Download Differential sensitivity of medium- and large

European Journal of Neuroscience, Vol. 14, pp. 1577±1589, 2001
ã Federation of European Neuroscience Societies
Differential sensitivity of medium- and large-sized striatal
neurons to NMDA but not kainate receptor activation in
the rat
Carlos Cepeda, Jason N. Itri, Jorge Flores-HernaÂndez, Raymond S. Hurst,* Christopher R. Calvert and
Michael S. Levine
Mental Retardation Research Center and Brain Research Institute, 760 Westwood Plaza NPI Room 58±258, UCLA School of
Medicine, Los Angeles, CA 90024, USA
Keywords: brain slices, electrophysiology, excitatory amino acid receptors, excitotoxicity, Huntington's disease
Abstract
Infrared videomicroscopy and differential interference contrast optics were used to identify medium- and large-sized neurons in
striatal slices from young rats. Whole-cell patch-clamp recordings were obtained to compare membrane currents evoked by
application of N-methyl-D-aspartate (NMDA) and kainate. Inward currents and current densities induced by NMDA were
signi®cantly smaller in large- than in medium-sized striatal neurons. The negative slope conductance for NMDA currents was
greater in medium- than in large-sized neurons and more depolarization was required to remove the Mg2+ blockade. In contrast,
currents induced by kainate were signi®cantly greater in large-sized neurons whilst current densities were approximately equal in
both cell types. Spontaneous excitatory postsynaptic currents occurred frequently in medium-sized neurons but were relatively
infrequent in large-sized neurons. Excitatory postsynaptic currents evoked by electrical stimulation were smaller in large- than in
medium-sized neurons. A ®nal set of experiments assessed a functional consequence of the differential sensitivity of mediumand large-sized neurons to NMDA. Cell swelling was used to examine changes in somatic area in both neuronal types after
prolonged application of NMDA or kainate. NMDA produced a time-dependent increase in somatic area in medium-sized neurons
whilst it produced only minimal changes in large interneurons. In contrast, application of kainate produced signi®cant swelling in
both medium- and large-sized cells. We hypothesize that reduced sensitivity to NMDA may be due to variations in receptor
subunit composition and/or the relative density of receptors in the two cell types. These ®ndings help de®ne the conditions that
put neurons at risk for excitotoxic damage in neurological disorders.
Introduction
The striatum is composed primarily of medium-sized spiny projection
neurons (Jiang & North, 1991; Gerfen, 1992). These cells make up
> 95% of the total number of striatal neurons and they have been
studied extensively. The other neuronal subtypes, medium- and largesized interneurons, are less numerous and because of their scarcity
they have not been extensively studied from a functional perspective.
Whilst there are several subpopulations of interneurons, one subtype,
the large cholinergic neuron, has been the subject of much interest
because it is the source of acetylcholine in the striatum (Bolam et al.,
1984) and it has been implicated in neurological disorders (Vonsattel
et al., 1985; Di Chiara et al., 1994).
Until recently it has been dif®cult to study large cholinergic
neurons electrophysiologically because they are scarce (Graveland &
DiFiglia, 1985) and rarely sampled with standard in vivo or in vitro
techniques (Wilson et al., 1990). The advent of infrared videomicroscopy and differential interference contrast optics (IR-DIC), allowing
visual identi®cation of different cell types during brain slice
Correspondence: Dr Michael S. Levine, as above.
E-mail: [email protected]
*Present address: Pharmacia Corporation, 7250±209±216, 301 Henrietta
Street, Kalamazoo, MI 49007, USA
Received 27 June 2001, revised 8 September 2001, accepted 26 September
2001
experiments (Dodt & ZieglgaÈnsberger, 1994), makes recording
from these neurons an easier task (Kawaguchi, 1993; Bennett &
Wilson, 1998, 1999). Brie¯y, in vivo these neurons display slow
spontaneous ®ring and low amplitude short latency excitatory
postsynaptic potentials (EPSPs) to cortical and thalamic stimulation.
In vivo and in vitro large cholinergic interneurons are more
depolarized than medium-sized projection neurons and show timedependent recti®cation (Wilson et al., 1990; Jiang & North, 1991;
Kawaguchi, 1993). Neuronal inputs from the cerebral cortex appear
to be considerably reduced compared to those on medium-sized
neurons, whereas a prominent thalamic projection which is likely to
be excitatory has been demonstrated (Lapper & Bolam, 1992). These
large interneurons express multiple subtypes of glutamate receptors
(Chen et al., 1996; Standaert et al., 1996). Excitatory postsynaptic
currents (EPSCs) induced by local intrastriatal stimulation can be
mediated by activation of both N-methyl-D-aspartate (NMDA) and
non-NMDA receptors (Kawaguchi, 1992; Bennet & Wilson, 1998).
Large cholinergic interneurons also express dopamine receptors
(Bergson et al., 1995; Yan et al., 1997). Dopamine, via activation of
D1 family receptors, produces an enhancement of excitability by
suppression of K+ conductances and by modulation of the afterhyperpolarization (Aosaki et al., 1998; Bennet & Wilson, 1998).
One important property of large cholinergic interneurons is that,
unlike medium-sized spiny striatal neurons, they are spared from
1578 C. Cepeda et al.
FIG. 1. (A) An IR image of a patch pipette attached to a large-sized striatal neuron (white arrow). A medium-sized striatal neuron is next to the large-sized
neuron (white double arrows). (B and D) IR images of a medium- (B) and a large-sized (D) neuron. The same cells are shown in C and E after biocytin
processing. Scale bar in A also applies to B and D; in E also applies to C.
neurodegeneration in Huntington's disease, a genetically based
neurodegenerative disorder (Ferrante et al., 1985; Kowall et al.,
1987). Large cholinergic interneurons also may be less vulnerable to
ischemic challenge (Chesselet et al., 1990; Calabresi et al., 1997;
Centonze et al., 2001). It has been hypothesized that glutamate
receptor excitotoxicity is responsible for cell death of medium-sized
neurons in Huntington's disease because some of the neurochemical
and neuropathological effects of this disorder are mimicked by
application of glutamate receptor agonists (McGeer & McGeer, 1976;
Beal et al., 1991). Potential explanations for sparing of large
interneurons have implicated altered expression of normal or mutant
huntingtin (Ferrante et al., 1997; Fusco et al., 1999) and/or
hyporesponsiveness to glutamate-containing inputs compared to the
medium-sized spiny neuron (DiFiglia, 1990). A previous study has
provided some support for this explanation by demonstrating that
large-sized striatal neurons are less responsive to glutamate receptor
agonists than are medium-sized neurons (Calabresi et al., 1998).
Our laboratory has studied extensively electrophysiological and
synaptic responses of medium-sized striatal neurons to activation of
excitatory amino acid receptors (Cepeda et al., 1991, 1993, 1998a;
Levine et al., 1996; Hurst et al., 2001). In addition, in our studies of
medium-sized neurons we have used cell swelling, an index of early
signs of excitotoxicity (Choi, 1992), to examine functional characteristics of excitatory amino acid receptors and their modulation by
dopamine and activation of metabotropic glutamate receptors
(Colwell & Levine, 1996; Colwell et al., 1996; Cepeda et al.,
1998b). The present study was designed to directly compare the
responsiveness of medium- and large-sized striatal neurons to
activation of excitatory amino acid receptors using several
approaches, whole-cell voltage-clamp analysis of current responses,
synaptic activation and cell swelling in response to application of
excitatory amino acid receptor agonists.
Methods
Animals
All procedures were carried out in accordance with the USPHS Guide
for Care and Use of Laboratory Animals and were approved by the
Institutional Animal Care and Use Committee at UCLA. SpragueDawley rat pups (12±18 days old; n = 60) were used in these
experiments. The choice of this age range was based on our
experience with NMDA receptor function as determined by
electrophysiological experiments or by cell-swelling experiments
from visualized cells in striatal slices (Cepeda et al., 1995, 1998a;
Colwell et al., 1996, 1998; Cepeda et al., 1998b). This age represents
a compromise between the presence of excitatory amino acid-evoked
responses, and the ability to visualize cells which becomes more
dif®cult in tissue from older rats. It is approximately the same age
range used in recent studies analysing electrophysiological responsiveness of large-sized striatal interneurons (Bennett & Wilson, 1998,
1999).
Preparation of slices
Procedures for tissue preparation, visualization, electrophysiology
and measurement of cross-sectional areas in cell-swelling experiments have been described (Cepeda et al., 1995; Colwell & Levine,
1996; Colwell et al., 1996; Cepeda et al., 1998b). Brie¯y, rats were
anaesthetized with halothane and then killed by decapitation. After
dissection, brains were placed in cold oxygenated arti®cial cerebrospinal ¯uid (ACSF1) containing (in mM) NaCl, 130; NaHCO3, 26;
KCl, 3; MgCl2, 5; NaH2PO4, 1.25; CaCl2, 1.0; glucose, 10 (pH 7.2±
7.4). Transverse striatal sections were cut (350 mm) and placed in
oxygenated (95%O2±5% CO2) ACSF2 (differing from ACSF1 as
follows: CaCl2, 2 mM; MgCl2, 2 mM; lactate, 4 mM) at 25±27 °C for
at least 1 h, and then transferred to a perfusion chamber attached to
ã 2001 Federation of European Neuroscience Societies, European Journal of Neuroscience, 14, 1577±1589
NMDA responses in striatal neurons 1579
the ®xed-stage of an upright microscope (Zeiss Axioskop,
Thornwood, New York, USA) in which the slice was submerged in
continuously ¯owing oxygenated ACSF2 (25 °C, 4 mL/min, lactate
removed). Cells were visualized with a 403 water-immersion
objective, illuminated with near IR light (790 nm, Ealing Optics,
Holston, MA, USA) and the image detected with an IR-sensitive
CCD camera. Digital images were stored for subsequent analysis
when necessary. Cells were typically visualized from 30 to 100 mm
below the surface of the slice. It was relatively easy to distinguish
medium- and large-sized cells. The somatic area of medium-sized
cells was » 100 mm2 whereas the somatic area of large cells was
typically > 150 mm2 (Fig. 1).
Whole-cell voltage-clamp
Patch electrodes (3±6 MW) were ®lled with one of the following
internal solutions, depending on the purpose of the experiment, (in
mM): K-gluconate, 140; HEPES, 10; MgCl2, 2; CaCl2, 0.1; ethylene
glycol-bis(b-aminoethyl ether)-N,N,N¢,N¢-tetraacetic acid (EGTA),
1.1; and K2ATP 2 or Cs-methanesulphonate, 125; NaCl, 4; KCl, 3;
MgCl2, 1; MgATP, 5; EGTA, 9; HEPES, 8; GTP, 1; phosphocreatine,
10 and leupeptin, 0.1 (pH 7.25±7.3; osmolality 280±290 mOsm).
Unless otherwise noted, tetrodotoxin (1 mM) was added to the
external solution to block Na+ currents after the whole-cell con®guration was obtained. In experiments in which responses to bath
application of excitatory amino acid receptor agonists during
injection of ramp command voltages were examined, a CsF-based
solution was used in order to block voltage-gated Ca2+ currents. It
contained (in mM): CsF, 125; NaCl, 4; KCl, 3; MgCl2, 1; EGTA, 9;
HEPES, 8; and K2ATP, 5. To block K+ currents, tetraethylammonium
(20 mM) was in the external solution.
Axopatch 200A or 1D ampli®ers were used for voltage-clamp
recordings. A 3-M KCl±agar bridge was inserted between the
extracellular solution and the Ag±AgCl indifferent electrode. Tight
seals (2±10 GW) from visualized large- and medium-sized cells were
obtained by applying negative pressure. The membrane was disrupted
with additional suction and the whole-cell con®guration was
obtained. The access resistances ranged from 8 to 15 MW. Series
resistances were compensated 60±85%.
Cell capacitance measurements as well as somatic area were used
to distinguish between large- and medium-sized neurons. Once the
whole-cell con®guration was obtained, the capacitance of the cell was
calculated by using a 10-mV hyperpolarizing pulse (40 ms duration).
In experiments in which excitatory amino acid receptor agonists were
bath-applied, the magnitude of the current was examined in response
to a ramp. The ramp consisted of a voltage command from ±70 to
±90 mV over 0.4 s, followed by a ramp to +40 mV over 6.4 s to
ensure that unblocked or poorly blocked Ca2+ channels were
inactivated. At +40 mV, the holding potential was followed by a
ramp command to ±90 mV over 0.9 s. Current responses in the
absence of excitatory amino acid receptor agonists were subtracted
from responses in the presence of bath-applied NMDA or kainate to
isolate speci®c agonist-induced currents. Isolated currents were
plotted against voltage to determine current±voltage relationships
(Figs 2 and 3). Currents were converted to current density by dividing
by cell capacitance to normalize responses between the different cell
sizes to better permit comparison of data across experimental
conditions (Alzheimer et al., 1993). Both upward (±90 to +40 mV)
and downward (+40 to ±90 mV) ramps were used to generate
current±voltage plots. Currents obtained from the upward and
downward ramps were similar and the downward ramp was used
when data were quanti®ed (Burgard & Hablitz, 1994). NMDA
(100 mM) and kainate (100 mM) were bath-applied for 3 min prior to
applying the ramp protocol. Each application of either NMDA or
kainate was followed by a 5±10 min washout. We used kainate
instead of a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid
(AMPA) because large interneurons have been shown to express
kainate but not AMPA receptor subunits (Chen et al., 1996), although
a more recent single-cell analysis indicated that AMPA receptor
subunit proteins are expressed (Richardson et al., 2000).
Whole-cell voltage-clamp data, especially quantitative estimates of
currents from neurons in slices, should be interpreted with caution
because the currents measured at the soma are undoubtedly distorted
as a result of the nonisopotentiality over the neuronal surface due to
space-clamp limitations (Armstrong & Gilly, 1992). To partially
control for this problem we also examined responses in acutely
dissociated neurons.
Electrical stimulation
A glass pipette ®lled with the external solution was placed » 200 mm
from the recording pipette. Single pulses (200 msec duration) were
delivered every 10±20 s. The stimulus intensity was adjusted to 23
the threshold to evoke synaptic responses. Four to six pulses were
applied at different holding potentials and averaged. Excitatory
postsynaptic currents (EPSCs) mediated by activation of glutamate
receptors were isolated by adding bicuculline methiodide (10 mM), a
GABAA receptor blocker, to the bath solution.
Acute neuron dissociation
Some slices also were used for acute dissociation of neurons. Slices
were incubated for 1±6 h at room temperature in NaHCO3-buffered
saline bubbled with 95% O2/5% CO2 (in mM except where otherwise
noted): NaCl, 126; KCl, 2.5; MgCl2, 2; CaCl2, 2; NaHCO3, 26;
Na2HPO4, 1; pyruvic acid, 1; glutathione 5 mM; NG-nitro-L-arginine,
1; kynurenic acid, 1; glucose, 10; HEPES, 15; pH 7.4 with NaOH,
300±305 mOsm/L. After 1 h incubation, a slice was placed in lowCa2+±isethionate solution (in mM): Na isethionate, 140; KCl, 2;
MgCl2, 4; CaCl2, 0.1, glucose, 23; HEPES, 15; pH 7.4, 300±
305 mOsm/L, and the dorsal striatum was dissected and placed in an
oxygenated cell-stir chamber (Wheaton, Inc., Millville, NJ USA)
containing papain (Calbiochem, La Jolla, CA USA; papain, 1±2 mg/
mL) in HEPES-buffered Hanks' balanced salt solution (HBSS, Sigma
Chemical Co., St. Louis, MO, USA) at 35 °C. After 20±40 min of
enzyme digestion, tissue was rinsed three times with the low-Ca2+±
isethionate solution and mechanically dissociated with a graded series
of ®re-polished Pasteur pipettes. The cell suspension was plated into a
35-mm NUNCLON dish containing HEPES-buffered HBSS saline on
the microscope stage.
Whole-cell recordings from dissociated neurons used standard
techniques (Hamill et al., 1981; Bargas et al., 1994). The internal
solution consisted of (in mM): N-methyl-D-glucamine, 180; HEPES,
40; MgCl2, 2; EGTA, 10; phosphocreatine, 12; Na2ATP, 2; Na3GTP,
0.2; leupeptin, 0.1; pH 7.2±7.3 with H2SO4, 265±270 mOsm/L. The
external solution consisted of (in mM): NaCl, 135; CsCl, 20; BaCl2, 5;
glucose, 10; HEPES, 10; tetrodotoxin, 0.001; glycine, 0.02; pH 7.3
with NaOH, 300±305 mOsm/L.
Recordings from acutely dissociated neurons were obtained with
an Axopatch 200A ampli®er (Axon Instruments, Foster City, CA,
USA). Electrode resistance was 2±4 MW in the bath. After seal
rupture, series resistance (4±10 MW) was compensated (70±90%) and
periodically monitored. Recordings were made only from medium- or
large-sized cells that had short (< 75 mm) proximal dendrites. NMDA
(1, 10, 50, 100, 200 or 1000 mM, 3 s duration every 20 s) was applied
with a gravity-fed `two-pipe' system. The array of application
capillaries (» 150 mm i.d.) was positioned a few hundred mm from the
ã 2001 Federation of European Neuroscience Societies, European Journal of Neuroscience, 14, 1577±1589
1580 C. Cepeda et al.
FIG. 2. Responses of medium- and large-sized neurons to NMDA application (100 mM). (A) Current±voltage plots for typical medium- and large-sized
neurons. A ramp command was used to change voltage from ±90 to +40 mV and back to ±90 mV. The plotted data were obtained from the downward ramp.
Control current±voltage plots were subtracted from plots in the presence of NMDA. (B) Current±voltage plots (mean 6 SEM) for medium- and large-sized
neurons. (C) Current density±voltage plots for medium- and large sized neurons. (D) Conductance±voltage plots for medium- and large-sized neurons. Drawn
curves represent best ®t functions according to the formula in the Results. In B, C and D asterisks indicate group differences were statistically signi®cant
(P < 0.05). See Results for further details.
cell under study. Solution changes were effected by changing the
position of the array with a DC drive system, controlled by a SF-77B
perfusion system (Warner Instruments Co., Hamden, CT, USA)
synchronized by pClamp. Solutions could be delivered within 5±
10 ms with this system.
Cell identi®cation
In most experiments, electrodes were ®lled with 0.2% Biocytin
(Sigma, St Louis, MO, USA) in the internal solution. After the
experiment, the slice was ®xed in 4% paraformaldehyde overnight,
then processed according to published protocols (Horikawa &
Armstrong, 1988).
Cell swelling
Preparation of brain slices for these experiments were the same as
those described for electrophysiology except tetrodotoxin and
tetraethylammonium were omitted from the bath. After equilibration
for 10 min, a baseline image of the cells was obtained and stored. The
slice was then exposed to different solutions depending upon the
purpose of the speci®c experiment. Images were obtained and stored
at 5-min intervals for the duration of each experiment (15 min
exposure to experimental treatment). In order to quantify changes in
response to excitatory amino acid receptor agonists, cross-sectional
somatic area was measured at each 5-min time point, before and
during experimental treatments. Each measurement was made two or
three times on each image and the average value recorded for each
cell at each time point. For each experimental group, data were
obtained from several animals. Typically, 3±7 medium-sized cells
were visualized within each slice whilst large cells were sparse so that
usually only one cell could be analysed in each slice. In slices
containing large-sized neurons, neighbouring medium-sized cells
were also measured. Cross-sectional areas at each time point were
converted to percentage change with respect to the baseline area for
each cell. For statistical analyses, data were pooled for all cells in
experimental and control groups. Concentrations of NMDA (50 and
100 mM) or kainate (100 mM) were based on our previously published
studies (Colwell & Levine, 1996; Cepeda et al., 1998b). Drugs were
applied in the bath and freshly prepared each day.
Statistics
Values in the tables, ®gures and text are presented as means 6 SEM.
Differences among group means were assessed with appropriate ttests or analyses of variance (ANOVAs). Welch's approximation to the
t-test for unequal variances was used when group variances were not
homogeneous (Welch, 1947). For post hoc evaluations using ANOVAs,
the Bonferroni t-test was used because this test is one of the more
conservative approaches using multiple comparisons. In the text, only
P-values and the type of test used are reported.
ã 2001 Federation of European Neuroscience Societies, European Journal of Neuroscience, 14, 1577±1589
NMDA responses in striatal neurons 1581
Results
Neuron identi®cation
Both medium- and large-sized neurons could be visualized easily in
the slices (Fig. 1). Cross-sectional areas were signi®cantly smaller for
the medium-sized neurons (Fig. 1, compare B vs. D and C vs. E)
[103 6 5 mm2 (n = 15) vs. 327 6 13 mm2 (n = 21) for medium- and
large-sized neurons, respectively; t-test, P < 0.001]. Dendritic processes were examined after ®lling the neuron with biocytin. Mediumsized neurons typically displayed dendritic ®elds with spine-like
varicosities (Fig. 1C), although spines were not abundant due to the
young age of the rats (Hattori & McGeer, 1973; Tepper et al., 1998).
Large-sized neurons exhibited dendritic branches with or without
scarce varicosities (Fig. 1E). Although several types of large neurons
have been described in striatum, the present study targeted the very
large, putative cholinergic, interneurons (Kawaguchi, 1993). These
cells more probably correspond to the so-called `Type 1' large
neurons (Bolam et al., 1984). Because we did not identify the large
cells immunohistochemically, we use the term large-sized neurons
rather than cholinergic interneurons to describe these cells.
NMDA- and kainate-induced currents during ramp voltage
commands
Whole-cell voltage-clamp recordings were obtained from 30 neurons
(15 medium- and 15 large-sized) exposed to bath application of
NMDA (100 mM). For both large- and medium-sized neurons ramp
voltage commands produced a typical current±voltage function to
NMDA application (Fig. 2A). Lower amplitude currents were
produced in large- compared to medium-sized neurons (Fig. 2A
and B). These differences were statistically signi®cant from ±40 to
±25 mV (ANOVA, post hoc t-tests, P < 0.05; Fig. 2B). Capacitance
measurements for medium- (48.3 6 3.1 pF) and large-sized
(83.8 6 5.7 pF) neurons re¯ected the difference in cross-sectional
area and were used to calculate current densities. It is important to
take into account cell size or capacitance when evaluating ionic
conductances in neurons of different sizes because the absolute
magnitude of the current can be a function of neuronal size and not
necessarily the density of channels (Alzheimer et al., 1993). The
differences between mean current densities for large- and mediumsized neurons were proportionately much greater than the differences
between current amplitudes (Fig. 2C). These differences were
statistically signi®cant from ±60 to ±10 mV (ANOVA, post hoc ttests, P < 0.05). In addition, the voltages at which peak currents were
obtained were signi®cantly shifted to more depolarized potentials in
the large-sized neurons (±27.0 6 1.0 mV vs. ±30.4 6 1.2 mV for
large- and medium-sized cells, respectively; t-test, P < 0.025;
Fig. 2A±C).
Because the slope of the current±voltage plots of the NMDAinduced currents in large- and medium-sized neurons appeared
different, we examined more closely the voltage-dependence of
NMDA receptor activation. The current±voltage relationship was
converted to a conductance±voltage relationship (G±V) according to
the formula G = I/(Vm ± Vrev), where Vm is the membrane potential
and Vrev is the potential where the current was observed to reverse
polarity. The midpoint potential (V0.5) and slope factor (b) of the G±V
relationship were calculated by ®tting the data obtained from
individual neurons by G/Gmax = 1/{1 + exp[(V0.5 ± Vm)/b]}, where
V0.5 is the potential at which G/Gmax = 0.5 and b is the number of mV
required to cause an e-fold change in conductance. According to this
function, the smaller the value of b, the fewer mV are needed to cause
an e-fold change in conductance. In other words, the smaller the value
of b, the steeper the slope of the G±V relationship. Mean
conductances (G) were signi®cantly greater for medium- than for
large-sized neurons from ±35 to ±20 mV (Fig. 2D) (ANOVA, post hoc
t-tests P < 0.05). The slope also was signi®cantly greater in mediumthan in large-sized neurons (13.1 6 0.8 vs. 11.0 6 0.7, respectively;
t-test, P < 0.05). The midpoint of the G±V relationship was » 10 mV
more negative in medium- than in large-sized neurons (±38.3 6 1.85
vs. ±30.5 6 2.1 mV, respectively, t-test, P < 0.05) indicating that
NMDA receptors of large-sized neurons required more depolarization
to remove the Mg2+ block.
Kainate (100 mM)-induced currents were measured in 10 large- and
10 medium-sized cells. For both large- and medium-sized neurons,
ramp voltage commands produced a typical linear current±voltage
function to kainate application (Fig. 3A and B). In contrast to NMDA
application, higher amplitude currents were produced in large- vs.
medium-sized neurons. These differences were statistically signi®cant from ±90 to ±30 mV (ANOVA, post hoc t-tests, P < 0.05;
Fig. 3B). Capacitance measurements for medium- (53.4 6 4.3 pF)
and large-sized (107.3 6 6.3 pF) neurons were used to calculate
current densities. The differences between mean current densities for
large- and medium-sized neurons were proportionately smaller and
were not statistically signi®cant (Fig. 3C).
Acutely dissociated neurons
Concentration±response relationships to activation of NMDA receptors were examined in more detail in acutely dissociated large- and
medium-sized neurons using a rapid perfusion system so that neurons
could be tested with a series of concentrations of NMDA. Data were
obtained from 12 medium- and 12 large-sized neurons (Fig. 4A). All
neurons were held at ±40 mV in Mg2+-free ACSF to remove the
voltage-dependence of the response. Neurons were exposed to a
series of ascending concentrations of NMDA (1, 10, 50, 100, 200 and
1000 mM, 3 s duration). Medium-sized neurons displayed a rapid
peak current followed by a steady-state response (Fig. 4B). Largesized neurons displayed a smaller peak response than did mediumsized neurons, and this was also followed by a steady-state response
(Fig. 4C). Concentration±current response relationships were sigmoidal and medium-sized neurons displayed greater peak currents at 50,
100, 200 and 1000 mM concentrations (Fig. 4D). However, because
not all neurons were tested at each concentration the difference was
only statistically signi®cant at the 100-mM concentration (t-test,
P < 0.05). The respective EC50s were 49.5 6 9.8 and 38.2
6 22.4 mM for medium- vs. large-sized neurons. Current densities
for peak responses were also computed by dividing by cell
capacitances (Fig. 4E, inset). There were much greater increases in
concentration±current density responses for medium- than for largesized neurons at 50, 100, 200 and 1000 mM concentrations (Fig. 4E).
The differences were statistically signi®cant for each of these
concentrations (t-tests, P < 0.05 to P < 0.01). The respective EC50s
were 33.3 6 14.6 and 14.7 6 14.1 mM for medium- and large-sized
neurons, respectively. Mean steady-state currents at the 100-mM
concentration were greater for medium- than for large-sized neurons
(±329 6 60 vs. ±196 6 26 pA, respectively), but the difference was
not statistically signi®cant. In contrast, steady-state current densities
at the 100-mM concentration were signi®cantly greater for mediumvs. large-sized neurons (±54 6 10 vs. ±12 6 2 pA/pF, respectively;
t-test, P < 0.001).
Spontaneous and evoked excitatory postsynaptic currents
Spontaneous EPSCs were examined in large- and medium-sized
neurons in the presence of bicuculline (10 mM), a GABAA receptor
blocker. Spontaneous currents were sampled from 30 s up to 15 min
in individual neurons at a holding potential of ±60 mV. These
ã 2001 Federation of European Neuroscience Societies, European Journal of Neuroscience, 14, 1577±1589
1582 C. Cepeda et al.
currents (> 5 pA in amplitude) occurred frequently in medium-sized
neurons (Fig. 5B, left trace). In marked contrast, few spontaneous
events were present in large-sized neurons (Fig. 5B, right trace). The
difference in mean frequency was statistically signi®cant
(1.64 6 0.42 vs. 0.05 6 0.03 events/s in medium- and large-sized
neurons, respectively; t-test, P < 0.05). Average amplitudes of events
were similar in medium- and large-sized neurons (11.5 6 0.89 and
9.2 6 0.58 pA, respectively; difference not statistically signi®cant).
The decrease in frequency of occurrence of spontaneous currents
probably re¯ects less afferent input to large- compared to the
medium-sized neurons (Bolam et al., 1984).
Electrical stimulation in close proximity to the cell was used to
evoke EPSCs in medium- and large-sized neurons. The mean currents
required to evoke EPSCs at 23 threshold in large- and medium-sized
neurons were similar (29.5 6 9.0 and 20.0 6 1.43 mA, respectively;
difference not statistically signi®cant). EPSCs were evoked at three
holding potentials: ±60, ±20 and +30 mV. To analyse the NMDA
receptor-mediated component 6-cyano-7-nitroquinoxaline-2,3-dione
(CNQX, 50 mM) to block non-NMDA receptors was added and the
membrane was held at ±20 mV. Peak currents at ±60 mV holding
potentials (in the absence of CNQX and mediated primarily by
activation of non-NMDA receptors) did not differ signi®cantly
between medium- and large-sized neurons (±169 6 42 and
±110 6 21 pA, respectively). There was a statistically signi®cant
difference between cell capacitances for medium- and large-sized
neurons (44 6 7.1 vs. 94 6 13.9 pF, respectively; t-test, P < 0.005).
To evaluate peak current densities, peak currents were divided by cell
capacitance. There was a statistically signi®cant decrease in current
density in the large- compared to the medium-sized neurons
(±1.4 6 0.3 vs. ±3.9 6 0.6 pA/pF, respectively; t-test, P < 0.001).
EPSCs mediated by activation of NMDA receptors (peak currents
measured at ±20 mV holding potential in the presence of CNQX) in
medium-sized neurons were not signi®cantly greater in amplitude
than those in large-sized neurons (±30.4 6 7.8 vs. ±16.4 6 3.3 pA,
FIG. 3. Responses of medium- and large-sized neurons to kainate (100 mM) application. (A) Current±voltage plots for typical medium- and large-sized
neurons. A ramp command was used to change voltage from ±90 to +40 mV and back to ±90 mV. The plotted data were obtained from the downward ramp.
Control current±voltage plots were subtracted from plots in the presence of kainate. (B) Current±voltage plots (mean 6 SEM) for medium- and large-sized
neurons. (C) Current density±voltage plots for medium- and large sized neurons. In B asterisks indicate groups differences were statistically signi®cant
(P < 0.05). See Results for further details.
ã 2001 Federation of European Neuroscience Societies, European Journal of Neuroscience, 14, 1577±1589
NMDA responses in striatal neurons 1583
FIG. 4. (A) Medium- and large-sized striatal neurons after acute dissociation and with the patch electrodes attached. Calibration in right panel refers to both
panels. (B and C) Current traces evoked by 3-s application of NMDA at 1, 10, 50, 100, 200 and 1000 mM concentrations in (B) medium- and (C) large-sized
neurons. (D) Concentration±peak current curves for medium- and large-sized neurons. (E) Concentration±peak current density curves for medium- and largesized neurons. Inset shows mean cell capacitances (6 SEM). In D and E, asterisks indicate groups differences were statistically signi®cant (P < 0.05 to
P < 0.01). See Results for further details. Scale bars in C also apply to B.
ã 2001 Federation of European Neuroscience Societies, European Journal of Neuroscience, 14, 1577±1589
1584 C. Cepeda et al.
FIG. 5. (A) Top left panel shows IR images of a pair of striatal cells; one is a medium-sized (double arrow) and the other is a large-sized neuron (single
arrow). Top right panel shows cells after being ®lled with biocytin. Single arrow points to the large neuron and the double arrow to the medium-sized neuron.
(B) Patch-clamp recordings were obtained from both cells. Spontaneous EPSCs were recorded in the presence of bicuculline (10 mM) at a holding potential of
±60 mV. Middle traces are slow-speed recordings illustrating the marked absence of spontaneous EPSCs in the large-sized neuron (right) compared to the
medium-sized cell (left). (C) A stimulating electrode was placed » 200 mm from the cells, thus approximately equidistant. Bottom traces are faster speed
recordings showing evoked EPSCs at three holding potentials: ±60, ±20 and +30 mV. The stimulation parameters were identical for both cells. In all cases the
evoked EPSCs were of greater amplitudes in the medium-sized neuron. Downward arrows indicate time of occurrence of stimulation pulse.
respectively). However, mean current densities for NMDA receptormediated responses were statistically signi®cantly greater in mediumvs. large-sized neurons (±0.64 6 0.10 vs. ±0.20 6 0.04 pA/pF,
respectively; t-test, P < 0.001). In selected experiments (n = 3) we
were able to record from pairs of large- and medium-sized neurons,
using the same stimulation placement and parameters (Fig. 5A).
Similar to the grouped data described above, spontaneous EPSCs
were more frequent and evoked EPSCs recorded at each holding
potential were of greater amplitude in the medium- than in the largesized neurons (Fig. 5B and C).
ã 2001 Federation of European Neuroscience Societies, European Journal of Neuroscience, 14, 1577±1589
NMDA responses in striatal neurons 1585
Cell swelling
Another approach to examining excitatory amino acid receptor
function in large- and medium-sized striatal neurons is to expose
slices to agonists and assess cell swelling. Cell swelling, induced
by activation of excitatory amino acid receptors, is presumably
the ®rst step in a cascade that may ultimately lead to cell death
(Choi, 1992). Previously, we showed that bath application of
NMDA or kainate produced dose- and time-dependent swelling of
medium-sized striatal cells (Colwell & Levine, 1996; Cepeda
et al., 1998b). In the present experiments we examined the ability
of large-sized striatal neurons to swell in the presence of NMDA
or kainate. Modal cross-sectional areas for large cells were
between 250 and 350 mm2 (Fig. 6B, inset). NMDA (50 or
100 mM) induced almost no change in cross-sectional area in
large-sized striatal neurons (Fig. 6A, top panels). Average percentage increases in area were < 5% over the 15-min assessment
period (Fig. 6B). When a group of medium-sized neurons (n = 15)
was exposed to the same concentration of NMDA, statistically
signi®cant cell swelling occurred. After 5 min exposure to NMDA
there was a 19 6 3% increase in somatic cross-sectional area
(ANOVA, post hoc t-test, P < 0.001). By 15 min the somatic crosssectional area increased by 43 6 6% (ANOVA, post hoc t-test,
P < 0.001). Because it is possible that large-sized cells may be
unable to increase somatic area, we tested the response to kainate
in another group of large-sized neurons. Kainate (100 mM)
produced a statistically signi®cant increase (» 40%) in largesized cell cross-sectional area 5, 10 and 15 min after application
(ANOVA, P < 0.003) (Fig. 6A, bottom panels, and 6B). Thus, the
lack of response to NMDA was not due to an inability of largesized cells to swell in the presence of excitatory amino acid
receptor agonists. In medium-sized neurons we have shown
previously that 100 mM kainate produces about a 30% increase
in cross-sectional area (Colwell et al., 1998).
We have shown previously that dopamine via activation of D1family receptors increases cell swelling induced by excitatory amino
acid receptor agonists in medium-sized striatal neurons (Cepeda et al.,
1998b). Thus, we examined whether NMDA in combination with
dopamine could produce cell swelling in large-sized neurons (n = 5
cells). In the presence of dopamine (50 mM), cell swelling was not
signi®cantly increased (Fig. 6B).
Discussion
The present experiments demonstrate, using a number of electrophysiological approaches, that large-sized striatal neurons were less
responsive to NMDA receptor activation than were medium-sized
neurons. First, whole-cell currents and to an even greater extent
current densities, induced by exogenous application of NMDA, were
decreased in large- compared to medium-sized neurons. Comparable
®ndings occurred in acutely dissociated neurons. Peak and steadystate current densities to NMDA application were reduced in largecompared to medium-sized neurons and this effect was consistent
across a series of concentrations. There was also a shift in the
voltage-dependence toward less negative values in large- compared
to medium-sized neurons and a less steep negative slope conductance.
In contrast, kainate-induced currents were greater in large- than in
medium-sized neurons whilst kainate-induced current densities were
similar in both cell types. In addition to decreased responsiveness to
exogenous application of NMDA, large-sized neurons received less
excitatory synaptic input than medium-sized neurons. Spontaneous
EPSCs were markedly reduced in frequency and evoked EPSCs
mediated by activation of NMDA or non-NMDA receptors were
reduced in amplitude. Finally, NMDA-induced cell swelling, a ®rst
step in an excitotoxic cascade, does not occur or is minimal in largesized neurons. This was true even in the presence of dopamine, a
treatment that enhances NMDA-induced cell swelling in mediumsized neurons (Cepeda et al., 1998b). In contrast, kainate-induced
swelling was robust in large-sized neurons.
It is clear from the present ®ndings as well as previous analyses
(Calabresi et al., 1998) that large-sized cholinergic neurons display
reduced responsiveness to activation of NMDA receptors compared
to medium-sized neurons. There are a number of mechanisms that
could underlie such reduced responsiveness. These include decreased
density of receptors and/or altered NMDA receptor subunit composition. The results of the present study clearly demonstrate that current
density, which is a measure of current per unit of membrane clamped,
is signi®cantly reduced in large-sized interneurons compared to the
medium-sized cells, suggesting lower density of receptors. Whilst
these values will be affected by differential space-clamp limitations
to a certain extent, the magnitude of the difference cannot be
accounted for by space-clamp errors.
Of at least equal importance is the difference in NMDA receptor
subunit composition between large- and medium-sized neurons
(Landwehrmeyer et al., 1995). Differences in subunit composition
will affect the agonist af®nity, Mg2+ sensitivity, voltage-dependence
and the kinetics of the responses. For example, the lack of a large
rapid peak response to NMDA application in dissociated large-sized
neurons is probably due to differences in subunit composition
(Huganir & Greengard, 1990). Large-sized cholinergic interneurons
express lower levels of NMDA-R1 and NMDA-R2B subunit mRNA
than do enkephalin-positive medium-sized neurons, and preferentially express the NMDA-R1 splice variant forms lacking one
alternatively spliced carboxy-terminal region (Landwehrmeyer et al.,
1995), which would change the sensitivity of protein phosphorylation
(Tingley et al., 1993). The large-sized cholinergic interneurons also
do not express NMDA-R2A subunits (Standaert et al., 1996). This
®nding is relevant because NMDA-R2 subunits impart different
pharmacological pro®les to NMDA receptors (Monaghan & Larsen,
1997), and residues on the NMDA-R2A subunit control glutamate
potency (Anson et al., 1998). Finally, the NMDA-R2D subunit is
selectively expressed in striatal interneurons and absent in mediumsized projection neurons (Standaert et al., 1996, 1999). Channels
expressing this subunit are unique in that they have a very long offset
decay and an altered Mg2+ blockade (Monyer et al., 1994) which may
explain, in part, the reduced negative slope conductance of the
NMDA current. Furthermore, cells expressing NMDA-R2D subunits
may have lower af®nity for agonists (Buller et al., 1994) which could
partially explain sparing of the large neurons after quinolinic acid
lesions. Recent studies using single-cell PCR have corroborated and
extended the initial analyses of NMDA receptor subunit composition
in large-sized cholinergic interneurons (Richardson et al., 2000).
Interestingly, the NMDA-R2A subunit was detected when the
sensitivity of the single cell assay was increased, suggesting low
levels of expression.
There are also fewer excitatory synaptic inputs to large- than to
medium-sized neurons. The present data con®rm and extend the
initial analyses demonstrating that these cells receive less excitatory
input than do medium-sized spiny neurons (Wilson et al., 1990;
Bennet & Wilson, 1999). We showed both reduced frequency of
spontaneous EPSCs and reduced amplitudes of evoked EPSCs.
Morphological evidence indicates fewer cortical excitatory synaptic
inputs on large-sized interneurons than on other striatal cell types,
although a thalamostriatal projection which is probably excitatory has
ã 2001 Federation of European Neuroscience Societies, European Journal of Neuroscience, 14, 1577±1589
1586 C. Cepeda et al.
been demonstrated (Lapper & Bolam, 1992). Based on electron
microscopic studies, synaptic input to the perikarya and proximal
dendrites of large cholinergic interneurons is very sparse and consists
predominantly of boutons forming putative inhibitory symmetrical
ã 2001 Federation of European Neuroscience Societies, European Journal of Neuroscience, 14, 1577±1589
NMDA responses in striatal neurons 1587
synaptic contacts. Asymmetric excitatory synaptic contacts are
infrequent near the soma, although their number increases in distal
dendrites (Bolam et al., 1984).
A previous study described a generalized decrease in responsiveness of large cholinergic interneurons to NMDA, AMPA and kainate
(Calabresi et al., 1998). Paradoxically, a large NMDA receptormediated EPSP was expressed in the cholinergic interneurons in the
presence of Mg2+ in the external solution, even at relatively
hyperpolarized membrane potentials (±75 mV). The electrophysiological data in the present study do not support an indiscriminate
hyposensitivity of large-sized neurons, at least for kainate receptor
activation. Neither do they support the presence of a large NMDA
receptor-mediated synaptic component at hyperpolarized potentials.
In fact, our results indicate that more depolarization is required to
remove the Mg2+ blockade in large interneurons and the NMDA
receptor-mediated synaptic current is reduced. Calabresi et al. (1998)
only examined peak voltages or currents and did not include
measurements of current density; this could signi®cantly alter the
interpretation of the outcomes because large- and medium-sized
neurons have markedly different surface areas. The present study has
examined, in more detail and more extensively, current density and
current±voltage relationships as well as directly comparing synaptic
responses using voltage-clamp techniques in which changes in other
variables would contribute less to the overall currents than in the
synaptic responses in the previously reported current-clamp study.
Calabresi et al. (1998) also did not initially block GABAA receptors
when examining the excitatory amino acid receptor-mediated EPSPs,
and consequently the electrical stimulation produced a mixed
depolarizing potential consisting of multiple components due to
activation of ligand- and voltage-gated channels. As pointed out
above, other studies have shown that, relative to medium-sized spiny
neurons, the EPSP in large interneurons is smaller (Wilson et al.,
1990) and, although it is mediated by activation of both NMDA and
non-NMDA receptors, the NMDA component is negligible at
hyperpolarized potentials (Kawaguchi, 1992). Although differences
in maturation of large- vs. medium-sized neurons may have
contributed to the disparity between the present ®ndings and those
of Calabresi et al. (1998), we believe this is unlikely because our
®ndings on large-sized neurons are in general agreement for NMDA
receptor function but contrast on kainate receptor function. We have
shown previously that non-NMDA receptor function actually
develops earlier in the striatum than do NMDA receptors (Colwell
et al., 1998; Nansen et al., 2000; Hurst et al., 2001). Thus, at the ages
used in the present study, kainate receptor function would be at least
as mature as or more mature than NMDA receptor function.
Some of the unique properties of large-sized neurons are relevant
for understanding their resistance to excitotoxic challenge, ischemia
and their preservation in Huntington's disease. For example, c-fos
induction after NMDA receptor activation is absent in large
interneurons but not in medium-sized neurons, suggesting less
coupling of NMDA receptor function to gene expression in the
large cells (Aronin et al., 1991). Large interneurons express kainate
receptor subunit proteins (Chen et al., 1996) and the present results
indicate that they respond to kainate application. It was originally
reported that large interneurons do not express AMPA receptor
subunits (Chen et al., 1996) although they respond to AMPA
application (Calabresi et al., 1998). More recently, single-cell PCR
analyses have demonstrated the presence of AMPA receptor subunit
proteins (Richardson et al., 2000). Unfortunately, little is known
about the subtypes of glutamate receptors activated by the
thalamostriatal pathway.
Differential effects of anoxia±hypoglycemia occur between
medium- and large-sized neurons in the striatum (Calabresi et al.,
1997). Cholinergic interneurons hyperpolarize in response to anoxia±
hypoglycemia whilst medium-sized neurons depolarize. Thus, they
may be protected from the deleterious effects of depolarization
induced by ischemic challenge. Whilst it was ®rst hypothesized that
activation of a K+ current that is insensitive to tolbutamide mediated
the hyperpolarization (Calabresi et al., 1997), subsequent studies
have demonstrated an ATP-sensitive K+ channel (Lee et al., 1997,
1998) that may provide a protective role in anoxia±hypoglycemia.
There are a number of reasons why large-sized cholinergic neurons
may be spared in Huntington's disease. First, there may be
differences in expression of normal or mutant huntingtin in largesized cholinergic interneurons (Ferrante et al., 1997). Selective
vulnerability of medium-sized spiny striatal neurons observed in
Huntington's disease has been associated with higher levels of
huntingtin expression, whereas the relative resistance of large- and
medium-sized aspiny neurons has been associated with low levels of
huntingtin expression (Ferrante et al., 1997; Kosinski et al., 1997).
However, a more recent study using a different antibody to identify
huntingtin expression indicates that large cholinergic interneurons
have higher levels of huntingtin than medium-sized neurons and
suggests that it is the local environment that ultimately decides
whether a neuron will be vulnerable to degeneration in Huntington's
disease (Fusco et al., 1999).
Early animal models of Huntington's disease produced excitotoxic lesions with kainate to duplicate human striatal cell loss
(Coyle & Schwarcz, 1976; McGeer & McGeer, 1976). However,
soon thereafter it was found that the excitotoxic model using
quinolinic acid, which has more selectivity for NMDA receptors,
better replicated the human ®ndings (Schwarcz et al., 1983; Beal
et al., 1986; Ferrante et al., 1993; Roberts et al., 1993). If it is
true that cholinergic interneurons are less sensitive to exogenous
application of NMDA but not kainate as demonstrated by the
present electrophysiological and cell swelling ®ndings, how can
we explain the sparing of these cells after kainate application?
One possibility is that excitotoxic lesions in the striatum require
two conditions, activation of NMDA receptors and a signi®cant
excitatory input (McGeer et al., 1978). The paucity of excitatory
inputs impinging upon cholinergic interneurons may explain their
relative resistance to kainate, whereas their resistance to NMDA
could be explained by the combination of reduced receptor
expression and different receptor subunit assembly, as well as
paucity of excitatory inputs (Ferrante et al., 1987, 1993).
FIG. 6. (A) IR images. Top left and right panels show NMDA application (100 mM, 15 min duration) failed to produce cell swelling in the large neurons
(white arrows). In the same slice, the medium-sized cells displayed signi®cant cell swelling (white arrowheads). Bottom left and right panels show that, in
contrast to the lack of swelling after NMDA, large neurons showed signi®cant increases in somatic area after kainate (100 mM, 15 min duration) (white
arrows). (B) Bar graphs showing lack of cell swelling in large-sized striatal neurons at 5, 10 and 15 min during application of NMDA at either 50- or 100-mM
concentrations. In the presence of dopamine, there was a slight increase in cell swelling to 100 mM NMDA. In contrast, kainate (100 mM, 15 min duration)
produced signi®cant cell swelling after 5-, 10- and 15-min applications in large neurons. Inset shows a histogram of cross-sectional areas of large neurons.
Asterisks in B indicate groups differences were statistically signi®cant (P < 0.003). See Results for further details.
ã 2001 Federation of European Neuroscience Societies, European Journal of Neuroscience, 14, 1577±1589
1588 C. Cepeda et al.
How are these results relevant for the understanding of
Huntington's disease or other neurodegenerative disorders? We
believe that the present results emphasize that multiple conditions
must be met before neurons are at risk for excitotoxic degeneration.
Large-sized cholinergic interneurons do not meet these conditions
and thus are spared both in excitotoxic models of Huntington's
disease and in the disease itself. In contrast, medium-sized spiny
neurons meet both conditions and are at risk in both the models and
the disorder.
In conclusion, the present study demonstrates that large interneurons in the striatum display unique responsiveness to NMDA receptor
activation, differing from medium-sized neurons. Inward currents are
signi®cantly smaller and a stronger membrane depolarization is
required to relieve the Mg2+ blockade. Cell swelling to NMDA, a ®rst
step in an excitoxic cascade, is practically nonexistent. In contrast,
large-sized neurons respond to kainate receptor activation in a
manner similar to medium-sized neurons. We also found that the
distribution of excitatory inputs onto large interneurons is relatively
sparse compared to medium-sized neurons. Thus, the reduced
responsiveness coupled with the altered NMDA receptor composition
or reduced receptor density may help explain the relative resistance of
large interneurons to neurodegeneration in neurological diseases.
Taken together the present ®ndings help de®ne the conditions that put
neurons at risk for damage in neurological disorders and they will aid
in the design of therapies that can reduce or prevent neurodegeneration.
Acknowledgements
The authors greatly acknowledge Donna Crandall and Carol Gray for the
preparation of the illustrations. This work was supported by USPHS grants
NS33538 and NS35649.
Abbreviations
ACSF, arti®cial cerebrospinal ¯uid; AMPA, a-amino-3-hydroxy-5-methyl-4isoxazole propionic acid; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione;
EGTA, ethylene glycol-bis(b-aminoethyl ether)-N,N,N',N¢-tetraacetic acid;
EPSC, excitatory postsynaptic current; EPSP, excitatory postsynaptic potential; HBSS, Hanks' balanced salt solution; IR-DIC, infrared-differential
interference contrast; NMDA, N-methyl-D-aspartate.
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ã 2001 Federation of European Neuroscience Societies, European Journal of Neuroscience, 14, 1577±1589