(+)-(S)-trujillon, (+)-(S)-4-(2,2-diphenyl-1,3,2-oxazabolidin-5-oxo) propionic acid, a novel glutamatergic analog, modifies the activity of globus pallidus neurons by selective NMDA receptor activation

Article abstract of DOI:10.1002/chir.20594

Decreased levels of glutamate and changes in several markers of glutamatergic function occur in movement disorders and chronic psychiatric illnesses. Ionotropic glutamate receptors have been implicated in neuronal cell death, and have, therefore, been related to the process of neurodegenerative diseases. Drugs that interact with the glutamatergic system are important tools for the development of better therapies. We examined the effect of a new glutamatergic analog, (+)-(S)-4-(2,2-diphenyl-1,3,2-oxazabolidin-5-oxo)propionic acid, (+)-(S)-Trujillon, on the spontaneous globus pallidus neuronal activity of the anesthetized rat. (+)-(S)-Trujillon excited most pallidal neurons in a dose-dependent manner. Furthermore, blockade of NMDA receptors (NMDARs) inhibited the (+)-(S)-Trujillon-induced excitation, whereas blockade of AMPA/kainate receptors did not. In addition, computational docking studies showed micromolar-range affinities of (+)-(S)-Trujillon for NR2A NMDARs. Our results indicate that (+)-(S)-Trujillon selectively activates NMDARs, an effect that could prove to be a useful tool in the analysis of motor, behavioral, and cognitive disorders, where NMDAR-mediated signaling is altered. Copyright

Full text of DOI:10.1002/chir.20594

CHIRALITY 23:429–437 (2011)
(
1)-(S)-Trujillon, (1)-(S)-4-(2,2-Diphenyl-1,3,2-oxazabolidin-5-
oxo)propionic acid, a Novel Glutamatergic Analog, Modifies
the Activity of Globus Pallidus Neurons by
Selective NMDA Receptor Activation
1
1
1
1
´
JUAN M. ARAUJO-ALVAREZ, JOSE G. TRUJILLO-FERRARA, DANIEL PONCE-FRANCO, JOSE CORREA-BASURTO,
2
1
ALFONSO DELGADO, AND ENRIQUE QUEREJETA *
1
Department of Physiology and Pharmacology, National Polytechnic Institute School of Medicine, M e´ xico D.F., M e´ xico
Department of Physiology and Neuroscience, Autonomous University of Chihuahua School of Medicine, Chihuahua Chih, M e´ xico
2
ABSTRACT Decreased levels of glutamate and changes in several markers of gluta-
matergic function occur in movement disorders and chronic psychiatric illnesses. Iono-
tropic glutamate receptors have been implicated in neuronal cell death, and have, there-
fore, been related to the process of neurodegenerative diseases. Drugs that interact
with the glutamatergic system are important tools for the development of better thera-
pies. We examined the effect of a new glutamatergic analog, (1)-(S)-4-(2,2-diphenyl-
1
,3,2-oxazabolidin-5-oxo)propionic acid, (1)-(S)-Trujillon, on the spontaneous globus pal-
lidus neuronal activity of the anesthetized rat. (1)-(S)-Trujillon excited most pallidal
neurons in a dose-dependent manner. Furthermore, blockade of NMDA receptors
(
NMDARs) inhibited the (1)-(S)-Trujillon-induced excitation, whereas blockade of
AMPA/kainate receptors did not. In addition, computational docking studies showed
micromolar-range affinities of (1)-(S)-Trujillon for NR2A NMDARs. Our results indicate
that (1)-(S)-Trujillon selectively activates NMDARs, an effect that could prove to be a
useful tool in the analysis of motor, behavioral, and cognitive disorders, where NMDAR-
mediated signaling is altered. Chirality 23:429–437, 2011. VV C 2008 Wiley-Liss, Inc.
KEY WORDS: basal ganglia; motor control; pallidal; spiking activity; glutamate
2
7
INTRODUCTION
1)-(S)-glutamate plays a key role in a great diversity of
processes from the monosynaptic reflex to cognitive func-
that act as a pacemaker for BG activity. Alterations in
this synchronized activity are associated with motor dys-
(
2
8
functions in patients with Parkinson’s disease and in ani-
2
9
1
–5
mal models of Parkinson’s disease. Accordingly, the
study of the glutamatergic transmission and drugs that
modify it, especially in the BG, has gained enormous
interest.
In this study, we analyzed the effect of intrapallidal infu-
sions of a new glutamatergic analog, (1)-(S)-4-(2,2-
diphenyl-1,3,2-oxazabolidin-5-oxo)propionic acid, named
(1)-(S)-Trujillon on GP spontaneous neuronal activity
in the anaesthetized rat. We found that (1)-(S)-Trujillon
increases the spontaneous firing rate of pallidal neurons
tions.
(1)-(S)-glutamate concentrations and markers of
glutamatergic function decrease in chronic psychiatric ill-
6
,7
nesses. Furthermore, (1)-(S)-glutamate is a critical ele-
8
ment in excitotoxic neuronal death, which presumably is
the process responsible for the genesis of many neurode-
9
,10
generative diseases.
(
1)-(S)-glutamate is spread throughout the globus pal-
3
0
1
1,12
13
lidus (GP)
and other brain regions. The GP is part of
the basal ganglia (BG), a circuit related to movement con-
1
4
trol. Through GABAergic projection fibers, the GP par-
1
5-17 by selectively activating NMDARs in a dose-dependent
manner, with micromolar-range affinities. Our results
ticipates in the regulation of the overall BG activity.
In turn, the activity of GP neurons depends on their
1
8,19
membrane properties,
on the incoming fibers from
2
0,21
other nuclei,
and on axon collaterals of its projection Contract grant sponsor: SIP-IPN; Contract grant numbers: 20061292,
2
2,23
20050757
neurons.
Contract grant sponsor: SEP-promeP; Contract grant numbers: NPTC-
The major glutamatergic input to the GP is the subtha- 2004
20,24
lamic nucleus (STN).
Communication between STN and *Correspondence to: Enrique Querejeta, Secci o´ n de Estudios de Posgrado
e Investigaci o´ n, Escuela Superior de Medicina del IPN. Plan de San Luis
pallidal neurons takes place in a dual process: (1) pallidal
y D ´ı az Mir o´ n Col. Casco de Santo Tom a´ s, C.P. 11340, M e´ xico D.F., M e´ x-
neurons release g-amino-butyric acid (GABA) onto subtha- ico. E-mail: equerejeta@ipn.mx
2
5
Received for publication 25 January 2008; Accepted 15 April 2008
DOI: 10.1002/chir.20594
Published online 20 June 2008 in Wiley Online Library
lamic neurons, and (2) subthalamic neurons release (1)-
(
2
6
S)-glutamate onto pallidal neurons. In both nuclei, this
dual phenomenon determines synchronized oscillations (wileyonlinelibrary.com).
VV C 2008 Wiley-Liss, Inc.

4
30
ARAUJO-ALVAREZ ET AL.
indicate that (1)-(S)-Trujillon could be a valuable tool in phonopentanoic acid (D-AP5), 6-cyano-7-nitroquinoxaline-
the study of cognitive and motor disorders with NMDARs 2,3-dione (CNQX), N-methyl-D-aspartate (NMDA), and
hypofunction and NMDA-related excitotoxic cell death.
(1)-(S)-glutamate were all from Sigma-RBI (St. Louis,
MO). Drugs were dissolved in saline (0.9%) and infused
into the GP using a 1-ll syringe. The piston of the syringe
was connected to a precision micrometer that allowed an
infusion rate of 50 nl/15 s. The total volume injected per
individual infusion of drug was 100 nl. Only neurons with
at least five minutes of stable baseline firing were chosen
for drug application. A 20% change relative to baseline dur-
ing the next minute after drug infusion was considered
significant. Vehicle/Drug application was performed only
once per recording, and at least 25 min were allowed to
pass before making another recording in the same track.
MATERIALS AND METHODS
Animals
Experiments were performed in 70 male Wistar rats
weighing 180–220 g. The animals were maintained and
handled according to the guidelines of the ESM-IPN Ani-
mal Care and Use Committee in compliance with the
National Institutes of Health Guide for the Care and Use
of Laboratory Animals. All efforts were made to minimize
animal suffering and the number of animals used. Animals
were housed in a temperature- and humidity-controlled
environment with a 12-h light/dark cycle and free access
to food and water.
Intrapallidal drug diffusion. To estimate the volume
within which applied drugs would diffuse in the GP, 100 nl
of 2% methylene blue dye solution were injected in six rats
at the coordinates mentioned earlier. Five minutes after
intrapallidal injections, animals were sacrificed and their
brains rapidly removed. Three brains were sliced in the
coronal plane and the other three in the sagital plane (all
slices were 30-lm thick). In both coronal and sagital brain
slices, the largest diameter of the dye-stained region was
measured (only regions with homogenous staining were
considered as drug diffusion areas). Then, for each brain,
diameters were superimposed rostrocaudally (coronal cut)
or mediolaterally (sagital cut) to form a sphere in the cen-
ter of which the tip of the injection cannula would be
located. Recordings outside this sphere were discarded.
(
1)-( S)-Trujillon Preparation
Synthesis. All reagents were reactive grade. (1)-(S)-
glutamic acid (1.0 g, 6.78 mmol) was dissolved in a mini-
mal quantity of distilled water and the pH was adjusted to
4
.0 with HCl 0.1 N (Solution A). Diphenylborinic acid etha-
nolamine ester complex (1.53 g, 6.78 mmol) was dissolved
in 100 ml of ethanol; later, 2 ml of concentrated HCl (37%)
were used to liberate diphenylborinic acid, and then dis-
tilled water was added until a milky suspension appeared
(
Solution B). Diphenylborinic acid was immediately and
quickly extracted by using ethylic ether and this extract
was mixed with Solution A. The extraction of diphenylbor-
inic acid was repeated until the milky suspension of Solu-
tion B disappeared. The mixture was left to reflux for 2 h
and the solvent was extracted with a Dean Star trap at 60–
Data Acquisition and Analysis
Standard extracellular recordings were performed using
glass electrodes (2–6 MX) filled with 2 M NaCl solution
containing 2% pontamine sky blue dye. GP recording
tracks were aimed vertically at stereotaxic coordinates as
7
08C. The white product was washed with distilled water,
filtered and dried.
Characterization. The compound was characterized follows: 0.9–1.2 mm posterior to bregma, 3.0–3.3 mm lat-
1
13
11
by H, C, and B nuclear magnetic resonance (NMR) eral, and 4.95–5.90 mm ventral to the brain surface. Extrac-
studies in a Jeol GSX spectrophotometer at 270, 68.7, and ellular signals were amplified 10,000X, bandpass filtered
9
0 MHz, respectively. The compound was soluble in dis- between 0.3 and 3 kHz (DAM-80 amplifier; WPI Saratosa,
tilled water, ketone, and ethanol, and its melting point was FL) and stored on an audiocassette device. Single-unit ac-
determined in an Electrothermal 9300 apparatus.
tivity was isolated using a window discriminator WPI-121
WPI Saratosa). Spike times were preprocessed online and
further analyzed offline using the INF-386 program for
(
Electrophysiology
3
2
Surgery and drug application. Rats were anaesthe- spike data analysis. Statistical comparisons between or
tized with chloral hydrate (300 mg/kg, i.p.; supplemented among groups were determined with Student’s t-test and
as needed) and gently positioned in a stereotaxic appara- one-way ANOVA (with Newman-Keuls post hoc test) by
tus. Body core temperature was maintained at 36–388C commercial software (GraphPad Prism 3, San Diego, CA).
with a heating pad and rectal thermometer system. A 4- The data are expressed as the mean 6 standard error of
mm burr hole was drilled in the skull to stereotactically the mean or as percent of control baseline.
guide glass electrodes and a steel double cannula system
Histology
(
30-gauge syringe needles) into the right GP. The tips of
both cannulas had a 0.1-mm separation. The recording
electrodes were placed 0.2 mm posterior to bregma, 3.0– studied unit was marked by administering pontamine sky
.3 mm lateral, and 4–6 mm ventral to the brain surface blue from the electrode by passing 10 lA constant nega-
all coordinates were according to the atlas of Paxinos and tive current for 20 min. After experiments, rats were given
At the end of each recording session, the level of the
3
(
3
1
Watson). The injection cannulas were placed at an angle a lethal overdose of pentobarbital and perfused with 4%
of 708 in the latero-medial direction to the following coordi- formaldehyde, then the brain was quickly removed and
nates: 1.1 mm posterior to bregma, 5.7 mm lateral, 5.6 mm placed in formaldehyde; following overnight incubation,
ventral to the brain surface. Drugs: D(2)-2-amino-5-phos- the brain was sliced to verify the positions of the recording
Chirality DOI 10.1002/chir

(
þ)-(S)-TRUJILLON EFFECT ON PALLIDAL ACTIVITY
431
Spontaneous Electrical Activity in GP Neurons
In this study, we recorded Type-I (initially negative
biphasic waveform) and Type-II (initially positive biphasic
4
1
waveform) neurons that were pooled together and given
that (1) there was not any statistically significant differ-
ence between the spontaneous firing rates of both types
(
26.43 6 1.90 spikes/s of Type I, n 5 102, vs. 29.75 6 2.35
spikes/s of Type II, n 5 168), (2) the local application of
00 pmol (1)-(S)-glutamate was found to significantly
increase the spontaneous firing rate of both types (49 6
7% for Type I, 73 6 55% for Type II) in a randomly
selected subset of six Type-I and six Type-II neurons, and
3) micropipette electrical resistance was a major factor for
neuron type discrimination (micropipettes with 2.5 MX or
less electrical resistance generally detected Type I,
whereas micropipettes with 4.0 MX or higher electrical re-
1
2
(
Fig. 1. Chemical structure of (1)-(S)-4-(2,2-diphenyl-1,3,2-oxazabolidin-
-oxo)propionic acid: (1)-(S)-Trujillon.
5
electrode and the injection cannulas. When either the re- sistance mostly detected Type II).
cording electrode or the injection cannulas were not posi-
Effect of Local Application of (1)-( S)-Glutamate
tioned within the GP, the experiment was discarded.
The main effect of the local application of 100 pmol (1)-
S)-glutamate was an increase in the spontaneous firing
Molecular Docking Studies
(
rate. In 77% of the recorded neurons (14/18), (1)-(S)-glu-
tamate produced a robust (89 6 16%) increase in the firing
rate (Figs. 2A and 2B). Only two neurons (11%) displayed
(
1)-(S)-Trujillon was geometrically optimized at the
3
3
B3LYP/6-31G* level (Gaussian 98, Gaussian, Walling-
ford, CT). Boron atom parameters were taken from Tafi
et al. To identify the recognition site and to determine
NMDARs-ligand affinity, docking simulations were done
on the one-monomer rat’s NR2A 3D protein structure
3
4
3
5
(
PDB code: 2A5S ). Before docking analysis, the protein
was minimized in 2000 steps using the CHARMM22 pa-
rameters implemented in the NAnoscale molecular dynam-
3
6
ics (NAMD) program. All possible flexible bonds of the
ligands, the partial atomic charges (Gasteiger-Marsili for-
malism), and the Kollman charges for all atoms in the
3
7
NR2A were assigned using AutoDock Tools. Then, polar
hydrogens were maintained on the amino acids at ꢀ7.4
pH. All other parameters were kept at their default values.
Binding was modeled using AutoDock 3.05 because its
3
7
algorithm allows full flexibility for small ligands. The
input preparations of the protein, ligand structures, and
binding site definitions were carried out under a GRID-
3
8
3
˚
based procedure. An 80 3 80 3 80 A point grid with
0
˚
.375 A spacing was constructed over the NR2A glutamate
binding site. All simulations used the hybrid Lamarckian
Genetic Algorithm, with an initial population of 100 ran-
domly placed individuals and a maximum of 10 million
energy evaluations. Docking orientations within a root-
˚
mean-square deviation of 0.5 A were clustered together.
The lowest energy cluster returned for each compound
was used for further analysis. Docking visualizations were
3
9
prepared using VMD.
Fig. 2. Local application of (1)-(S)-glutamate increases the firing rate
of globus pallidus neurons in a dose-dependent manner. (A, B) Spike rate
histograms of two globus pallidus neurons. (A) Application of 100 pmol
RESULTS
Chemicals
(
2)-(R)-glutamate did not evoke changes in the firing rate, whereas the
same dose of its stereoisomer, (1)-(S)-glutamate, considerably excited
the neuron. (B) The firing frequency of this neuron was increased after
(
1)-(S)-Trujillon (Fig. 1) was obtained at 608C with a
9
5% yield. Its melting point was 182–1858C and its optical 100 pmol (1)-(S)-glutamate infusion. (C) Statistics of the effect of intrapal-
2
5
lidal infusion of 10, 100 pmol, and 1 nmol (1)-(S)-glutamate, as well as
100 pmol and 1 nmol (2)-(R)-glutamate and saline. Notice the dose-de-
pendent effect of (1)-(S)-glutamate. *P < 0.01, **P < 0.001; one-way
ANOVA, Newman-Keuls, post hoc test.
activity was [a] 1 10, c 1.0 in water. The new compound
was characterized by B, C, and H NMR studies and
compared with compounds reported in the literature.
D
1
1
13
1
4
0
Chirality DOI 10.1002/chir

4
32
ARAUJO-ALVAREZ ET AL.
a reduction in their spiking rate; one of these neurons
came to a complete silence during (1)-(S)-glutamate infu-
sion and the other showed a 29% decrease from baseline.
Two out of 18 neurons (11%) did not show a significant
response to (1)-(S)-glutamate. The excitatory response to
(1)-(S)-glutamate was dose-dependent (Fig. 2C). Applica-
tion of 10 pmol (n 5 16) increased the spiking frequency
by 73 6 16%, whereas the infusion of 1 nmol augmented
the firing rate by as much as 280 6 37% (n 5 16). Three
neurons of the 1-nmol pool underwent an apparent depola-
rization block (i.e., a progressive broadening of the action
potential duration and a reduction of the amplitude with an
ever greater rate increase, until the action potential disap-
4
2
peared). One of them returned to baseline firing rate
within a minute of (1)-(S)-glutamate application, whereas
the other two did not recover even after 10 min. Further-
more, local application of similar volumes of saline solution
did not significantly modify the pallidal neuron discharge
rate (n 5 10), and local infusion of different doses of (2)-
(R)-glutamate, the (1)-(S)-glutamate stereoisomer, did not
cause statistically significant changes in the spontaneous
neuronal activity. Infusion of 100 pmol and 1 nmol (2)-(R)-
glutamate produced only a small decrease in neuronal fir-
ing (4.74 6 4.80% and 3.53 6 3.90% for 100 pmol and 1
nmol, respectively). There was a significant difference in
the effect of 1 nmol (1)-(S)-glutamate when compared to
Fig. 3. Local infusion of NMDA increases the spiking rate of globus
pallidus neurons in a dose-dependent manner. (A, B) Spike rate histo-
grams of two globus pallidus neurons under the effect of 100 pmol
its other doses (P < 0.01), to the cell responses after sa- NMDA. Both neurons suffered a depolarization block after the excitation
induced by NMDA. Thin horizontal lines represent the zero baselines.
line solution application (P < 0.01), and to the effect of 100
pmol and 1 nmol (2)-(R)-glutamate (P < 0.001).
(
C) Statistics of the effect of 1, 10, and 100 pmol NMDA. **P < 0.001;
one-way ANOVA, Newman-Keuls, post hoc test.
Effect of Local Application of NMDA
NMDA (100 pmol) increased the spontaneous discharge duced an 83.0 6 0.3% decrease in the spontaneous firing
rate by 275.11 6 40.40% in 20/28 (71.4%) recorded neu- rate. Eighteen percent (4/22) of the recorded neurons
rons (Fig. 3). Twelve out of the 20 (60%) neurons that dis- did not significantly respond to (1)-(S)-Trujillon. The
played an excitatory response to NMDA also showed an response to (1)-(S)-Trujillon was dose-dependent as fol-
apparent depolarization block. Most neurons (7/12) that lows: local infusion of 1 pmol, 10 pmol, and 1 nmol
suffered this phenomenon recovered to their baseline fir- increased the firing rate by 7.3 6 3.9% (n 5 11), 50.93 6
ing rates within 158 6 48 sec after drug application (Fig. 18.30% (n 5 9), and 276.0 6 69.5% (n 5 13), respectively
3
B). Although to a lesser extent, NMDA also decreased (Figs. 4A–4C).
the pallidal neurons spontaneous firing rate. NMDA
The effect of 100 pmol (1)-(S)-Trujillon was statistically
reduced the electrical activity of five out of 28 neurons significant when compared with the effects of 1 nmol (2)-
17.8%) by 39.97 6 6.67%; two of these neurons showed a (R)-Trujillon, 1 and 10 pmol (1)-(S)-Trujillon, and saline
(
complete firing arrest that lasted for up to 30.5 6 3.46 sec. solution (P < 0.01, Fig. 4C). Additionally, the effect of 1
Three out of 28 neurons (10.7%) did not significantly nM (1)-(S)-Trujillon was significant when compared with
respond to 100 pmol NMDA applications. Both the excita- the effects of the same dose of its stereoisomer, (2)-(R)-
tory and the inhibitory responses to NMDA were present Trujillon (Fig. 4C) and equal volumes of saline solution (P
during the infusion time. The effect of NMDA was dose- < 0.001). The effect of 1 nmol (1)-(S)-Trujillon was also
dependent (Fig. 3C). Local application of 1 pmol (n 5 15) significantly different when compared with its effects at 10
and 10 pmol (n 5 16) NMDA caused a 9.3 6 5.1% and a and 100 pmol (P < 0.01). Two out of 13 neurons (15%) that
2
3.6 6 8.2% increase in the firing rate, respectively. Lower were excited by 1 nmol (1)-(S)-Trujillon and 2/16 (12%)
doses of NMDA did not significantly modify pallidal firing neurons that were excited by 100 pmol (1)-(S)-Trujillon
frequency. The response to 100 pmol NMDA was statisti- displayed a depolarization block.
cally significant when compared with all other doses
Effect of Local Blockade of Ionotropic
tested (P < 0.001).
Glutamate Receptors
Effect of Local Application of (1)-( S)-Trujillon
To determine the presence of active glutamatergic
Sixteen out of 22 (77.2%) neurons responded to the 100 fibers within the GP and to analyze their effect on pallidal
pmol (1)-(S)-Trujillon application with a 180.8 6 31.6% neurons electrical activity, we proceeded to locally apply
increase in their firing rate. In only two neurons (9%), the the NMDAR-selective antagonist, D-AP5, and the AMPA/
local infusion of the same dose of (1)-(S)-Trujillon pro- kainate receptor-selective antagonist, CNQX. Local applica-
Chirality DOI 10.1002/chir

(
þ)-(S)-TRUJILLON EFFECT ON PALLIDAL ACTIVITY
433
Fig. 5. Local infusion of D-AP5 blocks the excitatory effect of (1)-(S)-
Trujillon on globus pallidus neurons. (A, B) Spike frequency histograms
of two different pallidal neurons. (A) The excitatory effect of 100 pmol
(
1)-(S)-Trujillon was reversed by the local application of 100 pmol D-AP5,
Fig. 4. Local (1)-(S)-Trujillon infusion increases the spontaneous fir-
ing frequency of globus pallidus neurons in a dose-dependent manner. (A,
B) Spike frequency histograms showing the effect of 1 nmol (1)-(S)-Tru-
jillon on two different neurons. (B) (1)-(S)-Trujillon caused a depolariza-
tion block after a strong initial excitation period. (C) Statistics of the effect
of 1, 10, 100 pmol, and 1 nmol of (1)-(S)-Trujillon, and 1 nmol of (2)-(R)-
Trujillon. *P < 0.01, **P < 0.001, one-way ANOVA, Newman-Keuls, post
hoc test.
a selective NMDA antagonist. Note that D-AP5 alone did not change the
firing rate. (B) The excitatory response induced by (1)-(S)-Trujillon is
inhibited by the concurrent application of D-AP5. (C) Statistics of the
blockade of the (1)-(S)-Trujillon-induced excitation by D-AP5. P < 0.05,
Student’s t-test.
#
tion of 100 pmol D-AP5 induced a 46.92 6 8.52% spiking
rate reduction in 24.5% (5/17) of the neurons tested (P <
0
.001, Fig. 5A). There was not any significant modification
in the firing rate of the remaining 12 (75.5%) neurons. On
the other hand, none of the neurons tested showed signifi-
cant changes in their basal firing rate after the local appli-
cation of 100 pmol CNQX (n 5 11, Fig. 6A).
Effect of NMDARs Blockade on the
(
1)-( S)-Trujillon-Induced Excitation
To investigate whether the excitatory response elicited
by (1)-(S)-Trujillon was mediated by ionotropic glutamate
receptors, we tested the simultaneous application of (1)-
(S)-Trujillon with a NMDA or an AMPA/kainate antago-
nist. In our preparation, most recorded neurons did not
have tonically active glutamatergic inputs (see the earlier
section). However, 24.5% of them displayed a firing rate
reduction in the presence of 100 pmol D-AP5. Conse-
quently, only neurons that did not respond to D-AP5 were illustrating that the concurrent application of (1)-(S)-Trujillon and CNQX,
used to test for (1)-(S)-Trujillon–NMDARs interactions.
Fig. 6. Local blockade of the AMPA/kainate receptors does not modify
the excitatory effect of (1)-(S)-Trujillon. (A) Spike frequency histogram
an AMPA/kainate selective antagonist, cause the same response as (1)-
(
S)-Trujillon alone. (B) There was not significant difference between the
Application of 100 pmol (1)-(S)-Trujillon caused a 142
41% spiking rate increase (n 5 4). Further application of pmol CNQX.
effect of 100 pmol (1)-(S)-Trujillon alone and its co-application with 100
6
Chirality DOI 10.1002/chir

4
34
ARAUJO-ALVAREZ ET AL.
ligand-receptor recognition. The ligand-binding sites of
4
3
NMDARs are located in two extracellular domains
1
8
(
(
NR1/NR2A-D ); the selective activation of NMDARs by
1)-(S)-Trujillon led us to further explore its interaction
with the glutamate-binding monomer NR2A. The dock-
ing studies showed that (1)-(S)-Trujillon binds to NR2A
close to its glutamate-binding site (Fig. 8), with the same
type of interactions, but with very different affinity, as evi-
4
4
denced by their DG and K values. The DG for (1)-(S)-
d
Trujillon was 27.61 kcal/mol and 24.72 kcal/mol for glu-
tamate. The K for (1)-(S)-Trujillon was 2.63 lM and 342
d
lM for glutamate; these values are in agreement with our
experimental data and suggests that the high energy liber-
ated by the interaction of (1)-(S)-Trujillon with the
NMDAR can activate the signal transduction system of the
receptor (Fig. 8).
DISCUSSION
The five-membered heterocyclic ring of (1)-(S)-Trujil-
lon eliminates the charge of the alpha-amino and carboxyl
groups, but conserves the charge of the omega-carboxilate
side chain, which in addition to the presence of phenyl
groups bonded to the boron atom, makes (1)-(S)-Trujillon
Fig. 7. NMDARs blockade inhibits the (1)-(S)-glutamate-induced exci-
tation in globus pallidus neurons. (A) Local application of D-AP5 (a selec-
tive NMDARs antagonist) attenuates the effect of (1)-(S)-glutamate. (B) more lipophilic (Log P 5 1.19 vs. 0.79 of (1)-(S)-glutamate).
There was no statistically significant difference between the effect of 100
pmol (1)-(S)-glutamate alone and its co-application with 100 pmol D-AP5.
We previously confirmed (1)-(S)-Trujillon lipophilic-
3
0
ity; the systemic application of (1)-(S)-Trujillon induced
convulsions in mice, whereas (1)-(S)-glutamate adminis-
tered by the same route did not have any effect. Addition-
ally, (1)-(S)-Trujillon has high hydrolytic stability at vari-
(
C) The firing frequency increase evoked by NMDA is significantly
#
blocked by D-AP5. P < 0.05, Student’s t-test.
1
00 pmol D-AP5 significantly reverted the (1)-(S)-Trujillon- ous pH, which is a valuable feature because this novel
induced increase to baseline values (0.67 6 5.47% of base- agonist could be used in pH-varying conditions.
line, P < 0.05, n 5 4; see Figs. 5A–5C). On the other
hand, the CNQX application did not significantly alter the
excitatory effects of (1)-(S)-Trujillon (Fig. 6A). The local
infusion of 100 pmol (1)-(S)-Trujillon increased the dis-
charge rate by 153.33 6 59.40%, but, in the same neurons,
a concurrent application of 100 pmol CNQX and 100 pmol
(
1)-(S)-Trujillon caused a 140.42 6 65.5% increase (n 5 4,
Fig. 6B).
In addition, local application of 100 pmol (1)-(S)-gluta-
mate increased pallidal neurons firing rate by 69.3 6 27.6%
n 5 5), whereas in the same neurons, concurrent applica-
(
tion of 100 pmol D-AP5 caused a 9.3 6 5.6% firing rate
increase, an effect that was not statistically different from
that of (1)-(S)-glutamate alone (Figs. 7A and 7B). Further
confirmation of NMDAR-mediated excitation was assessed
by local co-application of NMDA and D-AP5 (Fig. 7C). Infu-
sion of 100 pmol NMDA caused a 149 6 7% firing fre-
quency increase (n 5 4), which was subsequently blocked
by further application of 100 pmol D-AP5 (Fig. 7C, P <
0
.05).
Fig. 8. Docking of (1)-(S)-Trujillon into NR2A NMDAR: The ligand is
displayed in a ball-and-stick model, whereas the amino acids involved in
ligand binding are color-coded as capped sticks. The (1)-(S)-Trujillon side
chain and its chyral carbon make electrostatic bonds at Lys 41 and at Lys
(
1)-( S)-Trujillon Docking into NMDARs
Using the NR2A crystalline 3D structure available at the 187 and Glu 15 backbones, respectively. The aromatic moiety of (1)-(S)-
Protein Data Bank (PDB, http://www.rcsb.org), docking
studies were conducted to find the ligand-binding sites for
Trujillon makes p-cation interactions with the guanidine of Arg 195.
Ligand binding affinity: 27.18 kcal/mol; 5.39 lM (glutamate: 24.72 kcal/
mol; 342 lM). [Color figure can be viewed in the online issue, which is
the NMDARs and the amino acids that are related to available at wileyonlinelibrary.com.]
Chirality DOI 10.1002/chir

(
þ)-(S)-TRUJILLON EFFECT ON PALLIDAL ACTIVITY
435
We selected the GP nucleus to analyze the effects of NMDARs activation, an idea supported by other
4
8,50,57
(1)-(S)-Trujillon because it is the final stage of a circuit reports.
The activation of NMDARs is required to
involved in the initiation, execution, and termination of reach a depolarization block, a phenomenon that is
4
5
motor programs at precise timing, most probably deter- thought to be involved in the therapeutic effect of deep
2
7
46
mined by the GP-STN pacemaker. Furthermore, the GP brain stimulation. However, extended neuronal depolari-
5
8
plays a key role in therapy for Parkinson’s disease and (L)- zation might cause delayed exitotoxicity. In this context,
dopa-induced dyskinesias and has a great diversity of our data suggest that activation of NMDARs by (1)-(S)-
4
6
2
2,26,47,48
49,50
ionotropic
and metabotropic
glutamate recep- Trujillon does not cause a significant depolarization block
tors. Besides, its dimensions in the rat brain allow for the or persistent NMDARs activity, thus avoiding cytotoxic
5
9
recording of 2–3 neurons per track and the placement of actions.
an injection cannula without touching the micropipette
tip.
We did not find a tonic AMPA/kainate-mediated gluta-
matergic discharge in the GP, and the local infusion of
5
1
A previous report of the differential response of Type-I CNQX did not modify the spontaneous spiking rate of GP
5
2
and Type-II neurons to dopamine receptor activation sug- neurons. These results are in agreement with previous
gests that these two types of neurons cannot be used studies showing that local infusion of low doses of NBQX,
indistinctively. However, we pooled them together an AMPA/kainate antagonist similar to CNQX, does not
because the response to (1)-(S)-glutamate was equal in modify GP spontaneous activity in the ketamine-anesthe-
2
6
both neuron types. Furthermore, we observed that micro- tized rat. However, local infusion of NBQX decreases the
2
2
pipette electrical resistance is a major factor for neuron pallidal firing rate in the unanesthetized monkey.
type discrimination; results that are in complete agree-
ment with other reports.
We also found that blockade of NMDARs by D-AP5
diminishes the spontaneous firing rate in 25% of pallidal
5
3,54
In a previous study, we reported that the intravenous neurons. However, previous studies show that application
application of (1)-(S)-Trujillon caused excitation in GP of MK-801, a noncompetitive NMDA antagonist, does not
60
neurons, whereas the application of (1)-(S)-glutamate by change the discharge rate in the accumbens nucleus or
3
0
26
the same route did not. Here, the local infusion of (1)- in the GP of ketamine-anesthetized rats. These differ-
S)-Trujillon caused excitation in most pallidal neurons ences are likely due to the different anesthetics and gluta-
(
tested and, furthermore, this excitatory effect was dose-de- mate receptors antagonists employed. Our results give
pendent. These findings show that the excitation evoked support to the possibility that some NMDA-mediated
2
6
by (1)-(S)-Trujillon on GP neurons is a local effect.
responses are dampened in ketamine-treated animals.
Equimolar infusions of (1)-(S)-glutamate, NMDA, and Our data also suggest that, in the GP, different mecha-
1)-(S)-Trujillon increases GP neurons firing rate. The nisms mediate the glutamatergic tone through AMPA/kai-
(
excitatory effect of (1)-(S)-glutamate is significantly differ- nate- and NMDA receptors.
ent from that of NMDA (Figs. 2C and 3C). Interestingly,
The excitatory effect of (1)-(S)-Trujillon is blocked by
the effect of (1)-(S)-Trujillon is equal to that of NMDA the selective NMDA antagonist AP-5, which also blocks
(Figs. 3C and 4C). The smaller effect of (1)-(S)-glutamate the excitatory effect of the local infusion of NMDA. In con-
is most likely due to the activation of glutamatergic recep- trast, the selective blockade of AMPA/kainate receptors
tors that could negatively modulate the basal firing rate of by CNQX does not modify the excitatory effect of (1)-(S)-
5
5
GP neurons, an effect that does not occur with NMDA
Trujillon. Thus, an important conclusion of our study is
or (1)-(S)-Trujillon. In this context, the activation of pre- the selective activation of NMDARs by (1)-(S)-Trujillon.
3
5
synaptic kainate receptors in STN-GP fibers diminishes These receptors have been the targets for drug design
glutamate release.
4
8
Moreover, activation of group I because of their central role in neural plasticity, long-term
metabotropic glutamate receptors (mGluRs) uncouples the potentiation and learning, and the generation of rhythmic
5
0
excitation evoked by other mGluRs. Furthermore, gluta- motor activity, among other functions. Furthermore, the
mate-mediated inhibition has been demonstrated in retinal coactivation of NMDARs and mGluRs has been related to
5
6
bipolar cells.
the reduction of excitotoxic cell death because of a
It has been reported that the same dose of NMDA that decrease of the inappropriate stimulation of the sys-
6
1,62
was used in this study increases the GP neurons firing tem.
Accordingly, (1)-(S)-Trujillon has a high affinity
2
6
rate, whereas lower doses do not have any effect. In our (5.39 lM) for NR2A. (Fig. 8). The carboxyl oxygen atom
study, NMDA, (1)-(S)-glutamate, and (1)-(S)-Trujillon of the (1)-(S)-Trujillon side chain makes electrostatic
diminishes the spontaneous activity of some pallidal neu- bonds with the NH group of Lys 41, which in addition to
3
rons. This inhibition could be explained by the activation p-cation interactions between the aromatic moieties of
2
3
of intrapallidal collaterals : neighboring GP neurons (1)-(S)-Trujillon and the guanidine groups of Arg 195 of
release GABA into the neuron being recorded. On the NR2A, allows for a stable binding. There are also electro-
other hand, 100 pmol NMDA induces a depolarization static bonds between the Lys 87 and Glu 15 backbone and
block in some neurons; whereas (1)-(S)-glutamate and the carbonyl group close to the chyral carbon of (1)-(S)-
(
1)-(S)-Trujillon induces a depolarization block in fewer Trujillon.
neurons, even with the highest dose of (1)-(S)-Trujillon (1 In conclusion, the study of drugs interacting with the
nmol). This suggests that the activation of non-NMDA glutamatergic transmission within the BG and other brain
but not AMPA/kainate) receptors by (1)-(S)-glutamate nuclei is a subject of major interest because it could gener-
(
or (1)-(S)-Trujillon attenuates the excitatory effect of ate new therapeutic alternatives for some motor and cogni-
Chirality DOI 10.1002/chir

4
36
ARAUJO-ALVAREZ ET AL.
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NMDARs, and therefore a valuable tool to study the CNS
glutamatergic transmission. The interaction of this novel
compound with mGluRs is yet to be determined, and fur-
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Chirality DOI 10.1002/chir