Neuroscience 151 (2008) 155163

NONGENOMIC REGULATION OF GLUTAMATERGIC

NEUROTRANSMISSION IN HIPPOCAMPUS BY THYROID HORMONES
G. LOSI, G. GARZON AND G. PUIA*

the hippocampus (Gilbert et al., 2003; Gilbert, 2004).

Changes in circulating THs may also modify brain functions in adults. The link between altered THs levels and
mental disorders such as cognitive impairment, depression
and anxiety is well documented (Burmeister et al., 2001;
Haggerty et al., 1990; Simon et al., 2002; Gulseren et al.,
2006) but the underlying mechanism is not well understood
yet. It is known that THs regulate noradrenergic and serotonergic function by modulating the expression of -adrenergic receptors and 5-HT1 autoreceptors (Lifschytz et al.,
2006).
In the last decade several groups have shown that THs
exert also important nongenomic effects (Bassett et al.,
2003; Davis et al., 2005) such as the modulation of Na,
K and Ca2 channels, the activation of several types of
protein kinases and extracellular signal-regulated/mitogen
activated kinases (ERK/MAPK) pathways via V3 integrin
(Bergh et al., 2005). There is substantial evidence also for
a complex interaction between GABAergic system and
THs, as recently reviewed by Wiens and Trudeau (2006).
THs modulate GABA production, metabolism, release and
uptake. Importantly THs can also regulate GABAA receptor
function (Martin et al., 1996; Chapell et al., 1998) in analogy with some structurally similar sulfonated neurosteroids
(Martin et al., 2004).
THs were shown to act on glutamatergic system: they
inhibit L-(3H) glutamate binding (Oguro, 1989) and regulate N-methyl-D-aspartate (NMDA) receptor subunit expression in hippocampus (Lee et al., 2003). Hippocampus,
that is a substrate for some forms of learning and memory,
is particularly sensitive to THs (Madeira et al., 1992).
Gerges and Alkadhi (2004) showed that hypothyroidism
reduces long-term potentiation (LTP) at Schaffers collateralCA1 synapses in hippocampus while T4 administration induces LTP (Alzoubi et al., 2005).
Because of the link between THs and brain excitability
we investigated the direct nongenomic effect of these hormones on ionotropic glutamate receptors expressed in
hippocampal neurons grown in culture and in acutely dissociated hippocampal slices.

Department of Biomedical Science, Pharmacology Section, University

of Modena and Reggio Emilia, via Campi 287, 41100 Modena, Italy

AbstractThyroid hormones (THs) are well known for their

genomic effects but recently attention has focused also on
their nongenomic actions as rapid modulators of membrane
receptors. Here we show that thyroxine (T4) and 3,3=,5=-Ltriiodothyronine (T3) rapidly decrease N-methyl-D-aspartate
(NMDA)-evoked currents in rat hippocampal cultures with
potency in the micromolar range. The effect is not mediated
by glutamate or glycine binding sites as an increase in agonist or glycine concentration does not alter TH potencies.
Furthermore THs effect on NMDA receptors is independent
of voltage and of subunit composition. The mechanism of
THs antagonistic effect does not involve PKC phosphorylation of NMDA receptors since neither blocking nor stimulating PKC changed THs modulation. T3, but not T4, inhibits
also kainate-evoked currents in hippocampal neurons in culture. In hippocampal pyramidal neurons in slice, T3, but not
T4, significantly reduced the frequency of miniature excitatory postsynaptic currents (mEPSCs) without affecting their
amplitude and decay.
In cultured rat cortical neurons THs prevented glutamateinduced neuronal death at concentrations similar to those
effective on glutamatergic receptors. Taken together our data
show for the first time that THs can rapidly affect ionotropic
glutamatergic receptors in hippocampal neurons, an effect
that could have an important role in their modulation of brain
function in physiological and pathological states. 2008
IBRO. Published by Elsevier Ltd. All rights reserved.
Key words: thyroxine, 3,3=,5=-L-triiodothyronine, NMDA receptors, AMPA receptors, patch-clamp.

Brain development and maturation are profoundly affected

by thyroid hormones (THs). Indeed TH deficiency during
development, even for short periods, may cause permanent modifications of brain morphology and functions (reviewed by Bernal, 2002). These changes, that depend on
the time and duration of THs deficit (Zoeller and Rovet,
2004), may produce altered behavior and impaired cognition, which correlate with a reduced synaptic plasticity in
*Corresponding author. Tel: 39-592055135; fax: 39-592055376.
E-mail address: puja@unimore.it (G. Puia).
Abbreviations: BME, basal Eagles Medium; DIV, days in vitro; DME,
Dulbeccos Modified Basal Eagles medium; IC50, agonist concentration eliciting the half-maximal response; KA, kainic acid; LTP, longterm potentiation; mEPSC, miniature excitatory post-synaptic current;
MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; NB, Neurobasal medium; NMDA, N-methyl-D-aspartate; PD,
postnatal day; PMA, phorbol 12-myristate 13-acetate; Ra, access
resistance; rev-T3, 3,3=,5=-L-triiodothyronine; SYM 2081, (2S,4R)-4methylglutamic acid; TH, thyroid hormone; TR, thyroid receptor; TTX,
tetrodotoxin.

EXPERIMENTAL PROCEDURES
Primary cultures of hippocampal neurons
Primary culture of hippocampal neurons were prepared from newborn (postnatal day (PD) 1) SpragueDawley rats as previously
described (Luo et al., 2002). Briefly, cells from hippocampi were
dispersed with trypsin (0.24 mg/ml; Sigma Aldrich, Milan, Italy)
and plated at a density of 0.8106 cells/ml on 35 mm Falcon
dishes coated with poly-L-lysine (10 g/ml, Sigma Aldrich). Cells
were plated in basal Eagles Medium (BME; Celbio, Milan, Italy),

0306-4522/08$32.000.00 2008 IBRO. Published by Elsevier Ltd. All rights reserved.

doi:10.1016/j.neuroscience.2007.09.064

155

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G. Losi et al. / Neuroscience 151 (2008) 155163

supplemented with 10% fetal bovine serum (Celbio), 2 mM glutamine (Invitrogen, Milan, Italy), 25 mM KCl and 100 g/ml gentamycin (Sigma Aldrich) and maintained at 37 C in 5% CO2. After
24 h in vitro, the medium was replaced with 1:1 mixture of BME
and Neurobasal medium (NB, Celbio) containing 2% B27 supplement (Celbio), 1% Pen-Strep (Celbio) and 0.25% glutamine. At 5
days in vitro (DIV 5), cytosine arabinofuranoside (Ara-C; Sigma
Aldrich) was added at final concentration of 10 M. Thereafter,
half of the medium was replaced twice a week with NB containing
2% B27 supplement, 1% Pen-Strep and 0.25% glutamine.

JM4C cells culture

Mouse connective tissue fibroblasts JM4C cells stably transfected
with NR1a/NR2A or NR1a/NR2B human NMDA inducible receptors were kindly donated by Dr. Paul Whiting, Merck Sharp &
Dohme Laboratories (Harlow, Essex, UK). They were grown in
Dulbeccos Modified Basal Eagles medium high glucose (DME)
containing 4500 mg/l glucose, sodium pyruvate 110 mg/l and
0.25% L-glutamine that was supplemented with geneticin 1 mg/ml
(Sigma Aldrich) and fetal bovine serum 10%. One day before the
experiment fibroblasts confluent dishes were split by using 0.05%
trypsinEDTA (Euroclone, Milan, Italy) and plated at the density of
0.5106 cells/ml in dishes containing DME with 0.015% ketamine
(Gellini International Milan, Italy) and 25 nM dexamethasone
(Sigma Aldrich) to induce NMDA receptor expression.

Hippocampal slices
Hippocampal slices were cut from PD1519 male rats. Briefly,
animals were anesthetized with halothane, brains were dissected
and mounted on a vibratome (Campden Instruments, Loughborough, UK) in ice cold cutting extracellular solution containing (in
mM): NaCl (120), KCl (3.1), CaCl2 (1), MgCl2 (2), KH2PO4 (1),
NaHCO3 (26), glucose (2.5), sucrose (30). Transverse hippocampal slices (350 m) were cut and transferred on a grid immersed
in the recording solution (same as cutting solution but CaCl2 was
2 mM and MgCl2 1 mM) continuously bubbled with O2/CO2 at
35 C for 30 min. Slices were then kept at room temperature.
Whole-cell recordings from pyramidal neurons in the CA1-subiculum were obtained under visual control with a Nikon Eclipse
600FN microscope (Nikon, Tokyo, Japan) equipped with Nomarski IR optics and an electrically insulated water immersion 40
objective.
All procedures involving animals were performed in accordance with the guidelines of the national (D.L. 116/1992) and
European legislation (EEC 86/609) on the use and care of laboratory animals. All efforts were made to minimize the number of
animals used and their suffering.

Electrophysiological recordings
Recordings were performed at room temperature, under voltageclamp in the whole-cell configuration of the patch-clamp technique
(Hamill et al., 1981). Electrodes were pulled from borosilicate
glass (Hidelberg, FRG) on a vertical puller (PB-7, Narishige) and
had a resistance of 57 M (23 M for recordings in slices).
Currents were amplified with an Axopatch 1D amplifier (Axon
Instruments, Foster City, CA, USA), filtered at 5 kHz, digitized at
10 kHz. Access resistance (Ra) was monitored throughout the
recordings. Cells with Ra greater than 20 M or with an RA
variation of more than 15% during the recording were not taken
into account.

Solutions and drugs

The recording chamber for cultured cells was continuously perfused at 5 ml/min with an artificial extracellular solution composed
of (mM): 145 NaCl, 5 KCl, 1 CaCl2, 5 Hepes, 5 glucose, 20

sucrose, pH 7.4 with NaOH. Electrode intracellular solution contains (mM): 140 KCl, 3 MgCl2, 5 EGTA, 5 Hepes, 2 ATP-Na, pH
7.3 with KOH. THs, NMDA and glycine were purchased from
Sigma Aldrich. THs were dissolved in 0.1 N NaOH (stock solution
at 10 mM). After dilution to the final concentration the pH was
adjusted with 0.1 N HCl. NMDA and glycine were dissolved from
10 mM frozen stocks in water. All drugs were applied directly by
gravity through a Y-tube perfusion system (Murase et al., 1989).
Drug application had a fast onset and achieved a complete local
perfusion of the recorded cell.
Miniature excitatory post-synaptic currents (mEPSCs) were
recorded in presence of 0.5 M tetrodotoxin citrate (TTX; Ascent,
UK) and pharmacologically isolated with 20 M bicuculline methiodide (BMI, Sigma Aldrich). Phorbol 12-myristate 13-acetate
(PMA) and chelerythrine were purchased from Sigma Aldrich.
Chelerythrine (5 M) was extracellularly perfused and PMA
(80 nM) was applied inside the patch pipette. Similar results were
observed after perfusion of PMA in the bath (200 or 400 nM) for
more than 15 min.
(2S,4R)-4-Methylglutamic acid (SYM 2081) was from
Tocris, UK.

Toxicity experiments
For neurotoxicity experiments cortical neurons were plated on 24
wells multiplates. At the day of the experiment wells were washed
once with phosphate buffered saline (PBS), then incubated for 1 h
in sterile Lockes buffer (154 mM NaCl, 5.6 mM KCl, 2.3 mM
CaCl2, 3.6 mM NaHCO3, 5.5 mM D-glucose, 5 mM Hepes, pH 7.4)
containing 100 M glutamate and 10 M glycine with or without
THs. Control group was treated with buffer solution only. Twentyfour hours later neuronal cell death was assessed by the MTT
assay (Ankarcrona et al., 1995). MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, 3 mg/ml) was dissolved in serum-free medium and added to the wells. MTT is
converted to an insoluble purple formazan by mitochondrial dehydrogenases of living cells. Formazan was solubilized in a solution
containing 333 l of HCl 37% in 50 ml of isopropanol. Absorbance
was measured with Labsystem Multiskan MCC/30 at 570 and
630 nm.

Data analysis
Data were expressed as the meanstandard error of mean (S.E.M.).
Fitting of the concentration-response curve was performed using the
equation %Imax100/Imax(1(IC50/[ANTAGO]nh)) where Imax is the
maximal current elicited by the antagonist, IC50 is the agonist concentration eliciting the half-maximal response, nh is the Hill coefficient. Fitting of the decay phase of mEPSCs was performed using a
simplex algorithm for least squares exponential fitting routines. Decay times of averaged currents derived from fitting to double exponential equations of the form: I(t)Ifexp(t/f)Isexp(t/s), where If
and Is are the amplitudes of the fast and slow decay components,
and f and s are their respective decay time constants used to fit the
data. To compare decay time between different conditions we used
a weighted mean decay time constant: w(If /(IfIs))f(Is/(IfIs))s.
Off-line data analysis, curve fitting, and figure preparation
were performed with Clampfit 8 (Axon Instruments), Origin 6.1
(Microcal, Northampton, MA, USA) and Minianalysis (Synaptosoft,
Decatur, GA, USA).

RESULTS
T3 and T4 inhibit NMDA-evoked currents
NMDA-evoked currents (100 M NMDA and 10 M glycine) were recorded in hippocampal neurons in culture at
DIV 8 10. Application of T3 and T4 reduced NMDA
steady-state currents in a dose dependent way. Hormone

G. Losi et al. / Neuroscience 151 (2008) 155163

157

Fig. 1. THs inhibit NMDA-evoked currents in hippocampal neurons in culture. (A) Whole cell recordings showing the effect of THs (10 M) on
NMDA (100 M plus glycine 10 M)-evoked currents from a neuron at DIV 8 in culture. (B) Concentration response curves of TH effect (filled
squares for T4 and empty squares for T3) on NMDA currents expressed as percentage of control current. Each data point is the meanS.E.M.;
n8.

response was immediate and almost fully reversed after

60 s of washout (Fig. 1A).
The IC50 was 83 M and 155 M and the maximal
effect 674% (n16) and 645% (n10) for T4 and
T3 respectively (Fig. 1B). Reverse-T3 (3,3=,5=-L-triiodothyronine; rev-T3), the genomically inactive isomer of T3, also
reduced NMDA-activated currents (483% at 50 M).
To investigate the nature of THs antagonism we tested
their effect on currents evoked by increasing concentrations of NMDA. TH reduction of NMDA current was similar
at all agonist concentrations tested (NMDA from 10 to
500 M; Fig. 2A) suggesting a noncompetitive mechanism
of the effect.
We then tested the possible interaction with the coagonist
glycine site by performing concentration response curves of
THs at different glycine concentrations. As shown in Fig. 2B,
T3 potency was the same at all glycine concentration tested,
the IC50s being 143 M at glycine 0.1 M, 142 M at
glycine 1 M and 155 M at glycine 10 M. Similar results
were observed for T4 (not shown).
Next we studied NMDA inhibition by T3 and T4 at
different holding potentials (Fig. 2C) and compared it to
that of memantine, a well-known voltage dependent NMDA
antagonist. Unlike memantine, T3 and T4 antagonism was
not significantly different at positive vs. negative holding
potentials (Fig. 2D).
It was reported that THs can modulate protein phosphorylation. Since phosphorylation plays an important role in the
regulation of NMDA receptor function we tested T3 effect in
the presence of the PKC activator PMA and of the selective
PKC inhibitor chelerythrine (Fig. 3A). In the presence of PMA
(80 nM) T3 modulation was not different than in control conditions (373% PMA vs. 435% control) and also after
perfusion with chelerythrine (5 M) the effect of T3 was

unchanged (393% chelerythrine vs. 435% control)

(Fig. 3B).
To evidentiate a subunit selectivity of TH effect, T3 was
tested on fibroblast JM4C cells expressing NMDA receptors
containing NR1a-NR2A or NR1a-NR2B subunits. T3 at
10 M reduced NMDA-evoked currents to the same extent in
cells expressing NR2A or NR2B subunits (257% in
NR1a-NR2A and 195% in NR1a-NR2B subunits).
T3, but not T4, inhibits AMPA-receptor-mediated
currents
We then tested in hippocampal cultures T3 and T4 on
currents activated by kainic acid (KA, 100 M) that are
mediated by AMPA and kainate receptors (Fig. 4A). T3
reduced KA-evoked steady state currents in a dose dependent way with an approximate IC50 of 317 M. The
maximal effect (at 80 M) was a reduction of 685%. T4 and
rev-T3 were inactive at all concentrations used (Fig. 4B).
Similar results were obtained in the presence of SYM
2081 (3 M), a compound that selectively blocks kainate
receptors through a process of agonist-induced desensitization (Zhou et al., 1997) suggesting that the effect of T3
was mainly at the level of AMPA receptors.
THs on mEPSCs in hippocampal slices
mEPSCs (in the presence of 0.5 M TTX and 20 M
bicuculline) were recorded in CA1-subiculum area of hippocampal slices from P1519 rats in the absence of added
Mg2 to allow both AMPA and NMDA receptor activation.
However the NMDA component was very small, as indicated
by the fast decay time of the currents. As reported in Table 1,
T3 application (20 m) did not alter peak amplitude or decay
time constants but significantly reduced mEPSCs frequency.

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G. Losi et al. / Neuroscience 151 (2008) 155163

Fig. 2. TH effects are not competitive and voltage independent. (A) Summary of T3 and T4 effects (both at 10 M) on currents evoked by increasing NMDA
concentrations. Each bar is the meanS.E.M.; n6. (B) Concentration response curves of T3 effect on NMDA currents at three different glycine
concentrations. (C) Whole cell recordings showing the effect of voltage on T3 (10 M) modulation of NMDA currents. (D) Histogram of THs and memantine
effects (all at 10 M) on NMDA currents at negative (60 mV) and positive (60 mV) holding potentials (each bar is the meanS.E.M.; n6; ** P0.01
Student t-test vs. 60 mV).

The lack of Mg2 however increased background noise,

making it difficult to detect small events. For this reason we
then tested THs effect on mEPSCs in the presence of 1 mM
Mg2. Also in these conditions T3 reduced mEPSCs average

frequency without changing peak amplitude and decay time

constant (Table 1). Perfusion of slices with high K (13 mM)
increased the number of mEPSCs that were again reduced
by T3 (Fig. 5). Not all cells we recorded from were sensitive

Fig. 3. T3 modulation of NMDA receptors does not involve PKC. (A) Whole cell recordings of T3 effect (20 M) on NMDA-evoked currents (NMDA
100 Mgly 10 M) in the presence of the PKC activator PMA (80 nM) or of the PKC blocker chelerythrine (CHEL; 5 M). (B) Summary of T3 effect
on NMDA currents in absence or presence of PMA or CHEL (each bar is the meanS.E.M.; n8; ** P0.01 Student t-test vs. CTRL).

G. Losi et al. / Neuroscience 151 (2008) 155163

159

Fig. 4. T3 inhibits KA-evoked currents in hippocampal neurons in culture. (A) Whole cell recordings showing the effect of T3 (30 M) on KA
(100 M)-evoked currents from a neuron at DIV 8 in culture (Vh60 mV). (B) Concentration response curves of TH effects on KA-evoked steady
state (ss) currents. Each point is the meanS.E.M.; n4 8 cells.

to T3 modulation (about 65%). T4 application (20 M) failed

to alter any mEPSC parameters analyzed.
T3 and T4 reduce glutamate- and NMDA-induced
toxicity
It is well known that antagonists of NMDA receptor have
neuroprotective effects, for this reason we tested the acTable 1. Summary of TH effect (both at 20 M) on mEPSC parameters
recorded in hippocampal neurons in slice

mEPSCs (n17)
T3
(No Mg2)
mEPSCs (n6)
T3
(Mg2)
mEPSCs (n11)
T3
(high K; Mg2)
mEPSCs (n9)
T4
(Mg2)

Peak (pA)

Freq (Hz)

Decay (ms)

263
273

0.160.03
0.090.02*

113
92

122
101

0.150.03
0.100.02*

71
71

132
121

0.860.19
0.530.15*

50.4
60.5

101
101

0.110.03
0.110.03

81
71

Mg21 mM; high K13 mM. Each data point is the meanS.E.M.
of the number n of cells indicated in parentheses.
* P0.05, Student t-test vs. control.

tivity of T3 and T4 against glutamate-induced toxicity in

cultured cortical neurons. Glutamate (see Experimental
Procedures) decreased cell viability to 645% of control in
eight different experiments. T3, T4 and rev-T3 prevented
the effect of glutamate at high concentrations restoring cell
viability to control levels (Fig. 6). T3 and T4 applied alone
(at 50 M) did not alter neuronal viability being 935% and
1003% of control respectively.

DISCUSSION
THs regulate genes expression by binding to specific nuclear thyroid receptors (TRs) but several rapid effects of
THs are not mediated by this mechanism and involve other
than nuclear TRs (Basset et al., 2003; Davis et al., 2005).
In the CNS TH genomic effects are particularly important
during development (Anderson et al., 2003; Bernal, 2002)
while in adult life they are relatively few (Bernal, 2002;
Calza et al., 1997). However several neurological and
psychiatric disorders are related to TH imbalances in adult
life, and in contrast to developmental effects most of these
CNS-related changes are reversed with the adjustment of
hormone levels.
Here we show for the first time that i) THs modulate
nongenomically NMDA receptor activity ii) T3 inhibits KAevoked currents and reduces mEPSCs frequency and
iii) THs protect from glutamate-induced neuronal death.

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G. Losi et al. / Neuroscience 151 (2008) 155163

Fig. 5. T3 reduces mEPSCs frequency in hippocampal neurons in slices. (A) Whole cell recordings showing the effect of T3 (20 M) on mEPSCs
(bicuculline 20 M, TTX 0.5 M, Mg2 1 mM, K 13 mM). (B) Average of mEPSCs aligned to their rise time in control and after T3 from the same
neuron as in A, with indication of the weighted time constant of the decay (w). (C) Cumulative fraction of the inter-event intervals of mEPSCs in control
and after T3 perfusion (P0.01, Kolgomorov-Smirnov test).

T4, T3 and even rev-T3 exert a negative modulation on

NMDA channel function. The antagonism of NMDA receptors is independent of agonist and coagonist concentration
and is not voltage or subunit dependent. The effect is very
rapid, less than 1 s, and reversible, suggesting a direct
interaction with the receptor channel.
Importantly our data show that also receptors activated
by kainate are modulated by T3, but not by T4 or rev-T3,

with potency in the micromolar range (105M) similar to

that measured on NMDA receptor current. Further experiments are needed to better characterize the effect of THs
on AMPA and KA receptors.
Our data on the effect of THs on synaptic transmission in hippocampal slices show that only T3 is able to
modulate mEPSCs. The frequency of the events was
significantly reduced by T3 while peak and decay of the

Fig. 6. THs protect against glutamate-induced neuronal damage. Histogram showing cortical neuron viability, measured with the MTT assay (see
Experimental Procedures) and expressed as percentage of control (meanS.E.M.; n 8), after glutamate treatment and in the presence of different
TH concentrations (expressed in M). ** P 0.01 Student t-test vs. glutamate group.

G. Losi et al. / Neuroscience 151 (2008) 155163

currents were unchanged. A drug that affects mEPSC

frequency acts preferentially through a presynaptic
mechanism: it is possible that T3 modulates presynaptic
glutamatergic receptors (autoreceptors) or heteroreceptors (for example nicotinic 7). In support of this hypothesis is the work of Vara et al. (2002) that showed an
increase in probability of transmitter release in slices
from hypothyroid rats, an increase that was reversed
when TH was administered.
In our experiments T3 does not reduce mEPSCs amplitude or decay while it is decreasing NMDA- and KAevoked currents. The lack of effect on mEPSCs peak
amplitude, that is mainly mediated by AMPA receptors, is
expected since also in hippocampal cultures T3 reduced
mostly AMPA-mediated steady state currents, with little or
no effect on peaks.
Compounds that block or increase desensitization of
NMDA receptors exert important physiological effects
among these the protection from glutamate-induced neuronal damage (Stone and Addae, 2002; Kemp and McKernan, 2002; Villarroel et al., 1997). We observed that THs
decrease glutamate-induced neuronal death at concentrations that blocked NMDA-evoked currents in electrophysiological experiments. THs are known to activate metabolism and thus improve cell vitality per se. Nevertheless in
our experiments we believe that the protection from glutamate toxicity was due to the antagonism on glutamatergic
ionotropic receptors because THs applied alone did not
change cell viability.
In our experiments THs were active at micromolar
concentrations while TH brain levels are in the nanomolar
range. It was reported however that these hormones can
accumulate in nerve endings (Dratman et al., 1976; Dratman and Crutchfield, 1978; Kastellakis and Valcava, 1989)
reaching high concentrations in synaptosome, especially
during hypothyroidism (Sarkar and Ray, 1994). Mason et
al. (1993) demonstrated that T3 can be released from
synaptosome with a Ca2 dependent mechanism, supporting the hypothesis of a neurotransmitter role for T3, as
developed by Dratman et al. (1976). Thus it is conceivable
that in the synaptic cleft THs could reach concentrations
high enough to directly modulate excitatory neurotransmission. Furthermore the concentrations of THs in the CNS,
that are determined by the activity of deiodinase type II
(Bernal et al., 2003), may vary substantially after pharmacological intervention (i.e. administration of antidepressive
drugs such as fluoxetine) (Eravci et al., 2000) or in conditions of chronic stress (Gerges et al., 2001).
Previous observations have pointed out the influence
of THs on neuronal excitability: TH deficiency lowers seizure threshold in animals (Sandrini et al., 1992), enhances
synaptic neurotransmission, impairs long term and short
term synaptic plasticity (Sui and Gilbert, 2003). The majority of these studies though are performed in animal
models of hypo- or hyper-thyroidism while in our experiments the effect of an acute administration of THs was
analyzed on neurons from euthyroid rats.

161

Through which mechanism do THs modulate

ionotropic glutamate receptor function?
THs nongenomically activate protein kinases directly (Lawrence, 1989) or through a second messenger pathway
linked to integrin V3 (Bergh et al., 2005). Since NMDA
are known targets for protein kinases especially for PKC
(for review see MacDonald et al., 2001) we tested the
hypothesis that T3 effect on NMDA receptors could occur
via PKC-mediated phosphorylation of the channel. Our
data show that blocking or stimulating PKC activity does
not affect T3 modulation of NMDA currents, suggesting
that this kinase is not implicated in THs mechanism of
action.
Interestingly T3 structure is very similar to that of the
neurosteroid pregnenolone sulfate (Martin et al., 2004)
that, although with an opposite effect, is also active on
NMDA receptor at micromolar concentrations (Bowlby,
1993).
Which are the possible implications of a
nongenomic regulation of glutamatergic
neurotransmission by THs?
During brain development changes in glutamatergic responsiveness induced by THs could be involved in the
altered radial migration of glutamatergic neurons observed
in pups of euthyroid hypothyroxinemic dams (Lavado-Autic
et al., 2003; Aus et al., 2004) and in the neurological
alterations (low IQ, ADHD) found in children of hypothyroxinemic women (Haddow et al., 1999; Pop et al., 1999,
2003; Vermiglio et al., 2004). In the adult the implications
of changes in brain TH levels are more difficult to predict
because THs exert their effects also on other neurotransmitter systems (Martin et al., 1996; Chapell et al., 1998)
and their actions and levels are different in the diverse
brain regions (Gerges and Alkadhi, 2004; Broedel et al.,
2003).
Since NMDA and AMPA receptors play a role in TSH
release (Arufe et al., 2002), this effect could also be involved in the negative feedback loop between TH levels
and TSH release.

CONCLUSION
In conclusion our finding of the regulation of glutamatergic
neurotransmission by THs adds new insight into the pleiotropic effects of these hormones and provides a better
understanding of their regulation on brain excitability.
AcknowledgmentWe thank Dr. Paul Whiting (Merck Sharp &
Dohme Laboratories, Harlow, Essex, UK) for kindly providing us
with the JM4C cell line stably expressing NMDA receptor subunits.

REFERENCES
Alzoubi KH, Gerges NZ, Alkadhi KA (2005) Levothyroxin restores hypothyroidism-induced impairment of LTP of hippocampal CA1: electrophysiological and molecular studies. Exp Neurol 195(2):330341.
Anderson GW, Schoonover CM, Jones SA (2003) Control of thyroid
hormone action in the developing rat brain [review]. Thyroid 13(11):
10391056.

162

G. Losi et al. / Neuroscience 151 (2008) 155163

Ankarcrona M, Dypbukt JM, Bonofoco E, Zhivotovsky B, Orrenius S,

Lipton SA, Nicotera P (1995) Glutamate-induced neuronal death: a
succession of necrosis or apoptosis depending on mitochondrial
function. Neuron 15:961973.
Arufe MC, Duran R, Perez-Vences D, Alfonso M (2002) Endogenous
excitatory amino acid neurotransmission regulates thyroid-stimulating hormone and thyroid hormone secretion in conscious freely
moving male rats. Endocrine 17(3):193197.
Aus E, Lavado-Autric R, Cuevas E, Del Rey FE, Morreale De Escobar G, Berbel P (2004) A moderate and transient deficiency of
maternal thyroid function at the beginning of fetal neocorticogenesis alters neuronal migration. Endocrinology 145(9):4037 4047.
Bassett JH, Harvey CB, Williams GR (2003) Mechanism of thyroid
hormone receptor-specific nuclear and extra nuclear actions. Mol
Cell Endocrinol 213:111.
Bergh JJ, Lin HY, Lansing L, Mohamed SN, Davis FB, Mousa S, Davis
PJ (2005) Integrin alphaVbeta3 contains a cell surface receptor
site for thyroid hormone that is linked to activation of mitogenactivated protein kinase and induction of angiogenesis. Endocrinology 146(7):2864 2871.
Bernal J (2002) Action of thyroid hormone in brain [review]. J Endocrinol Invest 25(3):268 288.
Bernal J, Guadano-Ferraz A, Morte B (2003) Perspectives in the study
of thyroid hormone action on brain development and function [review]. Thyroid 13(11):10051012.
Bowlby M (1993) Pregnenolone sulfate potentiation of N-methyl-Daspartate receptor channels in hippocampal neurons. Mol Pharmacol 43:813 819.
Broedel O, Eravci M, Fuxius S, Smolarz T, Jeitner A, Grau H, Stoltenburg-Didinger G, Plueckhan H, Meinhold H, Baumgartner A (2003)
Effects of hyper- and hypothyroidism on thyroid hormone concentrations in regions of the rat brain. Am J Physiol Endocrinol Metab
285(3):E470 E480.
Burmeister LA, Ganguli M, Dodge HH, Toczek T, DeKosky S, Nebes
RD (2001) Hypothyroidism and cognition: preliminary evidence for
a specific defect in memory. Thyroid 11:11771185.
Calza L, Aloe L, Giardino L (1997) Thyroid hormone-induced plasticity
in the adult rat brain [review]. Brain Res Bull 44(4):549 557.
Chapell R, Martin J, Machu TK, Leidenheimer NJ (1998) Direct channel-gating and modulatory effects of triiodothyronine on recombinant GABAA receptor. Eur J Pharmacol 349(1):115121.
Davis PJ, Davis FB, Cody V (2005) Membrane receptors mediating
thyroid hormone action. Trends Endocrinol Metab 16(9):429 435.
Dratman MB, Crutchfield FL, Axerold J, Colburn RW, Thoa N (1976)
Localization of triiodothyronine in nerve ending fractions of rat
brain. Proc Natl Acad Sci U S A 73:941944.
Dratman MB, Crutchfield FL (1978) Synaptosomal [125I]triiodothyronine
after intravenous [125I]thyroxine. Am J Physiol 235:E638E647.
Eravci M, Pinna G, Meinhold H, Baumgartner A (2000) Effects of
pharmacological and nonpharmacological treatments on thyroid
hormone metabolism and concentrations in rat brain. Endocrinology 141(3):10271040.
Gerges NZ, Stringer JL, Alkadhi KA (2001) Combination of hypothyroidism and stress abolishes early LTP in the CA1 but not dentate
gyrus of hippocampus of adult rats. Brain Res 922(2):250 260.
Gerges NZ, Alkadhi KA (2004) Hypothyroidism impairs late LTP in
CA1 region but not in dentate gyrus of the intact rat hippocampus:
MAPK involvement. Hippocampus 14(1):40 45.
Gilbert ME, Paczkowski C (2003) Propylthiouracil (PTU)-induced hypothyroidism in the developing rat impairs synaptic transmission
and plasticity in the dentate gyrus of the adult hippocampus. Dev
Brain Res 145:19 29.
Gilbert ME (2004) Alterations in synaptic transmission and plasticity in
area CA1 of adult hippocampus following developmental hypothyroidism. Brain Res Dev Brain Res 148(1):1118.
Gulseren S, Gulseren L, Hekimsoy Z, Cetinay P, Ozen C, Tokatlioglu
B (2006) Depression, anxiety, health-related quality of life, and

disability in patients with overt and subclinical thyroid dysfunction.

Arch Med Res 37(1):133139.
Haddow JE, Palomaki GE, Allan WC, Williams JR, Knight GJ, Gagnon
J, OHeir CE, Mitchell ML, Hermos RJ, Waisbren SE, Faix JD, Klein
RZ (1999) Maternal thyroid deficiency during pregnancy and subsequent neuropsychological development of the child. N Engl
J Med 341(8):549 555.
Haggerty JJ Jr, Garbutt JC, Evans DL, Golden RN, Pedersen C, Simon
JS, Nemeroff CB (1990) Subclinical hypothyroidism: a review of neuropsychiatric aspects. Int J Psychiatry Med 20(2):193208.
Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch
391:85100.
Kastellakis A, Valcana T (1989) Characterization of thyroid hormone
transport in synaptosomes from rat brain. Mol Cell Endocrinol
67:231241.
Kemp JA, McKernan RM (2002) NMDA receptor pathways as drug
targets [review]. Nat Neurosci 5 (Suppl):1039 1042.
Lavado-Autric R, Aus E, Garca-Velasco JV, Arufe Mdel C, Escobar
del Rey F, Berbel P, Morreale de Escobar G (2003) Early maternal
hypothyroxinemia alters histogenesis and cerebral cortex cytoarchitecture of the progeny. J Clin Invest 111(7):10731082.
Lawrence WD, Schoenl M, Davis PJ (1989) Stimulation in vitro of
rabbit erythrocyte cytosol phospholipid-dependent protein kinase
activity. A novel action of thyroid hormone. J Biol Chem 264(9):
47664768.
Lee PR, Brady D, Koenig JI (2003) Thyroid hormone regulation of
N-methyl-D-aspartic acid receptor subunit mRNA expression in
adult brain. J Neuroendocrinol 15(1):8792.
Lifschytz T, Segman R, Shalom G, Lerer B, Gur E, Golzer T, Newman
ME (2006) Basic mechanism of augmentation of antidepressant
effects with thyroid hormone. Curr Drug Targets 7(2):203210.
MacDonald JF, Kotecha SA, Lu WY, Jackson MF (2001) Convergence
of PKC-dependent kinase signal cascades on NMDA receptors
[review]. Curr Drug Targets 2(3):299 312.
Madeira MD, Sousa N, Lima-Andrade MT, Calheiros F, Cadete-Leite
A, Paula-Barbosa MM (1992) Selective vulnerability of the hippocampal pyramidal neurons to hypothyroidism in male and female
rats. J Comp Neurol 322:501518.
Martin JV, Williams DB, Fitzgerald RM, Im HK, Vonvoigtlander PF
(1996) Thyroid hormonal modulation of the binding and activity of
the GABAA receptor complex of brain. Neuroscience 73:705713.
Martin JV, Padron JM, Newman MA, Chapell R, Leidenheimer NJ,
Burke LA (2004) Inhibition of the activity of the native gammaaminobutyric acid A receptor by metabolites of thyroid hormones:
correlations with molecular modeling studies. Brain Res 1004(12):
98107.
Mason GA, Walker CH, Prange AJ (1993) L-Triiodothyronine: is this
peripheral hormone a central neurotransmitter? Neuropsychopharmacology 8:253258.
Murase K, Ryu PD, Randic M (1989) Excitatory and inhibitory amino
acids and peptide-induced responses in acutely isolated rat spinal
dorsal horn neurons. Neurosci Lett 103:56 63.
Oguro K, Ito M, Tsuda H, Mutoh K, Shiraishi H, Shirasaka Y, Mikawa
H (1989) Interactions of thyroid hormones with L-(3H) glutamate
binding sites, with special reference to N-methyl-D-aspartate receptors. Res Commun Chem Pathol Pharmacol 65(2):181196.
Pop VJ, Kuijpens JL, van Baar AL, Verkerk G, van Son MM, de Vijlder
JJ, Vulsma T, Wiersinga WM, Drexhage HA, Vader HL (1999) Low
maternal free thyroxine concentrations during early pregnancy are
associated with impaired psychomotor development in infancy.
Clin Endocrinol (Oxf) 50(2):149 155.
Pop VJ, Brouwers EP, Vader HL, Vulsma T, van Baar AL, de Vijlder JJ
(2003) Maternal hypothyroxinaemia during early pregnancy and
subsequent child development: a 3-year follow-up study. Clin Endocrinol (Oxf) 59(3):282288.

G. Losi et al. / Neuroscience 151 (2008) 155163

Sandrini M, Marrama D, Vergoni AV, Bertolini A (1992) Repeated administration of triiodothyronine enhances the susceptibility of rats to isoniazid- and picrotoxin-induced seizures. Life Sci 51(10):765770.
Sarkar PK, Ray AK (1994) Synaptosomal T3 content in cerebral cortex
of adult rat in different thyroidal states. Neuropsychopharmacology
11(3):151155.
Simon NM, Blacker D, Korbly NB, Sharma SG, Worthington JJ, Otto
MW, Pollack MH (2002) Hypothyroidism and hyperthyroidism in
anxiety disorders revisited: new data and literature review. J Affect
Disord 69(13):209 217.
Stone TW, Addae JI (2002) The pharmacological manipulation of glutamate receptors and neuroprotection. Eur J Pharmacol 447:285296.
Sui L, Gilbert ME (2003) Pre- and postnatal propylthiouracil-induced
hypothyroidism impairs synaptic transmission and plasticity in area
CA1 of the neonatal rat hippocampus. Endocrinology 144(9):
4195 4203.
Vara H, Martinez B, Santos A, Colino A (2002) Thyroid hormone
regulates neurotransmitter release in neonatal rat hippocampus.
Neuroscience 110(1):19 28.

163

Vermiglio F, Lo Presti VP, Moleti M, Sidoti M, Tortorella G, Scaffidi G,

Castagna MG, Mattina F, Violi MA, Crisa A, Artemisia A, Trimarchi
F (2004) Attention deficit and hyperactivity disorders in the offspring of mothers exposed to mild-moderate iodine deficiency: a
possible novel iodine deficiency disorder in developed countries.
J Clin Endocrinol Metab 89(12):6054 6060.
Villarroel A, Regalado MP, Lerma J (1997) Desensitization of NMDA
receptors as a mechanism of neuroprotection. Methods Find Exp
Clin Pharmacol 19(Suppl A):5153.
Wiens SC, Trudeau VL (2006) Thyroid hormone and gamma-aminobutyric acid (GABA) interactions in neuroendocrine systems.
Comp Biochem Physiol A Mol Integr Physiol 144(3):332344.
Zhou LM, Gu ZQ, Costa AM, Yamada KA, Mansson PE, Giordano T,
Skolnick P, Jones KA (1997) (2S,4R)-4-Methylglutamic acid (SYM
2081): a selective, high-affinity ligand for kainate receptors. J Pharmacol Exp Ther 280(1):422 427.
Zoeller RT, Rovet J (2004) Timing of thyroid hormone action in the
developing brain: clinical observations and experimental findings.
J Neuroendocrinol 16(10):809 818.

(Accepted 4 October 2007)

(Available online 5 October 2007)