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Vol. 302, Issue 3, 1135-1145, September 2002
Department of Neuropharmacology, The Scripps Research Institute, La Jolla, California
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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In the course of studying
N-methyl-D-aspartate (NMDA) receptors of the
nucleus accumbens (NAcc), we found that 20% of freshly isolated medium
spiny neurons, as well as all interneurons, responded in an unexpected
way to long (5-s) coapplication of NMDA and glycine, the coagonist of
NMDA receptors. Whereas the reversal potential of the peak NMDA current
of this subset of neurons was still around 0 mV, the desensitizing
current became outward at hyperpolarized potentials around 
30 mV. A
Cl
-free solution shifted the equilibrium potentials of
the desensitized currents to around 0 mV. This outward current was not
blocked by a Ca2+-free, Ba2+-containing
solution, suggesting that the anionic conductance was not activated by
Ca2+ influx through NMDA receptor channels. Interestingly,
glycine alone also evoked a current with a similar hyperpolarized
reversal potential in this subset of neurons. The glycine current
reversed around 50 mV, rectified outwardly, and inactivated strongly. Its desensitization was best fitted with a double exponential. Only the
slow desensitization showed clear voltage dependence. The glycine
current was not blocked by 200 µM picrotoxin and 10 µM zinc, was
weakly antagonized by 1 µM strychnine, and was not enhanced by
1 µM zinc. In addition, 1 mM taurine, but not GABA, inactivated
glycine currents, and 1 mM glycine occluded 10 mM taurine-mediated
currents. These data indicate that a subset of nucleus accumbens
neurons expresses glycine receptors and that either glycine or taurine
could be an endogenous agonist for these receptors.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The
nucleus accumbens (NAcc), an interface region between limbic structures
(hippocampus, amygdala, and prefrontal cortex) and the extrapyramidal
motor system, modulates cognitive and motivational aspects of behavior
that are translated into motor activity (Dunah et al., 1996
).
Projecting GABAergic neurons, also containing mostly either substance
P or enkephalin, account for 90% of the neuronal population of
NAcc (Meredith, 1999). These neurons receive massive dopaminergic input
from the ventral tegmental area, glutamatergic input from cortical and
subcortical regions, and GABA innervation from interneurons. The
physiology of the receptors for these transmitters is relatively well
known, because they have been heavily studied with regard to addiction
to psychostimulants, opiates, and alcohol (Koob et al., 1998).
NAcc-projecting neurons also receive inputs from acetylcholine- and
somatostatin-containing interneurons (Groenewegen et al., 1991;
Meredith, 1999), but relatively little is known about the physiology of
receptors for these ligands in NAcc. Furthermore, some data suggest
that other receptors, including glycine receptors, remain to be
identified and characterized.
Glycine receptors, along with GABA receptors, represent the primary
fast inhibitory mechanisms in the central nervous system. Activation of
glycine receptors opens anionic channels that hyperpolarize neurons.
Until recently, it was generally believed that glycine receptors were
almost exclusively found in the spinal cord and brainstem of adult rats
(Rajendra et al., 1997). However, recent findings suggest that these
receptors may also be expressed in upper brainstem regions (Rampon et
al., 1996). Recent studies also reported the presence of glycine
receptors in forebrain structures (Yoon et al., 1998; Mori et al.,
2002).
Glycine receptors are heteromultimeric receptors. Molecular approaches
have revealed a certain diversity of glycine receptor subunits. To
date, four different 
and one 
subunit have been identified
(Legendre, 2001). Examination of the organization of glycine receptors
revealed that glycine receptors are pentameric with an invariant
stoichiometry of three and two subunits (Langosch et al.,
1988). Although the expression of the subunit matches that of subunits in spinal cord, pons, and midbrain, there is evidence that mRNA expression does not fully match that of subunits in the brain
(Fujita et al., 1991; Malosio et al., 1991). Thus, it was established
that forebrain regions such as nucleus accumbens and striatum express
subunits, although in moderate levels (Fujita et al., 1991; Kirsch
and Betz, 1993; Kirsch et al., 1993), but lack the subunit (Sato et
al., 1991; Malosio et al., 1991) that carries the binding site for
glycine (Pfeiffer et al., 1982).
This absence of a systematic overlap between and subunit
expression has prompted some investigators to suggest that additional subunits, which could confer different properties for glycine receptors, remain to be discovered (Betz et al., 1999). It was also
suggested that subunits may form part of another receptor complex.
Herein, we demonstrate for the first time in a subpopulation of NAcc
neurons the existence of a functional receptor with
electrophysiological properties reminiscent of glycine receptors but
with a distinctive pharmacological profile.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Animals, Slice Preparation, and Experimental Solutions.
We
used male Sprague-Dawley rats (100-200 g) to prepare NAcc slices. The
rats were anesthetized with 4% halothane, decapitated, and the brains
rapidly transferred into a cold (4°C) oxygenated, low-calcium
HEPES-buffered salt solution: 234 mM sucrose, 2.5 mM KCl, 2 mM
NaH2PO4, 11 mM glucose, 4 mM MgSO4, 2 mM CaCl2, and 1.5 mM HEPES. We glued a tissue block containing NAcc to a Teflon chuck
and cut it transversally with a Vibroslicer (Campden Instruments, Loughborough, UK). Then we incubated the slices (400 µm in thickness) for up to 6 h at room temperature (20-22°C) in a gassed (95%
O2 and 5% CO2)
NaHCO3-buffered saline solution: 116.4 mM NaCl,
1.8 mM CaCl2, 0.4 mM MgSO4,
5.36 mM KCl, 0.89 mM
NaH2PO4, 5.5 mM glucose, 24 mM NaHCO3, 100 mM glutathione, 1 mM
nitro-arginine, and 1 mM kynurenic acid. pH was adjusted to 7.35 with
NaOH (osmolarity 300-305 mOsM/l). After 1 h of incubation, we
dissected out the region of the nucleus accumbens with the aid of a
dissecting microscope. We incubated the tissue for 25 min in an
oxygenated (100% O2 and constant stirring)
HEPES-buffered solution in the inner chamber of a Cell-Stirr flask
(Wheaton, Millville, NJ) that contained papain (1 mg/ml) and 136 mM
NaCl, 0.44 mM KH2PO4, 2.2 mM KCl, 0.35 mM NaH2PO4,
5.5 mM glucose, 10 mM HEPES, 100 mM glutathione, 1 mM nitro-arginine, 1 mM kynurenic acid, and 1 mM pyruvic acid (pH 7.35 with NaOH, osmolarity
300-305 mOsM). The temperature of this solution was kept constant
(36°C) by a circulating water bath in the outer chamber of the flask
(Stefani et al., 1994; Martin et al., 2002).
Whole-Cell Recordings.
We used standard whole-cell recording
methods (Hamill et al., 1981). Briefly, we pulled patch electrodes from
borosilicate capillary glass (Sutter Instruments, Novato, CA) on a
Flaming-Brown puller (Sutter Instruments) to a final resistance of 1.8 to 2.2 M
. We filled the electrodes with a solution that consisted of 120 mM CsF, 10 mM CsCl, 11 mM EGTA, 10 mM HEPES, and 0.5 mM
CaCl2 (pH 7.35 with CsOH; osmolarity 270-275
mOsM). The capillaries were first filled through the tip and then
backfilled with the recording solution. We recorded in voltage-clamp
mode with an Axopatch 1D amplifier and a DAC Digidata 1200 interface
from Axon Instruments (Union City, CA). The signal was filtered at 5 kHz and digitized at 1 kHz. The series resistance was compensated (70-80%) only when the ramp protocol was used. We subtracted the NMDA
current obtained with the ramp from the membrane response recorded in
absence of NMDA and glycine. Potentials were not corrected for the
liquid junction potential but are estimated to be +4 mV.
Superfusion and Drug Application. Control and drug-containing solutions were applied by gravity at a rate of 1.5 ml/s, using a rapid three-barrel capillary superfusion device (Warner Instrument, Hamden, CT) with the pipette tips placed about 200 µm from the recorded cell. The flow of solutions was controlled by solenoid valves. Each capillary had a tip diameter of 500 µm, and the distance from center to center was 700 µm. The barrel was attached to a motor, allowing fast lateral motions controlled by pClamp 6 (Axon Instruments), our data acquisition program. A drug onset time of 20 ms for the application system was determined by measuring the changes in the tip potential of the recording pipette filled with intracellular solution as the perfusion was switched from a normal to a 1:2 dilution of the extracellular recording solution.
Our standard drug-testing protocol was as follows. After recording a stable control current in ACSF alone, we superfused the cells with ACSF containing the drug for 2 to 3 min before recording. This was followed by a washout with the control ACSF. We examined the reversal potential of early NMDA currents by measuring the peak NMDA current amplitudes at various holding membrane potentials that were set manually between70
and +20 mV with voltage steps of 10 mV. We also used a ramp protocol to
determine the reversal potential of NMDA/glycine and
glycine-desensitized currents. The membrane potential was held at 100
mV for 8 s, whereas the cells were superfused with the agonists
and depolarized at a rate of 10 mV/s up to +40 mV.
Effects of zinc, bicuculline, and picrotoxin were tested by exposing
the neurons with these drugs for at least 1 min before applying either
GABA or glycine. Strychnine usually was coapplied with glycine although
in some cells strychnine was applied before glycine. We purchased
glycine, picrotoxin, taurine, ZnCl2,
BaCl2, bicuculline, strychnine, and GABA from
Sigma-Aldrich (St. Louis, MO). CGP 55845 was a gift from Novartis
Pharma (Basel, Switzerland).
We fitted the glycine dose-response curve with a Hill equation as
follows: I = Imax/[1 + (EC50/C)n],
where I, Imax,
C, EC50, and n are
glycine-activated current, maximal glycine-activated current, glycine
concentration, the concentration for 50% of maximum response, and the
Hill coefficient, respectively. We measured the voltage-dependent
glycine current desensitization using a linear regression formula in
DeltaGraph 4.5 (SPSS, Chicago, IL).
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Freshly Isolated NAcc Neurons.
Mechanical trituration after
enzymatic treatment of NAcc slices yielded cells of various sizes. Most
neurons had a small soma (Fig. 1, A-C)
and only a small population had a larger cell body (Fig. 1, D and E).
Neuronal shape also varied markedly; some neurons showed multipolar
processes (Fig. 1B), whereas others were bipolar and ovoid (Fig. 1, A
and C). Nevertheless, most of the small neurons recorded were likely to
be medium spiny cells, because they represent up to 90% of the total
number of cells in NAcc (Meredith and Totterdell, 1999). The larger
neurons (Fig. 1D) are likely to be interneurons due to their size; few
of them are seen after an incubation of 30 min with papain (Bargas et
al., 1994; Stefani et al., 1994). Only neurons presenting a smooth and
shiny membrane were recorded, because these criteria usually correlate
with healthy cells.
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Currents Evoked by Coapplication of NMDA and Glycine.
For 80%
of medium spiny neurons tested (class I; n = 54),
coapplication of 200 µM NMDA and 100 µM glycine (the NMDA receptor coagonist) evoked characteristic NMDA currents that were inward at
negative holding potentials and outward at positive ones (Fig. 2A), with the reversal potentials of both
peak and desensitizing currents near 0 mV (Fig. 1B). Interestingly, in
the remaining medium spiny neurons (class II; Fig. 2C), as well as in
all large interneurons (Fig. 2E, arrow), an equivalent coapplication of NMDA and glycine evoked a transient inward current that reversed around
0 mV, followed by a desensitizing current that became outward around
30 mV. To determine the nature of this desensitized outward current,
we examined its sensitivity to various ions. Because NMDA receptors can
be coupled to calcium-dependent chloride channels (Scott et al., 1995),
we studied the NMDA effects in a Cl-free
solution in class II neurons. In control solution, coapplication of
NMDA and glycine onto these neurons evoked an outward current around
20 mV. One minute after switching to a
Cl-free solution the outward current was almost
entirely blocked, revealing the underlying NMDA current, whose
amplitude increased at negative holding potentials, possibly because
the shunt exerted by the chloride conductance was blocked (Fig.
3A). In addition, both peak and
desensitized currents reversed around 0 mV, and the NMDA current seemed
similar in shape to that of class I neurons (Fig. 2A). Because
Ca2+-dependent Cl
channels are blocked by Ba2+ (Scott et al.,
1995), we substituted Ba2+ for
Ca2+. Surprisingly, Ba2+
did not prevent the outward current (Fig. 3A). Averaged over six cells,
the Cl-free solution clearly shifted the
reversal potential of the outward current to around 0 mV, whereas
Ba2+ had no effect (Fig. 3B), suggesting the
absence of Ca2+-dependent
Cl channels in class II NAcc neurons.
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26 to 30 mV; Fig. 3B).
This small effect is probably due to a decrease of the inward NMDA
current amplitudes, clearly visible in Fig. 3, A and B, at negative
membrane potentials, that would weaken the NMDA inward currents
opposing the outward current and thus shift the apparent reversal
potential of the latter to the left. Although glycine currents were
generally very stable, as shown by the experiments on current
inactivation (see below), it is possible that exposure to
Ba2+, which sequentially preceded the
Na+-containing solution, may prevent glycine
currents from fully recovering.
Effects of Glycine Alone on Class II NAcc Neurons.
We asked
whether glycine alone could activate a similar outward current. At 70
mV, application of 100 µM glycine evoked an inward current that
decreased at more depolarized holding potentials and reversed around
50 mV (Fig. 4A). Using a ramp protocol
in 10 cells, the mean reversal potential of the glycine current was 50 ± 2 mV (54 mV corrected for the liquid junction
potential), a value close to the theoretical value for
Cl given by the Nernst equation (55.4 mV)
with [Cl]o of 145 mM
and estimated [Cl]i of
10 mM. This current rectified outwardly at both depolarized and
hyperpolarized holding potentials (Fig. 4B). It is possible that part
of the rectification may be due to poor space clamping or to the
participation of unblocked voltage-gated channels. It is also possible
that fluoride, used in the recording pipette, passing through glycine
receptors may contribute to some extent to the rectification. However,
because the permeability of glycine receptors for this anion is the
lowest of the six tested by Fathima-Shad and Barry (1989), its
contribution is probably minimal.
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5
(Fig. 5B), suggesting that at least two glycine molecules are needed to
activate each receptor. Although not systematically studied, we
observed that the desensitization of the current was profoundly altered
as the concentration of glycine increased (Fig. 5A). To characterize
these receptors further, we measured the desensitization (decay of the
current measured in continued presence of glycine) of glycine currents
at several holding potentials from 70 to +10 mV. At all potentials
(Fig. 6A;
Vh = 0 mV), glycine-mediated currents
desensitized very rapidly then returned close to baseline levels within
the 5-s application. The desensitization was best fitted with a double
exponential (Fig. 6A), as also shown for hypothalamic neurons and
Mauthner cells (Faber and Korn, 1987; Akaike and Kaneda, 1989). Between
80 and +10 mV, the mean fast and slow decay taus were 0.381 ± 0.23 and 1.705 ± 0.26 s, respectively (n = 8; Fig. 6B). Interestingly, the fast tau did not seem to be
voltage-dependent, unlike the slow desensitization that showed a clear
voltage dependence between 80 and +10 mV when fitted by linear
regression. A similar voltage dependence was reported by Akaike and
Kaneda (1989) and Faber and Korn (1987).
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), by measuring the responses of test glycine currents
after conditioning glycine applications at various intervals. Such
conditioning applications strongly inactivated test glycine currents at
2-s intervals. As the interval increased, the inactivation clearly
diminished (Fig. 6C): the mean average amplitude (n = 5) of the test glycine current at 2-s intervals was 48 ± 9% of
the conditioning current (Fig. 6D), increasing to 95 ± 4% at
14-s intervals.
Pharmacology of NAcc Glycine-Like Receptors.
We first examined
the effects of strychnine on the glycine current because this compound
is a highly selective and potent (IC50 = 5-10
nM) glycine receptor antagonist (Young and Snyder, 1973, 1974).
Surprisingly, glycine currents were strongly antagonized only with high
strychnine concentrations (10-100 µM) (Fig.
7A), whereas 1 µM strychnine, coapplied
with glycine decreased the mean glycine current amplitudes by only
18 ± 1.8% (n = 8; Fig. 7B), an equivalent
strychnine-insensitivity was found in three neurons where strychnine
was applied before glycine. The apparent strychnine
IC50 was 12 µM. We also examined the effects of
picrotoxin, an antagonist of both GABAA and
glycine receptors in some preparations (Rajendra et al., 1997) on
glycine currents at 0 mV. A 1-min application of 50 µM picrotoxin
moderately decreased glycine current amplitudes (Fig.
8A). At a greater picrotoxin
concentration (200 µM) glycine current amplitudes were slightly
increased (data not shown). On average, 50 µM picrotoxin decreased
mean glycine current amplitudes by 15 ± 4%, whereas 200 µM
increased it by 22 ± 6% (Fig. 8C). We also examined the effects
of Zn2+, known to potentiate and to block glycine
currents at a low (1 µM) and high (10 µM) concentrations,
respectively (Bloomenthal et al., 1994; Laube et al., 1995); we found
that 1-min preincubation with 1 and 10 µM zinc only slightly
decreased glycine current amplitudes, by 8 ± 2 and 6 ± 2%,
respectively (Fig. 8C).
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What Is the Endogenous Ligand for NAcc Glycine-Like Receptors?
Because glycine-immunoreactive cells and fibers have not been
identified to date in forebrain structures such as NAcc (Rampon et al.,
1996), we further examined the possibility that GABA could be the
endogenous transmitter for the glycine-like receptor. GABA applications
(500 µM) onto class II NAcc neurons evoked currents with strong
similarities to the glycine-evoked currents in that they displayed a
strong desensitization (Fig. 9A) and were
also strongly inactivated by a conditioning GABA application (data not
shown). Bicuculline (40 µM) moderately decreased the GABA currents
and altered the time to peak of the current, with a recovery on washout
(Fig. 9A). Averaged over four neurons, bicuculline decreased mean GABA
current amplitude by only 17 ± 6% (Fig. 9B). Because of the
surprising ineffectiveness of bicuculline we also tested picrotoxin
(200 µM), another potent GABAA receptor antagonist. As
with bicuculline, picrotoxin markedly altered the kinetics of the
currents, with recovery on washout (Fig. 9C). However, picrotoxin also
failed to fully antagonize the response (mean GABAA current
amplitudes decreased by 66 ± 12%; Fig. 9D). The persistence of a
sizable GABA current in the presence of a high concentration of
picrotoxin suggests that GABA may activate a conductance different from
that of GABAA receptors, although we cannot totally rule
out that the concentration of GABA used in our experiments may also
partially account for this effect due to competitive agonist/antagonist
interactions. This current did not involve GABAB receptors
because it was not altered by the presence in the recording solution of
a GABAB receptor antagonist (CGP 55845) or
Cs+, known to block potassium currents. Despite the fact
that the glycine current was strongly inactivated by a conditioning
glycine pulse, currents evoked by 500 µM GABA did not alter glycine
current amplitudes at any interapplication interval (Fig. 9E),
suggesting little interaction between GABAA and
glycine-like receptors in NAcc.
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In these experiments on acutely isolated NAcc neurons we found that all NAcc interneurons and 20% of medium spiny cells expressed glycine-like receptors. Although their biophysical properties are similar to those of other glycine receptors, their pharmacological properties set them apart. In addition, our data support the possibility that taurine is an endogenous transmitter for NAcc glycine-like receptors.
Identity of NAcc Glycine-Like Receptors.
These newly described
NAcc receptors exhibited a set of properties reminiscent of
strychnine-sensitive glycine receptors. Thus, NAcc glycine-evoked
currents rectified outwardly in both the hyperpolarizing and
depolarizing range, a phenomenon observed at voltages negative to 50
mV in cultured (Bormann et al., 1987; Fatima-Shad and Barry, 1992,
1993) and acutely isolated hypothalamic (Akaike and Kaneda, 1989)
neurons, as well as in recombinant glycine receptors in expression
systems (Gundersen et al., 1984; Akagi et al., 1991; Morales et al.,
1994). The affinity of glycine for NAcc glycine-like receptors was 110 µM, a value similar to those obtained from glycine receptors in
hyothalamic, cerebellar, and ventral cochlear neurons (Akaike and
Kaneda, 1989; Virginio and Cherubini, 1997; Harty and Manis, 1998).
NAcc glycine currents desensitized rapidly with a biphasic course, in
agreement with glycine receptors in other studies (Akaike and Kaneda,
1989; Lewis et al., 1991). However, in contrast to neurons in
hypothalamus (Akaike and Kaneda, 1989), only the slow desensitization
was clearly voltage-dependent in NAcc neurons. NAcc glycine currents
also showed a strong inactivation, as reported for other glycine
receptors (Akaike and Kaneda, 1989; Virginio and Cherubini, 1997; Harty and Manis, 1998).
and subunits (Legendre, 1997; Yoon et al., 1998), further
suggests the presence of a glycine receptor in NAcc. However, the
apparent presence of glycine receptors in NAcc is somewhat at odds with anatomical data. For example, glycine binding sites are extremely abundant in spinal cord and to a lesser extent in the midbrain, hypothalamus, cerebellum, and thalamus and are almost absent in forebrain structures such as cortex, NAcc, or dorsal striatum (Rajendra
et al., 1997). An in situ hybridization study by Sato et al. (1991),
showing that forebrain regions lack 1 subunits, supported the
absence of glycine receptors in the forebrain. However, the same
authors (Sato et al., 1992) showed that 2 subunits were expressed,
although at moderate levels, in the NAcc. Interestingly, a recent study
by Racca et al. (1998) confirmed the presence of 2 subunits in the
forebrain (striatum) but also found the expression of 1 subunit
mRNA. It would be interesting to extend such immunohistochemical study
to the NAcc, to determine the kind of glycine receptor subunits expressed there and whether a similar expression in a subset of neurons
mirrors our electrophysiological data and the Racca et al. (1998)
findings in the dorsal striatum.
Although these findings do not directly demonstrate the presence of
functional glycine receptors, they may explain the ability of glycine
to evoke currents in some NAcc neurons. Indeed, subunits, known to
carry glycine binding sites, are usually associated with the presence
of functional glycine receptors. The study by Racca et al. (1998) also
may help explain the fact that glycine currents were partly restricted
in our study to interneurons, which represent only 5 to 10% of the
NAcc neuronal population. Although Racca et al. (1998) reported only
weak-to-moderate levels of 1 mRNAs in striatum, they found a few
scattered, highly positive cells that could represent NAcc interneurons
and/or the fraction of medium spiny neurons that did show glycine
currents. The expression of 2 mRNA and more specifically the
presence of 2* subunit, if confirmed, may explain the surprisingly
weak strychnine effect we observed as these subunits are usually
considered to be less sensitive to strychnine (Vanderberg et al,
1992a,b). The method of coapplication of strychnine and glycine cannot
account for the relative ineffectiveness of strychnine compared with
published data (Young and Snyder, 1973, 1974), as preincubation of a
subset of cells with 1 µM strychnine (data not shown) also failed to block glycine currents as reported in other studies. We obtained a
similar result when testing the effects of 10 µM zinc (Fig. 8B).
Finally, although there are no data that would support the idea that
heteromeric glycine receptors composed of 1 and 2 subunits may be
combined to form functional NAcc glycine receptors, this hypothesis
cannot be ruled out given the extraordinary ability of subunits that do
not belong to the same class of receptors to form new entities (see below).
An alternative to the necessity for 1 and/or 2 proteins was
recently formulated by Betz et al. (1999), who proposed that subunits, widely expressed in the forebrain (Fujita et al., 1991;
Malosio et al., 1991; Kirsch et al., 1993; Racca et al., 1997), along
with a protein yet to be identified, may form a novel complex that
retains only some of the properties of glycine receptors. Recent
studies indicate that receptors of different families can interact to
alter their individual properties. Thus, dopamine D2 and somatostatin
SST5 receptors, two structurally related G protein-coupled receptors,
can form a novel hetero-oligomer with pharmacological properties
different from those receptors alone (Rocheville et al., 2000). A
similar heterodimerization was also reported for 
- and 
-opioid
receptors (Jordan and Devi, 1999). With respect to ionotropic
receptors, the 
subunit of GABAC receptors interacts with a novel splice variant of the glycine transporter GLYT-1
(Hanley et al., 2000) that is also found in NAcc (Zafra et al., 1995).
These findings challenge the dogma of strict boundaries between
receptors. Several studies (for review, see Rajendra et al., 1997) have
established unequivocally that the primary sequence of glycine receptor
and subunits share a considerable homology with subunits of
other multimeric receptors such as nicotinic (Noda et al., 1982),
serotonin type 3 (Maricq et al., 1991), and GABAA
receptors (Schofield et al., 1987). Such similarities could support the
presence of receptors displaying distinct properties such as described herein.
Endogenous Agonist of NAcc Glycine-Like Receptors.
Regardless
of the exact identity of the subunits forming the NAcc glycine-like
receptors, the nature of their endogenous agonist remains to be
determined. Although glycine evoked the ionic conductance, this amino
acid may not be the endogenous ligand for the NAcc receptors because no
glycine-containing terminals have been detected in this region (Rampon
et al., 1996). Because the primary structure of
GABAA and glycine receptors shares significant
sequence homologies, we examined the possibility that GABA could
activate the glycine-like receptors in NAcc. We found that picrotoxin,
a potent GABA receptor antagonist, did not totally block the
GABA-mediated currents, suggesting the involvement of either a GABA
receptor resistant to picrotoxin or a glycine receptor insensitive to
picrotoxin. The latter choice has some credibility, because the ability
of picrotoxin to block glycine receptors varies dramatically with the
subunit composition of the receptor. Thus, homomeric glycine receptors are much more sensitive to picrotoxin block than heteromeric plus glycine receptors (Pribilla et al., 1992). In addition, Fucile et al. (1999) reported that GABA activates homomeric 1 glycine receptors from zebrafish. Similarly, in rat olfactory bulb
neurons both glycine and GABA can bind to either glycine or
GABAA receptors (Trombley et al., 1999). However,
the fact that GABA failed to inactivate or occlude glycine currents
supports the idea that GABA does not bind to the glycine-like receptors.
-alanine and taurine as having a strong affinity for
glycine receptors (Curtis et al., 1968; Young and Snyder, 1973).
Taurine is a good candidate because it is the most abundant amino acid,
after glutamate, and is widely, although unevenly, distributed in the
brain (Palkovits et al., 1986), including the NAcc (Madsen et al.,
1987). Both taurine (Ottersen and Storm-Mathisen, 1986; Madsen et al.,
1987) and cysteine sulfinate decarboxylase, the enzyme responsible for
taurine biosynthesis (Legay et al., 1987a,b), have been found
predominantly in forebrain structures, including the striatum.
The presence of taurine in NAcc medium spiny neurons and its apparent
absence in terminals pose the question of its release. An interesting
parallel can be drawn between GABA and taurine in the NAcc. GABA, also
a nonvesicular transmitter, can be released into the extracellular
space from the soma by prolonged activation of excitatory amino acid
receptors (for review, see Attwell et al., 1993), presumably by
an influx of Na+ through NMDA receptor channels
in neurons of striatum and NAcc (Schoffelmeer et al., 2000). This would
explain why, in NAcc slices, we rarely observed
GABAA receptor antagonist-insensitive inhibitory postsynaptic potentials, because we evoked synaptic events only with
single stimulations; a train of stimuli would have been required for
nonvesicular release. It might also suggest that glycine receptors may
be silent under normal conditions. Furthermore, it is likely that these
glycine- and taurine-like receptors are the same entity because the
existence of a specific taurine receptor has yet to be demonstrated.
This idea is supported by the recent findings of Hussy et al. (2001)
who reported that taurine-mediated osmotic regulation in the
neurohypophysis is exerted through glycine receptors. To date, no
protein that could unequivocally account for this receptor has been
identified and characterized. In addition, our occlusion results
indicating that taurine acts on the same receptor as glycine further
strengthen the idea that taurine is an endogenous glycine receptor
agonist. It seems unlikely that taurine-mediated currents are the
result of GABAA receptor activation, because the
affinity of taurine for GABAA receptors is low.
In addition, if the response evoked by taurine reflected an effect on
GABAA receptors, the likelihood of
taurine-occluding glycine responses would be very small.
Furthermore, the presence of glycine receptor subunits as well as
the newly reported subunits, further strengthens the case for
existence of functional glycine receptors in the NAcc. Finally, these
NAcc glycine receptors present no apparent similarities to a newly
described excitatory glycine receptor (Chatterton et al., 2002).
In conclusion, glycine elicits a current in some medium spiny neurons
and in all interneurons in the rat NAcc. Although many of the
biophysical properties of this current are similar to those of glycine
currents found in spinal cord and brain stem, its pharmacological characteristics set it apart. The activation of this receptor, as with
GABA receptors in most medium spiny neurons, should selectively inhibit
a subset of NAcc neurons.
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Acknowledgments |
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We were indebted to Dr. J. Surmeier for considerable assistance in setting up the acutely isolated neuron preparation. We also thank Drs. N Hussy, P. Schweitzer, and M. Tallent for helpful comments.
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Footnotes |
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Accepted for publication May 13, 2002.
Received for publication January 23, 2002.
This research was supported by National Institutes of Health Grants DA-03665 and AA-06420 (to G.R.S.) and DA-08301 (to S.J.H.).
DOI: 10.1124/jpet.102.033399
Address correspondence to: Dr. Gilles Martin, University of Massachusetts School of Medicine, Department of Neurobiology, 364 Plantation St., Worcester, MA 01605-2324. E-mail: gilles.martin{at}umassmed.edu
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Abbreviations |
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NAcc, nucleus accumbens; ACSF, artificial cerebrospinal fluid; NMDA, N-methyl-D-aspartate CGP 55845, 2S-3-[[(1S)-(3,4-dichlorophenyl)ethyl]amino-2-hydropropyl]phosphonic acid.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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