1Department of Pharmacology and 2Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0615
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ABSTRACT |
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Partridge, John G., Ka-Choi Tang, and David M. Lovinger. Regional and Postnatal Heterogeneity of Activity-Dependent Long-Term Changes in Synaptic Efficacy in the Dorsal Striatum. J. Neurophysiol. 84: 1422-1429, 2000. High-frequency activation of excitatory striatal synapses produces lasting changes in synaptic efficacy that may contribute to motor and cognitive functions. While some of the mechanisms responsible for the induction of long-term potentiation (LTP) and long-term depression (LTD) of excitatory synaptic responses at striatal synapses have been characterized, much less is known about the factors that govern the direction of synaptic plasticity in this brain region. Here we report heterogeneous activity-dependent changes in the direction of synaptic strength in subregions of the developing rat striatum. Neurons in the dorsolateral region of the anterior striatum tended to express LTD after high-frequency afferent stimulation (HFS) in slices from animals aged P15-P34. However, HFS in dorsolateral striatum from P12-P14 elicited an N-methyl-D-aspartate (NMDA) receptor-dependent form of LTP. Synapses in the dorsomedial anterior striatum exhibited a propensity to express an NMDA-receptor dependent form of LTP across the entire developmental time period examined. The NMDA receptor antagonist (±)-2-amino-5-phosphopentanoic acid (APV) inhibited evoked excitatory postsynaptic potentials recorded in striatum obtained from P12-P15 rats but had little effect in striatum from older animals. The expression of multiple forms of synaptic plasticity in the striatum suggests mechanisms by which this brain region plays pivotal roles in the acquisition or encoding of some forms of motor sequencing and stereotypical behaviors.
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INTRODUCTION |
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The striatum serves as the
entryway to the basal ganglia in assisting voluntary motor behaviors,
motor memory, and retrieval mechanisms, as well as habit learning and
other aspects of cognition (Graybiel et al. 1994;
Knowlton et al. 1996
). Synaptic plasticity has been
suggested to play an important role in cognitive functions in several
models of striatal function (Beiser and Houk 1998
). At
least two forms of long-lasting synaptic plasticity can be induced by
high-frequency synaptic activation of striatal neurons: long-term
depression (LTD) and long-term potentiation (LTP) of glutamatergic
transmission (Calabresi et al. 1992a
,b
; Charpier and Deniau 1997
; Lovinger et al. 1993
;
Walsh 1993
; Wickens et al. 1996
).
However, these forms of plasticity have rarely been observed in the
same laboratory under identical pharmacological and physiological
conditions. It is not clear why some investigators observe a depression
while others detect an enhancement of synaptic responses following
high-frequency afferent stimulation (HFS).
Striatal LTD induction and maintenance is well characterized and is
dependent on the activation of a variety of receptor molecules and ion
channels (Calabresi et al. 1992a; Choi and
Lovinger 1997a
,b
; Lovinger et al. 1993
;
Tang and Lovinger 2000
). In adult rat striatum, metabotropic glutamate receptor, but not
N-methyl-D-aspartate (NMDA) glutamate receptor,
activation is necessary for LTD induction. In contrast, striatal LTP
induction in the adult rat is dependent on the activation of NMDA receptors.
The striatum matures from ganglionic eminences in the telencephalic
neural tube. Postmitotic cells migrate into the developing ventricles
in a lateral to medial direction during pre- and postnatal development
(Hattori and McGeer 1973). Ultrastructural studies have
shown that synapses also develop across a lateral-medial gradient in
early postnatal rat striatum. For example, the number of asymmetric and
symmetric synapses as determined by electron microscopy is roughly
doubled in dorsolateral regions of the striatum in comparison to
dorsomedial regions during the third week of postnatal development
(Butler et al. 1998
). In addition, Tepper and associates
have shown that the third week of postnatal maturation in
Sprague-Dawley rat striatum is an intense period of
electrophysiological and morphological change (Tepper et al.
1998
). This discriminating pattern of synaptic development may
give rise to functional differences in integrating afferent input
during this stage of striatal development.
The data presented in this report demonstrate that activity-dependent long-term changes in synaptic efficacy are expressed heterogeneously as a function of both postnatal age and subregion in the rat dorsal striatum. In addition to previously described striatal LTD, an NMDA receptor-dependent form of LTP also can be evoked in this preparation. These studies reinforce the notion that the NMDA receptor plays a critical role in aspects of striatal development and plasticity. Our findings suggest that different subregions of the striatum process afferent input differentially during early postnatal development. Furthermore, this study provides possible explanations for the variable results obtained by different groups examining activity-dependent plasticity at excitatory striatal synapses.
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METHODS |
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SLICE PREPARATION. Brain slices were prepared from 12- to 34-day-old Sprague-Dawley rats. Coronal brain slices (400 µm) were cut in ice-cold modified artificial cerebrospinal fluid (aCSF) containing the following (in mM): 94 sucrose, 30 NaCl, 4.5 KCl, 1 MgCl2, 26 NaHCO3, 1.2 NaH2PO4, and 10 D-glucose and adjusted to pH 7.4 by bubbling with 95% O2/5% CO2. A hemislice containing the cortex and the anterior striatum was completely submerged and continuously superfused with aCSF containing the following (in mM): 124 NaCl, 2 CaCl2, 4.5 KCl, 1 MgCl2, 26 NaHCO3, 1.2 NaH2PO4, and 10 D-glucose. Normal aCSF and drug-containing external solutions were delivered to slices via superfusion by gravity flow. Superfusate was passed through a heated water jacket and flow rate (1.8-2.8 ml/min) was held constant with a corresponding chamber temperature of 32-33°C for an individual experiment. (±)-2-amino-5-phosphopentanoic acid [(±)-APV] was purchased commercially from RBI. All other chemicals were of reagent grade or higher.
WHOLE CELL RECORDING.
Ruptured-patch (i.e., traditional) whole-cell voltage-clamp recordings
were obtained using pipettes made from borosilicate glass capillaries
pulled on a Flaming-Brown micropipette puller. Pipette resistance
ranged from 3.0-5.5 M, when filled with an internal solution
containing the following (in mM): 120 cesium methane sulfonate, 5 NaCl,
10 tetraethylammonium chloride, 10 HEPES, 4 lidocaine
N-ethyl bromide, 1.1 EGTA, 4 Mg-ATP, and 0.3 Na-GTP, pH
adjusted to 7.25 with CsOH. Cesium-based solutions were used to reduce
dendritic filtering of synaptic responses. Recordings were made from
medium-sized cells with the aid of a differential interference
contrast-enhanced visual guidance system (Olympus) from neurons three
or four layers below the surface of 400-µm-thick slices. Cells were
voltage-clamped at
70 mV during test periods prior to and subsequent
to HFS. Test stimuli were delivered at a frequency of 0.05 Hz through a
bipolar-twisted Tungsten wire placed in the white matter dorsal to the
region of interest within the striatum. Synaptic currents or potentials were recorded with an Axopatch 1D amplifier (Axon Instruments, Foster
City, CA), filtered at 5 kHz, digitized at up to 6 kHz, and stored on a
microcomputer. Some experiments required the use of the perforated
patch-clamp technique. In these studies, amphotericin B (Sigma, St.
Louis, MO) was used at a working concentration of 200 µg/ml in normal
internal solution. Electrical access to the cell was monitored as an
increase in the capacitive transients. Stable openings under these
conditions required 10-30 min for the ionophore to equilibrate with
the solution at the tip of the pipette and subsequent electrical access
to allow whole-cell voltage-clamp recording. Access resistance was
monitored throughout the duration of each experiment. For some
experiments, the following internal solution was used and composed of
(in mM): 130 potassium methylsulfate, 1 MgCl2,
0.1 CaCl2, 10 HEPES, 1 EGTA, 2 Mg-ATP, and 0.4 Na-GTP, pH adjusted to 7.25 with KOH to test if postsynaptic firing
patterns are important in synaptic plasticity. Under these conditions, passive and active neuronal electrophysiological parameters were examined. All cells used for data analysis from experiments using the
potassium-based internal solution did not fire spontaneous action
potentials and had resting membrane potentials ranging from
65 to
76 mV.
FIELD POTENTIAL RECORDING.
Extracellular recordings were obtained using pipettes made from
borosilicate glass. Pipette resistance ranged from 0.5 to 1.0 M when
filled with a 0.9% NaCl solution. Signals were amplified with the use
of a differential AC amplifier (A-M Systems, model 1700, Seattle, WA).
During the testing periods before and after HFS, the stimulus intensity
was set to the level at which an evoked population spike (PS) was
approximately one half the amplitude of the maximal obtainable response
prior to HFS. Stimulus intensity was adjusted to a level evoking a
maximal response during HFS. Stimulus intensity ranged from 15 to 120 V
while the stimulus duration ranged from 0.01 to 0.2 ms using a Grass
S-88 Stimulator. The majority of the slices were also subjected to
maximal stimulation at the end of an experiment to ensure that no
significant deterioration of the slices occurred during the experiment,
as determined by the presence of a maximal PS in response to such stimulation.
LTD/LTP INDUCTION.
LTD or LTP were induced by the following high-frequency stimulation
protocol: four 1-s duration, 100-Hz trains, delivered at a frequency of
one train every 10 s. In voltage-clamp experiments, this protocol
was simultaneously paired with a 1-s depolarization of the neuron to
10 mV.
DATA ANALYSIS. The amplitude of excitatory postsynaptic currents (EPSCs) was measured using peak detection software using pCLAMP (Axon Instruments, Foster City, CA). PS amplitude was measured using identical software protocols. The magnitude of LTD or LTP was normalized on a single-recording basis and defined as the ratio of the average response from 20-30 min (30 episodes) following HFS to the average response amplitude during a 10-20 min baseline pre-HFS recording period (30-60 episodes). This time window was chosen due to the stability of the response at this time following HFS. Evoked synaptic responses in a small percentage of the slices (<5%) were not stable during this time period following HFS and were not included in the data analysis. Nonlinear curve fitting to estimate EPSP decay rates was calculated by fitting the decay phase with a single exponential function from the time point defining the peak amplitude to the time point defining the return to the resting potential. Data points in the figures showing the time course of LTP and LTD are plotted as the means ± SE normalized to values during a stable period before HFS. A one-way within-region analysis of variance (ANOVA) was used to determine if HFS-induced changes in response amplitude differed across postnatal age. A posthoc Tukey-Kramer test was then used to determine if changes in efficacy varied between different individual ages. To determine if statistically significant changes in transmission were produced by HFS at different time points during development in the different striatal subregions, we grouped all of the data within a particular time period based on the week of postnatal development for each region. Thus, determination of expression of significant LTP or LTD was performed for ages P12-P14 (week 2), P15-P21 (week 3), and P22 and up (weeks 4 and 5) for both dorsolateral and dorsomedial striatum. A repeated measures t-test was used for this analysis because we wanted to test the hypothesis that slices in a given age range showed a significant change in response amplitude relative to the pre-HFS baseline. This analysis is appropriate because we compare the post-HFS and the pre-HFS responses in each slice to determine if such changes take place in a large group of slices.
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RESULTS |
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SYNAPTIC PLASTICITY FOLLOWING HIGH-FREQUENCY STIMULATION IN DORSOLATERAL AND DORSOMEDIAL STRIATUM. The expression pattern of activity-dependent and persistent changes in synaptic efficacy at striatal synapses was examined. Identical tetanus protocols were capable of evoking both LTP and LTD of excitatory neurotransmission at striatal synapses in studies performed using field potential recording. Activity-dependent changes in synaptic efficacy varied as a function of postnatal age in dorsolateral striatum (Fig. 1). Synaptically driven population spikes with measurable and stable amplitudes could be evoked in slices from the dorsolateral striatum at ages P12 and older using the recording location shown in Fig. 1A. Attempts were made to record at earlier ages; however, the maximal amplitude of the evoked population spike was approximately 0.3 mV and unstable in response to low-frequency stimulation. Therefore, synaptic plasticity could not be investigated at these ages. In the dorsolateral striatum, a statistically significant difference in the type of HFS-induced changes in synaptic strength was observed when comparing different ages (one-way ANOVA, F(13,84) = 7.122, P < 0.05). The change in synaptic efficacy at P12 and P13 was significantly different from at later ages (P < 0.05, Tukey-Kramer multiple comparisons test). In the dorsolateral striatum from P12-P13 animals, there was a high probability for LTP to occur following HFS (Fig. 1B). LTP was observed in 13 of 14 slices during this period of postnatal development, and the increase in synaptic response was statistically greater than control at postnatal days P12 and P13 (P < 0.05, repeated measures t-test). At postnatal day P15, approximately equal numbers of slices showed LTP and LTD. In 13 slices examined at this postnatal day, five showed LTP, six showed LTD, while two slices showed no significant change in the population spike amplitude following HFS. In P17-P34 dorsolateral striatum, LTD was observed following HFS in the majority of the slices (Fig. 1, C and D). The average HFS-induced change in synaptic responses from the dorsolateral striatum as a function of postnatal age is shown in Fig. 1D. Statistically significant decreases in PS amplitude were observed for postnatal weeks 3 and 4 + 5 when comparing baseline to post-HFS responses (P < 0.05, repeated measures t-test). It is important to note that no change in the presynaptic fiber volley occurred following HFS, as shown by the amplitude and shape of the first negativity (N1) in the field potential. Examples of this are shown in the traces plotted in Fig. 1, B and D.
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STRIATAL LTP IS DEPENDENT UPON THE ACTIVATION OF NMDA-TYPE
GLUTAMATE RECEPTORS.
Previous studies indicate that striatal LTP in a mature preparation is
dependent on the activation of NMDA-type glutamate receptors in vitro
under conditions in which the voltage-dependent block of the NMDA
receptor has been removed by eliminating Mg2+
(Calabresi et al. 1992b). Because LTP in the developing
striatum has not been characterized pharmacologically, we wished to
determine if NMDA receptors are involved in LTP at striatal synapses
during the second and third postnatal week.
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BASELINE SYNAPTIC TRANSMISSION IN DORSOLATERAL STRIATAL NEURONS
EARLY IN DEVELOPMENT IS SENSITIVE TO NMDA-RECEPTOR ANTAGONISTS.
In 5 slices out of 10 challenged with 100 µM APV application during
recordings from the dorsolateral striatum from P12-P14 animals, the
pre-HFS baseline transmission as measured by the PS amplitude was
inhibited by an average of 31 ± 4% (see Fig. 4A
inset). This inhibition was reversed on washout of APV. The other five slices were insensitive to the application of the NMDA receptor antagonist during low-frequency stimulation baseline recording. In both dorsolateral and dorsomedial striatum from P16-P26
animals, 100-µM APV had no significant effect on the baseline transmission in all slices examined at these ages (n = 6 dorsolateral, n = 7 dorsomedial). APV inhibited
transmission by an average of 3 ± 3 and 2 ± 3% in
recordings from the more mature dorsolateral and dorsomedial striatum,
respectively. These findings are consistent with the pharmacological
profile of excitatory postsynaptic responses from medium spiny neurons
under resting conditions in adult preparations (Cherubini et al.
1988; Jiang and North 1991
; Lovinger et
al. 1993
).
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DISCUSSION |
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Our findings demonstrate two important factors that help to
determine the direction of changes in synaptic efficacy at striatal synapses, namely the region of the striatum examined and the age of the
animal. In past studies, both striatal LTP and LTD have been observed
(Calabresi et al. 1992a,b
; Charpier and Deniau
1997
; Lovinger et al. 1993
; Walsh
1993
; Wickens et al. 1996
). This work has been
performed using either in vivo or brain slice recordings from adult rat
striatum. However, LTD and LTP have rarely been observed in the same
preparation under similar recording conditions. In striatal slices, LTD
is the predominant form of plasticity observed after high-frequency
afferent stimulation (Calabresi et al. 1992a
;
Lovinger et al. 1993
; Walsh 1993
;
Wickens et al. 1996
). In vitro, LTP has been observed
when HFS is paired with pharmacological manipulation or genetic
modification. For example, LTP can be elicited by HFS when
Mg2+ is omitted from the extracellular solution
(Calabresi et al. 1992b
), in mice genetically deficient
in dopamine D2-type receptors (Calabresi et al. 1997
),
or when HFS is accompanied by pulsatile coapplication of extracellular
K+ and dopamine (Wickens et al.
1996
). In contrast, studies performed in vivo indicate that
stimulation of cortical afferents results in LTP even when HFS patterns
were similar to those used to elicit LTD in vitro (Charpier and
Deniau 1997
). There has been little effort to bridge the gap
between these studies. Our data indicate that LTP can be observed in
striatal slices at certain ages and in certain subregions without
removal of Mg2+ or addition of exogenous dopamine.
Several findings presented in this study indicate that LTP and LTD
involve changes in synaptic responses at striatal synapses. We observed
an increase in EPSC amplitude after HFS in perforated patch recordings.
In this study, and in past studies, we have observed a decrease in EPSC
amplitude after HFS under conventional whole-cell recording conditions
at several stages in early striatal development (Choi and
Lovinger 1997a,b
). These observations clearly demonstrate that
synaptic changes contribute to LTP and LTD. Second, no change in the N1
fiber volley field potential is observed following HFS even in slices
where the synaptically driven PS shows evidence of LTP or LTD. This
rules out several possible explanations of the lasting change in PS
amplitude. For example, these changes cannot be due to differences in
the spread of stimulus current through the tissue, the flow of current
around the recording electrode, or the recording conditions. Any such
change would impact the N1 component to the same extent as the
synaptically driven PS. The lasting changes are not likely due to a
change in presynaptic excitability because this would be reflected by a
change in N1 amplitude. Third, a lack of change in postsynaptic
excitability during expression of LTD has been well documented as
measured using intracellular current injection (Calabresi et al.
1992a
). Furthermore, we have characterized the excitability of
neurons using current injection during whole-cell recording and have
never detected a change in excitability of neurons on tetanus
(Lovinger et al. 1993
). Finally, the observation that
striatal LTP is NMDA receptor-mediated strongly indicates that a change
in synaptic transmission contributes to this form of plasticity because
NMDA receptor-dependent forms of LTP have been shown to involve a
change in the synaptic response (Artola and Singer 1987
;
Collingridge et al. 1983
; Huang and Kandel
1998
). These findings do not rule out the possibility that a
change in postsynaptic excitability could contribute to LTP but
strongly indicate that a change in synaptic transmission contributes to
striatal LTP.
The heterogeneity in synaptic plasticity in different subregions of the
striatum suggests varying roles for these subregions in information
processing or development of the circuitry of the basal ganglia.
Indeed, recent studies suggest that the dorsomedial and dorsolateral
striatum have important differential roles in different learning and
memory paradigms (Devan and White 1999). The striatal
regions analyzed in this study receive topographical projections from
different areas of the cortex and thalamus. The dorsolateral striatum
receives a much greater number of input fibers from the primary motor,
sensorimotor, and sensory cortex. Conversely, the dorsomedial striatum
receives less motor input and more input from the visual and auditory
regions of the cortex (Cospito and Kultas-Ilinsky 1981
;
McGeorge and Faull 1989
). There is also a significant
amount of converging cortical input to both subregions of the striatum.
Furthermore, disparate regions of the thalamus project differentially
to the dorsomedial and dorsolateral striatum (Berendse and
Groenewegen 1990
). Thus, different sorts of information may be
processed by medium spiny neurons in the functionally segregated areas
of the caudate and putamen at different times, and therefore, synaptic
efficacy may need to be modified heterogeneously in the distinct
subregions of the neostriatum.
That subregional differences in plasticity reflect differential
development of synapses is an intriguing possibility. Striatal neuronal
function, reflected by measurements of active and passive membrane
properties as well as evoked synaptic responses, matures during the
third week of postnatal development (Tepper et al. 1998). Synapses within the striatum develop along a
lateral-medial gradient with the synapses in lateral striatum maturing
earlier than those in medial striatum (Butler et al.
1998
). Two findings in the present study may relate to these
developmental patterns. First, we observed that synaptic responses
could more readily be elicited from lateral than from medial striatum
in slices from postnatal day P12-P15 animals. Second, we observed a
larger change in the forms of plasticity early in development in
lateral than in medial striatum. Lateral striatal synapses switch from
predominant LTP to predominant LTD during a time period when the medial
striatal synapses are just becoming functionally viable. The medial
striatal synapses generally exhibit a smaller magnitude LTP throughout this developmental time period. These observations suggest that LTP is
the predominant form of plasticity when synapses are just beginning to
come on-line. This is consistent with the development of hippocampal
synapses where early predominance of NMDA receptor-mediated responses
produces LTP and
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
"(AMPA)-fication" of synapses (Isaac et al. 1995
;
Liao et al. 1995
; Petralia et al. 1999
).
Indeed, earlier studies in striatum suggest that NMDA receptor-mediated
responses increase dramatically in the first two weeks of postnatal
development (Colwell et al. 1998
).
The heterogeneity of plasticity is not entirely surprising if one
considers that studies involving single-unit recordings from cells
during a striatal-based learning task indicate that some neurons
increase their firing rate while others decrease their firing rate
(Jog et al. 1999). Assuming modulation of synaptic efficacy at excitatory striatal synapses can change the firing pattern
of striatal neurons, the observation that LTD and LTP can occur in this
brain region in vitro suggests mechanisms underlying these firing rate
changes. Medium spiny neurons exhibit relatively hyperpolarized resting
potentials and depend strongly on coordinated excitatory synaptic input
to fire action potentials (Wilson and Kawaguchi 1996
).
Thus, it is likely that changes in striatal synaptic transmission
contribute to learning-related changes in firing rates.
Our findings also indicate that LTD may be expressed later in
development, at least within the dorsolateral striatum. It is reasonable to hypothesize that this occurs after synapses have been
undergoing potentiation for prolonged periods. The emergence of LTD may
help to fine-tune synaptic efficacy to refine movement and behavioral
sequencing. This is consistent with the fact that LTD predominates
during a time period when movement patterns are changing from more
neonatal to more adult-like. In past studies, we have found that paired
pulse facilitation, a change in synaptic function that is the
consequence of LTD, begins to appear during the same time period when
LTD is readily elicited by HFS (Choi and Lovinger
1997a). This finding suggests that a decrease in neurotransmitter release probability due to an LTD-like process is
indeed taking place during this developmental time period. Of course,
both LTD and LTP can occur in fully adult animals, as discussed
above. This is not unexpected because animals will have to
acquire new behaviors and habits and refine these behaviors in response
to environmental input throughout their lifetime. It will be important
to determine the cellular and molecular mechanisms that underlie these
developmental changes in synaptic plasticity. Our finding that the role
of NMDA receptors in transmission is quite strong early in development
of lateral striatum suggests that the ability to activate these
receptors is one important factor governing the direction of plasticity
in the striatum.
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ACKNOWLEDGMENTS |
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The authors thank Dr. Danny G. Winder for generously providing helpful comments for the manuscript. The authors are grateful to G. Gerdeman, Dr. Gabriella Stocca, and Dr. Ki-wug Sung for insightful discussion of the observations presented.
This research was supported by the Tourette Syndrome Association and National Institute of Neurological Disorders and Stroke Grant NS-30470.
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FOOTNOTES |
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* J. G. Partridge and K.-C. Tang contributed equally to this research.
Address for reprint requests: D. M. Lovinger, Vanderbilt University Medical Center, Dept. of Molecular Physiology and Biophysics, 724 Medical Research Building 1, Nashville, TN 37232-0615 (E-mail: david.lovinger{at}mcmail.vanderbilt.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 2 February 2000; accepted in final form 25 May 2000.
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REFERENCES |
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