Electrophysiological and pharmacological evidence for the existence of distinct subpopulations of nigrostriatal dopaminergic neuron in the rat

NeuroscienceVol. 27, No. 2, pp. 537-546, 1988 Printed in Great Britain

0306-4522/88$3.00+ 0.00 Pergamon Press plc 0 1988 IBRO

ELECTROPHYSIOLOGICAL AND PHARMACOLOGICAL EVIDENCE FOR THE EXISTENCE OF DISTINCT SUBPOPULATIONS OF NIGROSTRIATAL DOPAMINERGIC NEURON IN THE RAT P. D. SHEPARD*$ and D. C. GERMAN*t$ Departments of Physiology* and Psychiatry,t University of Texas Southwestern Medical Center, Dallas, TX 75235-9070, U.S.A. Ah&met--The electrophysiological and pharmacological properties of dopaminergic neurons were systematically examined throughout the anterior-posterior extent of the substantia nigra zona compacta in the rat. Cells were characterized in terms of their (1) firing pattern, (2) firing rate, (3) antidromic response properties, and (4) inhibition in firing rate following dopaminergic agonist administration. These properties were then related to the cell’s position within one of four anterior-posterior segments of the nucleus. There were three types of neuronal discharge pattern encountered; irregular, burst and regular. Cells which exhibited different firing patterns exhibited different firing rates and anatomical locations within the substantia nigra zona compacta. All neurons were antidromically activated from the striatum, however, the burst- and regular-firing cells exhibited significantly faster estimated conduction velocities than irregular-firing cells. The irregular-firing cells were most sensitive to dopaminergic autoreceptor agonists whereas the burst-firing cells were most sensitive to an indirect-acting dopaminergic agonist. These experiments provide both electrophysiological and pharmacological evidence to indicate that nigrostriatal dopaminergic neurons are composed of distinct subpopulations which are characterized by their firing pattern.

The dopaminergic (DA) neurons of the substantia nigra zona compacta (SNc) have been traditionally regarded as a homogeneous population of cells,

conduction velocity and sensitivity to autoreceptor stimulation which differs from irregular-firing cells? Also, are these neuronal properties related to the cell’s anatomical position within the SNc? Preliminary results of these experiments have been published previously.424

perhaps because most of them project to the striatum (caudate-putamen). 12,32,47 However, recent data suggest that these cells may be. subdivided on the basis of structure and functional differences. For example, EXPERIMENTAL PROCEDURES anatomical studies have identified two morphologically-distinct subpopulations of SNc DA cell~.‘~*~” Surgical procedures Functional studies also suggest that there are subOne-hundred-and-fifty male, Sprague.-Dawley rats populations of DA cells within the SNc since: (a) (Charles River) weighing approximately 25Og were used. Animals were housed in group cages, given ad libitum food some neurons are excitated and others are inhibited and water, and maintained on a 12 h light-dark cycle. in response to sensory stimuli, and these two cell Animals were anesthetized with chloral hydrate (400 mg/kg types are located in different regions of the nucleus;” i.p.) and the femoral vein cannulated for intravenous admin(b) some neurons exhibit a burst-firing pattern and istration of supplementary anesthetic and drugs. Tissues others exhibit an irregular-firing pattern;5.‘9.20and (c) surrounding the ear canals and all wound margins were these neurons possess “autoreceptors” which, when infiltrated with 2% mepivacaine HCl and the rat was mounted in a stereotaxic apparatus (Kopf Instruments). stimulated by dopamine or DA-agonists, cause an Body temperature was monitored and maintained between inhibition in cell firing, ‘,9~‘8*45,48 but the slow-firing 3637°C. The head was positioned in the same plane as in cells are inhibited to a greater extent than the fast- the Konig and Klippel atlas”‘. Following a midline incision and reflection of underlying tissue, a burr hole was drilled firing cell~.~*~~ exposing the cortex overlying the SNc (2.2-3.7 mm anterior The present experiments were undertaken to deterto lambda and 0.52.5 mm lateral to the midline) and the mine whether there are correlations among the cell’s dura carefully removed. In some experiments an additional structural and functional properties. For example, do burr hole was drilled over the region corresponding to the striatum to allow insertion of a stimulating electrode(s) burst-firing cells exhibit a specific firing rate, axonal $To whom correspondence should be addressed §Present address: Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510-8066, U.S.A. A6breviution.s: DA, dopaminergic; ISI, interspike-interval; SNc, substantia nigra zona compacta.

(anterior, 6.8-8.9 mm; 3.06.0 mm).

1.0-2.5 mm;

ventral

Extracellular recording Tungsten microelectrodes (Fredrick Haer), mately 1 pm in tip diameter, were used for extracellular action potentials. In oitro impedance sured at 135 Hz and ranged from 2.0 to 4.0 Mf2.

approxirecording was meaElectrode

537

lateral,

538

P. D.

SHEPAKUand

potentials were amplified and filtered using a high-impedance amplifier (Grass P511) and monitored visually (Tektronix 5115 oscilloscope) and aurally (Grass AM-8 audio monitor). Single unit activity was recorded on magnetic tape (Hewlett-Packard 3896A). Cells were tentatively identified as DA cells by their electrophysiological characteristics.5.‘7,24 All cells chosen for study exhibited long duration action potentials (2.5.-3.5 ms), bi- or triphasic waveforms and tiring rates between I.0 and 8.0 impulses/s. Standard microiontophoretic techniques were used as previously described.35 Five barrel micropipettes, obtained from R & D Opticai (Spencerville, MD), were pre-loaded with several strands of fine fiberglass filaments and pulled to a thin taper using a Narishige vertical pipette puller. Pipette tips were broken back under microscopic control to a diameter of 3-6pm. The center barrei was filled with 2.0 M NaCl saturated with Fast Green dve (Sigma) and used to record extracellular neuronal signals {impezande = 2.2 to 4.0 MR). Three side barrels were filled with dopamine HCI (0.2 M, pH 4.0; impedance = 90-120 MQ Sigma) so that alternative drug barrels were available should one become blocked during the experiment, The remaining barrel was tilled with a solution of 3.0 M NaCl and used for automatic neutralization of tip currents.90 A retaining current of - 10 nA was applied to all drug barrels to prevent leakage. Dopamine was ejected using anodal currents ranging from 5 to 30nA. Microionotophoretic retention, ejection and balancing currents were controlled automatically by a constant current apparatus (Medical Systems, Model BH-2). One to three cells were recorded per animal. Intravenous drug administration Single units were observed and recorded for a period of 5-I 5 min to establish baseline firing pattern and rate. As the level of chioral hydrate anesthetic can influence the firing properties of these neurons,$ care was taken to test each cell only at relatively fixed times after anesthetic administration. Subsequently, animals received either a single intravenous, autoreceptor-selective dose of apomorphine (5 gg/kg; Sigma) or multiple doses of the indirect-acting dopamine agonist, ~-amphetamine (0.5,0.5, 1.0 mg/kg; Smith, Klein & French Laboratories). In some experiments the dopamine receptor antagonist, haloperidol (0.1 or 0.5 mg/kg; McNeil Pharmaceutical), was used to reverse the agonist-induced inhibition in firing rate. Drug dosages were calculated in terms of free base weight. Only one cell was tested per animal. Electrical stimulation

Stainless steel concentric bipolar stimulating electrodes (Kopf SNEX-100) were used to antidromically activate SNc neurons projecting to the ipsilateral striatum. Electrical stimulation was provided by a Grass s48 stimulator coupled to constant current and stimulus isolation units. Square wave, cathodal pulses were used (500 ps, 0.1-4.5 mA). Neurons were considered antidromically activated when (a) stimulation resulted in a single spike of constant latency, and (b) the stimulus-induced spike could be collided with a spontaneous action potential. The conduction velocity was estimated by dividing the straight line distance between recording and stimulating electrodes by the conduction latency. Data analysis

Electrophysiolo~cal data analysis was conducted using a DEC LSI 1l/O3 ~ni~mputer. Action potentials were discriminated with a triggering circuit which provided square wave pulses coincident with each action potential. Care was taken in triggering from each spike, especially in the case of the small amplitude spikes which sometimes occur at the end of a burst. Three types of analyses were performed: (I) cumulative rate histograms were compifed on-tine by summing action potentiats across consecutive 10s bins; (2) interspike-interval (ISI) histograms were constructed from

D. C.

GERMAN

1000 action potentials using 10 ms bin width resohmon. imd (3) an analysis of burst firing. similar to that described by Grace and Bunney.*’ was employed. The computer was programmed to recognize the onset of a burst as the occurrence of 2 spikes with an ISI less than or equal to 80 ms. Once detected, the number of subsequent spikes was counted until a spike pair with an IS1 greater than or equal to 160ms was encountered which signaled burst termination. Cells which exhibited a minimum of 6 three-spike bursts were o~rationally defined as burst-firing neurons. Determination of these parameters as well as validation of the operational definition of the bursting pattern of discharge are given by Grace and Bunney.‘” Drug-induced alterations in firing rate were computed by averaging unit activity obtained after the first 1.5 min following drug administration and comparing it with the average basal firing rate computed across an equaf time interval prior to drug administration. Differences between groups were analysed using either Students [ (6,) or Welch’s t (t,), and ANOVA. Post-hoc comparisons were made using NewmanKeuls (N--K) multiple comparison tests. To be considered statisticaily significant. differences between groups had to exhibit a P value ~0.05, two-tailed trsi Histological procedures At the conclusion of each experiment, the positions of the stimulating and recording electrode tips were marked. Tungsten electrode tips were marked by passing a f I.5.uA current through the electrode for 30-45 s. In those experiments in which micropipettes were employed, a dye spot was deposited at the tip of the recording barrel by iontophoretic ejection of Fast Green dye using - 20 PA current for I h. The positions of the stimulating electrode tips were marked by passing +0.5 mA current through the electrodes for 10 s. Following its removal from the stereotaxic apparatus. the animal was-deeply anesthetized and perfused iranscardially with saline followed bv 10% neutral buffered formalin. Each animal was decapitated and the head mounted in the stereotaxic apparatus. The brain was blocked stereotaxically in the same plane as used in the recording experiment. Following removal from the skull, brains were sectioned at 5Opm thickness in the same coronal plane as used in the Konig and KlippePO atlas. The sections were mounted on slides and stained with Cresyl Violet. Microlesions and dye spots were localized using a projection microscope. The position of each single unit recording site was plotted onto corresponding sections from the Konig and KiippeP rat brain atlas. In order to facilitate comparison of the electrophysiological and pharmacological characteristics of DA neurons with their position within the SNc, the nucleus was partitioned into four segments along the anterior~sterior axis. These segments, each approximately 4OOpm tong, spanned the entire extent of the nucleus (from 1200 to 2800 pm anterior to the interaural line). The position of several prominent midbrain structures was used to differentiate adjacent segments. The most anterior segment (2351-28OOpm) extended into the diencephalon and was characterized by the presence of the mammilia~ bodies. In the second, or mid-anterior, segment (200@-2350pm) the interpeduncular fossa and fasciculus retroflexus were positioned ventrally along either side of the central gray. The third segment (161 I-1999ym) contained the internucleus. The most posterior segment peduncular (12~~6lO~m) encompassed the rostra1 pons and continued anteriorly to include the posterior border of the red nucleus.

RESULTS All cells’ which charge properties

exhibited associated

the characteristic diswith DA dls were

539

Types of nigrostriatal DA neurons

Fig. 1. Photomicrographs illustrating sections through each of the four segments of the SNc in which DA cells were characterized. Sections are arranged from anterior (A) to posterior (D). The arrow indicates the location of a microlesion produced at the tip of the recording electrode. Abbreviations: FR, fasciculus retroflexus; IP, interpenduncular nucleus; MB, mammillary bodies; ML, medial lemniscus.

located within the SNc, between 1.1 and 2.4 mm lateral to the midline and from 1200 to 28OOpm anterior to the interaural line (see Fig. 1). Electrophysiological data Firing rate. Baseline firing rates among the entire population of cells (n = 113) ranged from 1.0 to 7.2 impulses/s (3.8 + 0.1, mean + S.E.M.). As shown in Fig. 2, the firing rates of cells within each segment were not the same (ANOVA, F = 10.41, P < 0.001). Neurons located within the mid-anterior segment of the SNc exhibited the slowest firing rates (2.6 f 0.2 impulses/s) and differed significantly from neurons within the posterior half of the nucleus (> 4.2 impulses/s), but did not differ significantly from cells in the most anterior segment (3.7 + 0.3 impulses/s). Firingpattern. Cell firing pattern was characterized using two methods. First, cells were categorized as burst or non-burst according to the method of Grace and Bunney2’ (Table 1). Of 113 cells studied, 42% met the criteria associated with a bursting discharge pattern, and these cells exhibited significantly faster firing rates than non-bursting cells (t, = 8.02, P < 0.001).

The DA neuronal discharge pattern was also characterized using IS1 histograms. IS1 histograms were constructed from the same segment of data used to perform the burst analysis. The histograms were typically unimodal and either positively skewed to-

izoe-l6lc (N-351

IMI-ls%l

lN=34)

2000-2330 (N.Z?‘l

!2351-2300 (N=i7)

Distance from lnteroural Line (pm)

Fig. 2. Baseline DA neuronal firing rates differed within the four anterior-posterior segments of the SNc. Each segment is defined in terms of its distance from the interaural line (in pm). Each point represents the firing rate of a single DA cell. The dashed line connects the mean discharge rate for cells in each segment.

540

P. D. SHEPAKD and D. C. GERMA":

Table 1. Dopaminergic

neuronal

discharge

characteristics-I

48 4.9 * 31.3 + 12.8 + 24.5 k

n

Firing rate (impulses/s) Percent mikes in bursts Percent spikes in doublets Percent spikes in bursts of 3+ spikes Values = mean f S.E.M. *P < 0.001, Welch’s r-test,

0.2 3.2 1.1 3.1

65 3.0+0.1* 2.5 + 0.4* 2.1 i 0.4* 0.3 +0.1*

burst

ISI pattern

n

Firing rate*

65 48

4.4 + 0.2 3.0 f 0.2**

+ Skewed Normal

*Mean + S.E.M.

discharge

characteristics-

Percent burst

Percent non-burst

66 8

33 92

impulses/s.

vs non-burst

70.0 A IRREGULAR

35.0

52.5 6 5 $ 35.0 IL 17.5

70.0

C BURST

*o*

neuronal II

**P < 0.001, Welch’s r-test, normal vs + skewed

ward long intervals or normally distributed (Fig. 3). The symmetry of each IS1 distribution was determined by comparing the number of 10ms bins on either side of the mode. Histograms with more than 70% of the distribution to the right of the mode were

525

Table 2. Dopaminergic

Non-burst

Burst

operationally defined as positively skewed. The remaining histograms were regarded as normally distributed. Cells which exhibited bimodal ISI distributions (15%) were assigned to one of these two groups on the basis of the shape of the primary peak of the histogram. Of 113 cells tested, 57% were positively skewed and 43% were normally distributed. The firing patterns were quite stable over time. For example, in several experiments it was observed that the shapes of IS1 histograms constructed from the first 500 consecutive spikes following cell isolation were not different from those compiled from a second 500 spikes, after 15 min of recording had elapsed. The cell’s firing pattern was defined by combining characteristics from both burst and IS1 analyses. As shown in Table 2, the majority of cells with normal IS1 distributions were found to exhibit a non-bursting discharge pattern (92%), whereas cells with positively skewed distributions exhibited both bursting (66%) and non-bursting (34%) firing patterns. Thus, three pattern categories were identified: (1) cells exhibiting a bursting discharge (i.e. burst pattern); (2) nonbursting cells which exhibited positively skewed IS1 histograms (i.e. regular pattern); and (3) non-bursting cells characterized by normally distributed IS1 histograms (i.e. irregular pattern). The terms burst, regular and irregular refer to the firing pattern as revealed in spike trains (see inserts in Fig. 3). The three cellular firing patterns were not uniformly distributed throughout the anterior-posterior extent of the SNc. As shown in Fig. 4, burst-firing

35.0 h

400 Interspike

860 Interval

12bo

1600

(ms)

Fig. 3. Interspike interval (ISI) histograms and corresponding spike trains (inserts) obtained from three d&rent DA cells illustrating discharge patterns most frequently encountered in the SNc. Top panel: non-bursting cells, characterized by normal ISI distributions, exhibited irregu-

lar discharge patterns and constituted 38% of the cells encountered. Middle panel: non-bursting cells, characterized by positively-skewed ISI histograms, exhibited a regular discharge pattern and constituted 20% of the cells encountered. Bottom panel: burst-firing cells, characterized by positively-skewed IS1 histograms, constituted 42% of the cells encountered. Each IS1 histogram is constructed from 1000 spikes, and the histogram bin width is IOms.

lmD-161l-EmD-p511610 1999 2350 2600 Dirtonce

from

lnhrouml

Line

(pm)

Fig. 4. Histogram illustrating the frequency of occurrence of regular-, burst- and irregular-firing cells within the four anterior-posterior segments of the SNc. Each segment is defined in terms of its distance from the interaural line (in pm). The number of cells recorded in each segment is indicated within the bars. There was not a uniform distribution of firing patterns throughout the anterior-posterior extent of the nucleus.

Types of nigrostriatal DA neurons Table 3. Axonal properties of nigrostriatal neurons Firing pattern

n

Conduction latency (ms)*

Burst Regular Irregular

13 4 12

12.9 + 1.2 11.0+ 1.3 14.6 + 0.6

541

dopaminergic

Conduction velocity (m/s) Average Range 0.260.72 0.36-0.78 0.29-0.41

0.51 * 0.04 0.57 * 0.10 0.35 f 0.01**

*Conduction latency and average conduction velocity = mean k S.E.M. **P < 0.05, Welch’s ANOVA with Newman-Keuls comparisons, irregular vs burst and regular.

cells were most frequently observed in the posterior half of the SNc. Of the 48 burst-firing cells recorded, 85% were located in the posterior half of the nucleus. There was a significantly greater probability of encountering burst-firing cells than regularor irregular-firing cells in the posterior half of the nucleus (x2 = 7.9, P = 0.005 between 1200-1610 pm; and x2 = 11.9, P < 0.001 between 161 l-1999 pm). In the anterior half of the nucleus, the frequency of occurrence of burst-firing neurons decreased. This was particularly evident at the mid-anterior segment, where only 1 of 27 cells exhibited this discharge pattern. In addition to a lower incidence of burstfiring neurons within this segment (x2 =9.4, P = 0.002), the observed frequency of irregular-firing neurons was found to be significantly greater than expected (x2 = 22.0, P < 0.001). There was an equal frequency of occurrence of cells in each of the three pattern groups at the most anterior segment of the nucleus (x2 = 0.12, P = 0.9). Antidromic

activation

of dopaminergic

neurons.

Twenty-nine DA neurons were antidromically activated from the striatum (Table 3). Antidromicallyelicited spikes usually consisted of the initial segment component of the action potential (Fig. 5). Both initial segment spikes and full action potentials which occurred as a result of striatal stimulation met the criteria for antidromic activation. As a group, DA neurons exhibited slow estimated conduction velocities (0.45 + 0.03 m/s) and a high excitation threshold (1.8 f 0.24 mA). Thirteen antidromically-activated cells exhibited a burst-firing pattern, 4 exhibited a regular-firing pattern and 12 cells exhibited an irregular-firing pattern. There was a significant difference between the estimated conduction velocities of cells in the three pattern groups (ANOVA, F = 12.6, P = 0.005). Cells characterized by an irregular discharge pattern exhibited slower estimated conduction velocities than cells characterized by other discharge patterns (Newman-Keuls tests, P = 0.05). Pharmacological

data

Direct-acting dopaminergic agonists. Seventy-eight of the 113 cells analysed in the previous experiment were tested for their sensitivity to a single intravenous, autoreceptor-selective dose of apomorphine (5 fig/kg). Apomorphine significantly decreased the

Fig. 5. Antidromic activation of an SNc DA neuron. Top panel: an oscilloscope trace in which a shock (*; 1.5mA, 500 ps duration) to the striatum, 40 ms following a spontaneous action potential (s), resulted in an antidromic initial segment spike (A) occurring at a latency of 15 ms. Bottom panel: when the shock was applied 5 ms following the spontaneous spike, no antidromic spike was observed, demonstrating collision. Calibration bars = 200 pV, 10 ms.

firing rates of all cells tested (Fig. 6), and there was a significant inverse relationship between basal firing rate and the percent decrease in rate following apomorphine (r = -0.60, P < 0.001). However, only a small amount of the variance (36%) in the cells’ sensitivity to the drug could be accounted for by the cells’ firing rate. Cells with similar baseline firing rates were not

12004610

16414933

2000-2360

2351~2E00

Distance from Intomural Line (pm) Fig. 6. Apomorphine (5 pg/kg Lv.) disproportionately decreases the firing rates of DA neurons within each of the four anterior-posterior segments of the SNc. Each segment is defined in terms of its distance from the interaural line (in pm). Open bars indicate basal firing rates and cross-hatched bars indicate post-apomorphine firing rates (lines represent S.E.M.). The number of cells recorded in each of the four segments, from caudal to rostral, are 26, 22, 18, and 12.

P. D.

542 A. 5.0

,

APO (5+g)

D. C.

GERMAN

HALO (0.5)

1

I.

4.0

\”

SHEPARD and

I

8.0

12.0

a 1? B. 5.0

,

APO @UJ) I

6.6

HALO (0.5) I

13.3

19.9

TIME (mid

Fig. 7. Apomorphine decreases the firing rate of SNc cells exhibiting a burst-firing pattern (A) less than cells exhibiting an irregular-firing pattern (B). Apomorphine (APO, 5 rg/kg) and haloperidol (HALO, 0.5 mg/kg) were administered intravenously (arrow). The DA antagonist, haloperidol, reversed the APO-induced inhibition of cell firing.

uniformly inhibited by apomorphine (Fig. 7). This observation suggested the possibility that neuronal firing rate was not the sole determinate of the cell’s responsiveness to apomorphine. Therefore, we examined the relationship between firing pattern and sensitivity to apomorphine. Irregular-firing cells were significantly more sensitive to apomorphine inhibition than burst- and regular-firing cells (percent inhibition 56.7k4.9, n =30, vs 33.8f2.3, n = 31, and 40.2 f 3.7, n = 17, respectively). Because burstfiring cells exhibited significantly faster basal firing rates than cells in the other two groups regular = 3.3 + 0.3 (burst = 4.9 + 0.2 impulses/s; impulses/s; irregular = 2.7 f 0.2 impulses/s), and percent inhibition scores make it appear that slowerfiring cells (irregular pattern) are most sensitive to apomorphine, we compared the sensitivity of a subpopulation of cells which, although characterized by different discharge patterns, exhibited similar baseline firing rates. Despite nearly identical basal firing n = 26; rates (irregular = 2.5 + 0.2 impulses/s, burst + regular = 2.8 k 0.2 impulses/s, n = lg), cells characterized by an irregular discharge pattern still exhibited a significantly greater inhibition following apomorphine than cells characterized by the other two discharge patterns (irregular = 60.1 + 5.2%; t, = 2.5, burst + regular patterns = 42.4 f 3.5%; P < 0.02). Irregular-firing cells within the mid-anterior segment of the nucleus were significantly more sensitive to the rate-decreasing effects of apomorphine than irregular-firing cells in the posterior-most segment of the SNc (post-apomorphine firing rates between 2000--2350 pm = 0.5 + 0.1 impulses/s and between However, 1200-1610 pm = 2.2 + 0.5 impulses/s). burst- and regular-firing cells exhibited similar postapomorphine firing rates throughout the anterior-

I

OO

I

I

I

I

IO Ejection Current (nA

20

1

Fig. 8. Microiontophoresed dopamine decreases the firing rates of SNc cells exhibiting an irregular-firing pattern more than cells exhibiting a burst-firing pattern. Data points represent mean + S.E.M. percent inhibition of 4-16 cells/point.

posterior extent of the SNc. These data indicate that the sensitivity of SNc DA neurons to apomorphine varies as a function of both the cell’s discharge pattern and its position in the nucleus. Microiontophoresed dopamine produced a dosedependent decrease in the firing rates of SNc DA neurons. Among the 33 cells that were studied, there was a highly significant positive correlation between dopamine ejection current and the percent inhibition of cell firing in both burst and irregular pattern groups (burst r = 0.98, n = 18; irregular r = 0.86. n = 15). As shown in Fig. 8, the slopes of the regression lines were similar for cells in both pattern groups. However, significant differences were found between the intercepts of these lines [F( 1,88) = 47.7, P < O.OOl]indicating that the extent of the inhibition was significantly greater among irregular-firing cells than among bursting cells. Since the baseline firing rates differ between the burst- and irregular-firing groups (4.8 f 0.4 vs 2.8 f 0.3 impulses/s, P < 0.05) a subpopulation of irregular- and burst-firing cells with similar basal firing rates were compared. The irregular-firing cells (n = 7) were still significantly more sensitive to iontophoresed dopamine than burst-firing cells (n = 13) at both 5 nA and 10 nA ejection currents (5 nA, t, = 3.6, P < 0.005; IOnA. t, = 2.2, P < 0.05). The sample size was too small to adequately test this relationship at other iontophoretic currents. Indirect-acting dopaminergic agonist. d-Amphetamine produced a dose-dependent decrease in the firing rates of SNc cells (Fig. 9). Twenty-four cells were tested, 11 exhibiting an irregular-firing pattern, 10 exhibiting a burst-firing pattern and 3 exhibiting a regular-firing pattern. The irregular-firing cells exhibited the usual slower basal firing rate compared to cells exhibiting the other two patterns (2.9 If: 0.3 vs 4.3 k 0.4 impulses/s. P < O.Ol), but they exhibited

Types of nigrostriatal DA neurons

I

I

1

1

OO D-Amphetamine

I

2

( mg/ kg I.V. )

Fig. 9. d-Amphetamine decreases the firing rates of SNc DA cells exhibiting both irregular and other firing patterns (i.e. burst+regular patterns). At the 0.5 mg/kg dose, however, the irregular-firing cells are inhibited less than the other patterns cells (P
DISCUSSION

The present experiments provide electrophysiological and pharmacological evidence for the existence of distinct subpopulations of nigrostriatal DA neuron. Using burst- and W-analysis techniques, the firing patterns of SNc DA neurons were segregated into three types: irregular, burst and regular. The irregular- and burst-firing cells appear to constitute two subpopulations of nigrostriatal DA neurons since they exhibit significant differences in their: (a) baseline firing rates; (b) axonal conduction velocities; (c) DA agonist sensitivities; and (d) anatomical localizations within the SNc. Because of the low incidence of regular-firing cells, they were not as extensively studied as the irregular- and burst-firing cells, and therefore no firm conclusion can be drawn as to whether they constitute a third type of nigrostriatal DA neuron. D@erences among nigrostriatal dopaminergic neurons The irregular-firing SNc cells exhibit slower conduction velocities than either burst- or regular-firing cells. As a group, DA neurons exhibit slow estimated conduction velocities (mean = 0.45 m/s) and a high

543

threshold for excitation (mean = 1.8 mA), as previously observed by others.“‘* These data are consistent with the observation that these fibers are small and unmyelinated. 25*26 In as much as fiber diameter has been positively correlated with conduction velocity,” it is conceivable that the conduction velocity differences are related to differences in axonal morphologies. Alternatively, it is possible that the estimated conduction velocities are biased due to the tortuous course of the nigrostriatal axons3* The fact that the irregular- and burst-firing cells have the same conduction latencies but different conduction distances (irregular = 5.0 f 0.1 mm, and burst = 6.0 + 0.1 mm) suggests a morphological difference either in fiber size or in axonal trajectory, between these two firing pattern groups. Other studies have also reported differences among nigrostriatal DA neurons. Nigral DA neurons project to both patch and matrix compartments in the striatum Is but there are anatomical differences in the locations of the neurons which innervate these two compartments. 28,29Furthermore, dopamine turnover is lower in striatal patch compartments than in matrix compartments. ” Interestingly, in the present study, irregular-firing cells exhibited significantly slower firing rates than burst- and regular-firing cells. Further research will be needed in order to determine whether cells exhibiting different firing patterns exhibit different innervation patterns within the striatum (i.e. whether irregular-firing cells innervate patch and burst-firing cells innervate matrix compartments). Relationship betweenfiring rate and dopamine-agonist sensitivity In agreement with previous studies,‘8.45,48systemic administration of a single low dose of apomorphine significantly decreased the firing rate of all cells which met the electrophysiological criteria associated with DA neurons. The rate-decreasing effects of low doses of apomorphine are believed to be mediated via selective stimulation of somatodendritic autoreceptors located on the SNc DA neurons.2*,34*45. An inverse relationship exists between the cell’s baseline firing rate and the percent inhibition to apomorphine in agreement with a previous study.36 Other studies have also reported an inverse relationship between basal DA neuronal firing rate and the percent inhibition to DA agonists. For example, slow-firing SNc DA cells are more sensitive to the rate-decreasing effects of the autoreceptor-agonist dl 3-[3-hydroxyphenyll-N-n-propylpiperidine than fastfiring cells? and slow-firing mesocortical and other ventral tegmental area DA neurons are more sensitive to the effects of DA agonists than fast-firing ~~11s.~~ Relationship between jiring pattern and dopaminergic sensitivity Although the cell’s firing rate is inversely correlated with

the

magnitude

of apomorphine-induced

in-

544

P. D. SHEPARD and D. C.

hibition, there is a stronger relationship between firing pattern and sensitivity to apomorphine. Thus, in addition to exhibiting slower firing rates, 78% of the cells in the mid-anterior segment of the SNc were characterized by an irregular discharge pattern. To determine whether the cell’s discharge pattern influenced its sensitivity to autoreceptor stimulation, the magnitude of the rate-decreasing effects of apomorphine were compared among a subgroup of cells which, although characterized by differences in pattern, exhibited similar basal firing rates. If basal firing rate were the sole determinate of the cell’s sensitivity to autoreceptor stimulation, then no differences would be expected between pattern groups in the sensitivity to apomorphine. However, the inhibition was greater among irregular-firing cells (60%) than among cells characterized by other discharge patterns (42%). Likewise, microiontophoresed dopamine produced a significantly greater inhibition among irregular-firing cells than among burst-firing cells. These data suggest that the cell’s firing pattern is correlated with its sensitivity to autoreceptor stimulation. Additionally, irregular-firing cells within the midanterior segment of the SNc were most sensitive to apomorphine, followed by irregular-firing cells in other segments of the nucleus, followed finally by burst-firing cells within the nucleus in general. These data suggest that the cell’s position within the SNc is also correlated with its sensitivity to autoreceptor stimulation. One possible explanation for the enhanced responsiveness of irregular-firing cells to autoreceptor stimulation is that these cells possess a greater number, or a more sensitive population, of somatodendritic autoreceptors than cells characterized by other discharge patterns. This hypothesis has been proposed by other investigators to explain the increased responsiveness of slow-firing monoaminecontaining cells to the rate-decreasing effects of their agonists.27,49Since dopamine is released locally from DA cell dendrites7,‘4.31 and is, presumably, free to interact with autoreceptors present on these processes, it is conceivable that a greater number (or more sensitive population) of autoreceptors would predispose irregular-firing cells toward slower firing rates than other cells equipped with a smaller (or less sensitive) population of autoreceptors. On the other hand, irregular-firing cells are less sensitive than burst-firing cells to the rate-decreasing effects of low doses (0.5 mg/kg) of the indirect-acting DA agonist, d-amphetamine. Electrophysiological data indicate that low doses of d-amphetamine decrease the activity of SNc DA neurons via an influence on the striatonigral feedback pathway whereas higher doses additionally release dendritic dopamine onto somatodendritic autoreceptors to decrease impulse flow. 2,3.23These data suggest that the burst-firing cells receive a stronger influence from the striatonigral pathway than the irregular-firing cells.

GERMAN

The present data indicate that cells which exhihic the greatest inhibition to direct-acting agonists (irregular-firing cells) exhibit the least inhibition to low doses of d-amphetamine, whereas the opposite 1s true for burst-firing cells. These data are consistent with the hypothesis that compared to the burst-tiring cells, the irregular-firing cells: (a) possess more autoreceptors, or autoreceptors with greater sensitivity; and (b) are less sensitive to striatonigral influences. Importance of nigral a&rents in regulating ceil Jiring pattern

Perhaps differences in afferent inputs to SNc DA neurons are, in part, responsible for the regional variation in discharge pattern. The primary source of afferents to the SNc arises from projection neurons within the striatum.‘6.22,“.46 The striatonigral pathway, like the nigrostriatal pathway, is topographically organized. Recent anatomical studies provide evidence for two distinct types of organization within the striatonigral projection systhese separate striatonigral tem.16 Collectively, efferent systems result in a complex and non-uniform pattern of innervation which may reflect convergence from striatal areas upon some nigral loci but not upon others. Lesions of the striatonigral pathway markedly influence the firing pattern of SNc neurons. Such lesions increase the incidence of cells exhibiting an irregular discharge pattern” and block the changes in DA neuronal discharge pattern which normally follow chronic neuroleptic treatment.4.50 Likewise, SNc cells typically exhibit irregular- or regular-firing patterns in the in vitro preparation,41 and burst-firing is produced only when cell hyperpolarization precedes depolarization (vs depolarization alone).33 One explanation for these findings is that a functional inhibitory input, like the striatonigral GABAergic pathway, is required for cells to exhibit a bursting discharge pattern.

CONCLUSION

The present data indicate that the DA neurons within the SNc are not a homogenous population of cells. At least two types of cells, based upon their discharge pattern, make up the nucleus; irregularand burst-firing cells. These two cell types exhibit both functional and structural differences. Functionally, the cells exhibit different basal firing rates and sensitivities to direct- and indirect-acting dopamine agonists. Structurally, the cells exhibit different axonal morphologies (as indicated by the antidromic activation experiments) and neuronal locations within the SNc. Acknowledgements-The authors wish to thank MS Kathy McDermott for histology. MS Laura Boynton for secretarial assistance. Dr Alvin North for consultation on statistical

Types of nigrostriatal DA neurons analyses, and Dr B. S. Bunney for computer software. The authors also wish to thank McNeil Pharmaceutical for their gift of haloperidol and Smith, Klein & French Laboratories

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for their gift of d-amphetamine. This research was supported by the Dallas Area Parkinsonism Society and United States Public Health Service grant MH-30546.

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14 April 1988)