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Dose-Response, 9:416-433, 2011
Formerly Nonlinearity in Biology, Toxicology, and Medicine
Copyright © 2011 University of Massachusetts
ISSN: 1559-3258
DOI: 10.2203/dose-response.10-041.Marini

CELLULAR AND MOLECULAR RESPONSES OF CULTURED NEURONS TO

STRESSFUL STIMULI

Jun Chen, Hongna Pan 䊐 Uniformed Services University of the Health Sciences,
Department of Neurology and Program in Neuroscience, 4301 Jones Bridge Road,
Bethesda, Maryland 20814

Robert H. Lipsky 䊐 Department of Neurosciences, Inova Health System, Inova

Fairfax Hospital, 3300 Gallows Road, Falls Church, VA 22042 and the Department
of Molecular Neuroscience, Krasnow Institute for Advanced Study, George Mason
University, Fairfax, VA 22030

Anabel Pérez-Gómez, David Cabrera-Garcia, Maria Teresa Fernández-Sánchez 䊐

University of Oviedo, Department of Biochemistry and Molecular Biology, and

Institute of Biotechnology, 33006 Oviedo, Spain

Antonello Novelli 䊐 University of Oviedo, Department of Psychology and

Institute of Biotechnology, 33006 Oviedo, Spain

Ann M. Marini 䊐 Uniformed Services University of the Health Sciences,

Department of Neurology and Program in Neuroscience, 4301 Jones Bridge Road,
Bethesda, Maryland 20814
䊐 Synaptic function is critical for the brain to process experiences dictated by the envi-
ronment requiring change over the lifetime of the organism. Experience-driven adapta-
tion requires that receptors, signal transduction pathways, transcription and translational
mechanisms within neurons respond rapidly over its lifetime. Adaptive responses commu-
nicated through the rapid firing of neurons are dependent upon the integrity and func-
tion of synapses. These rapid responses via adaptation underlie the organism’s ability to
perceive, learn, remember, calculate and plan. Glutamate, the endogenous neurotrans-
mitter required for physiological excitation in the brain, is critically involved in neuronal
adaptive responses and in the pathophysiology of neurodegenerative disorders. Using
neuronal experimental systems, we will discuss how compounds with low dose effects
mediated via glutamate receptors can result either in a neuroprotective or neurotoxic
response. Because the brain has evolved to respond rapidly to environmental cues, expo-
sure of neurons to stressful stimuli can result in a pivotal response toward either synaptic
adaptation or dysfunction and neuronal cell death. Understanding how neurons adapt to
stressful stimuli will provide important clues toward the development of strategies to pro-
tect the brain against neurodegeneration.

Key words: rat, neurons, BDNF, palytoxin, neuroprotection, basic helix loop helix B2

Abbreviations: AMPA, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate; CNQX, 6-cyano-7-

nitroquinoxaline-2,3-dione

Address correspondence to Ann M. Marini, Department of Neurology and Program in

Neuroscience, 1036A, Uniformed Services University of the Health Sciences, 4301 Jones Bridge
Road, Bethesda, Maryland 20814; Telephone: 301-295-9686; Telefax: 301-295-1471

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 Modulation of neuronal function by the environment

INTRODUCTION

Brain-derived neurotrophic factor is widely distributed in brain and

exhibits early immediate gene kinetics. BDNF exerts diverse function in
brain including neuronal cell maintenance, survival, neurogenesis, mat-
uration and plasticity (Kaplan and Miller, 2000; Miller and Kaplan, 2001;
Lee et al., 2002; Blondeau et al., 2009; Greenberg et al., 2009). BDNF plays
a critical role in long-term potentiation (LTP) presynaptically (Xu et al.,
2000) and postsynaptically (Kovalchuk et al., 2002) and in the mainte-
nance of high frequency stimulation-induced LTP in the adult rat
(Gärtner and Staiger, 1992). Many of these effects are mediated by the
high affinity receptor TrkB, which are localized on cell bodies and den-
drites as well as postsynaptically (Yan et al., 1997).
The human BDNF gene spans more than 70 kb and is composed of
seven 5' exons that are differentially spliced to a single 3' terminal exon
(Liu et al., 2005). Each 5' exon is transcribed from a unique promoter.
This complex transcriptional regulation leads to the creation of at least
three pre-pro-BDNF isoforms, the function of which is not completely
understood (Marini et al., 2004; Liu et al., 2005). This overall gene archi-
tecture is highly conserved in mammals. In rat, four promoters from
BDNF were characterized by Timmusk et al. (1993). The well-known BDNF
5' flanking region of exon 4 (previously known as exon 3 [Timmusk et al.,
1993]) is activated by membrane depolarization in cultured cortical and
hippocampal neurons. Other activators of BDNF exon 4–specific tran-
scription have been described, including glutamate via NMDA receptors
(Lipsky et al., 2001), kainate via non-NMDA receptors (Nakayama et al.,
1994), and dopamine (Fang et al., 2003), with at least three transcription
start sites mapped within an 80-bp region of DNA known to contain acti-
vation-dependent start sites (Timmusk et al., 1993; Nakayama et al., 1994;
Tao et al., 1998). Expression patterns of exon-specific BDNF transcripts in
the brain are developmentally regulated, but the mechanisms remain
largely unknown. However, activity-dependent neuronal plasticity result-
ing in the modulation of the synaptic strength of existing synaptic con-
nections and in the formation of new synaptic contacts, are regulated in
part by BDNF (Thoenen, 1995; Katz and Shatz, 1996; Lu and Figurov,
1997; Chao, 2003). These functions may require the synthesis of exon-
specific BDNF transcripts. Thus, differential BDNF promoter use may be
targeted by different signal transduction pathways to promote the diverse
functions of BDNF (Timmusk et al., 1994; McAllister et al., 1995; Ringstedt
et al., 1998; Kafitz et al., 1999; Poo, 2001; Pattabiraman et al., 2005; Wang
et al., 1995).
Transcription factors regulate gene expression by binding to specific
promoter sites most frequently encountered preceding the protein cod-
ing region of a gene. Our group discovered that basic helix loop helix B2
(BHLHB2) regulates promoter IV of the bdnf gene, a major promoter
417
 A. M. Marini and others

that drives activity-dependent BDNF expression (Jiang et al., 2008).

BHLHB2 is a transcriptional repressor and a nuclear protein (Jiang et al.,
2008). One of the most interesting features of BHLHB2 is the fact that
this protein is induced by hypoxia in cancer cell lines (Ivanova et al.,
2001). Thus, regulation of promoter IV of the bdnf gene by BHLHB2 may
occur under hypoxic as well as normoxic conditions and differences in
oxygen level may dramatically affect exon 4-specific-mediated BDNF
expression and possibly function.
Neuronal function is critically dependent upon receptors and signal
transduction, which impact transcriptional and translational processes
that are influenced by genetic and environmental (epigenetic) mecha-
nisms. Dysfunction in any one or a combination of these molecular
processes can lead to neuronal cell death by increasing the vulnerability
of neurons to ion fluxes, increased energy requirement via oxidative
stress, a reduction in existing intrinsic survival pathways, DNA damage,
and/or alterations in chromatin remodeling. Environmental factors may
also affect glutamate receptor-mediated neurotransmission. The out-
break of encephalopathy due to eating mussels contaminated with the
toxin domoic acid from the diatom Nitzschia pungens (Perl et al., 1990) is
an example of how food consumed by humans can affect the brain and
specifically alter glutamate receptor function (Debonnel et al. 1989,
Tasker et al. 1991, Novelli et al., 1992, Tasker and Strain 1998; Novelli et
al., 2005). Domoic acid is a tricarboxylic excitatory amino acid that binds
to and activates AMPA as well as kainic acid receptors with low and high
affinity respectively (Novelli et al., 2000). Moreover, the human neu-
ropathology caused by domoic acid in the hippocampus is similar to that
found in animals injected with kainic acid (Teitelbaum et al. 1990).
Attention has been raised recently on the potential risk for human health
that other compounds found in oceans may represent. One of the marine
compounds most toxic to mammals is Palytoxin, a polyalcohol molecule
synthesized by microalgae of the genus Ostreopsis (Moore and Scheuer,
1971; Usami et al., 1995), and represents a human health hazard (Alcala
et al., 1988; Onuma et al., 1999). Palytoxin selectively binds to and alter
the Na+/K+-ATPase pump (Bottinger et al. 1986, Habermann 1989,
Scheiner-Bobis et al. 1994, Artigas and Gadsby, 2003) and recently paly-
toxin has been shown to facilitate excitotoxicity via non-NMDA receptors
at subtoxic concentrations (Pérez-Gómez et al. 2010).
In this paper, we investigated whether alterations in oxygen levels may
affect BHLHB2 expression in cultured hippocampal neurons and modu-
late exon 4-specific BDNF mRNA levels, thus linking environmental
changes to either neuroprotection or excitotoxicity. Furthermore, we also
investigated whether palytoxin, a compound synthesized in the marine
environment, activates intracellular pathways leading to neuroprotection
as opposed to excitotoxicity, similar to the results found for the activation

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 Modulation of neuronal function by the environment

of glutamate receptor subtypes (Marini and Novelli, 1991; Marini and

Paul, 1992; Wu et al., 2004).

METHODS

Preparation of hippocampal neurons.

Hippocampi were prepared from embryonic day 20 fetuses obtained

from Sprague-Dawley dams (Taconic Farms, Germantown, NY). Tissue
from 8-10 fetuses was dissociated by mild trypsinization and trituration as
described (Cheng et al., 1995; Jiang et al., 2005; Jiang et al., 2008).
Hippocampal cells were then plated onto poly-D-lysine-coated culture
dishes. Hippocampal neurons were maintained under serum-free condi-
tions throughout the cultivation period. One-fifth of the medium was
removed and replaced with fresh medium every third day to replenish
nutrients. All experimental treatments were performed on neurons on
day in vitro (DIV) 7–8, which contained 90-95% neurons. Hippocampal
neurons cultured in serum-free medium have been extensively charac-
terized using neuronal and glial antibodies (Perry et al., 2003; Fu et al.,
2002 and references therein; Jiang et al., 2008). This research was con-
ducted according to the principles set forth in the National Institutes of
Health (NIH) Publication No. 85-23, Guide for the Care and Use of
Laboratory Animals, and the Animal Welfare Act of 1986, as amended.
Every effort was made to minimize the number of animals.

Cerebellar neuron cultures and neurotoxicology.

Primary cultures of rat cerebellar neurons were prepared as previ-

ously described (Novelli et al., 1988). Cerebellar neuronal survival was
maintained for more than 40 days in culture by replenishing the culture
medium with glucose every four days and replacing evaporative losses
with sterile water. The animal procedures used were in accordance with
the protocols approved by the Institutional Animal Care and Use
Committee of the University of Oviedo, Oviedo, Spain. Neurons were
used between 14 and 20 days in culture. Drugs were added into the
growth medium or the incubation buffer at the indicated concentrations,
and neuronal cultures were observed for signs of neurotoxicity thereafter
by phase contrast microscopy.

cGMP determination.

Intracellular cGMP concentration was determined as previously

reported (Novelli and Henneberry, 1987). Briefly, cultures were washed
with 1 ml prewarmed (37°C) incubation buffer containing (in mM): 154
NaCl, 5.6 KCl, 5.6 glucose, 8.6 HEPES, 1 MgCl2, 2.3 CaCl2, pH 7.4. Dishes
were incubated at 37°C for 10 min. with 1 ml new incubation buffer and
for an additional 20 min. with a second 1 ml new incubation buffer. Drugs
419
 A. M. Marini and others

were added at the end of the 20 min. incubation period for the indicated
times. Incubation was stopped by aspiration of the solution and addition
of 1 ml HClO4 (0.4 N). After neutralizing the perchlorate extract, cGMP
content was determined by radioimmunoassay (125I). Protein content was
determined on the membrane pellet from the same sample as described
previously (Novelli and Henneberry, 1987).

Determination of neuronal survival.

To quantify neuronal survival, cerebellar granule cell cultures were

stained with fluorescein diacetate and ethidium bromide (Novelli et al.,
1988), photographs of three randomly selected culture fields were taken
and live (green) and dead (red) neurons were counted. Results are
expressed as percentage of live neurons. Total number of neurons per
dish was calculated considering the ratio between the area of the dish and
the area of the pictures (~3000).

Drug treatments.

A subtoxic concentration of palytoxin (PTX, 100 pM) was added 3h

prior to the addition of a subtoxic concentration of domoic acid (DOM,
5 µM). 6-cyano-7-hydroxy-5-methylisoxazole-4-propionate (CNQX, 15
µM), a non-NMDA receptor antagonist, or MK-801 (1 µM), an NMDA
receptor antagonist, was added 5 min prior to DOM. Neuronal survival
was quantified 24h after addition of DOM and expressed as the percent-
age of live neurons.

Preparation of nuclear extracts.

Nuclear extracts were prepared according to the manufacturer’s

instructions (Active Motif Inc., Carlsbad, CA) with minor modifications.
Primary hippocampal neurons at day in vitro (DIV) 8 (7-8 x 105 neurons)
were washed with 0.7 ml ice-cold PBS plus Phosphatase Inhibitors (Active
Motif, Carlsbad, CA) for each 35 mm dish and replaced with 0.5 ml ice-
cold PBS/Phosphatase Inhibitor. Neurons were removed from the dish by
gentle scraping and the cells from three dishes pooled. The suspension
was centrifuged for 2 min at 100 x g, 10 min, 4°C. The cell pellet was gen-
tly resuspended in 250 μl Hypotonic Buffer (Active Motif, Carlsbad, CA )
and the suspension incubated at 4° C for 15 min followed by the addition
of Triton-x100 ( 12.5 μl) to each tube followed by brief vigorous shaking
by vortex ( 10 sec at the highest setting). The suspension in each tube was
centrifuged at 14,000 x g for 1 min in a microcentrifuge pre-cooled at
4°C. The supernatant (cytoplasmic fraction) from each tube was trans-
ferred into a clean pre-chilled microcentrifuge tube. The remaining
nuclear pellet was then resuspended in 50 μl Complete Lysis Buffer
(Active Motif, Carlsbad, CA) followed by vigorous shaking on a Vortex for

420
 Modulation of neuronal function by the environment

10 sec at the highest setting. The suspension in each microfuge tube was
incubated for 30 min on a rocking platform at 4°C followed by vigorous
shaking by vortex for 30 sec at the highest setting. The samples were cen-
trifuged for 10 min at 14,000 x g at 4°C. The supernatants from each
microfuge tube (nuclear fraction) were placed in a pre-chilled microcen-
trifuge tube and stored at -80°C. Protein concentration was determined
by the Bradford assay (BioRad, Hercules, CA).

Western blot analysis.

Western blot analyses were performed using forty μg of nuclear

extract protein. Proteins were first fractionated on 10% SDS-PAGE gels
and transferred to nitrocellulose membranes (BioRad, Hercules, CA).
The blots were then blocked with Tris-buffered saline containing 0.2%
Tween 20 (TBST) and 5% non-fat dry milk followed by three washes in
TBST. The blots were incubated with anti-BHLHB2 antibody (1:200,
Aviva Systems Biology, San Diego, CA) or anti-TATA box binding protein
(TBP, 1:1000, Sigma-Aldrich, St. Louis, MO) overnight at 4°C. TBP was
used as a nuclear protein marker to ensure equal loading of protein.
After three washes with TBST containing 5% non-fat dry milk, mem-
branes were incubated with the appropriate secondary antibody linked to
horseradish peroxidase for 1h. Immunoreactive bands were visualized
using the enhanced chemiluminescence detection system according to
the manufacturer’s recommendations (SuperSignal West Femto
Maximum Sensitivity Substrate, Thermo Scientific) [Wu et al., 2004].
Immunoactive bands were quantified by image analysis using a scanner
(Hewlett-Packard) and Image J analysis software (NIH, Bethesda, MD).
The fold of induction is the quotient of the density of the band divided
by the density of the band at time zero and normalized to TBP density. All
experiments were performed in triplicate using two different neuronal
preparations.

Oxygen deprivation/hypoxic conditions.

Hippocampal cultures were seeded in 35 mm Nunc® culture dishes as

described above. At the time of the experiment, serum-free medium con-
taining all nutrients (Jiang et al., 2008) was exchanged for deoxygenated
(60 min nitrogen aeration) serum-free medium (Skaper et al., 1991).
Cultures for hypoxia time-course experiments were placed in a humidi-
fied incubator containing 1% oxygen/5% CO2/94% N2 whereas control
cultures were harvested immediately after exposure to deoxygenated cul-
ture medium.
Quantitative real-time PCR. Hippocampal primary neurons from rat
cultured in 35 mm Nunc® culture dishes were lysed directly in the culture
dish by removing the culture medium and adding 0.5ml RNA STAT-60
(Qiagen, Germantown, MD). The cell lysates were transferred to new
421
 A. M. Marini and others

sterile Eppendorf tubes and allowed to stand at room temperature for 5

min. The samples were placed at -80°C for 1 h, followed by addition of
0.1ml chloroform per 1 ml of RNA-STAT-60, and the samples were shak-
en vigorously for 15 sec and allowed to stay at room temperature for 2-3
min. The cell lysates were centrifuged at 12,000 x g for 15 min at 4°C. The
upper aqueous phase was placed in a fresh Eppendorf tube and absolute
ethanol added such that the final volume was 53% ethanol v/v. Each sam-
ple was placed on an RNeasy spin column (Qiagen, Germantown, MD)
and centrifuged for 15 sec at 8000 x g. The column was washed first with
700 μl Buffer RW1 then twice of 500 μl Buffer RPE for spun for 1 min at
8000 x g. RNase-free water (30–50 μl) was added directly to the spin col-
umn membrane and centrifuged for 1 min at 8000 x g to elute the RNA.
The purified RNA was subjected to RNase-free DNase digestion (Ambion,
Austin, TX) and re-extracted prior to being stored at -20°C. The first
strand cDNA synthesis was prepared using Cloned AMV First –strand
cDNA Synthesis kit (Invitrogen, Carlsbad, CA) using 120 ng of total RNA
for each reaction. Levels of specific mRNAs were determined using real-
time fluorescence 5’-nuclease (TaqMan) assays. The TaqMan (Applied
Biosystems, Foster City, CA) assay using fluorogenic detection probes to
BHLHB2, exon 4 and exon 7 of BDNF were performed as described pre-
viously (Jiang et al., 2008). Levels of specific mRNA from hippocampal tis-
sue were normalized to endogenous Gapdh mRNA levels (rodent Gapdh
mRNA assay; Applied Biosystems, Foster City, CA). Fold differences of
specific mRNAs relative to control were calculated by an algorithm that
determines the threshold cycle (Ct) when an increase in reporter fluo-
rescence above a baseline signal is first detected during PCR. The
sequence detection software generates a calibration curve of Ct versus
amount of reference cDNA and then determines unknown amounts by
interpolation. Real-time fluorescence detection was performed using a
7700 Sequence Detector (Applied Biosystems, Foster City, CA). RT-PCR
assays were performed in duplicate or triplicate from two different neu-
ronal preparations.

Statistical analysis.

All values are presented as mean ± SD or percent of control (untreat-

ed). The Student t test was used for analysis; p < 0.05 is considered sig-
nificant.
Materials. 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX), AMPA,
amino-phosphono-valeric acid (APV) and domoic acid were purchased
from Tocris Cookson (Bristol, UK). NS102 was a gift from Dr. J. Drejer
(Neurosearch, Glostrup, Denmark), and palytoxin was obtained from
Calbiochem (Merck, UK). All other chemicals were obtained from com-
mercial sources.

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 Modulation of neuronal function by the environment

RESULTS

Hypoxia increases BHLHB2 protein expression in hippocampal neurons.

BHLHB2 is a protein expressed in brain (Rossner et al., 1997) and a

hypoxia-inducible gene in tumor cell lines (Ivanova et al., 2001) and can-
cer cells (Turley et al., 2004). However, the effect of hypoxia on this pro-
tein in primary neurons is unknown. We first investigated the effect of
oxygen deprivation/hypoxia on BHLHB2 protein expression by exposing
primary rat hippocampal neurons to hypoxic conditions (1% oxygen).
BHLHB2 protein expression is low following brief exposure of the neu-
rons to deoxygenated serum-free medium (control). We performed a
standard time course to determine whether BHLHB2 protein levels
increased under hypoxic conditions. Hippocampal neurons were subject-
ed to oxygen deprivation/hypoxia for various times (see Methods), con-
ditions that do not affect their viability throughout the duration of the
experiment (data not shown). BHLHB2 protein expression was quanti-
fied and compared to the TATA box binding protein, a nuclear protein
that remains constant over time. BHLHB2 protein expression increases
and peaks at 3h followed by a return to baseline levels within 24h
(Figure 1).
Our recent study showed that BHLHB2 represses promoter IV activi-
ty of the BDNF gene under normal (95% air/5% CO2) conditions (Jiang
et al., 2008). To determine the transcription expression levels of BHLHB2
and exon-specific BDNF levels under hypoxic conditions, cultured rat
hippocampal neurons were subjected to oxygen deprivation/hypoxic
conditions for various times; BHLHB2 and BDNF exon 4- and exon 7-spe-
cific mRNA levels were quantified by real-time PCR. BHLHB2 mRNA lev-
els increase and reach a peak of 5.5-fold at 6h while exon 4-specific BDNF
transcripts increase rapidly to 5.6-fold at 2h but then decrease steadily
over the remainder of the experiment. Coincidently, the decline in exon
4-specific BDNF transcripts occurs when BHLHB2 mRNA levels increase
over the same time course. Exon 7-specific BDNF transcripts, the termi-
nal exon in rat that synthesizes mature BDNF protein (Marini et al., 2004)
rises more slowly reaching a maximum of 2.6-fold at 5h and then decreas-
es over the next three hours during the same time that BHLHB2 mRNA
levels are maximal (Figure 2).

Palytoxin evokes neuroprotection or excitotoxicity in cultured cerebellar

granule cells.

Palytoxin modulation of non-NMDA glutamate receptors activated by

domoic acid is one means by which chemicals synthesized by oceanic
plants affect neuronal function. Thus, exposure of cultured cerebellar
neurons to domoic acid (DOM) elicits a potent concentration-dependent
increase in intracellular cGMP with an EC50 of ≈ 4µM and an EC100 of

423
 A. M. Marini and others

FIGURE 1. Hypoxia increases BHLHB2 protein expression in cultured hippocampal neurons.

Cultured hippocampal neurons were exposed to hypoxia in a humidified incubator for various times.
At the indicated time, the neurons were harvested, nuclear extracts prepared followed by a standard
western blot to detect BHLHB2 and TBP. Results are expressed as the mean ± SD (n = 6).

7.5µM (Figure 3a). The increase in cGMP elicited by DOM (10 µM) is
fully blocked by the non-NMDA receptor antagonist CNQX but not by
the NMDA receptor antagonists APV or MK-801 (Figure 3b).
Furthermore, DOM stimulation of cGMP can be reduced by preventing
activation of either AMPA or kainate receptors with either the desensitiz-
ing agonist AMPA or with NS-102, an antagonist of kainate receptors
(Figure 3b) (Gouaux 2004, Lerma et al. 1993). A twenty four hour expo-
sure of cultured cerebellar neurons to 5 µM DOM fails to elicit excito-
toxicity (Figure 4). However, when cultures were pretreated with a
subtoxic concentration of palytoxin (PTX, 100 pM), the exposure to
DOM (5µM) results in a dramatic reduction in neuronal survival after
24h that can be prevented by the prior addition of CNQX but not MK-801
(Figure 4). The facilitation of DOM toxicity by palytoxin is dependent
upon the length of time the cultures are pretreated with this marine com-
pound. A significant facilitation is obtained after 5 min, and a maximal
facilitation is achieved after a 5h pretreatment time. Longer pretreatment
times with palytoxin results in a progressive reduction of excitotoxic facil-

424
 Modulation of neuronal function by the environment

FIGURE 2. Hypoxia-induced changes in exon 4- and exon 7-specific BDNF and BHLHB2 mRNA lev-
els in cultured hippocampal neurons. Cultured rat hippocampal neurons were exposed to hypoxic
conditions for various times. At the indicated time, neurons were harvested and total RNA isolated.
Exon 4- and exon 7-specific BDNF and BHLHB2 mRNA levels were quantified by the 5’ nuclease
assay as described elsewhere (Jiang et al., 2008). The fluorescence of the mean of each experiment
was compared with glyceraldehydes phosphate dehydrogenase (GAPDH) mRNA and is expressed as
relative fluorescence. Each experiment was performed in triplicate using two different preparations
of neurons (n = 6).

FIGURE 3. Domoic acid stimulates intracellular cGMP formation in a concentration-dependent

manner via non-NMDA receptors. Panel a: Intracellular concentration of cGMP was measured in
cerebellar cultures at 12-14 day in culture (DIC) following 1 min stimulation with increasing con-
centrations of domoic acid (DOM). Intracellular cGMP content was determined by radioimmunoas-
say. Represented values are the mean ± SD of duplicate values from three independent experiments.
Panel b: Pharmacological characterization of cGMP formation following domoic acid stimulation was
measured at 12-14 DIC. All drugs were added 10 min before domoic acid (DOM 10 µM) and cultures
were exposed to DOM for 1 min. Concentrations were: CNQX 15µM, MK801 (MK) 2µM, APV 1mM,
AMPA 200µM, NS102 10µM, Quisqualic acid (QUIS, 100µM). Represented data are the mean ± SD
of duplicate values from three independent experiments. *p < 0.01 by student t test vs. DOM alone.

425
 A. M. Marini and others

FIGURE 4. The non-NMDA receptor antagonist, CNQX, blocks domoic-induced neuronal cell death
enhanced by palytoxin. A subtoxic concentration of palytoxin (PTX, 100 pM) was added to cultured
rat cerebellar granule cells 3h prior to the addition of a subtoxic concentration of domoic acid
(DOM, 5 µM), The percentage of viable and degenerating neurons quantified. The addition of
domoic acid (5 µM) to neurons previously exposed to palytoxin reduces neuronal survival by 70%.
Addition of CNQX, a non-NMDA receptor antagonist, but not with MK-801, an NMDA receptor
antagonist, completely blocks the excitotoxic effect. No effect on neuronal survival was observed with
domoic acid or palytoxin alone compared with untreated neurons.

itation by DOM and disappears after a 48h palytoxin pretreatment time

(Figure 5). To exclude that this unexpected effect could be due to the
inactivation or degradation of the marine compound, palytoxin (100 pM)
was added to the culture medium of a group of neuronal cultures for 48h.
Then culture medium from naïve sister cultures was removed and
replaced with either that of 48h palytoxin-treated cultures (48h PTX-con-
taining 48h-CM) or with culture medium from untreated cultures to
which palytoxin was freshly added (PTX). Three hours later, either a vehi-
cle solution or a subtoxic concentration of domoic acid (5 µM) was added
to the medium of each culture group and neuronal survival was quanti-
fied 24 hours later. The addition of DOM in neuronal cultures where the
culture medium was replaced with 48h PTX-containing 48h-CM results in
a reduction in neuronal survival identical to that observed in cultures
where palytoxin was freshly added whereas exchanging and replacing sis-
ter culture medium from naïve cultures followed by the addition of only
DOM did not affect neuronal viability (Figure 6). These results support
the idea that effects other than degradation or inactivation of palytoxin
are responsible for the increase in neuronal survival following non-
NMDA receptor stimulation by DOM when neurons are exposed to
subtoxic concentrations of palytoxin over prolonged exposure times.

426
 Modulation of neuronal function by the environment

FIGURE 5. Palytoxin enhancement of domoic acid-induced excitotoxicity is biphasic and depends

upon the length of exposure time. Cultured rat cerebellar granule cells were treated with a subtoxic
concentration of palytoxin (100 pM, open circles) for various times (5 min- 48h) prior to the addi-
tion of a subtoxic concentration of domoic acid (DOM, 5 µM). Addition of palytoxin alone (100 pM)
does not affect neuronal survival (closed circles). Represented data are the mean ± SD of duplicate
values from three independent experiments (n = 6).

FIGURE 6. Palytoxin is not inactivated following long-term incubation with cultured neurons. A
subtoxic concentration of palytoxin (PTX, 100 pM) was added to cultured rat cerebellar granule cells
for 48h. Palytoxin-containing media (palytoxin-containing 48h-CM [conditioned media]) was trans-
ferred to naïve cultures followed by the addition of a subtoxic concentration of domoic acid (DOM,
5 µM) and neuronal survival was quantified 24h later. Addition of medium from control cultures
alone (control 48h-CM) to naïve cultures in the presence or absence of a subtoxic concentration of
domoic acid (DOM, 5 µM) served as the control. Addition of a subtoxic concentration of palytoxin
for 3h followed by the addition of a subtoxic concentration of domoic acid (DOM, 5 µM) freshly
added resulted in an excitotoxic response and served as the positive control (n = 6).

427
 A. M. Marini and others

DISCUSSION

The results in this paper show that BHLHB2 is regulated by hypoxia

in cultured hippocampal neurons. The apparent inverse relationship
between BHLHB2 mRNA and exon 4-specific BDNF mRNA levels is an
interesting association because BHLHB2 binds to and represses promot-
er IV of the bdnf gene, a major promoter that drives activity-dependent
BDNF expression (Jiang et al., 2008). Additional experiments will deter-
mine the role of BHLHB2 in regulating exon 4-specific BDNF mRNA lev-
els in cultured neurons under hypoxic and preconditioning conditions.
Taken together, modulation of oxygen is one of many mechanisms
responsible for changes in neuronal gene and protein expression activat-
ed by ionotropic glutamate receptors. Elucidating the role of BHLHB2 in
models of preconditioning (Marini et al., 2008) may contribute to delin-
eating additional neuroprotective pathways and toward more efficacious
drugs to protect the brain.
Organisms in the oceans synthesize a vast array of compounds, some
of which may alter the function of ionotropic glutamate receptors
through synergistic mechanisms that determine cell fate. Palytoxin is a
compound synthesized by oceanic microalgae and transforms the Na+/K+
ATPase pump into a non-selective channel permeable to monovalent
cations (Scheiner-Bobis et al., 1994). The results presented in this paper
show that modulation of the Na+/K+-ATPase activity by palytoxin plays a
crucial role in determining neuronal susceptibility to non-NMDA recep-
tor-mediated excitotoxicity, confirming and expanding previous results
by Pérez-Gómez et al. (2010). Subtoxic concentrations of palytoxin or
domoic acid alone do not affect neuronal survival. Addition of a subtox-
ic concentration of domoic acid three hours after the addition of a
subtoxic concentration of palytoxin results in a rapid excitotoxic
response. The excitotoxic effect is mediated by non-NMDA receptors
because excitotoxicity is completely blocked by prior treatment with
CNQX, a non-NMDA receptor antagonist. It is worth noting that the
subtoxic concentration of domoic acid used (5µM) potently stimulates
the intracellular formation of the second messenger cGMP, and that the
reduction to a similar extent of cGMP stimulation during exposure to a
toxic concentration of DOM (10 µM) did not prevent excitotoxicity
(Figure 3b and data not shown), suggesting that the pathway leading to
the formation of this messenger is not involved in excitotoxicity (Novelli
et al. 2005). Curiously, palytoxin does not affect susceptibility to NMDA
receptor-mediated excitotoxicity (Figure 3 and Pérez-Gómez et al. 2010).
In addition, palytoxin exerts its own dose effect depending upon the
incubation time. That is, the longer palytoxin is exposed to the neurons
the lower the excitotoxicity following domoic acid. Longer exposure
times of palytoxin do not result in the inactivation of the compound
because a prompt excitotoxic effect is observed when the palytoxin-con-
428
 Modulation of neuronal function by the environment

taining medium is added to naïve neuronal cultures followed by the addi-

tion of domoic acid (Figures 5-6). The mechanism of this differential
effect remains elusive but it is possible that cellular adaptation at the level
of the Na+/K+-ATPase or downstream factors may be responsible for the
reduced effect on excitotoxicity.
Modulation of NMDA receptors or Na+/K+-ATPase may result in alter-
ations in brain function. It has been shown that activation of NMDA
receptors is required to protect vulnerable brain regions against hypoxic-
ischemic insults in models of preconditioning (Kirino, 2002; Chazot et al.,
2002) and against traumatic brain injury (Ikonomidou et al., 2000).
Activity-dependent release of BDNF through direct activation of NMDA
receptors (Marini et al., 1998) or in a model of memory known to require
NMDA receptors (Gartner and Staiger, 2002) may provide important
clues as to how low dose effects modulate neuronal function to protect
the brain against neuropathological conditions known to involve gluta-
mate as a final common pathway. Thus, activity-dependent processes via
NMDA receptors and neurotrophins such as BDNF may be required for
neurons to promote their own survival such that in the absence of oxygen
and glucose, overactivation of NMDA receptors leads to neuronal cell
death. Likewise, a reduction in Na+/K+-ATPase function either through
decreases in activity or density is known to occur in hypoxic-ischemic neu-
ronal injury and following the administration of glutamate receptor ago-
nists (Mintorovitch et al., 1994; Lees, 1991; Tavalin et al., 1997). In con-
trast, an increase in Na+/K+-ATPase is a crucial event in ischemic precon-
ditioning (Tian et al., 2008). The results showing that palytoxin modu-
lates non-NMDA receptor-mediated neuronal cell death may have impor-
tant implications in neuronal function and dysfunction in the brain.
Because palytoxin and domoic acid are seafood contaminants, ingestion
of low concentrations (possibly below those actually allowed in seafood)
of both toxins may have opposing effects on human health depending
upon the amounts and the kinetics of the drugs crossing the blood brain
barrier.
In summary, low dose effects may have profound effects on brain
function. Delineating the mechanisms of low dose effects in the brain
may provide important insights on neuronal function and lead to com-
pounds that enhance efficacy and reduce unwanted side effects.

ACKNOWLEDGMENT

This work was supported by the Defense Threat Reduction Agency,

grant numbers CBM.NEURO.01.10.US.019 (AMM), CBM.NEURO.01.10.
US.012 (AMM) and by CICYT, Grants AGL2004-08241-C04-04/ALI,
AGL2005-07924-C04-03/ALI and CTQ2008-06754-C04-03/PPQ (AN and
MTFS). A. Pérez-Gómez was a fellow of FICYT.

429
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REFERENCES
Alcala AC, Alcala LC, Garth JS, Yasumura D, and Yasumoto T. 1988. Human fatality due to ingestion
of the crab Demania reynaudii that contained a palytoxin-like toxin. Toxicon 26, 105-107.
Artigas P and Gadsby DC. 2003. Na+/K+-pump ligands modulate gating of palytoxin-induced ion
channels. Proc.Natl.Acad.Sci. USA 100, 501-505.
Blondeau N, Nguemeni C, DeBruyne DN, Piens M, Wu X, Pan H, Hu X, Gandin C, Lipsky RH,
Plumier J-C, Marini AM, and Heurteaux C. 2009. Subchronic alpha-linolenic acid treatment
enhances brain plasticity and exerts and antidepressant effect: A versatile potential therapy for
stroke. Neuropsychopharmacology 34:2548-2559.
Bottinger H, Beress L, and Habermann E. 1986. Involvement of (Na++K+)-ATPase in binding and
actions of palytoxin on human erythrocytes. Biochem.Biophys. Acta 861, 165-176.
Chao MV. 2003. Neurotrophins and their receptors: a convergence point for many signaling path-
ways. Nat. Rev. Neurosci. 4:299–309.
Chazot PL, Godukhin OV, McDonald A, Obrenovitch TP. 2002. Spreading depression-induced pre-
conditioning in the mouse cortex: differential changes in the protein expression of ionotropic
nicotinic acetylcholine and glutamate receptors. J Neurochem 83:1235-1238.
Cheng B, Furukawa K, O’Keefe JA, Goodman Y, Kihiko M, Fabian T, and Mattson MP. 1995. Basic
fibroblast growth factor selectively increases AMPA-receptor subunit GluR1 protein level and
differentially modulates Ca2+ responses to AMPA and NMDA in hippocampal neurons. J
Neurochem 65:2525-2536.
Debonnel G, Beauchesne L, and de Montigny C. 1989. Domoic acid, the alleged “mussel toxin,”
might produce its neurotoxic effect through kainate receptor activation: an electrophysiologi-
cal study in the dorsal hippocampus. Can J Physiol Pharmacol 67:29–33.
Fang H, Chartier J, Sodja C, Desbois A, Ribecco-Lutkiewicz M, Walker PR, and Sikorska M. 2003.
Transcriptional activation of the human brain-derived neurotrophic factor gene promoter III by
dopamine signaling in NT2/N neurons. J. Biol. Chem. 278:26401–26409.
Fu W, Lu C, and Mattson MP. 2002. Telomerase mediates the cell survival-promoting actions of brain-
derived neurotrophic factor and secreted amyloid precursor protein in developing hippocam-
pal neurons. J Neurosci 22:10710-10719.
Gärtner A, and Staiger V. 1992. Neurotrophin secretion from hippocampal neurons evoked by long-
term-potentiation-inducing electrical stimulation patterns. Proc Natl Acad Sci 99:6386-6391.
Gouaux E. 2004. Structure and function of AMPA receptors. J. Physiol. 554: 249-253.
Greenberg ME, Xu B, Lu B, and Hempstead B. 2009. New insights in the biology of BDNF synthesis
and release: implications in CNS function. J Neurosci 29:12764-12767.
Habermann E. 1989. Palytoxin acts through Na+,K+-ATPase. Toxicon 27, 1171-1187.
Ikonomidou C, Stefovska V, and Turski L. 2000. Neuronal death enhanced by N-methyl-D-aspartate
antagonists. Proc Natl Acad Sci USA 97:12885-12890.
Ivanova AV, Ivanov SV, Danilkovitch-Miagkova A, Lerman MI. 2001. Regulation of STRA13 by the von
Hippel-Lindau tumor suppressor protein, hypoxia, and the UBC9/ubiquitin proteasome degra-
dation pathway. J Biol Chem 276:15306-15315.
Jiang X, Tian F, Mearow K, Okagaki P, Lipsky RH, and Marini AM. 2005. The excitoprotective effect
of N-methyl-D-aspartate is mediated by a BDNF autocrine loop in cultured hippocampal neu-
rons. J Neurochem 94:713–722.
Jiang X., Tian F, Du Y, Copeland NG, Jenkins NA, Tessarollo L, Wu X, Pan H, Xu K, Kenney H, Egan
SE, Turley H, Harris AL, Marini AM., Lipsky RH. 2008 BHLHB2 controls Bdnf promoter 4 activ-
ity and neuronal excitability. J. Neurosci 28:1118-1130.
Kafitz KW, Rose CR, Thoenen H, and Konnerth A. 1999. Neurotrophin-evoked rapid excitation
through TrkB receptors. Nature 401:918–921.
Kaplan DR, and Miller FD. 2000. Neurotrophin signal transduction in the nervous system. Curr Opin
Neurobiol. 10:381-391.
Katz LC, and Shatz CJ. 1996. Synaptic activity and the construction of cortical circuits. Science
274:1133–1138.
Kirino T. 2002. Ischemic tolerance. J Cereb Blood Flow Metab 22(11):1283-1296.
Kovalchuk Y, Hanse YE, Kafitz KW, and Konnerth A. 2002. Postsynaptic induction of BDNF-mediat-
ed long-term potentiation, Science 295:1729-1734.

430
 Modulation of neuronal function by the environment

Lee J, Duan W, and Mattson MP. 2002. Evidence that brain-derived neurotrophic factor is required
for basal neurogenesis and mediates, in part, the enhancement of neurogenesis by dietary
restriction in the hippocampus of adult mice. J Neurochem 82:1367-1375.
Lees GJ. 1991. Inhibition of sodium-potassium-ATPase: a potentially ubiquitous mechanism con-
tributing to central nervous system neuropathology. Brain Res Brain Res Rev. 16:283-300.
Lerma J. Paternain AV, Naranjo JR, Mellstrom B. 1993. Functional kainate-selective glutamate recep-
tors in cultured hippocampal neurons. Proc. Natl. Acad. Sci. USA 90: 11688-11692.
Lipsky RH, Xu K, Zhu D, Kelly C, Terhakopian A, Novelli A, and Marini AM. 2001. Nuclear factor
kappaB is a critical determinant in N-methyl-D-aspartate receptor-mediated neuroprotection. J.
Neurochem. 78:254–264.
Liu QR, Walther D, Drgon T, Polesskaya O, Lesnick TG, Strain KJ, de Andrade M, Bower J.H,
Maraganore DM, and Uhl GR. 2005. Human brain derived neurotrophic factor (BDNF) genes,
splicing patterns, and assessments of associations with substance abuse and Parkinson’s Disease.
Am. J. Med. Genet. B Neuropsychiatr. Genet. 134:93–103.
Lu B, and Figurov A. 1997. Role of neurotrophins in synapse development and plasticity. Rev
Neurosci 8:1–12.
McAllister AK, Lo DC, and Katz LC. 1995. Neurotrophins regulate dendritic growth in developing
visual cortex. Neuron 15:791–803.
Marini AM, and Novelli A. 1991. The glutamate uptake blocker DL-threo-3-hydroxyaspartate reduces
NMDA receptor activation by glutamate in cultured neurons. European J. Pharm. 194:131-132.
Marini AM, and Paul SM. 1992. N-Methyl-D-aspartate receptor-mediated neuroprotection in cerebel-
lar granule cells requires new RNA and protein synthesis. Proc. Natl. Acad. Sci. USA 89:6555-
6559.
Marini AM, Rabin SI, Lipsky RH, and Mocchetti I. 1998. Activity-dependent release of brain derived
neurotrophic factor underlies the neuroprotective effect of N-methyl-D-aspartate. J. Biol. Chem.
1998 273(45):29394-29399.
Marini A.M, Jiang X, Wu X, Tian F, Zhu D, Okagaki P, and Lipsky RH. 2004. Role of brain-derived
neurotrophic factor and NF-kappaB in neuronal plasticity and survival: From genes to pheno-
type. Restor. Neurol. Neurosci. 22, 121–130.
Marini AM, Popolo M, Pan H, Blondeau N, Lipsky RH. 2008. Brain adaptation to stressful stimuli: A
new perspective on potential therapeutic approaches based on BDNF and NMDA receptors.
CNS and Neurological Disorders – Drug Targets 7:382-390.
Miller FD, and Kaplan DR. 2001. Neurotrophin signalling pathways regulating neuronal apoptosis.
Cell Mol Life Sci. 58:1045-1053.
Mintorovitch J, Yang GY, Shimizu H, Kucharczyk J, Chan PH, Weinstein PR. 1994 Diffusion-weighted
magnetic resonance imaging of acute focal cerebral ischemia: comparison of signal intensity
with changes in brain water and Na+,K(+)-ATPase activity. J Cereb Blood Flow Metab. 14:332-
336.
Moore RE, and Scheuer P. J. 1971. Palytoxin: a new marine toxin from a coelenterate. Science
172:495-498.
Nakayama M, Gahara Y, Kitamura T, and Ohara O.1994. Distinctive four promoters collectively direct
expression of brain-derived neurotrophic factor gene. Brain Res. Mol. Brain Res. 21, 206–218.
Novelli A, and Henneberry RC. 1987. cGMP synthesis in cultured cerebellar neurons is stimulated by
glutamate via a Ca2+-mediated, differentiation-dependent mechanism. Dev. Brain. Res. 34: 307-
310.
Novelli A, Reilly JA, Lysko PG, Henneberry RC. 1988. Glutamate becomes neurotoxic via the N-
methyl-D-aspartate receptor when intracellular energy levels are reduced. Brain Res. 451:205-
212.
Novelli A, Kispert J, Fernández-Sánchez MT, Torreblanca A, Zitko V. 1992. Domoic acid-containing
toxic mussels produce neurotoxicity in neuronal cultures through a synergism between excita-
tory amino acids. Brain Research 577:41-48.
Novelli A., Fernández-Sánchez M.T., Doucette T.A., and Tasker RAR. 2000. Amnesic toxic episodes:
chemical and biological detection methods, in: Seafood and freshwater toxins. Pharmacology,
physiology, and detection, (Botana, L M., ed), pp. 383-400. Marcel Dekker, Inc., New York, N.
Y., USA.
Novelli A, Perez-Basterrechea M, Fernández-Sánchez MT. 2005. Glutamate and Neurodegeneration.
In: Dopamine and Glutamate in Psychiatric Disorders. Schmidt WJ, Reith MEA, Eds. Humana
Press Inc. Totowa, NJ, USA, pg. 447-472, 2005.

431
 A. M. Marini and others

Onuma Y, Satake M, Ukena T, Roux J, Chanteau S, Rasolofonirina N, Ratsimaloto M, Naoki H, and

Yasumoto T. 1999. Identification of putative palytoxin as the cause of clupeotoxism. Toxicon
37:55-65.
Pattabiraman PP, Tropea D, Chiaruttini C, Tongiorgi E, Cattaneo A, and Domenici L. 2005. Neuronal
activity regulates the developmental expression and subcellular localization of cortical BDNF
mRNA isoforms in vivo. Mol Cell Neurosci 28:556–570.
Pérez-Gómez A, Novelli A, Fernández-Sánchez MT. 2010. Na+/K+-ATPase inhibitor palytoxin
enhances vulnerability of cultured cerebellar neurons to domoic acid via sodium-dependent
mechanisms. J Neurochem 114:28-38.
Perl TM, Bédard L, Kosatsky T, Hockin JC, Todd ECD, and Remis RS. 1990. An Outbreak of Toxic
Encephalopathy Caused by Eating Mussels Contaminated with Domoic Acid. New Engl J Med
322:1775-1780.
Perry T, Lahiri DK, Sambamurti K, Chen D, Mattson MP, Egan JM, and Greig NH. 2003. Glucagon-
like peptide-1 decreases endogenous amyloid-beta peptide (Abeta) levels and protects hip-
pocampal neurons from death induced by Abeta and iron. J Neurosci Res 72(5):603-612.
Poo M.-M. 2001. Neurotrophins as synaptic modulators. Nat. Rev. Neurosci. 2:1–9.
Ringstedt T, Linnarsson S, Wagner J, Lendahl U, Kokaia Z, Arenas E, Ernfors P, and Ibanez CF. 1998.
BDNF regulates reelin expression and Cajal-Retzius cell development in the cerebral cortex.
Neuron 21:305–315.
Rossner MJ, Dörr J, Gass P, Schwab MH, and Nave K-A. 1997. SHARPs: Mammalian enhancer-of-split-
and hairy-related proteins coupled to neuronal stimulation. Mol Cell Neurosci 9, 460–475.
Scheiner-Bobis G, Meyer zu Heringdorf D, Christ M, and Habermann E. 1994. Palytoxin induces K+
efflux from yeast cells expressing the mammalian sodium pump. Mol Pharmacol 45:1132-1136.
Skaper SD, Leon A, and Facci L. 1991. Death of cultured hippocampal pyramidal neurons induced
by pathological activation of N-methyl-D-aspartate receptors is reduced by monsialoganglio-
sides. J Pharmacol Exp Ther 259:452-457.
Tao X, Finkbeiner S, Arnold DB, Shaywitz AJ, and Greenberg, ME. 1998. Ca2+ influx regulates BDNF
transcription by a CREB family transcription factor-dependent mechanism. Neuron 20:709–726.
Tasker RA, Connell BJ, Strain SM. 1991. Pharmacology of systemically administered domoic acid in
mice. Can J Physiol Pharmacol. 69(3):378-382.
Tasker RA, Strain SM. 1998. Synergism between NMDA and domoic acid in a murine model of behav-
ioural neurotoxicity. Neurotoxicology.19(4-5):593-597.
Tavalin SJ, Ellis EF, Satin LS. 1997. Inhibition of the electrogenic Na pump underlies delayed depo-
larization of cortical neurons after mechanical injury or glutamate. J Neurophysiol. 77:632-638.
Teitelbaum JS, Zatorre RJ, Carpenter S, Gendron D, Evans AC, Gjedde A, Cashman NR. 1990.
Neurological sequelae of domoic acid intoxication due to the ingestion of contaminated mus-
sels. N Engl J Med 322; 1781-1787.
Tian D, Dmitrieva RI, Doris PA, Crary JF, Sondhi R, Sacktor TC, Bergold PJ. 2008. Protein kinase M
zeta regulation of Na/K ATPase: a persistent neuroprotective mechanism of ischemic precon-
ditioning in hippocampal slice cultures. Brain Res. 5;1213:127-139.
Timmusk T, Palm K, Metsis M, Reintam T, Paalme V, Saarma M, and Persson H. 1993. Multiple pro-
moters direct tissue-specific expression of the rat BDNF gene. Neuron 10:475–489.
Timmusk T, Belluardo N, Persson H, and Metsis M. 1994. Developmental regulation of brain-derived
neurotrophic factor messenger RNAs transcribed from different promoters in the rat brain.
Neuroscience 60:287–291.
Thoenen H. 1995. Neurotrophins and neuronal plasticity. Science 270:593–596.
Turley H, Wykoff CC, Troup S, Watson PH, Gatter KC, and Harris AL. 2004. The hypoxia-regulated
transcription factor DEC1 (Stra13, BHLHB2) and its expression in human tissues and tumours.
J Pathol 203:808-813.
Usami M, Satake M, Ishida S, Inoue A, Kan Y, and Yasumoto T. 1995. Palytoxin analogs from the
dinoflagellate Ostreopsis siamensis. J. Am. Chem. Soc. 117, 5389-5390.
Wang T, Xie K, and Lu B. 1995. Neurotrophins promote maturation of developing neuromuscular
synapses. J. Neurosci. 15:4796–4805.
Wu X, Zhu D, Jiang X, Okagaki P, Mearow K, Zhu G, McCall S, Banaudha K, Lipsky RH, and Marini
AM. 2004. AMPA protects cultured neurons against glutamate excitotoxicity through a phos-
phatidylinositol 3-kinase-dependent activation in extracellular signal-regulated kinase to upreg-
ulate BDNF gene expression. J Neurochem 90:807-818.

432
 Modulation of neuronal function by the environment

Xu B, Gottschalk W, Chow A, Wilson RI, Schnell E, Zang K, Wang D, Nicoll RA, Lu B, and Reichardt
LF. 2000. The role of brain-derived neurotrophic factor receptors in the mature hippocampus:
modulation of long-term potentiation through a presynaptic mechanism involving TrkB. J
Neurosci 20:6888-6897.
Yan Q, Radeke MJ, Matheson CR, Talvenheimo J, Welcher AA, and Feinstein SC. 1997.
Immunocytochemical localization of TrkB in the central nervous system of the adult rat, J Comp
Neurol. 378:135-157.

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