P-MAPK 2 are significantly higher than levels of P-Akt throughout to 240 min (p <
0.000 - 0.043). Levels of P-CREB was significantly lower than those of PI-3 K at 5 (p = 0.001), 10 (p = 0.001), 30 (p = 0.008)
and levels of P-Akt at 30 min (p = 0.022), 60 (p = 0.001) and 120 (p < 0.000) min; levels of P-CREB were significantly higher
than those of both P-MAPK 1 and P-MAPK 2 up to 60 min inclusive (p = 0.009 - 0.017). (a) Compared to baseline (vehicle),
PI-3 K is significantly higher from 5 - 120 min (p < 0.001). P-Akt is significantly higher than that of baseline from 10 - 240
min (p < 0.01); PI-3 K is significantly higher than P-Akt at 10 (p = 0.05), 60 (p < 0.000) and 120 (p = 0.001) min. *, statistically
significantly different from zero time point at p < 0.05; one column of two *, both PI-3 K and P-Akt are statistically signifi-
cantly different from zero time point at p < 0.05. (b) BDNF is significantly higher than that of baseline from 5 - 30 min (p <
0.05, inset) and significantly lower than that of baseline vehicle at 60 and 120 min (p < 0.05); P-TrkB is significantly higher
than that of baseline vehicle at 10 (p = 0.043), 30 (p = 0.043), 120 (p = 0.045) and 240 (p = 0.012) min; P-CREB levels are sig-
nificantly higher than that of baseline at only 30 min (p = 0.026). BDNF is significantly higher than P-TrkB at 5 (p = 0.008), 10
(p = 0.011) and 30 (p = 0.035) min; BDNF is significantly higher than P-CREB at 5 (p = 0.01) and 10 (p = 0.015) min; and P-
TrkB is significantly higher than P-CREB at 120- (p = 0.016) and 240- (p = 0.009) min. Inset: B, T and C refer to BDNF, P-
TrkB and P-CREB, respectively, and indicate that levels of this molecule are significantly different from that at zero min at p
< 0.05. (c) Both P-MAPK1 and P-MAPK 2 levels are significantly lower than that of baseline from 30 - 120 min (p = 0.004 -
0.039). Levels of P-MAPK 1 and P-MAPK 2 did not significantly differ from each other throughout the entire duration. Data
are the mean ± SEM. (d) Representative immunoblots in the presence of the appropriate inhibitor for TrkB (K252a, 10 μM),
PI-3 K/Akt pathway (LY, LY 294002 (10 μM) or Wort, wortmannin (20 μM)) or MAPK (PD, PD 98059, 50 μM) pathway.
Each inhibitor was added at this final concentration 1 hr prior to the application of neurotransmitter. Each experiment was
conducted at least twice, with each sample measured in triplicate.
PI-3 K expression was significantly higher than at all
other time points, except that at 10 min and 120 min
(Figure 4(a)). Akt was significantly phosphorylated over
time [F (6, 14) = 72.07, p < 0.000]. Akt phosphorylation
was significantly higher at all time points than at zero
min (p < 0.000); Akt phosphorylation was significantly
higher at both the 5- and 10-min time points than at the
30- and 240-min time points (p < 0.000). At 60 min, Akt
was phosphorylated significantly higher than at all other
time points (p < 0.000 - 0.009), except those at 5 and 10
min. At 120 min, phosphorylation of Akt was signifi-
cantly higher than at 30 and 240 min (p < 0.000) statisti-
cally the same as that at 60 min (p = 0.009) (Figure
4(a)).
BDNF expression was significantly activated in re-
sponse to application of 5-HT and norepinephrine over
time [F(6, 14) = 17.97, p < 0.000], where the expression
was significantly higher at 30 (p < 0.000), 120 (p < 0.000)
and 240 (p = 0.001) min and significantly lower at 60
min (p = 0.000) than at zero min (Figure 4(b)). At both 5
and 10 min, BDNF expression was significantly lower
han that at 30 (p = 0.004, 0.018, respectively), 120 t
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Kinetics of Norepinephrine- and Serotonin-Induced BDNF Release in Cultured Embryonic Hippocampal Neurons 200
(a) (b)
(c)
Figure 4. Serotonin and norepinephrine administered together differentially affect various signaling intermediates and path-
ways. For ease of comparison, all 7 signaling molecules were divided into 3 graphs (a)-(c): (a) vehicle, PI-3 K and P-Akt, (b)
vehicle, BDNF, P-TrkB and P-CREB, and (c) vehicle, P-MAPK 1 and P-MAPK 2. These 3 graphs are displayed to provide
maximum clarity with respect to the ordinate and do not reflect the relative proportions of all 7 signaling molecules (P-Akt,
PI-3 K, BDNF, P-TrkB, P-CREB, P-MAPK1, P-MAPK 2). Vehicle represents the mean of the responses of all 7 signaling
molecules in response to application of vehicle, which serves as baseline; its small error bars reveal that there are no statisti-
cally significant differences among all 7 molecules and are displayed as such to help minimize complexity of the graphs. For
such direct comparisons among all 7 molecules, a 2-way ANOVA was conducted (molecule x time, see Methods). Activity of
both PI-3 K and P-Akt are significantly higher than that of both P-MAPK1 and P-MAPK 2 (p = 0.003 - 0.028), even at 30 min.
P-Akt levels are significantly lower than that of BDNF (p = 0.022) and P-TrkB (p < 0.000), but is statistically the same as that
as P-CREB at 30 min; then, at 60 min, P-Akt levels are significantly higher than that of BDNF (p < 0.000) and P-CREB (p =
0.033), but not statistically different from that of P-TrkB; at 240 min, P-Akt is significantly lower than that of BDNF (p =
0.012) and P-TrkB (p = 0.034), and significantly higher than that of P-CREB (p = 0.037). Finally, starting at 30 min, P-TrkB
levels are significantly higher than that of both P-MAPK 1 (p = 0.004 - 0.039) and P-MAPK 2 (p < 0.000) throughout to 240
min; at 60 min, BDNF levels are significantly lower than that of P-MAPK1 (p = 0.029), but significantly higher than that of P-
MAPK 2 (p < 0.000); but at 120 to 240 min, BDNF levels are significantly higher than that of both P-MAPK 1 (p < 0.001) and
P-MAPK 2 (p < 0.001). (a) Expression and activity levels of both P-Akt and PI-3 K, whose time courses parallel each other,
are significantly higher than that of baseline (vehicle) from 5 - 240 min (p < 0.001). *, statistically significantly different from
zero time point at p < 0.05; one column of two *, both PI-3 K and P-Akt are statistically significantly different from zero time
point at p < 0.05. (b) BDNF is significantly higher than that of baseline vehicle from 5 - 30 min (p < 0.05, inset) and statisti-
cally the same as that of baseline at 60 min, but is significantly higher than that of baseline at 120 (p = 0.019) and 240 (p =
0.024) min; P-TrkB is significantly higher than that of baseline throughout from 5 - 240 min (p = 0.013 - 0.041); P-CREB is
significantly higher than that of baseline throughout from 5 - 240 min (p = 0.001 - 0.018). BDNF is significantly higher than
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Kinetics of Norepinephrine- and Serotonin-Induced BDNF Release in Cultured Embryonic Hippocampal Neurons 201
that of P-CREB at 30 (p = 0.039), 120 (p = 0.041) and 240 (p = 0.02) min, but is significantly lower than that of the phos-
phorylated transcription factor at 60 min (p = 0.013); BDNF is significantly lower than that of P-TrkB at 30 (p = 0.026), 60 (p
= 0.009), 120 (p = 0.045) and 240 (p = 0.038) min; P-TrkB levels are significantly higher than those of P-CREB starting at 30
(p < 0.001), 60 (p = 0.006), 120 (p = 0.005) and 240 (p = 0.002) min. Inset: B, T and C refer to BDNF, P-TrkB and P-CREB,
respectively, and indicate that levels of this molecule are significantly different from that at zero min at p < 0.05. (c) P-MAPK
1 levels are significantly higher than that of baseline at 10 (p < 0.001), 60 (p = 0.021), 120 (p = 0.031) and 240 (p = 0.048), but
not at 5 nor 30 min; P-MAPK 2 levels are significantly lower than that of baseline starting at 10 min throughout to 240 min (p
< 0.001); likewise, P-MAPK1 levels are significantly higher than those of P-MAPK 2 starting at 10 min throughout to 240 min
(p < 0.001). *, statistically significantly different from zero time point at p < 0.05. Data are the mean ± SEM. Each experiment
was conducted at least twice, with each sample measured in triplicate.
(p = 0.000, 0.000, respectively) and 240 (p = 0.013, 0.05,
respectively). At 60 min, BDNF expression was signifi-
cantly lower than at all other time points (p = 0.000 -
0.004). At 240 min, BDNF expression was significantly
higher than that at zero and at 60 min (p = 0.001) (Figure
4(b)).
Differences in phosphorylation of TrkB was signifi-
cant over time [F(6, 21) = 36.86, p < 0.000]: all time points,
except at 5 min, saw a statistically significant increase in
TrkB phosphorylation (p < 0.000) compared with that at
zero min. However, starting at 10 min, TrkB phosphory-
lation was not significantly different from all subsequent
time points.
CREB phosphorylation was significantly different
over time [F(6, 14) = 30.59, p < 0.000] in response to ap-
plication of both 5-HT and norepinephrine. Starting at
zero min, all time points were significantly higher (p <
0.000); at 5 min, phosphorylation was significantly lower
than at all other subsequent time points (p < 0.000 -
0.014), except at 30 min; at 10 min, phosphorylation was
significantly higher (p < 0.000 - 0.014), except at 60 and
120 min; at 30 min, phosphorylation was significantly
higher than that at zero and significantly lower than that
at 10 (p = 0.008), 60 (p < 0.000) and 120 (p = 0.023) min
(Figure 4(b)).
Phosphorylation of MAPK 1 was significantly differ-
ent over time [F (6, 14) = 25.63, p < 0.000] in response to
application of both 5-HT and norepinephrine. Phos-
phorylation of MAPK 1 was significantly higher at the
10-(p = 0.012), 60-(p = 0.42) and 120-(p = 0.039) min
time points than that at zero min (Figure 4(c)). Likewise,
phosphorylation of MAPK 2 was significantly different
over time in response to both neurotransmitters [F(6, 14) =
6.22, p = 0.0212]. MAPK 2 phosphorylation was signifi-
cantly higher at the 5-min (p = 0.039 - 0.009) than at all
subsequent time points (Figure 4(c)).
3.5. Kinetics of BDNF Concentrations in Lysates
and Media in Response to Norepinephrine,
Serotonin or both
In response to application of norepinephrine, levels of
BDNF between lysates and media were significantly
changed over time [F(6, 14) = 46.81, p < 0.001] (Figure
5(a)). BDNF levels in lysates were significantly higher at
120 min than that 30 (p = 0.002) and 480 (p = 0.008) min
(Figure 5(a)). BDNF levels in media were significantly
higher at 240 min (p < 0.001) than at all other time points
evaluated (Figure 5(a)).
In response to application of 5-HT, levels of BDNF
between lysates and media were significantly changed
over time [F(6, 14) = 29.44, p < 0.001] (Figure 5(b)).
BDNF levels in lysates were significantly higher at 10
min (p = 0.007) than at all other time points. BDNF lev-
els in media were significantly higher at 10 (p < 0.000)
and 30 (p < 0.000) min than at all other time points
evaluated (Figure 5(b)).
In response to application of both norepinephrine and
5-HT, levels of BDNF between lysates and media were
significantly changed over time [F(6, 14) = 18.76, p =
0.002] (Figure 5(c)). BDNF levels in lysates were sig-
nificantly higher at 10 min (p = 0.004) and lower at 30
min (p = 0.006) than at all other time points (Figure
5(c)). BDNF levels in media were significantly lower at
30 min (p = 0.039) than at all other time points evaluated
(Figure 5(c)).
4. Discussion
We have previously shown that hippocampal neurons in
culture respond to application of norepinephrine by in-
creasing expression of BDNF, its receptor, TrkB, activa-
tion of the PI-3 K and MAPK pathways, and the tran-
scription factor, CREB [24]. In that study, we demon-
strated that in a dose- and time-response experiment, 10−7
M norepinephrine for 2 hr was maximally effective in
eliciting BDNF expression; consistently, activation of
TrkB was sustained for 4 hr and that of PI-3 K and
MAPK pathways was maintained out to 8 hr [24,34]. In
the current study, we extend characterization of norepi-
nephrine-induced expression of BDNF, not only within
hippocampal neurons themselves (lysates via immunoblot-
ting, [24]), but also, to the kinetics of BDNF release into
the surrounding media (Figure 5(a)). We also character-
ized the kinetics of 5-HT-induced BDNF expression
(Figure 5(b)) just as we did earlier with norepinephrine
[24]. As expected, the time course expression of BDNF
in neuronal lysates precedes that released into the media
(Figures 5(a), (b)).
W
e show that although significant BDNF expression is
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Kinetics of Norepinephrine- and Serotonin-Induced BDNF Release in Cultured Embryonic Hippocampal Neurons 202
(a) (b)
(c)
Figure 5. Norepinephrine and/or 5-HT increase BDNF in neuronal lysates, followed by an increase into the surrounding me-
dia. (a) Norepinephrine increased BDNF in both lysates (p < 0001) and media (p < 0.001) significantly higher than that in
baseline vehicle controls at zero min. Lysate levels of BDNF are significantly higher than media levels at 120 min (p < 0.001)
(b) Serotonin increased BDNF in both lysates (p < 0.001) and media (p < 0.001) significantly higher than that in baseline vehi-
cle controls. Media levels of BDNF are significantly higher than lysate levels at 30 (p = 0.001), 60 (p = 0.034) and 120 (p =
0.043) min. (c) Both norepinephrine and 5-HT increased BDNF in both lysates (p = 0.003) and media (p = 0.0025) significantly
higher than that in baseline controls. Both neurotransmitters resulted in no significant differences in BDNF between lysates
and media, except at 60 min (p = 0.045). NE, norepinephrine; *, statistically significantly different from zero time point at p <
0.05. Data are the mean ± SEM. Each experiment was conducted at least twice, with each sample measured in triplicate.
indeed induced at the lowest concentration of 5-HT
evaluated (10−9 M), 5-HT induces maximal BDNF ex-
pression on a much shorter time scale [32], but less
potently (10−5 M, Figure 1) than that of norepinephrine
[24]. Moreover, we show that norepinephrine-induced
secretion of BDNF into the media manifests a delay of
about 2 hr (from 120 to 240 min) from which expression
of BDNF in neuronal lysates themselves was maximal at
120 min; following another 2 hr, BDNF levels in the me-
dia were decreased to those comparable at 120 min. A
similar pattern of BDNF secretion into the media was
observed with 5-HT; except that at 10 and 30 min, BDNF
was maximal and then decreased significantly 30 min
later (at 60 min), but was still higher than that at 5, 120,
and 240 min (Figure 2). Again, this represents a delay
between maximal BDNF expression observed in lysates
at 1 and 10 min, compared to that observed in the media
at 10, 30 and 60 min later.
Then, when norepinephrine and 5-HT are co-admin-
istered, BDNF secretion into the media is significantly
higher throughout the duration of this time scale, out to
240 min. Surprisingly, at both the beginning (5 min) and
at the end (240 min), BDNF secretion into the media
indicates a synergistic effect between the 2 neurotrans-
miters as neither one alone elicited such a large response
(Figure 2). Upon application of both neurotransmitters,
the time course of BDNF in lysates and media roughly
parallel each other (Figure 5(c)). Presented another way,
when norepinephrine and 5-HT are co-administered, ex-
cept for a momentary decline at 30 min in lysates, either
neurotransmitter alone or both together result in in-
creased BDNF expression (Figure 6(a)). This tendency
is even more pronounced in the media in which striking
synergistic effects are observed between norepinephrine
and 5-HT (Figure 6(b)).
At first glance of the data, it would seem that when
each neurotransmitter is added alone, 5-HT would in-
crease BDNF early, whereas norepinephrine would in-
crease it later. When co-administered, however, this tis-
sue culture system does not respond so simplistically,
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Kinetics of Norepinephrine- and Serotonin-Induced BDNF Release in Cultured Embryonic Hippocampal Neurons 203
(a)
(b)
Figure 6. Norepinephrine and/or 5-HT increase BDNF in
cell lysates and that secreted into the media over time. (a)
BDNF in cell lysates is significantly higher as a result of
norepinephrine or 5-HT administered separately or to-
gether, compared to that in baseline controls across all time
points (p = 0.001 - 0.022); starting at 60 min, BDNF levels
are significantly higher as a result of both neurotransmit-
ters than that as a result of only norepinephrine (p = 0.003 -
0.021) or only 5-HT (p < 0.001). Note the significant interac-
tion with time, at 30 (p = 0.014) and 60 (p = 0.022) min,
which shows BDNF levels increasing as a result of norepi-
nephrine application and decreasing as a result of 5-HT
application. one column of two *, both 5-HT and NE + 5-HT
are statistically significantly different from zero time point
at p < 0.05. (b) BDNF in media is significantly higher as a
result of 5-HT at 10 (p < 0.000), 30 (p < 0.000) and 60 (p <
0.000) min than that at baseline; BDNF in media is signifi-
cantly higher as a result of norephinephrine at 240 (p =
0.001) and 480 (p = 0.016) min than that at baseline; and
BDNF in media is significantly higher as a result of both
norepinephrine and 5-HT than that at baseline across all
time points (p < 0.000). NE, norepinephrine; *, statistically
significantly different from zero time point at p < 0.05.
resulting in an overlap during which the entire time
frame evaluated sees an increased expression of BDNF.
Rather, such a momentary decline at 30 to 60 min belies
a more complex mechanism, rather than simply the sum
of the effects of two different neurotransmitters on
BDNF expression and was so reliable, that the Westerns
and ELISAs were each performed at least 4 - 5 times to
confirm. This temporary decline is seen with BDNF in
lysates (60 min, Figure 4(b); 30 min, Figures 5(c), 6(a)),
BDNF in media (30 min, Figures 2, 5(c), 6(b)), P-TrkB
(60 min, Figure 4(b)), PI-3 K and P-Akt (30 min, Figure
4(a)), P-CREB (30 min, Figure 4(b)) and MAPK 1 (30
min, Figure 4(c)). It is conceivable that the co-presence
of both neurotransmitters entails that each bind their re-
spective GPCRs, resulting in production of cAMP and
subsequent activation of PKA, which then phosphory-
lates CREB, which, then regulates the transcription of
BDNF, which, in turn would be packaged into vesicles
and be released extracellularly, where it would bind its
TrkB receptors via an autocrine or paracrine diffusion
[35], resulting in a positive feed-back loop [24]; all of
this could occur within 5 - 10 min and certainly before 30
- 60 min. Then, after 30 - 60 min, this released BDNF
can then modulate a rapidly recycling pool of vesicles
storing norepinephrine and/or 5-HT by promoting the
entry of Ca2+ and subsequent activation of the SNARE
complex and consequent increased release of these neu-
rotransmitters [36-38]. Such a positive feedback loop [24]
is corroborated in Figure 3(d) in which PI-3 K/Akt and
MAPK inhibitors result in decreased expression of
BDNF and decreased phosphorylation of CREB.
In mixed hippocampal cultures (neurons and astro-
cytes), once they have begun establishing some rudi-
mentary connections, the application of neurotransmitter
may be considered as roughly equivalent to the applica-
tion of neurotransmitter-specific antidepressant, such as
reboxtine (norepinephrine-specific reuptake inhibitor) or
fluoxetine (serotonin-specific reuptake inhibitor). Indeed,
treating cultured astrocytes with fluoxetine [39], cultured
cortical neurons with imipramine or fluvoxamine [5], or
mixed cultured hippocampal neurons/astrocytes with
reboxetine or tranylcypromine [24] have all led to in-
creased expression of BDNF. Antidepressants that work
through both neurotransmitters, therefore, would not only
release BDNF faster, but for longer periods of time. To
resolve the apparent discrepancy between in vivo efficacy
and in vitro potency, Tsuruda et al. [40] characterized the
in vitro binding kinetics of several antidepressants in
HEK 293 cells and found a wide range of kinetics bind-
ing affinities among many of the currently used antide-
pressants, thereby underscoring the need for standard
assays and cell lines to screen and consistently charac-
terize antidepressants in terms of their ability to exert
long-term cellular and genetic effects. Even in our low-
density culture of 50 K/cm2, which is considerably lower
than those screened by others [35] at 80 K/cm2, BDNF
exhibits both autocrine and paracrine diffusive radii [35],
as evidenced by the activation of TrkB, PI-3 K and
MAPK pathways and CREB. Indeed, we have found that
the PI-3 K/Akt pathway can be activated as early as 10
min following application of 5-HT (Figure 3(a)). This is
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Kinetics of Norepinephrine- and Serotonin-Induced BDNF Release in Cultured Embryonic Hippocampal Neurons 204
consistent with earlier findings of 10−8 M 5-HT within as
little as 2 to 5 min [32] and with what others found, albeit
in cerebellar granule neurons, that direct application of
BDNF itself resulted in phosphorylation of Akt in as lit-
tle as 5 min and was sustained for 6 h [41]. To achieve
rapid and sustained activation of these survival signaling
pathways, therefore, if not applying BDNF directly
[32,41], but rather, using antidepressants or the neuro-
transmitters themselves, at least both the noradrenergic
and serotonergic systems must be targeted. This latter
indirect application of BDNF provides further evidence
for receptor/pathway crosstalk between GPCRs and Trk
receptors ([24], and references cited therein; [42,43]).
The time course for increased BDNF expression and
release into the media is consistent with earlier studies
that found increased BDNF kinetics on the order of sev-
eral minutes [44]. High K+ concentration has been shown
in cultured hippocampal neurons to secrete both pro- and
mature BDNF for 90 min following K+-induced depo-
larization [17], which is comparable to our findings in
response to norepinephrine-induced BDNF secretion
starting at 120 min. This suggests that depolarization-
driven BDNF release, when it occurs slowly (longer time
scale) may be mediated by noradrenergic receptor bind-
ing. Faster BDNF release (or shorter time scale) may be
mediated serotonergically as the current study shows.
Shen et al. [45] have also shown in vivo, that in as little
as 15 min of swim stress, hippocampal P-MEK is dra-
matically increased. Chen and Russo-Neustadt [8] have
shown that a single overnight bout of running exercise
significantly increased hippocampal BDNF and P-CREB.
Thus, although Aicardi et al. [46] found that in peripheral
cortical slices, theta burst stimulation at a frequency
known to induce LTP, the subsequent secretion of BDNF
was sustained 5 - 12 min beyond the stimulation period.
High K+ has also been shown to result in rapid BDNF
release (400 sec, [22]). It is possible that these few min-
utes beyond the stimulation interval are serotonergically
mediated.
Still, it is not clear why this discrepancy in kinetics of
release may exist between noradrenergically and sero-
tonergically mediated release of BDNF. Others have
suggested that the speed of BDNF release/secretion is
limited by the slow dissolution of the secretory granule
peptide core following opening of the fusion pore con-
necting the membranes of the granule with that of the
cell [47]. It is also possible that co-localization of BDNF
with other small peptides in the secretory granule in both
pre- and post-synaptic terminals [48], their respective
concentrations and molecular weights, their speed of
binding and unbinding from the core and their speed of
diffusion through the fusion pore all determine the rate of
secretion into the media [47]. Moreover, secretion of
BDNF is relegated to the regulated pathway of secretion
and co-localization with the PSD 95 in glutamatergic
synapses [49]. Those investigators found that depolariza-
tion-induced release of BDNF from secretory granules
typically takes the longest of all neurotrophins, on the
order of about 300 sec [13,49], following a 30-sec delay
onset and a half-life decay of about 230 sec [49]. Finally,
there is ample evidence that because BDNF is actually
secreted in its precursor form (pro-BDNF), which is then
cleared from the extracellular space via rapid uptake by
nearby astrocytes and neurons, which internalize pro-
BDNF via formation with a complex with the p 75 re-
ceptor and subsequent endocytosis. This endocytosed
pro-BDNF is then routed to a fast recycling pathway for
subsequent soluble NSF attachment protein receptor-
dependent secretion occurring on a 5-min [50] to 10-min
[16] time scale.
5. Conclusion
In conclusion, our hippocampal culture study shows that
norepinephrine and 5-HT differentially induce the ex-
pression and release of BDNF. Serotonin elicits an earlier,
but less sustained expression and release of BDNF,
whereas norepinephrine elicits more delayed and more
sustained release of the neurotrophin. Together, both
neurotransmitters cover the entire time scale evaluated.
In addition, for each neurotransmitter, the amount of
BDNF in cell lysates shortly preceded those in the media.
Consistent with their induced expression of BDNF, acti-
vation of TrkB, CREB and the PI-3 K/Akt, but not the
MAPK pathway was sustained throughout the time
course evaluated. These results, taken together along
with those of our earlier study [24], provide a further
insight into the possible mechanisms of neurotransmitter
action such as that which occurs with physical exercise
and/or antidepressant therapy.
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