Influence of dopamine on ventrolateral thalamic inputs in cat motor cortex

Brain Research 963 (2003) 178–189 www.elsevier.com / locate / brainres

Research report

Influence of dopamine on ventrolateral thalamic inputs in cat motor cortex Kadrul Huda a,b , *, Kenichi Matsunami b,c a

b

Department of Information Physiology, National Institute for Physiological Sciences, Myodaiji, Okazaki 444 -8585, Japan Department of Neurophysiology, Institute of Equilibrium Research, Gifu University School of Medicine, Tsukasamachi 40, Gifu 500 -8705, Japan c Science and Technology Promotion Center, Kakamigahara, 509 -0108, Japan Accepted 21 October 2002

Abstract Neuronal activity in several brain regions is modulated by dopaminergic inputs. When single neuronal activity / 20 trials of single-pulse ventrolateral thalamic (VL) stimulation was extracellularly recorded in the in vivo, anesthetized cat motor cortex, iontophoretic application of dopamine (DA) elicited either suppression or, in a fewer instances, facilitation of evoked unitary responses. The predominant inhibition exerted by DA appeared to be consistent for successive trials, and a D 1 , D 2 , and D 1 / D 2 receptor antagonist restored the effect, thereby reflecting a possible coexistence of two DA receptors. By contrast, only a fewer neurons’ response to DA displayed facilitation, which was not attenuated by DA antagonists. Moreover, subsequent trials with receptor agonist and antagonists induced inconsistent effects. Except for the jitters, single unit spikes showed invariant latency, which was constant during all recording parameters, and the mean latency remained unchanged. The modulatory effects mediated by DA did not reveal any substantial difference between short- and long-latency responses. Both pyramidal tract neurons and non-pyramidal tract neurons, determined on the basis of antidromic potentials from the pyramidal tract, responded to DA essentially in a similar manner. It appears that DA overall inhibits cat motor cortical neuronal activity in response to VL inputs. We propose that such DAergic inhibition of thalamocortical excitation in the motor cortex could be critical for ongoing sensorimotor transformation.  2002 Elsevier Science B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters, and receptors Topic: Catecholamines Keywords: Dopamine; Extracellular recording; Iontophoresis; Motor cortical neuron; Single unit activity; Ventrolateral thalamic nucleus

1. Introduction The motor cortex is thought to be like a module organized according to different functional hierarchies. Representations of incoming cortical information are modulated into functional assemblies by a variety of neurotransmitters and neuromodulators. Since there are many afferents of dopamine (DA) neurons in the motor cortex, it is strongly suggested that DA might have a considerable impact on motor cortical reorganization so as *Corresponding author. Department of Information Physiology, National Institute for Physiological Sciences, Myodaiji, Okazaki 444-8585, Japan. Tel.: 181-56-455-7851; fax: 181-56-455-7853. E-mail addresses: [email protected] (K. Huda), [email protected] (K. Huda).

to enable functional capabilities and shape sensory receptive fields [7,52,54]. Existing evidence indicates that dense DA trajectories originating in the ventral tegmental area (VTA, A 10 cells) traverse the cortex in a bimodal pattern; layers II, III, and V and layers V–VI are predominant with D 1 and D 2 receptors, respectively. At the ultrastructural level, DAergic and glutamatergic terminals form triadic complexes with pyramidal cells [17,28,52], which are highly interconnected to relay powerful intracortical communication essential for amplifying and encoding information [9]. Although the precise mechanism regulating the functional status of neuronal output is less understood, it is conceivable that the midbrain DA could modulate excitatory transmission at such triads, thereby controlling the temporal pattern of neuronal activity and neural integrity. Abnormalities in DA signaling disrupt cortical network

0006-8993 / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 02 )03969-0

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synchrony and integrity, and lead to many neuropsychiatric disorders [3,7,8,28,31,47,54]. Determining the role of DA on single neuronal activity in the motor cortex is, therefore, a crucial step towards understanding the cellular mechanism of higher order functions. DA circuits derived from the substantia nigra (SN, A 9 cells) impose an excitation–inhibition balance of basal ganglia output that is conveyed through the thalamus [3,16,51]. The bulk of projections from basal ganglia, as well as deep cerebellar nuclei, converge essentially via the ventrolateral thalamus (VL) into the motor cortex, where they form asymmetric synapses onto pyramidal cells, which are exclusively output neurons that represent different afferent inputs. A high convergence of fiber systems with extensive arborizations and terminal tufts in all cortical layers [24,26,50] implies that the motor cortex is a potential site for thalamocortical signal integration. Recent advances in neurophysiology have indeed provided ample evidence on interactions between the thalamus and cortex serving as the gateway for sensorimotor transformation in accord with sensory modalities or behavioral demands [24,34,45,49]. Therefore, the possibility for dynamic modulation of such thalamic signals in the motor cortex by mesocortically innervated DA is of great functional interest. We intend to explore this possibility in the cat in vivo, where internal neuronal circuits are preserved, and responses of pyramidal tract neurons (PTNs) and non-PTNs (nPTNs), the most representative forms of neocortical neurons related to posture and locomotion, are distinguishable [11,32]. Evidence is accumulating that the motor cortex is not just an ‘upper motor neuron’, but a functional node involved in spatio–temporal information processing crucial for perceptual and motor skill acquisition [15,45]. By exploring DAergic characteristics at the single neuronal level, we seek new knowledge about the motor cortex in relation to thalamocortical inputs, which use excitatory glutamate as the central neurotransmitter. Because no DA fibers have been reported in the VL so far, it seems unlikely that even a strong stimulation in the VL would release endogenous DA to interfere with the results. Both D 1 and D 2 receptors were pharmacologically manipulated considering their differential location depending on cortical layers and target cells [13,17].

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ketamine hydrochloride (10 mg / kg body wt, i.m.). Cannulation of the right cephalic vein and tracheotomy were done. Maintaining doses of diluted anesthetic (Na pentobarbital, 1 mg / kg body wt / h, i.v.) were infused through the cannula to keep a steady anesthetic state. Depth of the anesthesia was constantly assessed from the condition of slow waves (EEG) and background noise. Physical signs such as pinna and vibrissa reflexes were also checked to judge the anesthetic level. Trachea was intubated with a ‘Y’-shaped cannula for artificial ventilation. Suction was used to remove oropharyngolaryngeal secretions. Cut margins of the incised wounds and dissected pressure points were thoroughly infiltrated and lubricated with local anesthetics (Xylocaine Jelly and Xylocaine Spray). Cats were warmed using disposable heating pads, and a stable body temperature (36–38 8C) was maintained. Fluid therapy was ensured with the supplement of 5% dextrose in normal saline infusion through the veno-fixed cannula. Also, aseptic measures were taken.

2.2. Operative technique The skull was opened, and a small burr hole was drilled over the right pericruciate area (A523–27, L55–9) for recordings. An additional hole was made on the same side (A58–11, L53–5) to insert electrodes for the VL stimulation (Fig. 1a). Cerebrospinal fluid was drained by perforating the dorsal column with an incision at the base of the

2. Materials and methods

2.1. Animal preparation Experiments were conducted on twenty-four adult cats (2.8–4.2 kg body wt) in a manner rather similar to that described previously [21,22]. All procedures were designed to minimize animal discomfort in accordance with NIH guidelines. Cats were anesthetized initially with pentobarbital sodium (20 mg / kg body wt, i.p.) and

Fig. 1. Experimental setup. (a) Diagram of a cat brain showing sites of stimulation at the ventrolateral thalamus (VL stim.) and bulbar pyramid (pyramid stim.) while recording from the precruciate region. (b) Orthodromic response latency classified into group I (short latency, top) and group II (long latency, bottom). (c and d) Recording and stimulating regions in enlarged view. Points of recording sites are indicated by dots (c). Profuse dopamine (DA) projections from A 10 cell group overlap VL-cortical system in motor cortex. Two small bars (d) represent location of stimulating electrode.

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skull. All dural coverings of exposed surfaces were excised and removed very carefully. The cortex was protected from drying by adding frequent saline drops on the uncovered portions. For stimulation, an electrode was constructed from four acupuncture needles arranged in a row, spaced 2 mm apart, and insulated completely except at the tips. The stimulating electrode, oriented vertically under thorough visual control, was gently advanced downward through the exposed cortex and stereotaxically placed in the VL (centered at A59.0, L54.0, H52.0). A control on the appropriate pair of adjacent needles within the VL was guided by the consistency and pattern of antidromic potentials evoked by ipsilateral motor cortical stimulation. Once set to an accurate position, the electrode assembly was sealed to the cranium with dental acrylic.

2.3. Pyramidal tract stimulation A bipolar electrode was positioned in the bulbar pyramid (Fig. 1a), tilted backward and inclined at a 308 angle with the coronal plane to avoid bony tentorium. The electrode was then moved 2.0 mm anteriorly and 0.5 mm laterally before being inserted through the brain stem and aimed at the PT. An appropriate pair of needles was selected to evoke antidromic PT potentials. Activity of PTNs was identified using collision technique and firing characteristics such as: (1) Fixed response latency, (2) all-or-none response to suprathreshold stimulation, (3) straddle of response at the threshold stimulus intensity, and (4) ability to follow double or triple stimulation at high frequency (.300 Hz). In a few PTN activities, collision of the antidromic spike with spontaneous firing was observed. Latencies of the antidromic PT response were measured from the CRT of an oscilloscope (Dual-Beam Memory Oscilloscope, VC-10, Nihon Kohden, Tokyo, Japan). Conduction velocity was estimated to recognize ‘fast’ PTNs and ‘slow’ PTNs.

2.4. Extracellular recordings and drugs Conventional extracellular recording of single unit activity was carried out [21]. A five-barreled carbon fiber glass microelectrode was fabricated. The tip of a glass pipette electrode was broken back under microscopic control to a diameter of 2 mm (o.d.) with an in vitro impedance of 4–8 MV (DC). The central barrel of the electrode, filled with a fine carbon filament (7 mm diameter) and 2 M NaCl, was used for recordings. Each of four peripheral barrels was filled with one of the following drugs (10 mM in phosphate-buffered saline) for iontophoretic administration: dopamine hydrochloride, a dopamine agonist (DA, pH54.5); SCH 23390, a potent and selective D 1 receptor antagonist (SCH, pH55.0); sulpiride, a weak but specific D 2 receptor antagonist (SUL, pH54.5); and haloperidol, a broad spectrum D 1 / D 2

receptor antagonist (HAL, pH55.0). Drugs were stored at 225 8C as stock solution until used. At first, an electrode was placed on the cortical surface at the pericruciate area (A 5 25, L 5 7) to identify the area evoking maximum field potentials to VL stimulations. Thereafter, the tip of a recording pipette was positioned vertically at a place where the largest surface-evoked potentials were recorded. Using a micromanipulator (Narishige, MO-90), the glass pipette electrode was lowered very slowly to the cortical depth through a small hole made in the dura while unit activity was monitored on an oscilloscope. Although attempts were made to record from all cortical layers, most of the recorded neurons were located in the deeper layers; layer V being the most common. Current intensity for stimulation was usually kept less than 0.5 mA to minimize current spread, some of which is inevitable. Nevertheless, this would be unlikely to interfere with the results, since effects of current spread on neuronal excitability would be common for all recording conditions, and recordings were performed along the rostral and lateral border of the cruciate sulcus, which receive thalamic inputs principally from the VL. Neuronal activity was amplified using conventional pre- and main amplifiers (JB 101J and VC 10, Nihon Kohden, Tokyo, Japan).

2.5. Data collection and iontophoresis Raw spike units emitted by a single neuron during 20 trials of single-shock stimulation (pulse width50.1 ms) were superimposed (upper part of Fig. 2a), and displayed in the form of rasters on the CRT of a signal processor (7T17, NEC-San-ei, Tokyo, Japan). Recordings were sampled by 5 kHz and accumulated by 0.2 ms / bin. Peristimulus rastergrams were also constructed (lower part of Fig. 2a) with a signal processor. Spikes recorded in 20 successive stimulations prior to any drug application were treated as the control response. Pre- and post-stimulus periods were 30 ms and 70 ms, respectively (Fig. 2a). Contamination from spontaneous discharges was unlikely because only low levels of spontaneous activity were detected in many neurons. Moreover, the influence of spontaneous discharges from the number of spikes generated during the 30 ms pre-stimulus period could be determined [32]. Activation of recurrent collaterals of antidromically responding corticothalamic neurons, or recruitment of fibers-of-passage could not be excluded, but could hardly be the primary source of excitatory inputs [26,42]. Data were traced on a chart sheet and stored on a floppy disk. After acquiring control data, the effect of each drug was examined. If not mentioned otherwise, the order of drug application was DA, SCH, DA, SUL, DA, and HAL for each neuronal activity. For notational convenience, the first, second, and third approaches of DA ejection will be termed DA1st , DA2nd , and DA3rd , respectively. Each antago-

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Fig. 2. Representative records of a PTN activity from cat motor cortex along with a dotted line, which indicates the trigger level of spikes. Each panel above the line shows superimposed spikes in 20 trials of VL stimulation. (a) A sample trace shows the pre-stimulus period (30 ms) and post-stimulus period (70 ms). The number of spikes constructing rastergrams appeared below the line. Spike responses to different conditions are displayed in expanded time scale (truncated) and spike numbers are enclosed below the dotted line for each set of condition; (b) CONT (control); (c) DA (dopamine 1st); (d) SCH (SCH 23390); (e) DA (dopamine 2nd); (f) SUL (sulpiride); (g) DA (dopamine 3rd) and (h) HAL (haloperidol).

nist was delivered immediately after acquisition of data for DA, while DA (2nd and 3rd) was delivered at at least 5 min (usually 10 min) intervals following antagonist application. Drugs were ejected iontophoretically for 1 min using 30 nA positive current. In some instances, more current was applied, but it was limited to below 50 nA to avoid possible detrimental effects due to too strong drug concentration [54]. A negative 5 nA was also used as the retaining current. Neuronal activities / 20 VL stimulations for each drug condition were acquired and stored in a similar fashion. Anesthesia was deep enough to keep the cat calm and quiet throughout the recording procedures. It should be noted that anesthesia could depress the VL [6,48]. It is not clear whether an identical response would be valid in a natural state of wakefulness and vigilance.

2.6. Control of electrode location Orthodromic responses to thalamocortical activation were recorded from the precruciate area along the lateral part of the rostral border of the cruciate sulcus (Fig. 1a,c). Spikes triggered with orthodromic potentials showed little latency variation. Occasionally, thalamic activation elicited antidromic responses. A response was considered antidromic if it appeared at constant latency, expressed all-ornothing phenomenon, responded to high-frequency multiple stimulations, and / or collided with a spontaneous spike (tested in few cases). After stereotaxic implantation of the stimulating electrode (Fig. 1a,d), motor cortically-activated antidromic potentials served as an indicator for the position

of electrode tips within the VL, which was later verified histologically [32]. Recordings were confined usually to a depth of 1.5–2.0 mm measuring from the dura, a level likely to coincide with the cortical pyramidal cell layer. When the recording session was over, cats were given a lethal dose of Nembutal. Stimulating sites in the VL were marked by passing DC current. These lesions were carried out as reference tracks in the histological check to confirm the point of stimulation.

2.7. Data analysis and statistics For off-line analysis, data constructing the rasters were sampled again in an expanded time scale for 30 ms, instead of 70 ms, of the post-stimulus period. Reconstructed rastergrams were redrawn on the chart sheet and analyzed. Latency of the earliest spike was considered to be the orthodromic response latency for each neuron. Based on latency, we classified motor cortical neurons into two groups: Group I (Fig. 1b, top) comprised early or short latency (,10 ms) while group II (Fig. 1b, bottom) comprised late or long latency (.10 ms) neurons. Data showing too long latencies (.20 ms) were not included since VL-cortical volleys evoked usually short latency responses [11,22,24,48,50]. Experimental data values including histograms were expressed in mean6SD. All statistical comparisons were determined by comparing the firing rates (raw data, i.e., spikes / 20 trials of stimulation following drug administration), using ANOVA (Bonnferoni / Dunn) and Paired t-test (StatView, 4.02;

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ABACUS Concepts). The criterion taken for the statistically significant difference was P,0.05 in all tests.

Table 1 Dopaminergic effects on VL-cortical inputs onto motor cortical neurons Cell

3. Results

3.1. Unitary responses The single neuronal spike response to single-pulse VL stimulation was isolated, and the stimulus strength was set so that each control unit exhibited around 10 spikes / 20 trials. Evoked spikes (Fig. 2, upper part of each panel) were displayed in a 70 ms post-stimulus period while spontaneous activity could be observed in 30 ms prestimulus period. Iontophoretically applied DA predominantly suppressed the spike activity. To elucidate whether exclusively D 1 or D 2 receptor activation could account for DA mediated inhibition, we applied a specific DA receptor antagonist following DA induced inhibition. In the majority of cases, both D 1 and D 2 receptor antagonists blocked this inhibition. As shown in Fig. 2b, 20 trials of VL stimulations in control condition evoked 11 spikes with a latency of 2.2 ms. With the application of DA1st , the spike number decreased to seven, but the latency (2.2 ms) remained unchanged (Fig. 2c). Immediate application of SCH, a selective D 1 receptor antagonist, restored the effect and yielded 13 spikes (Fig. 2d). Ten minutes later, following SCH application, DA2nd induced seven spikes (Fig. 2e) while SUL, a specific D 2 receptor antagonist, induced eight spikes (Fig. 2f). When DA3rd was applied, the number of spikes reduced to five (Fig. 2g), and HAL, a mixed D 1 / D 2 receptor antagonist, increased the number to nine (Fig. 2h). Latencies for the rest of the conditions were measured, but failed to show any notable difference.

3.2. Dopamine effect on motor cortical neuronal activity

MCN PTN nPTN

Response pattern unit

Effect*

Decrease

Increase

No change

84 (57%) 35 (62%) 43 (52%)

53 (36%) 18 (31%) 34 (41%)

10 (7%) 4 (7%) 6 (7%)

MCN, motor cortical neuron; PTN, pyramidal tract neuron; nPTN, nonpyramidal tract neuron; Asterisk (*), firing rate of neurons decreased by DA application.

Effects of receptor-specific antagonists on the blockade of cellular responses to DA were tested, and contributions of the receptors have been summarized in Table 2 for PTNs, and in Table 3 for nPTNs. On many occasions, we failed to complete unit recordings with all drug administrations. The fact is that during the long recording procedure required for the series of drug administrations many unitary spike responses deteriorated, changed, or were accompanied by spontaneous and / or bursting activity. After excluding those responses, we selected 63 units recorded completely and successfully throughout the entire series of drug administrations. In a majority of these neurons as well, DA1st exerted predominantly suppression, which was restored by DA antagonists. These neurons showed consistent response patterns to the application of DA and DA antagonists. The neuronal response potentiated by DA1st , on the other hand, did not follow a similar trend, evidencing inconsistency

Table 2 Blocking effect of DA antagonists on DAergic inhibition of pyramidal tract neuron activity Drug Response pattern unit

More than 200 unit responses were investigated with DA. However, a substantial number of units that displayed too long latency, high levels of spontaneous discharges, bursting activity, or very short action potentials, were excluded. In 84 (57%) of recorded neurons, DA induced inhibition, which was usually abolished by DA antagonist. On the other hand, the remaining neurons exhibited facilitation or no response to DA, but without being significantly affected by DA antagonists. Table 1 shows the effect of DA on motor cortical neurons categorized into PTNs and nPTNs on the basis of antidromic PT potentials [11,22,50]. Antidromic volleys from the PT further differentiated PTNs according to axonal conduction velocity. Most of the recorded PTNs appeared to be fast PTNs because of their rapidly conducting axons characterized by very short latencies (,2 ms, mostly ,1 ms) [11,48]. PTNs showed preferential location in the deeper cortical layer (layer V) whereas nPTNs did not. The order of antagonist application was changed in some instances.

60% 59% 61%

Increase SCH 36 (68%) SUL 34 (72%) HAL 36 (75%)

Effect*

Decrease

No change

12 (23%) 7 (15%) 8 (17%)

5 (9%) 6 (13%) 4 (8%)

166% 172% 153%

SCH, SCH 23390; SUL; sulpiride; HAL, haloperidol; Asterisk (*), net firing rate of neurons disinhibited by DA antagonists.

Table 3 Blocking effect of DA antagonists on DAergic inhibition of nonpyramidal tract neuron activity Drug Response pattern unit Increase SCH 49 (65%) SUL 47 (66%) HAL 43 (68%)

Effect*

Decrease

No change

20 (26%) 15 (21%) 14 (22%)

7 (9%) 9 (13%) 6 (10%)

151% 161% 157%

SCH, SCH 23390; SUL; sulpiride; HAL, haloperidol; Asterisk (*), net firing rate of neurons disinhibited by DA antagonists.

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under successive applications of drugs. In this report, we will focus on the neurons that showed a suppressive response to DA1st application.

3.3. Changes in spike number Of 63 neurons presented here, 33 neurons responded to pyramidal tract stimulation (PTNs) and 30 neurons did not (nPTNs). The percentages of PTNs (52%) and nPTNs (48%) were similar, presumably because a large number of recorded neurons were located in the deeper cortical layers. DA1st reduced the neuronal activity in 36 neurons, of which 19 were PTNs (53%) and 17 were nPTNs (47%).

3.3.1. Pyramidal tract neurons Fig. 3a–g illustrates the spike activity obtained from these 19 PTNs. Results show that the mean value of spike numbers in control condition was 11.163.4 (Fig. 3a). With the application of DA1st , the mean spike number decreased to 6.763.8 (Fig. 3b). Immediate application of SCH blocked DA-mediated inhibition and increased the mean value of spikes to 12.365.1 (Fig. 3c). DA2nd also reduced the spike number with a mean value of 8.764.2 (Fig. 3d), which was restored to 13.364.2 by SUL (Fig. 3e). Similarly, DA3rd induced a reduction of spike rate and yielded a mean spike number of 9.165.0 (Fig. 3f). HAL abolished the effect and increased the mean value to 12.464.4 (Fig. 3g). Statistical analyses revealed a significant difference (P,0.05, ANOVA). We also calculated the

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following median values (Fig. 3a–g) of spike numbers for each condition: 11.0 (CONT), 6.0 (DA1st ), 12.0 (SCH), 8.0 (DA2nd ), 13.0 (SUL), 8.0 (DA3rd ), and 12.0 (HAL). A bar graph (Fig. 3h) was constructed from the mean6SD of spikes to display the level of differences. Since an ANOVA test revealed a significant difference in the change of spike numbers, a t-test was further conducted to detect the pairs of recording parameters that produced such differences. These pairs were: CONT versus DA1st (P,0.01), DA1st versus SCH (P,0.01), SCH versus DA2nd (P,0.01), DA2nd versus SUL (P,0.01), SUL versus DA3rd (P, 0.01), and DA3rd versus HAL (P,0.05). When compared with the control value, the effects of DA2nd and DA3rd also appeared to be significant (P,0.05).

3.3.2. Nonpyramidal tract neurons Similar results were obtained from 17 nPTNs. Under control conditions, the mean value of the spike number was 9.764.3 (Fig. 4a). Application of DA1st decreased the mean value to 6.364.5 (Fig. 4b) while SCH increased it to 9.864.5 (Fig. 4c). A reduction in the mean value to 6.963.5 after DA2nd application (Fig. 4d) was followed by an increase to 11.264.5 after SUL (Fig. 4e). DA3rd also reduced the mean spike number to 6.764.2 (Fig. 4f) while HAL restored it to 10.165.2 (Fig. 4g). Changes in the spike rate were statistically significant (P,0.05, ANOVA). The bar graph (Fig. 4h) constructed from the mean6SD of spike numbers shows the level of changes and statistical

Fig. 3. Frequency histograms of spike number for 19 PTNs; (a) CONT (control); (b) DA (dopamine 1st); (c) SCH (SCH 23390); (d) DA (dopamine 2nd); (e) SUL (sulpiride); (f) DA (dopamine 3rd) and (g) HAL (haloperidol). Two vertical lines in each panel denote the mean6SD (continuous) and median (dotted) values. (h) A bar graph represents the mean value of spikes in each condition. Error bars indicate the standard deviation (SD) of the mean. Asterisk (*), compared with the previous value; plus (1), compared with the value of DA1st . Single symbol (* or 1), P,0.05; double symbol (** or 1 1), P,0.01.

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Fig. 4. Spike histograms for 17 nPTNs; descriptions of different experimental parameters (a–g) and a bar graph (h) are similar to corresponding panels of Fig. 3.

significances between different pairs of conditions. Pairs that exhibited a significant difference in t-test were: CONT versus DA1st (P,0.05), DA1st versus SCH (P,0.05), SCH versus DA2nd (P,0.05), DA2nd versus SUL (P,0.01), SUL versus DA3rd (P,0.01), and DA3rd versus HAL (P,0.05). Differences in spike number were also significant for CONT versus DA2nd (P,0.05), and CONT versus DA3rd (P,0.05). Median values of spikes (Fig. 4a–g) in different conditions were 9.0 (CONT), 6.0 (DA1st ), 10.0 (SCH), 6.0 (DA2nd ), 12.0 (SUL), 6.0 (DA3rd ), and 9.0 (HAL). Median values were comparable to the mean

values for the corresponding experimental conditions and showed similar changes for both PTNs and nPTNs. Two stick diagrams have been plotted in Fig. 5 to display the change in spiking probability of individual neurons in relation to different recording conditions. The left frame represents the result of PTNs while the right frame indicates that of nPTNs. Each approach of DA reduced the spike rate in almost all neurons. However, with the application of SCH, SUL, and HAL, the spike numbers began to rise and regained the control level. On several occasions, the effect of antagonists exceeded the

Fig. 5. Stick diagram illustrating the trend of change in spike number for individual neuron by DA and DA antagonists: SCH (SCH 23390, a selective D 1 antagonist), SUL (sulpiride, a specific D 2 antagonist), and HAL (haloperidol, a non-selective D 1 / D 2 antagonist). Results from pyramidal tract neurons and nonpyramidal tract neurons were plotted in left and right frame, repectively. The number of neurons is enclosed in parentheses.

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control value, possibly due to blockade of the tonic inhibition. On average, all three antagonists exerted a similar efficacy in abolishing DA effects and restoring the firing probability. Both PTNs and nPTNs responded to DA and DA antagonists in a similar manner. In three PTNs and two nPTNs, DA2nd elicited a modest to moderate increase in the firing rate. A slight increase in the firing was also observed in two PTNs after DA3rd application. Nevertheless, the inhibition by the first, second, and third approaches of DA and restoration by DA antagonists tended to be virtually the same for both groups.

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very little regardless of latency response length, and fast or slow PTNs. However, drugs could modify the spike latency jitters to some extent, but without significance or clear correlation with receptor specificity. Latency distributions for PTNs and nPTNs in each recording condition have been shown in Fig. 6. There was no distinct difference in mean response latencies. Analyses of latencies showed no statistical significance either for PTNs or for nPTNs (P.0.05).

3.5. Other 3.4. Response latency Neurons in the motor cortex activated by VL stimulation discharged spikes with a wide range of latency determined from the earliest spike. Variations in response latencies could be attributed to a number of factors such as synaptic numbers, fiber widths, conduction velocities, somatodendritic size of postsynaptic cells, input resistance, and synaptic locations. The relative lack and abundance of spines on fast PTNs and slow PTNs, respectively, might underlie the biophysical difference between these two types of neurons. In fact, fast and slow PTNs are known to elicit different biophysical properties; for example, in the duration of action potentials and the form of afterpotentials. Neurons were initially divided into short and long latency groups. Since long latency neurons were few in number, and the response pattern was the same for the two groups, we arranged all data together. Latencies for each unit activity were apparently fixed in many neurons. Yet spikes showed only a very minor shift (0.3–1.5 ms) in latency distribution (jittering), supportive of orthodromic responses. Application of any drug changed the latency

We will briefly present the data that DA1st led to an increase of neuronal discharges. DA antagonists did not impose a potential effect to block facilitation. Moreover, subsequent DA administrations tended to induce inhibition in a substantial number of these neurons. In total, 27 neurons, i.e., 14 PTNs and 13 nPTNs, elicited facilitation of responses by DA1st . The mean values of spike numbers induced by DA agonist and antagonist for PTNs were: 11.663.1 (CONT), 14.663.9 (DA1st ), 13.764.7 (SCH), 13.464.7 (DA2nd ), 14.864.5 (SUL), 12.464.7 (DA3rd ), and 13.565.3 (HAL). The values for nPTNs were: 10.563.8 (CONT), 12.763.9 (DA1st ), 11.265.0 (SCH), 11.066.2 (DA2nd ), 11.065.8 (SUL), 11.365.9 (DA3rd ), and 10.465.5 (HAL). Changes in spike activity in these neurons did not differ much, and the mean spike number remained virtually unchanged. We also analyzed the latency of spike discharges for these neurons. Although occasional variation of latency could be seen, the mean response latencies showed no net difference. The mean response latencies obtained from PTNs were: 3.963.7 (CONT), 3.963.6 (DA1st ), 3.963.6 (SCH), 3.963.7

Fig. 6. Latency histograms constructed for 19 PTNs (upper panel) and 17 nPTNs (lower panel); Recording parameters for both panels are: (a) CONT (control); (b) DA (dopamine 1st); (c) SCH (SCH 23390); (d) DA (dopamine 2nd); (e) SUL (sulpiride); (f) DA (dopamine 3rd); and (g) HAL (haloperidol). Mean6SD of latency, indicated by a vertical line, is enclosed in each frame. Ordinates denote the number of neurons while abscissas indicate latency (ms).

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(DA2nd ), 3.763.5 (SUL), 3.863.5 (DA3rd ), and 3.963.6 (HAL). The mean latencies for nPTNs were: 4.965.4 (CONT), 5.065.6 (DA1st ), 4.865.3 (SCH), 5.065.5 (DA2nd ), 4.765.3 (SUL), 5.065.7 (DA3rd ), and 4.865.4 (HAL). Statistical comparison did not reveal any significant change in spike rates or response latencies either for PTNs or for nPTNs (P.0.05).

4. Discussion Our study suggests that DA predominantly suppressed VL-cortical inputs to the motor cortex. Neurons showing inhibition of spike activity by DA1st also exhibited consistency in successive DA applications, and D 1 , D 2 , and D 1 / D 2 receptor antagonists blocked the effect. Although DA1st exaggerated the response in a smaller population of neurons, subsequent administration of DA agonist and antagonists failed to display any clear-cut effect. The preponderance of inhibition in respect to excitation by DA corresponded well to the evidence assembled in most extracellular and intracellular studies conducted in vivo and in vitro.

4.1. Study with extracellular recordings DA-induced inhibition of excitatory drives in a group of PTNs was previously reported [22]. Inhibition of spontaneous and evoked activity by DA iontophoresis is consistent with the findings of extracellular recording in rat prefrontal [40], caudate [25] and amygdala [43], or cat caudate [35] and cortical [21] neurons. In a consciously behaving monkey performing Go / Nogo tasks, D 1 receptor agonist suppressed almost all recorded putamen neurons, and D 2 receptor agonist suppressed about half, while facilitating the other half of neurons, suggesting involvement of D 1 and D 2 receptors in behavioral control [23]. In vivo electrical stimulation of the VTA / SN, the main source of endogenous DA, reversibly suppressed the spontaneous and afferent-driven firing rate of prefrontal neurons due to stimulation-induced DA release [10,40], and the effect was abolished by 6-OHDA lesions of the specific mesocortical DAergic system [10]. Along with the VTA stimulation, systemically introduced DA agonist also inhibited spontaneous and trans-synaptically-induced unit activity [39,43]. This inhibition was blocked by cis-flupentixol, a mixed D 1 / D 2 DA receptor antagonist [39].

4.2. Study with intracellular recordings In intracellular recording, the effect of DA on spontaneous and evoked activity has most often been reported to be inhibitory. Applied iontophoretically, DA induced slow depolarization of the cell membrane in cat caudate nucleus, reduced the amplitude of sequential excitatory–inhibitory

postsynaptic potentials, and decreased the ongoing firing rate without significant modification of membrane conductance [20]. Reduction of firing activity was reportedly linked to hyperpolarization of the initial segment while slow depolarization of the distal dendrite. DA also elevated the threshold for spike generation [14], increased K 1 conductance [29,53], or blocked Ca 21 currents [55] to dampen membrane excitability. The most compelling evidence, moreover, is that mice lacking D 4 receptor, a member of D 2 receptor family, displayed neuronal hyperexcitability in frontal cortex [44], strongly suggesting that DA could serve as an inhibitory modulator of excitatory drive.

4.3. Presynaptic and postsynaptic inhibition Activation of D 1 , or D 2 , or both D 1 and D 2 receptor has been reported to be responsible for prevention of action potential generation and decrement of excitatory synaptic transmission by altering membrane voltage and the interrelationship between cations [19,29]. The role of DA, however, seems more complex since the excitatory effect of DA [2,36,38,46] has been reported as well. In prefrontal neurons, D 1 and D 2 block have differential effects in vivo and in vitro [12,54]. The duality of DAergic activity has led to further investigation into the pre- and postsynaptic locus for DA receptor. Suppression of EPSCs and spontaneous and miniature EPSCs via D 1 receptor [12,36] and D 2 receptor [27,37] was considered a presynaptic effect since the paired pulse ratio displayed facilitation and the postsynaptic cell showed no change in membrane potential or conductance. On the other hand, postsynaptic activation of D 1 receptor [1,53] and D 2 receptor [18,51] also mimicked DAergic inhibition on cellular excitation.

4.4. Both D1 - and D2 -mediated inhibition The similar effect exerted by both D 1 and D 2 is puzzling, but can readily be reconciled with most in vivo findings [21–23,43]. The cortical layer V has numerous D 1 and D 2 receptors. This is in agreement with the fact that activation of two DA receptors could generate similar effects, given that the mechanism of actions involves different routes and different neural substrates. DA inhibited not only the postsynaptic NMDA component of Schaffer collateral input, but also the presynaptic NMDA and AMPA components of perforant path input via D 1 and D 2 receptors [37]. The general view of our results is in accord with the concept of receptor colocalization, although such effect, in iontophoretic study, could indirectly be mediated via other neurons [23]. Nevertheless, recent data accomplished by in situ hybridization have demonstrated a high degree of colocalization of D 1 and D 2 receptor in the same cell, lending support to the idea that interactions between two receptors could be important to

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depress cellular excitability [1,4,51]. Suffice it to say that the activation of receptor subtypes, depending on their locations, plays a key role in synaptic targeting, which in turn can be equally important as a synaptic property.

4.5. Complexity in dopaminergic actions Synaptic modulation, efficacy, and direction mediated via DA could be determined by a number of parameters. Apart from pre- and postsynaptic interactions, variability in distribution, coexistence or segregation of two receptors [3,16,51], as well as differential sensitivity of receptors including autoreceptors [7], could account for the perplexing role of DA. Depending on neuronal subtypes [13], D 1 and D 2 receptors could show synergism [4,51] or antagonism [1]. In glutamate-receptor interaction, an excitatory amino acid determines the cellular excitability by activating D 1 receptor or D 1 / D 2 synergism [5]. Concentration could be a factor activating a different target receptors [2,37], or producing non-specific action such as the least potentiation of response by DA1st observed in the present study. Some difference between anesthetized and awake states may be relevant to DAergic effects. Very recently, DAergic effects have been explained in a state-dependent manner [30,33]. D 1 receptor potentiated membrane excitability when in a depolarized ‘upstate’, but diminished excitability in a hyperpolarized ‘downstate’. Transitions between states of membrane potentials could be a significant determinant in cellular response to DA. Terminals of DA-positive fibers synapse on the dendritic shafts and spines of pyramidal cells where they make symmetric, putatively inhibitory contacts. Spines that receive symmetric synaptic contact from DA terminals also invariably receive an asymmetric synapse, forming a socalled synaptic triad [17,28]. Considering such arrangements along with the bioaminergic network, it is conceivable that the motor cortex can express heterogeneity in sensitivity to DA. Inhibitory GABA, whose terminals preferentially impinge on or are close to pyramidal cell soma, could not be integrated easily with DA [36,38,40,56]. Recruitment of non-DA catecholamines [37] or ‘heteroreceptors’ is intriguing, and should be considered when interpreting in vivo animal studies.

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motor cortex [1,50] and recordings were obtained mainly from the layer of Betz cells, a variety of large pyramidal neurons found in the motor cortex. Furthermore, short spikes with high-frequency spontaneous firing, presumably triggered by interneurons, were excluded. Despite the debate on the role of DA, our study suggests that DA predominantly inhibits both short and long latency responses of motor cortical neurons as well as fast and slow PTNs evoked by VL activation. Inhibitory modulations of short and long latency responses are quite compatible with the results reported previously [21,25,43]. Although the mean latency did not change, few neurons displayed variability in latency after DA antagonist application. Alteration of membrane excitability following removal of tonic inhibition could account for such a latency variation.

4.7. Role of dopamine on VL-cortical systems DAergic modulation of thalamocortical activity can provide spatial buffering for redistribution of ions and neurotransmitters to a normal level that stabilizes cortical impedance and prevents cortical spreading depression. By changing VL-cortical synaptic potentials [24], DA can control the strength of such synaptic transmissions, which play a key role in several forms of synaptic plasticity and memory retrieval [54]. A change in the output discharge of

4.6. Response latency In our experiments, spike latencies were mostly short and invariant except for the jitters. The presence of spike latency jitter largely ruled out the possibility of contamination by antidromic invasions. Short latency spikes with minor jittering are likely to be monosynaptic, and thus are pyramidal cell responses because thalamic inputs to cortical interneurons are at least disynaptic [41]. Neurons with oligosynaptic responses are also likely to be pyramidal since the majority (.70%) of neocortical neurons are pyramidal cells, spiny stellate cells are few, if any, in the

Fig. 7. Schematic diagram shows thalamocortical (TC) inputs from ventrolateral nucleus (VL) to different motor cortical layers (layer III– VI). Left, mono- and / or polysynaptic inputs from VL relay neurons (R) excite pyramidal cells (P), which trigger feedforward and feedback activation of intracortical (IC) excitatory (continuous line) and inhibitory (broken line) networks. Dopamine afferents (dotted line) from ventral tegmental area (VTA) interact with TC inputs to modulate pyramidal cell outputs, as well as corticothalamic (CT) feedback pathways. Right, expanded diagram of a VL-cortical synapse showing possible modification of synaptic transmission of glutamate (Glu) by DA. CN, cerebellar nuclei; GPi, internal segment of globus pallidus; I, interneuron; SNr, substantia nigra pars reticulata.

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pyramidal cells, which project back onto thalamocortical relay neurons (Fig. 7), would alter thalamic transfer of information associated with the level of arousal, slow wave sleep, attention deficit disorders and cognition [26,34,49]. If, as our findings suggest, DA’s overall influence is suppressive, then a loss of DAergic tone could promote epileptogenesis. Conversely, excessive release of DA could suppress inhibitory control by higher centers, such as the prefrontal cortex, over maladaptive behaviors unrelated to current goals. We speculate that this phenomenon might also have implications in relation to schizophrenia.

Acknowledgements We thank Professor K. Imoto for his incisive comments and Miss Y. Nishijima for her assistance with the illustrations. This research was partially supported by a grant from the Japanese Ministry of Education, Culture, Sports, Science and Technology, and the ‘Research for the Future’ program of the Japan Society for the Promotion of Science.

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