Regional and Postnatal Heterogeneity of Activity-Dependent Long-Term Changes in Synaptic Efficacy in the Dorsal Striatum

John G. Partridge,1,* Ka-Choi Tang,2,* and David M. Lovinger1,2

 1Department of Pharmacology and  2Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0615


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 MOmega , 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 MOmega 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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1. Developmental heterogeneity of synaptic plasticity in the dorsolateral striatum. A: schematic diagram of a striatal hemislice and the placement of stimulating electrode (stim) and recording electrode (record) in dorsolateral (DL) subregion (circled area). Although the exact region would slightly vary from experiment, the recording location ranged from 3.0 to 3.5 mm from the midline of the hemislice. In addition, the recording location along the anterior-posterior axis would range from 2.0 to 3.0 mm anterior from the bregma. B: plot of normalized population spike (PS) amplitudes during experiments examining the effect of high-frequency afferent stimulation (HFS) in DL striatum at early developmental time points (n = 9, P12-P14). Long-term potentiation (LTP) was consistently induced at these ages. C: normalized PS amplitudes from P17-P23 DL striatum following identical HFS protocol (n = 10). Inset (average of 15 individual responses) in B and C show PSs from a single slice recorded at the indicated times during the experiment. D: ratio of post-HFS synaptic response to pre HFS response plotted as a function of age for all recordings performed in DL striatum (n = 4 slices at P12, 10 at P13, 7 at P14, 13 at P15, 10 at P16, 11 at P17, 14 at P18, 8 at P19, 4 at P20, 6 at P21, 3 at P22, 3 at P23, 3 at 33, and 3 at P34). * significant changes (P < 0.05, repeated measures t-test) in synaptic efficacy induced by HFS at a particular postnatal day.

In contrast to the dorsolateral striatum, the dorsomedial striatum exhibited a tendency to express LTP at all postnatal ages examined. Stimulation and recording locations used during examination of the dorsomedial striatum are shown in Fig. 2A. In this striatal subregion, field potentials evoked from animals younger than P16 were unreliable and relatively small in amplitude when input fibers were stimulated at low frequencies, and thus, plasticity could not be investigated. Following HFS to the white matter overlying the dorsomedial striatum, long-lasting potentiation of synaptically mediated events was detected in the majority of slices examined (Fig. 2, B and C) and this potentiation was significant for slices at postnatal weeks 4 and 5 when comparing pre- to post-HFS values (P < 0.05, repeated measures t-test). One-way ANOVA indicated no difference in the percent change in HFS-induced responses at different ages in the dorsomedial striatum [F(9,44) = 1.479, P > 0.05]. However, LTD was observed in some slices from this subregion of the striatum from animals aged P16-P22. For example, in P19 dorsomedial striatum, LTP was evoked in 7 of 17 slices, while LTD was observed in 6 of 17 slices and four slices showed no significant change. Following postnatal day 19, LTD expression declined significantly and was observed in only 3 of 20 slices. Analysis of the presynaptic fiber volley showed no change in this component of the field potential as shown in the traces in Fig. 2B. These observations, coupled with the observed tendency for LTD expression in dorsolateral striatum at postnatal weeks 3-5, indicate a subregional variation in the direction and magnitude of activity driven changes in synaptic efficacy in the dorsal striatum.



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Fig. 2. HFS-induced LTP in the developing dorsomedial striatum. A: schematic diagram of a striatal hemislice and the placement of stimulating electrode (stim) and recording electrode (record) in dorsomedial (DM) subregion (circled area). This region corresponded to an average of 1.5-2.0 mm from the midline. B: plot of normalized PS amplitudes during experiments examining the effect of HFS in DM striatum at ages P17-P23 (n = 10). LTP was often induced at these ages. Insets (average of 15 individual responses): PSs from a single slice recorded at the indicated times during the experiment. C: ratio of post-HFS synaptic response to pre-HFS response plotted as a function of age for all DM recordings (n = 4 at P16, 4 at P17, 9 at P18, 17 at P19, 5 at P20, 6 at P21, 4 at P22, 3 at P23, 3 at P33, and 3 at P34). *, significant changes (P < 0.05, repeated measures t-test) in synaptic efficacy induced by HFS at a particular postnatal day. The range of stimulus intensities used to evoke synaptic responses did not differ across age or region.

Subsequent experiments were designed to determine if differential plasticity could be observed within the same hemislice. In this experiment, HFS was first delivered with stimulation and recording on one side of the dorsal striatum. In 22 hemislices from P16-P34 rats, HFS was given and synaptic responses were recorded beginning on one side (medial or lateral) of the hemislice. After the effects of HFS were determined in this subregion of the slice, a second recording was then performed on the opposite side of the hemislice and the responses to HFS were examined. The order in which the different subregions were examined was varied such that in approximately equal numbers of experiments, stimulation was first given to the medial side, or first on the lateral side. We confirmed that stimulation to one side of a hemislice did not evoke a synaptic response on the opposite side of the dorsal striatum but did evoke a population spike in the region just ventral to that stimulation site. This suggests that activation of fibers in the white matter over a dorsolateral or dorsomedial subregion of the striatum results in significant activation of only local synapses. The changes in PS amplitude following HFS displayed a pattern similar to that observed in experiments on separate hemislices, with the dorsolateral region more prone to express LTD and the dorsomedial region likely to express LTP (Table 1). We observed LTD in 14 recordings from the dorsolateral striatum, and in eight of these slices we observed LTP in the dorsomedial subregion of the same hemislice. In the dorsomedial striatum, we observed LTP in 12 slices, and in eight of these hemislices we observed LTD in the dorsolateral portion of the striatum. Overall, we observed LTD in the dorsomedial striatum of only five slices, and we observed LTP in only three slices from dorsolateral striatum. These results were combined with the data that was collected from single recordings per hemislice in Figs. 1 and 2. These findings suggest that the subregional difference in LTD versus LTP expression does not reflect variation between striatal hemislices.


                              
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Table 1. Paired recordings of synaptic plasticity from same hemislice in postnatal striatum

We have previously shown that striatal LTD involves a change in evoked synaptic current in slices from young animals (Choi and Lovinger 1997a,b). However, we were not certain from the foregoing experiments if the LTP observed during field potential recording in P12-P14 dorsolateral striatum resulted from a change in the excitatory synaptic response. We thus measured the excitatory postsynaptic current produced by activation of glutamatergic synapses onto these neurons. However, after repeated attempts, we could not observe LTP following HFS during ruptured-patch whole-cell recordings from striatal medium spiny neurons in P12-P15 dorsolateral striatum. Instead, LTD was routinely observed. Figure 3A shows the results of a typical set of experiments in which the effects of HFS on the amplitude of EPSCs (n = 8) were examined in medium-sized neurons from P12-P15 dorsolateral striatum under ruptured-patch conditions. To test whether the firing pattern of the postsynaptic cell is important in LTP expression, some of these cells were placed in current clamp using a potassium-based internal solution during the HFS protocol. Whether the neuron was in voltage-clamp with a cesium-based internal solution or in current-clamp during the HFS, the evoked EPSC following HFS was reduced for the duration of the experiment. This finding is consistent with our past observations using the ruptured-patch whole-cell approach (Choi and Lovinger 1997a,b). However, cellular constituents crucial for LTP induction or expression might diffuse out of neurons or be inactivated during ruptured-patch recordings due to perfusion of the intracellular environment. This had been observed previously in hippocampal CA1 neurons (Malinow and Tsien 1990). We therefore utilized the perforated patch-clamp technique in subsequent experiments.



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Fig. 3. Expression of striatal LTP observed during perforated patch-clamp recordings from P12-P15 DL striatum. A: normalized excitatory postsynaptic current (EPSC) amplitudes plotted as a function of time before and after HFS when recorded in ruptured-patch standard whole-cell configuration (n = 8). LTD was observed using this protocol. B: normalized EPSC amplitude values for perforated patch-clamp recordings (n = 5). LTP was observed using this protocol.

Using whole-cell voltage clamp with the perforated patch-clamp technique, we observed LTP as evidenced by an increase in EPSC amplitude in P12-P15 striatal neurons following a pairing of HFS and depolarization of the membrane potential to -10 mV (Fig. 3B). LTP was observed in five of seven cells using the perforated patch technique. We were able to sustain recordings for at least 15 min post-HFS in all five of the cells showing LTP, and in two of these cells, recording duration lasted for more than 30 min after HFS. In all of the cells where LTP was evoked, the potentiation of the synaptic responses lasted throughout the extent of the recording following HFS. The series resistance measured during the perforated patch recording did not change significantly during the duration of these experiments, as can be seen in the trace shown in Fig. 3B. In one neuron from P15 striatum, we observed LTD using the perforated patch technique to monitor excitatory synaptic transmission. This suggests that LTD can occur in young striatum even when cellular constituents are well preserved using the perforated patch-clamp technique.

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.

The NMDA receptor competitive antagonist (±)-APV (100 µM), when applied during HFS, blocked the induction of LTP of synaptic responses from the P17-P21 dorsomedial and P12-P14 dorsolateral striatum (Fig. 4). Upon washout of this agent, subsequent HFS resulted in LTP (Fig. 4B inset). Thus, the HFS-induced potentiation is largely dependent on the activation of the NMDA receptor. The NMDA-receptor dependency of striatal LTP was observed in six of six recordings from P12 to P14 dorsolateral recordings striatum as well as five of five recordings in P17 to P21 dorsomedial slices. In two recordings from the dorsolateral striatum, LTD was evoked in the presence of 100 µM (±)-APV. This reinforces the NMDA receptor-independent nature of striatal LTD.



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Fig. 4. Striatal LTP is dependent on the activation of NMDA-subtype glutamate receptors. A: effect of 100 µM (±)-2-amino-5-phosphopentanoic acid [(±)-APV] on LTP induction in the DL striatum from P12-P14 animals. Superimposed are the averaged normalized responses obtained in the presence (filled circles) and absence (open circles) of (±)-APV. Inset: representative experiment demonstrating the NMDA receptor antagonist sensitivity of transmission in young (P12-P14) dorsolateral striatum. B: HFS-induced LTP in the DM striatum. Superimposed normalized PS amplitudes in the presence (filled circles) and absence (open circles) of (±)-APV. Traces are the average of 15-30 responses. Inset: representative single experiment showing the NMDA-receptor dependency of DM striatal LTP and reversibility of APV block. Each point within the insets are the peak PS amplitude (mV) evoked at a frequency of 0.05 Hz. Bar: time of APV application. Arrow: delivery of HFS.

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).

In nine neurons, we performed conventional whole-cell current clamp recordings of evoked excitatory postsynaptic potentials (EPSPs) to determine which glutamate receptor subtypes contribute to transmission at excitatory striatal synapses. In whole cell recordings of medium-sized neurons in the dorsolateral striatum, the time constant (tau ) of the decay phase of the evoked postsynaptic potential was 23.2 ± 3.3 ms in younger dorsolateral (P12-P13, n = 4) neurons and 19.4 ± 1.8 ms from more mature neurons (P22-P26, n = 5) (Fig. 5). However, in the presence of 100-µM APV, these tau  values changed to 13.9 ± 2.2 and 19.6 ± 1.0 ms in neurons recorded from immature and mature preparations, respectively. This apparent change in the EPSP kinetics was not due to differences in the resting membrane potential (Vrest) of the neurons. In six neurons from P12-P13 animals, the Vrest averaged -74.0 ± 1.6 mV, while from P22-P26 animals this value was -73 ± 1.3 mV. In addition, application of 100 µM APV inhibited the amplitude of the peak response from 14.6 ± 2.1 mV to 11.5 ± 2.1 mV in neurons from P12-P13 rats but had no effect on the EPSP amplitude in P26 neurons. These data indicate that slices from different aged animals exhibit differential contributions of NMDA receptor-mediated synaptic transmission in the developing striatum.



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Fig. 5. NMDA receptor-mediated component contributes differentially to glutamatergic transmission during postnatal striatal development. Top: averaged waveforms showing EPSP evoked by a single afferent stimulus while recording in the current clamp mode from a striatal neuron from a P13 rat before and during APV application (left and center), and superimposition of both traces (right). In this neuron, the amplitude was reduced from 19.5 to 16.9 mV and the time constant was reduced from 21.4 to 9.7 ms in the presence of 100 µM APV. Bottom: averaged EPSPs recorded under current clamp in a striatal neuron from P26 rat in the absence and presence of APV as above. Excitatory postsynaptic potential (EPSP) amplitude and time constant values in the absence and presence of APV were slightly reduced from 15.8 to 15.3 mV and from 19.4 to 18.9 ms. Each waveform is the average of 10-15 responses.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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.


    ACKNOWLEDGMENTS

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.


    FOOTNOTES

* 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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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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