Modulation of striatal neuronal activity by glutamate and GABA: iontophoresis in awake, unrestrained rats

Brain Research 822 Ž1999. 88–106

Research report

Modulation of striatal neuronal activity by glutamate and GABA: iontophoresis in awake, unrestrained rats Eugene A. Kiyatkin, George V. Rebec

)

Program in Neural Science, Department of Psychology, Psychology Building, Indiana UniÕersity, Bloomington, IN 47405, USA Accepted 22 December 1998

Abstract To examine the effects of glutamate ŽGLU. and g-aminobutyric acid ŽGABA. and their interactions in the striatum under behaviorally relevant conditions, single-unit recording was combined with microiontophoresis in awake, unrestrained rats. Iontophoretically applied GLU Ž0–40 nA, 20 s. excited all spontaneously active neurons in dorsal Žcaudate–putamen. and ventral Žaccumbens, core. striatum; phasic GLU-induced excitations Žmean threshold 19.7 nA. were dose-dependent, inversely correlated with rate of basal activity Žexcitation limit ; 65 imprs., and highly stable during repeated GLU applications. GLU also excited silent and sporadically active units, which greatly outnumbered spontaneously active cells, and enhanced neuronal excitations associated with movement. Both spontaneously active and GLU-stimulated striatal neurons were highly sensitive to GABA Ž0–40 nA, 20 s.; most showed short-latency inhibitions during GABA diffusion from the pipette Ž0 nA. and the response quickly progressed to complete silence with a small increase in current. The GABA-induced inhibition was current-dependent, equally strong on spontaneously active and GLU-stimulated units, and independent of neuronal discharge rate, but less stable than the GLU-induced excitation during repeated drug applications. Prolonged GABA application Ž0–20 nA, 2–4 min. reduced basal impulse activity, but was less effective in attenuating the neuronal excitations induced by GLU or associated with movement. Our data support the role of GLU afferents in the phasic activation of striatal neurons and suggest that the effects of GLU strongly depend on the level of ongoing neuronal activity. The ability of GABA to modulate both basal and GLU-evoked activity suggests that GABA, released from efferent collaterals and interneurons, plays a critical role in regulating neuronal activity and responsiveness to phasic changes in excitatory input. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Glutamate; GABA; Striatum; Integration; Modulation

1. Introduction Glutamate ŽGLU. and g-aminobutyric acid ŽGABA. play crucial, but opposing roles in striatal neuronal processing w9,14,31x. GLU, which originates primarily from cortical and thalamic structures w8,13,17,31x, provides the major excitatory input to striatal neurons w8,21,38,39x. In fact, striatal GLU release appears to be responsible for the phasic excitations that occur in response to somatosensory stimuli and during behavior w16,35,36x. GABA, in contrast, is contained in most striatal neurons, which release it locally via axonal collaterals to exert an inhibitory function w15,21,31,46x. Thus, whereas GLU directly activates striatal neurons in response to external events, GABA appears critical for coordinating local circuit activity.

) Corresponding author. Fax: q1-812-855-4520; E-mail: [email protected]

Most information on the role of GLU and GABA in regulating striatal activity is based primarily on electrophysiological data obtained either in vitro or from anesthetized animals w1,2,6,7,19,33,43 x. These preparations have been instrumental in establishing basic ionic and receptor mechanisms underlying the effects of GLU and GABA, but they shed little light on how these transmitters influence striatal neuronal functions under behaviorally relevant conditions. Thus, although it should be evident that GLU excites and GABA inhibits striatal activity, the patterns of these effects, their interactions, and their relationship with basal levels of neuronal activity in intact, fully functioning animals are entirely unknown. To address these issues, we combined single-unit recording with iontophoresis in awake, unrestrained rats. Because the neuronal response to afferent signals is dependent on membrane potential w5,9,45x, we first examined spontaneous impulse activity of striatal neurons under quiet resting conditions and during naturally occurring

0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 1 0 9 3 - 8

E.A. Kiyatkin, G.V. Rebec r Brain Research 822 (1999) 88–106

movement. Subsequent iontophoretic testing with GLU and GABA focused on several variables, including neuronal response thresholds, dose effects, relationships with basal activity, and response stability to repeated or continuous applications. Because GLU iontophoresis can be used to reveal an apparently large number of silent and sporadically active striatal neurons, we tonically activated these cells with continuous, low-current GLU applications and examined their activity and responsiveness to GABA. Finally, we examined GLU-GABA interactions by testing the effects of brief applications of one during continuous application of the other. Our neuronal sample included units recorded from both dorsal Žcaudate–putamen. and ventral striatum Žnucleus accumbens, core.. Although these two major striatal divisions differ in their source of dopamine ŽDA. innervation and presumed functional roles, they have generally similar cellular organization and afferent and efferent connections w31x.

2. Methods 2.1. Animals and surgery Data were obtained from male, Sprague–Dawley rats, weighing ; 400 g and bred in our animal colony from source animals supplied by Harlan Industries ŽIndianapolis, IN.. All animals were housed individually under standard laboratory conditions Ž12-h light cycle beginning at 0700. with free access to food and water. All protocols were performed in compliance with the Guide for the Care and Use of Laboratory Animals ŽNational Institutes of Health, Publication 865-23. and were approved by the Indiana University Institutional Animal Care and Use Committee. Rats were anesthetized with chloropent Ž0.33 mlr100 g, i.p.., mounted in a stereotaxic apparatus, and prepared for subsequent single-unit recording as described in detail previously w26,28x. For recording in the striatum, a hole was drilled unilaterally through the skull Ž1.2–1.4 mm anterior and 2.0 mm lateral to bregma. and the dura carefully retracted. A plastic, cylindrical hub, designed to mate with the microelectrode holder on the recording day, was centered over the hole and secured with dental cement to three stainless steel screws threaded into the skull. One screw served as both electrical ground and attachment for the head-mounted preamplifier. The hub was sealed with silicone rubber to prevent drying of the brain surface. After a 3–4 day recovery period, during which each animal was habituated to the recording chamber for a total of 4–6 h, the recording session began and resumed on each of the next 2–5 days. 2.2. Single-unit recording and iontophoresis Four-barrel, microfilament-filled, glass pipettes ŽOmega Dot 50,744; Stoelting., pulled and broken to a diameter of

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between 4 and 5 mm, were used for single-unit recording and iontophoresis. The recording and balance barrels contained 3 M and 0.25 M NaCl, respectively, and the remaining barrels were filled with 0.25 M solutions of l-GLU monosodium salt and GABA ŽSigma.. Retaining Ž; "8 nA. and ejecting Ž"5–80 nA. currents were applied with a constant current generator ŽDagan 6400.. GLU was dissolved in distilled water and GABA in 0.005 M NaCl. The in vivo resistance of the drug-containing barrels ranged between 20 and 40 M V Žmeasured at constant current., while the recording channel had an impedance of ; 4–10 M V Žmeasured at 1 kHz.. To prevent electric crosstalk between channels, the microfilaments were removed from the upper part of the pulled pipettes and the opening of each barrel was separated by 2–3 mm and covered with paraffin. The multibarreled pipette was filled with fresh solutions less than one hour before each recording session and fixed in a microdrive assembly, which allowed for 11 mm of dorsoventral travel w34x. The microdrive assembly was inserted into the skull-mounted hub and the electrode was advanced 4.0 mm below the brain surface to the starting point of unit recording. Neuronal discharges passed through a head-mounted preamplifier ŽLF 441CN, National Semiconductor., and all electrical connections from the microdrive assembly were fed outside the recording chamber via shielded cable through an electric swivel. Electrophysiological signals were amplified, filtered Žbandpass: 100–3000 Hz., and stored on an audio channel of a videocassette recorder ŽVCR.. Spike activity was monitored on-line with a digital oscilloscope and audioamplifier and counted in 2-s bin width by computer in conjunction with an amplitude-sensitive spike discriminator. A second audio channel of the VCR was used to mark iontophoretic applications and behavioral events. Behavioral activity was recorded on the video channel. All recordings took place during the day Ž1000–1800. in a plexiglass cage Ž35 = 35 = 40 cm3 . housed inside a sound-attenuating, electrically shielded chamber within view of a video camera. After the isolation of single-unit discharges Žsignal-to-noise ratio of at least 3:1., data collection for each neuron usually lasted for 20–30 min. Our protocol typically included brief Ž20 s. applications of GLU Ž0 to y80 nA. and GABA Ž0 to q80 nA. performed at 1–2 min intervals with the same or increasing currents. To detect silent cells, some electrodes were advanced during continuous or pulsatile ejection of GLU at low currents. These units were subject to GABA testing under conditions of continuous GLU application. In some cases, we tested the effects of relatively prolonged applications Ž2–5 min. of GABA at low currents on basal impulse activity and phasic excitations induced by pulsatile GLU ejections. All iontophoretic applications used for statistical analysis were performed when the animals rested quietly with no sign of overt movement, as in previous work w26x. Data

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obtained during spontaneous movements Žchanges in neuronal activity from the quiet resting baseline as well as responses to iontophoresis. were analyzed separately. 2.3. Histology After completion of the last recording session, rats were deeply anesthetized with cholorpent, and an epoxy-insulated tungsten electrode, inserted into one barrel of the pipette, was lowered into the recording area. Current was passed through the electrode Ž50 mA for 15–20 s. to produce microlesions at depths of 5.0 and 7.0 mm below the skull surface. Rats were then perfused transcardially with formosaline, and the brain was removed and stored for subsequent histological processing. Brain tissue was frozen, cut Ž50 mm sections., mounted on slides, and stained with cresyl violet. The location of each recording site was determined from histological data on the electrode track and depth information noted on the microdrive assembly at the time of each recording. The atlas of Paxinos and Watson w32x served as the basis for both electrode placement and histological analysis. 2.4. Data analysis Three parameters of impulse activity—mean rate Ž X ., standard deviation ŽS.D.. and coefficient of variation ŽCV s S.D.rX . —were calculated for each spontaneously active unit during a 30 s period of quiet rest Žbin width 2 s.. S.D. and CV provided a measure of the absolute and relative variability Žirregularity. of impulse flow, respectively. Iontophoretic responses were considered significant Ži.e., excitation or inhibition. when the mean firing rate during iontophoresis differed Ž p - 0.05; two-tail Student’s t-test. from the immediately preceding period. The same statistical comparisons were used to assess the effects of prolonged application of GABA on basal impulse activity and phasic GLU-induced excitations. Iontophoretic responses also were assessed in terms of onset and offset latencies, absolute and relative magnitude, the effect of ejection current Ždose–response relationships., and stability during repeated drug applications at the same current. For units tested at different GLU or GABA currents, response thresholds were calculated as the minimum current necessary to induce a significant and consistent change in firing rate. We also used a one-way, repeated measures analysis of variance ŽANOVA. to obtain group-response data w28x. Such information is useful because small, statistically nonsignificant changes in firing rate during individual iontophoretic applications may actually result in a significant group effect if these changes occurred consistently in many units. Because the duration of each neuronal recording varied from 6 to 90 min and the testing program for each unit was different, it was impossible to assess response thresholds and dose–response relationships

for all units, and our data are reported as numbers of both units and iontophoretic tests. Various relationships between impulse activity and iontophoretic responses were assessed with standard statistical techniques, including natural logarithmic Žln. transformations, ANOVA, and correlation and regression analyses. Off-line analysis of videotape records was used to assess changes in impulse activity during spontaneous behavior Že.g., grooming and locomotion. and the influence of such behavior on iontophoretic responses. Although individual animals often showed unique patterns of behavior and not all behavioral patterns were expressed during each neuronal recording, it was possible to make a general assessment of movement-related changes in neuronal activity by comparing firing rates during a particular movement episode with the resting, pre-movement baseline.

3. Results 3.1. Spontaneous impulse actiÕity Our sample of spontaneously active cells included 207 units recorded from 44 rats during 66 daily sessions. These units had typical somatic, biphasic or triphasic spikes with a duration of 0.8–2.0 ms, an irregular discharge pattern, and highly variable rates Žrange 0.1–49.5, mean 6.39 imprs.. Rates of 0.1 imprs or higher were used as a formal criterion for spontaneously active neurons. Although units with very low, sporadic activity Ž- 0.5 imprs. were frequently observed, most were not included in our sample of spontaneously active cells due to complications in recording and iontophoretic testing. As shown in Fig. 1A, spontaneously active units had a highly variable X and S.D.; both parameters were distributed asymmetrically with modal values sharply shifted toward the low end. The majority of our units had a low rate of activity: 31% at 0.1–2 imprs, 26.1% at 2–4 imprs, 15.0% at 4–6 imprs, and 9.7% at 6–8 imprs. Only 18.0% of our units had rates between 8 and 49.5 imprs. A ln transformation of X and S.D. values ŽFig. 1B. revealed that these parameters were distributed highly symmetrically with very similar mean and modal values calculated for ln derivatives of X and S.D. Žnormal distribution.. The same normal distribution was typical for CV, a relative measure of impulse flow irregularity Ždata not shown.. The results of this transformation indicate, that: Ž1. our population of spontaneously active striatal neurons is very homogeneous based on parameters of impulse activity; Ž2. these parameters are distributed according to ln normal law; and Ž3. the characterization of impulse activity in this neuronal population will be statistically accurate with the use of ln derivatives of X and S.D. Fig. 1C indicates that the X and S.D. of impulse activity of individual units were closely correlated Ž r s 0.789; p -

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Fig. 1. Spontaneous impulse activity: percent distribution of mean rates Ž X, impr2 s. and standard deviations ŽS.D., impr2 s. ŽA. and their natural-logarithmic transformation ŽB.; relationships between X and S.D. ŽC. and between X and coefficient of variation ŽCV, %. ŽD.. Vertical hatched lines in B indicate modal values. Regression lines and coefficients of correlation Ž r . are shown in C and D.

0.001, F-test., i.e., absolute variability of impulse flow directly depends on discharge rate. This correlation is well approximated by ln law wi.e., lnŽS.D.. s f lnŽ X .x. The same strong correlation was found between X and CV ŽFig. 1D; r s 0.791, p - 0.001; F-test., i.e., relative variability Žirregularity. of impulse flow negatively depends on discharge rate wln ŽCV. s Žy. f lnŽ X .x. Fig. 2A and B shows the X and S.D. distributions as a function of unit location in the dorsoventral direction. Note that most units in dorsal and ventral striatum had a low rate of activity, though units near the ventral border discharged at the highest rates. Dorsal and ventral units also had almost identical and equally strong correlations between X–S.D. and X–CV ŽFig. 2C and D.. Thus, units in both striatal areas were combined for most analyses of iontophoretic testing. As shown in Table 1, however, ventral units had a significantly higher X and lower CV than dorsal units, indicating that fast firing is associated with a more regular discharge pattern. The activity of 22 striatal units was recorded during episodes of spontaneous movement. Nineteen of these cells increased activity above the quiet, resting baseline in association with head or whole body movement. The magnitude of the increase varied among individual units from a weak and unstable change to a pronounced Žup to 40–60

imprs. phasic excitation, which was characteristic of slow-firing cells. In the three remaining units, all of which had a relatively high discharge rate Ž14–38 imprs., we observed biphasic changes in impulse activity: a brief phasic excitation during the initiation of movement followed by a more prolonged decrease below baseline rate. 3.2. GLU responses of spontaneously actiÕe neurons The effects of brief GLU applications were studied in 140 spontaneously active neurons Ž98 in dorsal and 42 in ventral striatum. recorded from 28 rats during 47 sessions. Each unit received from three to 12 GLU applications at the same or different currents, resulting in a total of 405 GLU tests for analysis. Table 2 summarizes these results and Fig. 3 depicts representative GLU responses. All units in our sample responded to GLU with an excitation. Although the number of GLU-sensitive units increased with ejection current, all three measures of the GLU excitation Žrange of absolute and relative response magnitude and the mean response magnitude. were comparable at different currents. The response threshold to GLU Ž n s 25 units. ranged from 2.5 to 35 nA with a mean of 19.7 " 1.75 nA. Although the number of units tested with low currents was relatively small, 1 of 3 cells was sensitive to spontaneous

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Fig. 2. Spontaneous impulse activity: relationships ŽA, B. between dorsoventral location of the recording site and mean rate Ž X, imprs. or standard deviation of impulse activity ŽS.D., impr2 s.; relationships ŽC, D. between X–S.D. and X–CV, shown separately for dorsal and ventral striatal units. n, Numbers of units; r, coefficients of correlation.

GLU diffusion Žcurrents 0 nA; a 14-c-6 in Fig. 3. and 11 of 14 units were sensitive to GLU at 5 nA current. An ANOVA revealed that GLU at any current had a strong

excitatory effect on neuronal activity Žsee F values in Table 2.. When an ANOVA was applied to GLU tests that show no significant changes in discharge rate during indi-

Table 1 Spontaneous and GLU-stimulated activity of striatal neurons in awake, unrestrained rats Parameters

Number of units X, imprs Mean Range Mean lnŽ X . " S.E. S.D., impr2 s Mean Range Mean lnŽS.D.. " S.E. CV, % Mean Range Mean lnŽCV. " S.E. r Žln X y ln S.D.. r Žln y ln CV.

Spontaneously active

GLU-stimulated

Dorsal striatum

Ventral striatum

All units

All units

141

66

207

69

4.53 0.1–36.94 1.78 " 0.08 a

10.35 0.4–49.5 2.32 " 0.15

6.39 0.1–49.5 1.95 " 0.07

14.54 2.57–58.46 3.17 " 0.08 c

4.57 0.45–17.34 1.31 " 0.06

5.46 0.97–14.87 1.50 " 0.08

4.86 0.45–17.34 1.37 " 0.05

11.24 1.60–51.77 2.25 " 0.07 c

75.31 8.8–197 4.16 " 0.05a 0.745 0.677

56.51 8.0–187.6 3.78 " 0.09 0.850 0.889

69.31 7.99–196.92 4.03 " 0.05 0.779 0.790

45.12 12.15–116.9 3.68 " 0.06 c 0.682 0.472 b

X, mean rate; S.D., standard deviation; and CV, coefficient of variation of impulse activity. All parameters were calculated based on 2 s rate values, but mean and ranges of X are shown as imprs. a indicates the difference between dorsal and ventral striatum at p - 0.001; b and c indicate difference between spontaneously active and GLU-stimulated conditions at p - 0.01 and 0.001, respectively. S.E., standard error of the mean.

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Table 2 Effects of brief GLU iontophoresis on striatal neurons Parameters

Number of units tested Total number of tests Units with excitations Percent Tests with excitations Percent F Excitation Range Žimprs. Range Ž%. Mean magnitude Ž%. " S.E.

GLU current ŽnA. 5

10–15

20–25

30–35

40

Total

15 26 12 80.0 20 76.9 22.4

51 109 41 80.4 91 83.5 83.5

78 152 68 87.2 138 90.8 183.8

42 78 40 95.2 75 96.2 240.9

20 31 20 100 31 100 44.7

140 405 140

5.45–67.84 164–2421 618.6 "146.1

1.94–58.17 129–8271 757.2 "130.2

1.28–58.17 116–22,300 1257.2 "214.3

2.25–64.45 106–6100 961.4 "145.2

2.4–61.15 118–5342 1082.3 "273.1

364

F values show the strength of the GLU effect on impulse activity estimated using a one-way ANOVA with repeated measures. Each value indicates a significant effect Ž p - 0.001., which, however, differs in strength at different currents. S.E., standard error of the mean.

vidual analysis Ž n s 14 at 20 nA., a significant and strong effect of GLU was found Ž F1,27 s 13.57, p - 0.003.. Similar results were obtained for other GLU currents.

GLU-induced excitations had relatively short onset and offset latencies Ž2–6 s. and appeared as uniform, strong, and sustained increases in discharge rate, although the

Fig. 3. ŽA. Rate-meter histograms showing impulse activity and responses of striatal neurons to glutamate ŽGLU, short open box.. Numbers below iontophoretic application indicate ejection current in nA, no numbers in subsequent applications indicate the same current as the last marked application. In all cases, neuronal activity is presented as imprs and each division on the ordinate of all histograms represents 10 imprs. Short solid lines below some histograms indicate periods of overt movement; at all other times, the animals were at quiet rest. Triple numbers above each histogram identify the rat, session, and unit, respectively. Ejection of current alone Ža 22-a-2. is indicated by long open boxes and Žy. or Žq. signs. NS identifies GLU tests with no significant changes in impulse activity; in all other cases, GLU significantly increased discharge rate. ŽB. Representative patterns of GLU responses in five different striatal cells. Each response was obtained as a sum of at least five individual responses and shown as changes in mean" S.E. rate Žimprs..

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pattern of this increase varied in different units Žsee examples in Fig. 3B.. The CV, however, decreased during GLU application Ž34.18 " 2.11% vs. 88.34 " 4.47% for basal activity; p - 0.0001. suggesting that GLU regularized impulse flow. The GLU-induced excitation was frequently accompanied by a decrease in spike amplitude Žas much as two to three times., but apparent depolarization inactivation Ži.e., a complete disappearance of discharges due to over-excitation. rarely appeared at the currents used. An example shown in Fig. 3 Ža 14-c-6. demonstrates the rare case of a presumed GLU-induced depolarization inactivation. Note that the excitation initially increased in magnitude with an increase in current, but decreased and eventually disappeared completely with further current increases. Off-line analysis of this and two similar cases revealed that

the disappearance of firing was always preceded by a marked increase in discharge rate Žup to 60–70 imprs. associated with a profound decrease in spike magnitude. When the pattern of the GLU-induced excitation was analyzed on a second-to-second scale Žsee examples in Fig. 3B., the response typically reached a maximal level of excitation or even slightly decreased before current off. In some units, the GLU-induced excitation was followed by a rebound-like inhibition Žlast GLU response in Fig. 3B., but these relatively rare cases were typical of fast-firing units and became evident only during superimposition of multiple responses. To determine the relationship between the GLU response and ejection current Ždose., we analyzed three repeated GLU tests performed on the same cells, in which

Fig. 4. Dependence of various parameters of the GLU responses ŽA–C. on ejection current ŽnA.. ŽA. Percent tests with excitations; ŽB. absolute magnitude of the GLU response Žimprs; open bars, mean basal activity; closed bars, mean GLU-induced activity.; and ŽC. relative magnitude of the GLU response Ž%.. Asterisks indicate level of statistical significance of differences Ž), p - 0.05; )), p - 0.01; and ))), p - 0.001; Student’s t-test. between current groups ŽB. or between mean rates before and during each application at given currents ŽC.. Relationships between basal activity and the effects of iontophoretic GLU ŽD, E.. Absolute ŽD, imprs. and relative ŽE, %. magnitudes of the GLU-induced excitation Ž20 nA. shown separately for dorsal and ventral striatal neurons. Points to the left of the hatched lines indicate units with a very slow rate, which were excluded from the correlative analysis. n, The number of responses in each group; r, the correlation coefficient. The point of intersection between the regression line and the angled line of no effect indicates the basal rate at which GLU would no longer elicit the activation. Common regression lines are shown for all striatal neurons.

E.A. Kiyatkin, G.V. Rebec r Brain Research 822 (1999) 88–106

current was progressively doubled Ž5–10–20, or 10–20–40 nA.. This analysis revealed that the excitatory effect of GLU strongly depends on ejection current ŽFig. 4A, B, C.. Note that the mean discharge rates before each test Žopen bars in B. are the same in all current groups. Dose–response relationships for each individual unit, however, varied in terms of the first appearance of the response and

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its magnitude during the next current increase. In some units, the GLU-induced excitation reached a limit and did not change with a subsequent increase in current Ža 12-a-7 in Fig. 3.. Fig. 4 depicts the relationships between discharge rate and absolute ŽD. or relative ŽE. magnitudes of the GLU response for dorsal and ventral striatal units at the same

Fig. 5. Variability of GLU and GABA responses during repeated applications. ŽA, D. Changes in impulse activity Žimprs. before repeated applications of GLU and GABA. ŽB, E. Changes in absolute magnitude of the GLU or GABA responses Žimprs. for each pair of repeated applications Ž1 test vs. 2 test.. In the case of GLU, the same numbers of decreases and increases were found for both parameters, the coefficients of correlation Ž r . were very high, and the regression lines were almost superimposed on the line of no effect Žnot shown.. Although almost equal numbers of increases and decreases were also typical of GABA Žfilled circles identify significant appearances or disappearance of the response., the coefficients of correlation Ž r . were smaller and the regression lines were shifted from the line Žhatched. of no effect. If the GABA response during the first application was strong, this response became weaker with the next application and vice versa. ŽC, F. the relationships between changes in the GLU or GABA responses and associated changes in basal impulse activity. In each graph, the difference in response magnitude for each pair of tests Ž1 vs. 2. is plotted against the difference in discharge rate before these tests. In both cases, no significant correlation emerged between the parameters suggesting the stochastic nature of their fluctuations.

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currents Ž20–25 nA.. Note that units in both striatal divisions have similar relationships between basal discharge rate and the parameters of GLU response. In ventral units, however, both the basal discharge rate Žrange 0.13–50.22; mean 7.36 " 1.78 imprs. and the absolute magnitude of the GLU-induced excitation Žrange 2.63–58.17; mean 17.89 " 2.11 imprs. were higher than in dorsal striatal cells Žrange 0–17.11; mean 2.50 " 0.30 imprs and range: 1.28–31.17, mean 10.34 " 0.62 imprs, respectively.. The GLU responses of very slow-firing units are shown separately Žleft of the hatched lines in both graphs.. As shown in Fig. 4D, the absolute magnitude of the GLU-induced excitation strongly and directly depends on the rate of basal activity Ždorsal striatum, r s 0.665; ventral striatum r s 0.657; one regression line is shown for all spontaneously active units except almost silent cells., i.e., fastfiring cells have a higher magnitude GLU response and vice versa. In contrast, the relative magnitude of the GLU-induced excitation ŽB. is negatively related to basal activity Ždorsal striatum r s 0.829; ventral striatum r s

0.915., i.e., is lower in fast-firing and higher in slow-firing units. The regression lines in both graphs intersect the line Žhatched. of no effect at approximately the same rates Ž; 65 imprs., indicating that the maximal possible response frequency Žexcitation limit or response saturation. is close to the upper level of spontaneous impulse activity Ž49.5 imprs, see Table 1.. The GLU response was very stable during repeated applications. Of 61 pairs of repeated tests at the same current Ž20–25 nA., only two qualitative changes in the GLU response were seen. In one case, the excitation disappeared and in another, it reappeared after a previous no response. Fig. 5B demonstrates that the GLU-induced excitation in units with different discharge rates varied with repeated application equally in both directions and within relatively narrow limits. In 34 cases of repeated tests, the response magnitude slightly increased; and in 27 cases, it slightly decreased without a significant change in means Žfirst test, 12.18 " 1.17; second test, 13.59 " 1.29 imprs.. Changes in magnitude during the first and second

Fig. 6. Impulse activity of GLU-stimulated units. ŽA, B. distributions of mean rate Ž X, impr2 s. and standard deviation ŽS.D., impr2 s. calculated separately for spontaneously active Žopen circles. and GLU-stimulated Žclosed circles. striatal units. Vertical hatched lines show distribution modal values. ŽC, D. The relationships between X–S.D. and X–CV calculated separately for spontaneously active Žclosed symbols. and GLU-stimulated units Žopen symbols.. Both neuronal groups had similar relationships between parameters of impulse activity Žsimilar locations of regression lines with respect to the line of no effect, similar coefficients of correlation., but GLU-stimulated and spontaneously active units significantly differed in X, S.D., and CV Žhatched horizontal and vertical lines..

E.A. Kiyatkin, G.V. Rebec r Brain Research 822 (1999) 88–106

applications were highly correlated Ž r s 0.917. and the regression line was superimposed on the line of no effect Žnot shown.. Similar small, bidirectional fluctuations Ž23 increases, 32 decreases and 6 no changes. were also typical of the resting discharge rate before GLU ŽFig. 5A.. The mean rates before the first and second GLU tests were similar Ž4.39 " 0.87 and 4.47 " 0.97 imprs. and the regression line was also superimposed on the line of no effect. When changes in the GLU-induced excitation were plotted against changes in discharge rate for each pair of GLU tests ŽFig. 5C., no correlation emerged Ž r s 0.158.. When neuronal activity before the second test decreased Ž n s 32., the GLU response either slightly decreased Ž n s 17. or increased Ž n s 12.. The same was true for increases in discharge rate Ž n s 23., which were accompanied by either increases Ž n s 15. or decreases Ž n s 17. in the GLU-induced excitation. Sixteen GLU responses, in which movement occurred, were analyzed separately. These responses were recorded from units with no or relatively slow discharge rates Žrange 0–3.9 imprs. at different GLU currents Ž5–25 nA.. In 14 cases, the magnitude of the GLU-induced excitation increased compared to either previous orrand subsequent application during quiet rest Žsee a 19-a-1 for 30 nA and 1-c-1 for 20 nA., and in two cases, when the initial GLU response was very large, it decreased. Group analysis revealed that concomitant movement has a significant en-

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hancing effect on the GLU response ŽGLU q movement: F1,31 s 47.21, mean rate s 13.4 " 1.7 imprs; GLU alone: F1,31 s 21.57, mean rate s 8.13 " 1.57; p - 0.05.. 3.3. ActiÕity of silent and sporadically firing units during continuous GLU application Along with spontaneously active units, we also encountered silent units, which discharged only during GLU application and returned to inactivity when the GLU current was turned off. Although the exact ratio between spontaneously active and silent neurons remains unknown, our results suggest that silent units greatly outnumber spontaneously active units. The activity of silent and sporadically active Žrate - 0.5 imprs. units Ž54 in dorsal and 15 in ventral striatum. was studied under conditions of continuous GLU ejection at relatively low currents Žrange 3–35 nA; mean 14.34 " 0.45 nA.. Some neurons failed to maintain stable activity during continuous GLU application: either discharge rate gradually increased to the point of presumed depolarization inactivation, including complete cessation of firing, or the recording was contaminated by discharges from a second or third silent unit. All such cases were excluded from our sample. As shown in Table 1 and Fig. 6, units tonically stimulated by GLU had a significantly higher X and S.D. and a lower CV than spontaneously active units. The distribu-

Table 3 Effects of GABA in spontaneously active and GLU-stimulated striatal units Parameters

Spontaneously actiÕe units Number of units Žtests. Units with inhibitions Tests with inhibitions F value Mean change Ž%. "S.E. Inhibition Ž%. Range Mean magnitude "S.E. GLU-stimulated units Number of units Žtests. Units with inhibitions Tests with inhibitions F value Mean change Ž%. "S.E. Inhibition Ž%. Range Mean magnitude "S.E.

GABA current ŽnA. 0

5

10

20

40

Total

19 Ž27. 12 16 8.04)) 58.33 "5.88

24 Ž32. 20 23 14.87)) 37.77 "5.93

16 Ž21. 15 19 9.96) 39.56 "5.43

11 Ž23. 10 18 6.34) 39.21 "5.68

5 Ž11. 5 11 10.69)) 38.8 "6.73

28 Ž117. 28 90

0–78 39.25 "5.97

0–68 25.62 "4.73

1.4–66 33.99 "4.83

0–85 28.92 "4.62

1–44 38.80 "6.73

31 Ž50. 23 30 44.78))) 54.96 "4.63

31 Ž71. 30 58 78.09))) 31.80 "3.27

27 Ž50. 25 45 82.60))) 33.79 "3.84

24 Ž43. 22 39 53.97))) 30.79 "3.97

17 Ž33. 17 33 32.09))) 23.58 "3.60

1.2–83 38.05 "4.24

0–71 24.17 "2.80

0–72 28.09 "3.18

0–69 24.85 "3.02

0–78 23.58 "3.6

35 Ž258. 35 216

), p - 0.05; )), p - 0.01; and ))), p - 0.001, significance level of the GABA effect evaluated using a one-way ANOVA with repeated measures; S.E., standard error of the mean. The total number of tests in the last column is slightly larger than the sum of the responses Ž0–40 nA. because of the inclusion of some tests with higher currents Ž60 nA..

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tions of X and S.D., however, did not change in form Žln normal curve., but their modes were shifted to higher values ŽA, B.. The range of X values was also smaller due to a lack of units with a low discharge rate Ž2.57–58.46 imprs under GLU vs. 0.1–49.5 imprs in spontaneously active units., but in both conditions the highest firing rates were close to the excitation limit Ž; 65 imprs. found for brief GLU applications Žsee Fig. 4 above.. In addition, under continuous GLU stimulation, discharge rate was more regular ŽCV s 45.12%. than in the spontaneously active state Ž69.31%; p - 0.01.. Spontaneously active and GLU-stimulated units had similar relationships for X–S.D. ŽC. and X–CV ŽD., although with GLU the mean CV was smaller. Thus, silent and sporadically active units under conditions of continuous GLU stimulation discharged quite similarly to spontaneously active cells. 3.4. GABA responses The effects of brief GABA applications were tested on 28 spontaneously active units Ž18 in dorsal and 10 in ventral striatum. with highly varying discharge rates Žfrom 0.1 to 65 imprs. and 35 silent and sporadically active units Ž27 in dorsal and eight in ventral striatum., which maintained relatively stable discharges during continuous GLU application Žfrom 2.6 to 59 imprs.. As shown in Table 3 and Fig. 7, units in both these groups were

exceptionally sensitive to GABA. When the retaining current was turned off Ž0 nA., significant inhibitions were found in 12 of 19 spontaneously active Ž63.2%. and in 23 of 31 GLU-stimulated Ž74.2%. units. The number of GABA-inhibited units further increased with relatively small increases in ejection current; 83.3% of spontaneously active and 96.8% of GLU-stimulated units were inhibited by GABA at 5 nA. A one-way ANOVA revealed that spontaneous diffusion of GABA Ž0 nA. had a strong inhibitory effect on impulse activity in both neuronal groups. Although GABA-induced inhibitions occurred more frequently with an increase in ejection current, the mean change in discharge rate, the range, and the mean magnitude of the inhibitions were comparable at different currents ŽTable 3.. GABA-induced inhibitions developed with relatively short onset latencies Ž2–6 s., quickly disappeared after the ejection current was turned off Ž- 2 s., and were usually manifest as a uniform and profound decrease in discharge rate Žsee examples in Fig. 7.. These results applied to both spontaneously active and GLUstimulated units. To assess dose–response relationships, we examined three repeated GABA tests performed on the same cells during progressive doubling of the ejection current Ž0–5– 10, 5–10–20, or 10–20–40 nA.. Because the inhibition in many units was evident at minimal current Ž0 nA. and the range of effective currents was relatively narrow, data

Fig. 7. Rate-meter histograms showing impulse activity and responses of striatal neurons to GABA Žlong, open boxes. and GLU Žshort, open boxes.. In some units, the effects of GABA were tested during both spontaneous activity and continuous GLU application. VS indicates unit location in ventral striatum Žaccumbens core.; all other units were recorded from dorsal striatum. NS indicates GABA tests with no significant change in impulse activity. In all other cases, GABA significantly decreased discharge rate. Other abbreviations are as in Fig. 3.

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from three currents were combined for each point. As shown in Fig. 8A, B, C, all parameters of GABA responses on either spontaneously active or GLU-stimulated units significantly increased with increases in current. Note that the mean discharge rate before GABA application in each current group was similar. The relationship between basal discharge rate and the GABA response was analyzed separately for spontaneously active and GLU-stimulated units tested with the same current Ž5 nA.. As shown in Fig. 8D, the absolute magnitude of the GABA-induced inhibition for both groups of units varied greatly, both had a relatively large number of either total or near-total Ž- 10% of initial baseline. inhibitions. Note that the regression line for GLU-stimulated units was shifted further from the line of no effect Žhatched. than for spontaneously active neurons, but both lines had similar slopes. In contrast, the relative magnitude

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of inhibition in both groups was larger in slow-firing and weaker in fast-firing units, although the coefficient of correlation for GLU-stimulated units was smaller than that for spontaneously active cells ŽE.. The regression lines for this response measure had similar slopes in both unit groups. Unlike the GLU-induced excitation, the GABA response was more variable with repeated applications. Qualitative changes were found in 17.5 " 6.0% Ž7r40. and 21.8 " 3.9% Ž24r110. of repeated GABA applications for spontaneously active and GLU-stimulated units, respectively. These changes occurred over a broad range of currents and were manifest as either the appearance Ž n s 17. or disappearance Ž n s 14. of inhibition during the next test. The changes in GABA responses, moreover, were related to changes in discharge rate. Most cases of response appearance Ž14r17. occurred when basal activity

Fig. 8. Dependence of various parameters of the GABA response on ejection current ŽnA. in spontaneously active and GLU-stimulated units ŽA–C.. Percent of tests with inhibitions ŽA.; change in rate induced by GABA Žopen bars, basal activity; closed bars, GABA-induced activity; imprs. ŽB.; and relative magnitude of the GABA response ŽC.. Asterisks indicate level of statistical significance as in Fig. 4. Relationships between basal activity and the effects of iontophoretic GABA ŽD, E.. Absolute ŽD, imprs. and relative ŽE, %. magnitudes of the GABA-induced inhibition Ž5 nA. shown separately for spontaneously active and GLU-stimulated striatal units. Other abbreviations as in Fig. 4D, E.

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before the second test increased, whereas most cases of response disappearance Ž12r14. were seen when basal activity decreased. Fig. 5 depicts the results of quantitative analysis of GABA-response variability in GLU-stimulated units at 5 nA when changes in the response were most frequent. As shown in E, the magnitude of the GABA-induced inhibition during the second GABA application either increased Ž n s 20. or decreased Ž n s 18. compared to the first application; the means were the same for both tests Ž22.58 " 3.42% and 22.54 " 3.65%.. The GABA-induced inhibition, moreover, was larger in fast-firing than slow-firing cells Ž r s 0.472, p - 0.05.. Neuronal discharge rates before the second GABA application also varied in both directions ŽFig. 5D; 14 increases, 19 decreases, four no changes. but their means remained the same for both tests Ž11.29 " 1.70 and 11.27 " 1.81 imprs.. Although both basal activity and GABA-induced changes fluctuated, no consistent relationship Ž r s 0.04. emerged ŽFig. 5F.. It is also interesting to note that a comparison of Fig. 5A and D

indicates that spontaneous impulse activity is more stable than GLU-evoked activity. Similar to GLU responses, the GABA response also depended on behavioral state. When spontaneous movement occurred during GABA ejection Ž n s 8., the inhibiting effect of GABA Ž0–40 nA. was greatly diminished Žinhibitions: 2r8, F1,15 s 2.10, p s 0.191. compared to either a previous or a subsequent test conducted during quiet rest Žinhibitions: 8r8, F1,15 s 9.36, p - 0.02.. The between-group difference was significant Ž p - 0.05; Student’s t-test.. Whereas at rest, GABA decreased the mean rate to 14.45 " 3.19% of baseline Žrange 2–29%., this effect was significantly weaker during movement Žrange 23–158; mean 76.53 " 15.5%; p - 0.05; Student’s t-test.. A significant, but weaker attenuating effect of movement on the GABA response was also found in GLU-stimulated units Žsee a 28-b-1 and 27-b-7 in Fig. 7.. It appears, therefore, that GABA, even at relatively large currents, cannot block the neuronal activation associated with movement.

Fig. 9. Rate-meter histograms Žimprs. showing changes in impulse activity and GLU responses Žshort, open boxes. during continuous GABA application Žlong, open boxes.. All other abbreviations are as in Figs. 3 and 7.

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3.5. Basal actiÕity and GLU responses during prolonged GABA application The effects of prolonged GABA application Ž2–4 min. on changes in basal activity and GLU-induced excitations were tested in 13 Ž10 dorsal and three ventral striatal. units, which had either a slow rate of spontaneous activity or were silent Žrange 0–3.54 imprs.. In most units, the effects of several GABA applications at the same or different currents Ž0–40 nA. were tested resulting in a total of 42 applications. Fig. 9 shows individual examples of the effects of continuous GABA application on basal activity and GLU-evoked responses. An ANOVA applied to all tests independently of dose and effect revealed that prolonged GABA application had

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a strong Ž p - 0.001. inhibitory effect on both basal discharge rate Ž F1,125 s 21.77. and the magnitude of the GLU-induced excitation Ž F1,125 s 49.62.. As shown in Fig. 10A, the mean values of both parameters significantly decreased during GABA application, but quickly returned to baseline after the GABA current was turned off. The inhibiting effect of GABA on both impulse activity and the magnitude of the GLU response increased with increases in GABA current, but varied with dose in different units Žsee examples in Fig. 9.. As shown in Fig. 10B and C, continuous GABA preferentially decreased both basal activity Ž35r42. and the magnitude of the GLU-induced excitation Ž40r42.. In 10 tests Ž24%., we observed a complete inhibition of basal activity, and in 13 other tests Ž31%., discharge rate de-

Fig. 10. Statistical analysis of changes in basal impulse activity and GLU responses during continuous GABA application. Time-course ŽA. of mean Ž"S.E.. changes in discharge rate Žimprs; open circles. and the magnitude of the GLU-induced excitation Žimprs; closed circles.. Both data sets are shown as logarithms. Onset and offset of GABA application is indicated by hatched vertical lines at 0 and 2.5 min, respectively. Asterisks indicate significantly different values Ž))), p - 0.001; Scheffe F-test. from the pre-GABA period. Effects of prolonged GABA application on basal impulse activity ŽB. and GLU-induced responses ŽC.. GABA had a strong inhibiting effect on both parameters; the effect was dependent on discharge rate Ž r, coefficient of correlation., but was virtually absent when basal activity was very slow and the initial magnitude of the GLU response was very small Ž- 5 imprs.. Relationships between GABA-induced changes in the GLU response and basal impulse activity ŽD.. The relative difference in the GLU response Žduring GABA vs. before GABA, %. is plotted against the relative difference in discharge rate Žbefore GLU tests during vs. before GABA.. Hatched lines indicate 0 and 100% for both effects. No significant correlation emerged between the parameters, but in many units the GLU response remained, albeit inhibited, while basal activity was totally or almost totally blocked Žpoints close to the 100%-line at the left..

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creased from 70 to 95% of baseline ŽB.. The inhibiting effect on the GLU-induced excitation ŽC. ranged from a very small decrease to a strong attenuation of the response Ž12 tests, 28.6%., but in contrast to a total blockade of basal activity, a total block of GLU-induced activity Ž0 spikes. was never seen. Usually, under continuous GABA at high currents, GLU induced a low-frequency sporadic bursting, which often occurred near the end of the GLU ejection Žincrease in onset and peak latencies; see a 26-c-5 and 24-b-5 in Fig. 9.. Both effects were strongly dependent on the initial discharge rate. The inhibition was stronger for units with a high basal and high-magnitude GLU-evoked activity and disappeared at low rates Ž0.1 and ; 5 imprs, respectively.. The relative effects of GABA on both parameters, however, were surprisingly similar: y63.78 " 6.74% for basal activity and y60.52 " 4.51% for GLU-induced activity Žsee Fig. 10D.. Although continuous GABA application in most cases did not result in consistent changes in the signal-to-noise ratio of the GLU response Ž16 weak increases and 16 weak decreases., in 10 cases Ž24%. the GLU-evoked excitation persisted, albeit at a greatly reduced level, while basal activity was completely blocked. Continuous GABA application even at high currents Ž20–40 nA. also could not completely block excitations associated with spontaneous movement and, if the movement occurred during GLU application, the GLU response actually increased Žsee a 27-a-5, the second GLU test under GABA at 20 and 40 nA.. Group analysis revealed that the mean magnitude of the GLU-induced excitation quickly returned to the pre-GABA level after the GABA current was turned off Žsee Fig. 10A.. In some cases, especially at high currents, however, a slower restoration Žup to three to six applications. or incomplete restoration of the GLU response occurred Žsee a 24-d-8 in Fig. 9.. Sometimes, the first GLU response after cessation of GABA application was also strongly attenuated, but in other cases, even at high currents, the GLU response was restored immediately Žcompare a 24d-8 for 20 nA with a 27-a-5 for 20 and 40 nA..

4. Discussion The activity of striatal neurons, and thus their filtering and integrative functions, depends on two opposing influences. GLU depolarizing inputs, which converge on striatal neurons mainly from multiple cortical structures and thalamus, are the primary driving force for neuronal activation, whereas GABA hyperpolarizing inputs, arising mainly from striatal axonal collaterals and interneurons, appear to modulate these excitatory influences. The interplay involving constantly fluctuating GLU and GABA inputs and the voltage-dependent conductances ŽKq, Naq, Cly and Ca2q . regulated by these transmitters w9x represents the basic substrate of striatal neuronal processing. This substrate, moreover, is modulated by other neurochemical inputs.

DA and acetylcholine ŽACh., for example, may change the activity of striatal neurons by directly acting on postsynaptic membranes and by modulating GLU and GABA inputs to these cells w10,14,31x. In vitro studies of striatal, including accumbal, neurons have revealed a high resting membrane potential and little or no activity at rest w18,33,42x. These studies also have shown that spontaneous oscillations from a ‘hyperpolarized’ to a ‘depolarized’ state w5x are induced by the phasic activation of GLU-containing cortical cells w8,12,45,46x. Presumably, therefore, the activity of striatal neurons reflects the integration of multiple excitatory and inhibitory inputs, among which GLU and GABA play a primary role. Our present data revealed that GLU-induced excitations strongly depend on the level of basal activity. Both the absolute and relative magnitudes of the GLU response were smaller in fast- than in slow-firing units, and the response saturated at ; 60–65 imprs. It is interesting that the upper limit of the GLU-induced excitation was close to the upper limit of spontaneous activity; even continuous GLU application could not drive firing rate above these limits. The GABA-induced inhibition was also dependent on the rate of impulse activity; the absolute magnitude of this response was larger in fast-firing and smaller in slow-firing cells. Unexpectedly, the relative magnitude of the GABA response Žpercent change. was less dependent on discharge rate and was maximal at relatively slow or moderate basal rates Ž2–10 imprs.. A strong dependence of neuronal responses on basal activity rates reported for DA, GLU, and ACh in striatal w26,27x, thalamic w22x, and midbrain units w28x suggests a commonality of this relationship for different neurons and transmitters in awake, unrestrained rats. In agreement with data obtained in anesthetized and awake animals w5,19,30,35,36x, we found that most spontaneously active striatal neurons discharged at relatively low rates. Distribution analysis of our sample revealed that the largest number of recorded units Ž; 31%. had discharge rates ranging from 0.1 to 2 imprs, although we detected units with rates up to 50 imprs. When a ln transformation was applied to the major parameters of impulse activity Ž X, S.D., and CV., it was found that their ln derivatives were distributed according to an ideal normal law ŽGaussian curve. with close matching of means and modes. The same distribution, but more narrow and with a higher mode, was also typical of silent units under conditions of continuous GLU stimulation. The ln normal distributions not only permit the use of parametric statistics for comparisons, but may have functional significance such that processing of incoming information by striatal neurons may include its ln transformation. This type of informational transformation occurs frequently in biological systems, as it allows for strong modulation of the energetic range of transmitted messages without loss of informational value. Interestingly, logarithmic transformation of transmitted information is well known in sensory systems, and in fact

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this regularity was substantiated more than 100 years ago as the Weber–Fechner law, which describes perceived sensation as a log function of physical stimulus intensity. A ln transformation of processed information may be a common feature of central neurons as ln distributions of X and S.D. in a neuronal population have been previously found in the medial thalamus, hypothalamus w23x and midbrain tegmentum w25,28x. Our statistical analysis also revealed that the major parameters of impulse activity are closely interrelated, suggesting that units with higher discharge rates have higher absolute but lower relative discharge variability. These relationships also may be a common feature of central neurons as the same correlations have been found in diencephalic and midbrain units w23,25,28x. It is important to note, however, that this dependence of impulse variability on rate is quantitatively different in each studied structure. Although most evaluations of striatal function are based on information obtained from spontaneously active cells, they appear to comprise a minority of units both in vitro w9x and in anesthetized preparations w19,30x. The appearance of multiple units during continuous or pulse-like GLU application suggests that silent or sporadically active units also greatly outnumber spontaneously active units in the awake state. Most silent and sporadically active cells, however, were physically activated during movement, showing responses similar to those induced by brief GLU application, and, when GLU was applied continuously, most maintained a stable discharge rate and showed GABA responses much like spontaneously active cells. Taking into account the importance of GLU in driving striatal neuronal activity, these data suggest that, in addition to a relatively small number of units that receive tonic GLU input under resting conditions, the striatum contains a large reserve of silent units capable of producing high-magnitude excitations in response to phasic GLU input. Although the exact number of such units remains unknown, they appear to represent a significant contribution to changes in striatal activity under behaviorally relevant conditions. Further experiments are underway to evaluate the ratio between spontaneously active and silent neurons as well as the contribution of GLU to basal activity and movement-related and other naturally occurring neuronal excitations. Our data also revealed that all GLU-sensitive striatal neurons are highly responsive to GABA. In fact, many spontaneously active and GLU-stimulated cells were inhibited by GABA diffusion from the pipette Ž0 nA current.. Differences in GLU and GABA sensitivity may reflect marked differences in the location of GABA and GLU receptors. Most corticostriatal and thalamostriatal GLU afferents, for example, make synaptic contacts on the head of dendritic spines on the distal part of the dentritic arbor w4,17,20x at some distance Ž25–35 mm. from the soma, whereas GABA synapses are located primarily on the soma and proximal dendrites w3x. Other contributing factors

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may include differences in sensitivity of receptors and reuptake. In the striatum of awake animals, for example, extracellular GLU is maintained at levels Ž; 1.5–3.5 mM w29,37x. much higher than those for GABA Ž; 0.2–0.3 mM w37x.. Both basal and behaviorally evoked GLU levels, moreover, are tetrodotoxin-independent w29x suggesting that in addition to its transmitter role, GLU may be released by glial cells in the extrasynaptic space to provide certain metabolic and energetic functions. GABA, in contrast, appears to be exclusively engaged in neurotransmission. Although individual units had highly varying thresholds and patterns of GLU and GABA responses, our data revealed that both the GLU-induced excitation and GABA-induced inhibition were dose-dependent. Whereas GLU could routinely increase discharge rate up to 20–50 imprs, or 5–20 times basal activity, and these effects were especially strong in silent or sporadically active units, the effects of GABA were most prominent in fast-firing cells, usually seen as a twofold to fivefold decrease in basal rate. Of course, the latter effects were not measurable in silent and very slow firing cells, but when these units were excited by GLU, GABA effectively inhibited their activity. Highly stable, sustained GLU-induced excitations were quite different from the rapidly sensitizing effects of GLU on spiny neurons reported in vitro Žsee Ref. w40x., but these GLU responses also differed from those seen in chloral hydrate anesthetized animals w19x. Although response thresholds were comparable or even lower in our preparation compared to that reported for anesthetized animals, the absolute magnitude of the GLU-induced excitation was notably higher Ž20–60 imprs vs. 10–20 imprs. and units reached ‘ceiling’ levels of excitation at much lower currents Ž20–30 vs. 128 nA.. Moreover, unlike anesthetized rats, in which GLU could induce depolarization inactivation in all tested accumbal units w19x, both dorsal and ventral striatal neurons in awake animals were quite resistant to this effect. Although we frequently observed marked decreases in spike amplitude during the GLU-induced excitation, most units maintained a relatively stable, bursting activity even at relatively high currents and during continuous GLU application without clear indices of depolarization inactivation. Thus, it appears that chrorale hydrate anesthesia has a marked effect on the GLU responsiveness of striatal units, making them less reactive to direct stimulation, but more sensitive to depolarization inactivation. This factor may explain a profound attenuation of striatal neuronal responsiveness to somatosensory stimuli found in anesthetized compared to freely moving animals w44x. Accumbal neurons in anesthetized animals also had lower rates and less variability of spontaneous activity than in freely moving animals Ž3.8 imprs w30x vs. 10.35 imprs in this study., suggesting that the awake state may be associated with a higher level of endogenous GLU input. The instability of afferent input characteristic of awake animals also may determine the variability of striatal activ-

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ity and responsiveness to GLU and GABA. When these transmitters were applied repeatedly to the same units at the same currents, both the respective excitations and inhibitions showed a certain degree of variability manifested as fluctuations in response magnitude and, occasionally, as changes in response quality Žperiodic disappearance or reappearance of the response.. These qualitative changes were very rare with repeated applications of GLU Ž; 3% of repeated tests. but were seen in ; 20% of all repeated GABA applications. In both cases, these changes in response were closely associated with spontaneous fluctuations in basal rate. Compared to the GLU-induced excitation, the magnitude of which was highly stable and varied during repeated applications within relatively narrow limits, the GABA-induced inhibition had a wider range of fluctuations and disappeared more frequently when basal rate decreased and reappeared when basal rate increased. Although iontophoretic responses are usually highly stable in anesthetized preparations Žsee Refs. w19,41x., spontaneous modulations in neuronal responses to different transmitters ŽGLU, GABA, DA, ACh, norepinephrine. have been described for striatal, thalamic, hypothalamic and midbrain units in awake animals w22,23,26–28x. Our neuronal responses to GLU and GABA were also dependent on the behavioral state of the animal. When spontaneous movements Žlocomotion, grooming, head movement. occurred during iontophoresis and most striatal neurons were physically activated, the GLU-induced excitation became more pronounced but variable, whereas the GABA-induced inhibition became weaker. Thus, the movement-related neuronal excitation was enhanced by concomitant GLU application, but inhibited, although not completely blocked, by GABA application. These data support the view that movement-associated phasic excitations of striatal neurons are mediated by phasic GLU release and indicate that GLU and GABA interact at the single-cell level. In fact, virtually all our units were simultaneously sensitive to both GLU and GABA with continuous application of one modulating the effects of the other. These as well as our previous data on differences in neuronal responses to ACh and norepinephrine between awake, freely moving and restrained animals w24x support a role for tonic afferent input in modulating unit responsiveness to phasic synaptic stimuli. The ability of iontophoretic GLU to stimulate spontaneously active neurons and to induce a similar excitation in silent units suggests that a difference in tonic GLU input is the primary factor determining the variability in both basal firing rates and responses induced by phasic GLU input. Depending on basal discharge rate, our units had progressively smaller responses to additional GLU stimulation up to a complete saturation of the response, whereas GABA responses became stronger with increases in basal rate. Silent and slow-firing units, in contrast, showed strong, high-magnitude GLU-induced excitations but

GABA was less effective in influencing their activity. Movement-related excitations, probably related to phasic increases in GLU release from cortical orrand thalamic units, were also dependent on basal activity rate. Most silent and slow-firing units showed a high-magnitude phasic excitation, quite similar to that induced by GLU, whereas much weaker activation and biphasic changes were typical of units with relatively high rates of activity. Although it is likely that our recordings were made from medium-spiny projection neurons, recent in vitro data indicate that these cells, which discharge at a low rate if at all, have higher responsiveness to GLU than tonically active cholinergic interneurons w11x. Thus, the level of basal activity appears to be a major factor in determining the neuronal response to phasic GLU stimulation. Although our data suggest that brief applications of GABA strongly inhibit both basal and GLU-stimulated activity, changes in GLU-induced and movement-related excitations caused by continuous GABA application may be more relevant for striatal functioning under behavioral conditions. Continuous GABA application, for example, had an inhibiting effect on both impulse activity and GLU-induced or movement-related excitations; these effects were directly dependent on both basal discharge rate and the initial magnitude of excitation. Thus, tonic GABA input inhibited both high-rate spontaneous discharges and strong excitatory responses more effectively than either low-rate activity or weak excitatory responses. In many units, moreover, both GLU-induced and movement-related excitations persisted, albeit at an attenuated level, when basal activity was completely blocked by continuous GABA. Thus, an increase in tonic GABA input, by inhibiting basal impulse activity Žnoise. may preserve, or even enhance phasic neuronal activation Žsignal. relative to background activity Žsignal-to-noise ratio.. Collectively, these data suggest a crucial role for GABA in regulating responsiveness of striatal neurons to phasic changes in excitatory input occurring under behaviorally relevant conditions. Because GABA input, densely converging on striatal neurons from numerous sources, partially depends on corticostriatal and thalamostriatal GLU input, this hierarchy of opposite but interdependent inputs appears to provide the basic mechanism for integrating incoming information and its modulation by other neurochemical inputs Že.g., DA and ACh. to play a role in behavioral regulation. Although it is conceivable that iontophoretically applied GLU or GABA acts via aphysiological mechanisms, this interpretation seems unlikely given our use of relatively low ejection currents. It is also difficult to believe that our rate-dependent effects, especially strong with respect to GLU, are aphysiological. Moreover, because we are recording from ambulant animals, we also can rely on behavior-related changes in striatal activity to confirm the relevance of our results. Movement-related activation of striatal neurons, for example, undoubtedly involves activation of corticostriatal GLU afferents w8,12,45x, and this

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activation is mimicked by brief GLU iontophoresis. That GABA at very low current is capable of inhibiting both movement-related and GLU-induced excitations also support a physiological basis of our observations. Finally, GLU continuously applied at low currents to silent and sporadically active units induced discharge patterns quite similar to those in spontaneously active units. In fact, one could argue that iontophoretic manipulations of behaviorrelated changes in striatal activity may make a better test of the physiological relevance of this technique than reliance on pharmacological techniques common to in vitro and anesthetized preparations. Of course, now that we have established the basic parameters of GLU and GABA neuronal effects in freely moving animals, further experiments can probe the involvement of specific receptor mechanisms underlying both the basal activity and neuronal activations and inhibitions occurring under behavioral conditions. Acknowledgements Supported by the National Institute of Drug Abuse 02451 and DA 00335.. We also greatly appreciate Langley for technical assistance and Faye Caylor for in preparing the manuscript.

ŽDA Paul help

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