Spontaneous bursting activity of dopaminergic neurons in midbrain slices from immature rats: role of N-methyl-d -aspartate receptors

Pergamon

PII:

Neuroscience Vol. 77, No. 4, pp. 1029–1036, 1997 Copyright ? 1997 IBRO. Published by Elsevier Science Ltd Printed in Great Britain 0306–4522/97 $17.00+0.00 S0306-4522(96)00474-5

SPONTANEOUS BURSTING ACTIVITY OF DOPAMINERGIC NEURONS IN MIDBRAIN SLICES FROM IMMATURE RATS: ROLE OF N-METHYL--ASPARTATE RECEPTORS G. MEREU,*† V. LILLIU,*‡ A. CASULA,* P. F. VARGIU,* M. DIANA,§ A. MUSA* and G. L. GESSAQ *Department of Experimental Biology, B. Loddo, University of Cagliari, Italy ‡Anni Verdi Foundation, Rome, Italy §Department of Pharmacological Sciences, University of Sassari, Italy QDepartment of Neurosciences, B. B. Brodie, University of Cagliari, Via Porcell 4, I-09124, Cagliari, Italy Abstract––Dopamine neurons in midbrain coronal slices from adult rats (40–70 days old) discharged only in pacemaker-like mode. Irregular or bursting mode was never observed. In contrast, dopamine neurons in slices from immature rats (15–21 days old) exhibited not only pacemaker-like firing (53.4% of neurons), but also irregular and bursting patterns (28.3 and 18.3%, respectively). Glutamate and kainate increased the firing rate but failed to induce bursts in dopamine neurons from either adult or immature rats. N-Methyl--aspartate augmented the firing rate in all neurons from adult rats and produced a modest increase of bursts in only three out of 18 cells. In slices from immature rats, N-methyl--aspartate activated the discharge rate in all neurons and also induced bursts in 37 and 53% of pacemaker and irregular neurons, respectively, and increased the occurrence of spikes in bursts in 76% of spontaneously bursting neurons. The selective N-methyl--aspartate receptor antagonist (&)2-amino,5-phosphonopentanoic acid prevented N-methyl--aspartate-induced changes and also reduced spontaneous bursts, suggesting that bursting discharge is mediated by N-methyl--aspartate receptor activation. While pacemaker neurons from immature and from adult rats exhibited the same sensitivity to N-methyl-aspartate-induced stimulation of firing rate, spontaneously bursting neurons were more sensitive than pacemaker neurons from either immature or adult rats. The present study indicates that spontaneous bursting, dependent on N-methyl--aspartate receptor activation, is present, and may be induced, in dopamine neurons in slices from immature rats. Its absence from cells in slices from adult rats may reflect a reduced sensitivity of N-methyl--aspartate receptors on dopamine or the loss of the N-methyl--aspartate-activated burst generator. ? 1997 IBRO. Published by Elsevier Science Ltd. Key words: kainate, glutamate receptor subtypes, N-methyl--aspartate, dopamine neurons, burst firing, ontogenesis.

Dopamine (DA) neurons in vivo display spontaneous activity characterized by both singlespiking, with irregular interspike intervals (ISIs), and bursting, with sequences of two to 12 action potentials occurring at very short ISI.5,9,21 Switching from one mode to the other is not casual but is associated with movements,6,7 cognitive behaviour,24 sensory responses28 and drug effects.13,17,20 The bursting pattern rather than firing rate of DA †To whom correspondence should be addressed at: Department of Neurosciences, University of Cagliari, Via Porcell 4, I-09124, Cagliari, Italy. Abbreviations: ANOVA, analysis of variance; AP-5, (&)2amino,5-phosphonopentanoic acid; CV, coefficient of variation; DA, dopamine; DMSO, dimethylsulfoxide; EAAs, excitatory amino acids; ISI, interspike interval; ISIH, interspike interval histogram; NMDA, N-methyl-aspartate; PD, postnatal day; PSB, percentage of spikes within bursts; SNc, substantia nigra pars compacta; VTA, ventrotegmental area.

neurons plays a relevant physiological role since it appears to determine the magnitude of DA29 and co-transmitters1,11 released in terminal areas. Indeed, Gonnon8 and Manley et al.16 found that the electrical stimulation of dopaminergic projections by bursting pulses leads to increased extracellular DA concentration by up to four times that elicited by the same number of pulses delivered in an equal time but at regular interpulse intervals. Therefore, it is likely that the effectiveness of a drug in inducing DA release may be dependent more on its effect on discharge pattern than on the firing rate,10 as traditionally believed. In contrast to their behaviour in vivo, DA neurons in slices show neither a bursting nor an irregular pattern but only a highly regular, pacemaker-like firing.9,12–14,18 The lack of burst firing in vitro has suggested that spontaneous bursting of DA neurons in vivo requires excitatory inputs mainly provided by glutamatergic afferents from

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prefrontal cortex29 and subthalamic nuclei27 to ventrotegmental area (VTA) and substantia nigra pars compacta (SNc). Since DA neurons in slices maintain the functionality of both N-methyl--aspartate (NMDA) and non-NMDA receptors,19 it is expected that bursting activity should be induced in vitro by the exposure of DA neurons to excitatory amino acids (EAAs). However, whereas the elevation of NMDA tone in vivo has been shown consistently to increase burst firing5,13,21,29 the addition of NMDA to DA neurons in slices has produced conflicting results. Thus, while the majority of investigations18,22,26,30,33 have shown that EAAs, including NMDA, increase the discharge frequency of DA neurons without disrupting the discharge regularity, some studies have reported that NMDA may induce bursting activity in a subset of cells.25,31 Moreover, Johnson et al.12 have observed that NMDA application converts pacemaker VTA-DA neurons to fire in bursts provided that apamine, a blocker of the a Ca2+-activated K+ outward current responsible for the delayed, after-spike hyperpolarization,2 is present in the bath solution. Recent studies have indicated that the sensitivity to NMDA of various neuronal populations is much higher during the early postnatal period than in adulthood,4,15 and therefore the present investigation was aimed at clarifying whether the occurrence of bursting in DA neurons in slices was related to the age of the animal donor. EXPERIMENTAL PROCEDURES

Slice preparation Male Sprague–Dawley CD rats (Charles River, Como, Italy) at postnatal day (PD) 15–21 and PD 40–70 were used. Colonies were housed at 22)C and 50% relative humidity, with the light on from 08.00 to 20.00, and free access to food and water. After 5 min of breathing in a 100% O2 atmosphere, rats were deeply anaesthetized with halothane (4.0% in O2) and decapitated. The brain was rapidly removed under ice-cold Ringer solution, and mesencephalic coronal slices, of 300–350 µm thickness, were prepared as described previously.19 Slices were incubated at 30&1)C for at least 30 min, then transferred to the recording chamber and completely submerged in artificial cerebrospinal fluid warmed to 35&1)C. Solutions and drug application The artificial cerebrospinal fluid contained (mM): NaCl 124, KCl 3.5, NaH2PO4 1.25, NaHCO3 22, dextrose 10.0, MgCl2 1, and CaCl2 2.0. The solution was maintained at pH 7.4 by continuous bubbling with 5% CO2+95% O2. All drugs were kept in stock solution in the dark, and were added to the Ringer solution to obtain their final concentration. Glutamate, NMDA, kainate, apomorphine, DA and GABA (all from Sigma, St Louis, MO, U.S.A.) were dissolved in H2O. (&)2-Amino,5-phosphonopentanoic acid (AP-5; from R.B.I, Natik, MA, U.S.A.) was suspended in dimethylsulfoxide (DMSO). The final concentration of DMSO (¦0.1%) did not produce any detectable electrophysiological effect on DA neurons per se. Haloperidol (Janssen, Beerse, Belgium) and (")-sulpiride (Ravizza, Muggio`, Italy) solutions were prepared from commercially available ampoules. The recording chamber had a volume

of 600 µl and was perfused at a rate of about 4 ml/min. A complete exchange of the medium was obtained in less than 30 s. A set of three-way electrovalves (LFAA 1200118H, Lee Comp, Frankfurt, Germany) allowed for a rapid switch in the solutions without flow perturbation. Single-cell recording Extracellular single unit recording from spontaneously active DA neurons was made with glass micropipettes (GC 150 F, Clark, Reading, U.K.), pulled using a two-stage puller (PE-2, Narishige, Tokyo, Japan) set to obtain a tip of about 1.0–1.5 µm (o.d.), and then filled with 0.5 M Na acetate. Their ohmic impedance at 256 Hz in the Ringer was 3.5–4.5 MÙ (610-C electrometer, Keithley Inst., U.S.A.). Extracellular action potentials were captured and processed using a conventional line of electronic amplification (Neurolog, Digitimer, Welwyn Garden City, U.K.) as described previously.20 Filters were set for a signal passband of 5–50 kHz, while an active notch filter (NL 125, Digitimer) at 50 Hz rejected line-frequency interference. Periods of interest were stored on magnetic cassette tapes (V-350 C, TEAC, Japan) for off-line analysis (see below). Identification of dopamine neurons Neurons were recognized on the basis of their wellknown electrophysiological and pharmacological characteristics.9,18,19,20 Briefly, all neurons included in the present study as dopaminergic exhibited a wide (2.5–5.0 ms), triphasic (+/"/+) extracellular action potential with an evident initial segment spike, discharged at a low rate (range 1.5–5.0 Hz in young and 2.5–6 Hz in adult rats), and were found within the visually identified SNc and VTA regions. Furthermore, only these neurons were completely inhibited by bath application of 5–10 µM concentrations of DA or apomorphine, an effect promptly eliminated by 10 µM of haloperidol or (")-sulpiride, two known DA-D2 receptor antagonists. Non-DA neurons displayed a narrow action potential (duration ¦2.2 ms), spontaneously fired at a high rate (range 5-50 Hz), and were excited or unaffected by DA agonists. Twelve and 35 neurons from adult and immature rats, respectively, were tested with DA agonist and antagonist either at the beginning or at the end of the experiments. Statistics and data analysis Values are given as the arithmetic means&S.E.M. Statistical significance of differences between groups was assessed by analysis of variance (ANOVA). The two-tailed t-test was used for differences between the means of two samples. Mean firing rate was obtained by counting (NL 601, Digitimer) the number of spikes generated over single periods of 10 s. In dose–response curves, the last four 10-s bins before each solution change were averaged and taken as the response to a given drug concentration. ISIs were measured using a digital oscilloscope (DL 1100, Yokogawa, Japan), while pattern analysis and distribution histograms of the ISIs (ISIH) were obtained using the authors’ own software.5 Neurons were classified as pacemaker, irregular or bursting on the basis of the criteria previously established for in vivo recording in rats.5,9,10,29 In brief, the degree of variability of the ISI was determined by the coefficient of variation (CV), defined as: CV=ó/ISIm #100, where ó is the standard deviation of the ISIs, in a sample of about 800 events, and ISIm is their arithmetic mean.32 A neuron was considered as pacemaker if CV <10, or irregular if CV §10. Moreover, an irregular neuron was considered as bursting if, in a sample of 800 events, it exhibited at least two bursts composed of three or more action potentials with ISIs between 90 and 250 ms, for burst onset and termination, respectively.5,9 Burst activity was estimated as the percentage of spikes within bursts (PSB), i.e. PSB=ç/N #100,

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Fig. 1. CR oscilloscope traces (left) of three representative DA neurons found in slices from immature rats, illustrating their characteristic spontaneous discharge modes: (A) pacemaker; (B) irregular, and (C) bursting. Each pattern generates the typically shaped ISIH shown in the right-hand panels. Although the cumulative frequency was around 3 Hz in all three cases, the most common ISI greatly differed among patterns. The CV was 2.5, 31.4 and 63.8 for the pacemaker, irregular and bursting neuron, respectively. Each analysis was performed on a sample of about 800 events. Bin width was set at 5 ms.

where ç is the number of action potentials occurring within bursts and N is the total number of action potentials counted. RESULTS

In slices taken from both young and adult rats, about 75% of the spontaneously firing DA neurons were encountered in the dorsomedial SNc, while the remaining ones were found within the VTA. Since no significant differences in firing rate, pattern, and drug responses were detected, results from these two neuronal populations have been pooled. Adult rats Firing pattern analysis of 64 DA neurons in slices from adult (PD 40–70) rats showed that these neurons discharged only in a single-spiking mode with a highly regular ISI, like the one illustrated in Fig. 1A. None of these neurons exhibited irregular or bursting patterns. The average firing rate of these neurons was 4.6&0.5 spikes/s (Table 1). As reported in previous studies,18,26,30,33 glutamate (n=18), NMDA (n=20)

and kainate (n=15) produced a concentrationdependent enhancement of the frequency discharge of all DA neurons tested (Fig. 2). The effect of each EAA started within a few seconds of the drug entry into the recording chamber (Fig. 3A), and was highly reproducible and readily washable, allowing the construction of a complete drug–concentration curve for different EAAs in the same neuron. The rank order of potency of EAAs in stimulating the firing rate was kainate>NMDA>glutamate. The highest concentration tested of kainate, NMDA and glutamate (10, 100 and 500 µM, respectively) produced complete firing inactivation after an initial stimulation. As expected,9,13 inactivation was reversed by hyperpolarizing agents such as apomorphine (10 µM, n=4), DA (10 µM, n=4,) or GABA (1 µM; n=6, data not shown). In spite of their powerful effect on the firing rate, EAAs did not change the regularity of the ISI, except for the highest concentration of NMDA (100 µM), which induced burst activity in three out of 18 pacemaker DA neurons tested (Fig. 3D). In these neurons the percentage of spikes in bursts (PSB) after NMDA was 10.4&3.5%.

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Table 1. Effect of (&)2-amino,5-phosphonopentanoic acid on the spontaneous and N-methyl--aspartate-induced discharge rate (spikes/s) of dopamine neurons in slices from immature and adult rats Drug (µM)

Immature (PN 15–21) Pacemaker (n=30) Irregular (n=18)

Baseline AP-5 (100) NMDA (50) NMDA (50)+AP-5 (50) NMDA (50)+AP-5 (100)

3.2&0.3 3.0&0.3 8.8&0.7** 5.2&0.6*† 3.9&0.4††

Adult (PN 40–70) Bursting (n=22) Pacemaker (n=20)

2.8&0.6 2.7&0.6 7.2&0.8** 4.7&0.7*† 3.2&0.6††

3.1&0.8 3.0&0.7 8.1&0.9** 6.3&0.6** 3.4&0.5††

4.6&0.5‡ 4.4&0.6 10.6&0.8** 6.6&0.6*† 4.8&0.4††

Each value is the mean&S.E. from the number of neurons (n) which were selected by their basal discharge pattern. *P<0.05, **P<0.01 with respect to baseline; †P<0.05, ††P<0.01 with respect to NMDA alone; ‡P<0.05 with respect to pacemaker neurons from immature rats.

Discharge rate (% of baseline)

300

250 B B B

200 B

150 B

Symbols: Open, young Closed, adult

B

100

B

B

Kainate 50

0.1

NMDA 1

Glutamate 10

100

1000

Concentration [mM] Fig. 2. EAA-induced stimulation of discharge rate of pacemaker dopaminergic neurons in slices from young rats (filled symbols) and adult rats (open symbols). The highest concentrations used were 10, 100 and 500 µM for kainate, NMDA and glutamate, respectively. These concentrations produced a complete depolarization of the neurons after their initial stimulation (see also Fig. 3). Maximal increment before inactivation was similar for the three drugs and ranged from about 150 to 70% over baseline. In none of the experiments was glutamate or kainate able to promote irregular or bursting pattern. There was no statistical difference in potency for each EAA between immature and adult rats (ANOVA, P>0.2).

Immature rats Firing pattern analysis of 322 DA neurons in slices from immature animals (PD 15–21) showed that about half of them (n=172) discharged in a singlespiking, pacemaker mode (Fig. 1A), at a rate of 3.2&0.3 spikes/s, a firing rate significantly lower than that shown by pacemaker neurons in slices from adult rats (P<0.05, t-test). Irregular and bursting patterns were exhibited by 59 (18.3%) and 91 (28.3%) neurons, respectively (Fig. 1). Bursts consisted of three to nine action potentials of progressively decreasing amplitude. Each burst was followed by a period of inactivity lasting up to 5 s (Fig. 1C). Firing frequency tended to decrease progressively within each burst so that the ISI increased from 78&9 ms for the first or second interval to 183&17 ms for the last of seven to nine intervals. In bursting neurons the PSB was, on average, 19.2&2.6%, a value significantly lower than that observed in vivo5,9,21 where this parameter usually ranges from about 40 to 50%.

The right-hand panels of Fig. 1 show the typical ISI of three representative DA neurons selected according to their discharge pattern. The mean baseline discharge rate (spikes/s) of pacemaker, irregular and bursting neurons was similar (around 3 Hz) indicating that, in agreement with in vivo studies,5,9 no correlation (r=0.28) exists between spontaneous discharge rate and the occurrence of bursts (Table 1). As observed for neurons from adult animals, glutamate kainate and NMDA produced a concentration-dependent, highly reproducible and readily washable stimulation of the firing rate (Figs 2 and 3). Also, the rank order of potency (kainate>NMDA>glutamate) was the same as in neurons from adult rats. Similarly, a neuronal inactivation after an initial stimulation was produced by the highest concentrations of these drugs. Each EAA was equally potent in stimulating the firing rate in pacemaker DA neurons from immature and adult rats (ANOVA, P>0.2; Fig. 2).

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Fig. 3. (A,B) Ratemeter response of two pacemaker DA neurons from immature rats to NMDA. (A) Bath application of increasing concentrations of NMDA elevated the discharge rate of this neuron until it inactivated after a few seconds of exposure to 100 µM NMDA. Note that during the NMDA (50 µM) application the drug efficacy declined, suggesting a time-dependent desensitization. (B) A different neuron, when exposed to 50 µM NMDA, not only increased its discharge rate but was also initiated to fire in bursts. Sustained application of 50 µM of the NMDA antagonist AP-5 led to a small (¦10%) reduction in the basal rate, but consistently attenuated the effect of 50 µM NMDA. Arrows indicate timing (90-s periods) and concentration of NMDA. (C,D) Concentration curves of the effects of NMDA on neurons selected on the basis of their spontaneous pattern and age of the animal donor. (C) Elevation of firing rate induced by different NMDA concentrations in bursting (B), irregular (I) and pacemaker (P) neurons from young rats and in pacemaker neurons from adult rats (Pad). Although all neurons responded to NMDA exposure, reaching a maximal activation of about 2.7 times the control value, bursting neurons were more sensitive with respect to pacemaker from immature (ANOVA, P<0.01) and pacemaker from adult (ANOVA, P<0.02) rats. The difference between pacemaker neurons from young and adult rats was not significant (ANOVA, P>0.2). The 50s of NMDA were: bursting 2.93&0.51 µM, pacemaker 6.45&0.95 µM, and irregular 7.25&0.92 µM for immature rats; and 9.86&0.98 µM for pacemaker from adults. Mean discharge rates before NMDA were as indicated in Table 1 (baseline). These values were normalized to 100. Each curve is the average of eight to 20 replicates. The number of cells used is indicated in Table 1. (D) Relation between NMDA concentration and burst production expressed as a percentage of spikes within bursts. Basal burst activity was obviously zero for pacemaker and irregular neurons and 19.2&2.6 for bursting neurons. Curves were calculated using data from neurons that responded to NMDA application with a burst increment, as indicated in the inset.

However, the qualitative effect of NMDA in pacemaker neurons from immature rats was quite different. While the low concentrations (0.1–5.0 µM) of the drug activated the discharge frequency but did not modify the firing pattern in all neurons tested (n=24, Fig. 3A), the higher concentrations (10–100 µM) either produced a further increase in the firing rate, and eventually inactivation, in 62.5% of neurons (15 out of 24; Fig. 3B), or induced bursting activity in 37.5% (nine out of 24) neurons (Fig. 3D, Table 2). The effect of NMDA on irregular and bursting neurons was more homogeneous. At concentrations higher than 1.0 µM, the drug increased the firing rate in all irregular (n=18) and bursting (n=22) neurons tested. Moreover, it induced bursts in about half of the irregular neurons (8/15; 53.3%) and increased burst production in the

majority (19/25; 76.0%) of the spontaneously bursting neurons (Fig. 3D, Table 2). As shown in Fig. 3D, the PSB in spontaneously bursting neurons were enhanced from an average of 19.2&2.6 to 84.5&9.3 after 30–40-s exposure to 100 µM NMDA, just before inactivation. Spontaneously bursting neurons exhibited a higher sensitivity than pacemaker or irregular DA neurons from either adult or immature rats to the stimulant effect of NMDA on the firing rate (Fig. 3C). The 50s of NMDA in activating the firing rate were 2.93&0.51, 6.45&0.95 and 7.25&0.92 µM for bursting, pacemaker and irregular neurons from immature rats, respectively, and 9.86&0.98 µM for pacemaker neurons from adult rats. In all tests, the predrug rate and pattern were reinstated within 60–120 s of washout.

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Table 2. Effect of (&)2-amino,5-phosphonopentanoic acid on the spontaneous and N-methyl--aspartate-induced burst firing of dopamine neurons in slices from rats at different ages Drug (µM)

Immature (PN 15–21) Pacemaker (n=24) Irregular (n=15)

Baseline AP-5 (50) AP-5 (100) NMDA (50) NMDA (50)+AP-5 (50) NMDA (50)+AP-5 (100)

0 0 0 22.6&4.5a 7.5&1.6†a 3.6&0.7††

0 0 0 39.5&8.0b 19.6&7.7††b 4.2&1.6††b

Adult (PN 40–70) Bursting (n=25) Pacemaker (n=18) 19.2&2.6 7.6&1.8 1.5&0.4 79.4&10.1c 51.4&7.1†c 21.4&4.5††c

0 0 0 8.6&2.8d 4.2&0.9†d 2.9&0.6††d

Values represent the percentage of spikes occurring within bursts. Each value is the mean&S.E. from the number of neurons (n) which were selected by their basal discharge pattern. Mean&S.E. calculated on anine, beight, c19, and dthree neurons responding to NMDA. †P<0.05, ††P<0.01 with respect to NMDA alone.

Effect of (&)2-amino,5-phosphonopentanoic acid The selective competitive NMDA receptor antagonist AP-5 was used to characterize further the role of NMDA receptors in the regulation of discharge rate and mode of DA neurons. In slices from both young and adult rats, changes in the firing rate and discharge pattern elicited by NMDA were partly or fully reversed by AP-5 (50 and 100 µM, respectively, Tables 1 and 2). In slices taken from young rats, 5–10 min application of AP-5 led 11 out of 19 irregular or bursting neurons to fire in pacemaker mode (Tables 1 and 2). The original pattern was reinstated within a few minutes of washing. AP-5 was completely ineffective in modifying the basal firing rate and mode of pacemaker DA neurons from both immature and adult rats (Tables 1 and 2).

DISCUSSION

In line with previous studies, we found that DA neurons in slices from adult rats discharge only in a pacemaker mode.9,13 However, we found that midbrain DA neurons from immature rats also exhibit irregular and bursting patterns. Since spontaneous bursting in vivo is considered to be sustained by NMDA receptor activation, its presence in DA neurons from immature rats may indicate that the burst generator, involving NMDA receptors, is preserved following the dissecting procedure used in this study. Accordingly, NMDA-mediated spontaneous miniature excitatory postsynaptic currents generated by NMDA channels have been detected in DA neurons in slices from immature rats19 and NMDA antagonists elicit outward currents in these neurons,19 as they do in CA1 pyramidal neurons in slices.23 The role of NMDA receptors in spontaneous and NMDAinduced bursting activity in DA neurons from immature rats is also supported by the fact that both phenomena were inhibited by the selective NMDA antagonist AP-5. The absence of spontaneous bursts and the modest response to NMDA observed in DA

neurons in slices from adult rats may reflect a reduced sensitivity of NMDA receptors and/or the loss of the burst generator. Indeed, an age-related decline in NMDA receptor sensitivity and NMDA channel gating properties has been described in different brain areas after the third week of life.4,15 Interestingly, spontaneous bursts have been observed in about 40% of DA neurons in 20-day primary cultures from neonatal rats.3 However, while spontaneously bursting neurons from immature rats were found to be more sensitive to NMDA than pacemaker neurons from adult or immature rats, no significant difference in sensitivity to the NMDA-induced firing rate activation was observed between pacemaker DA neurons from immature and adult rats. Since experimental evidence has indicated that burst firing in DA neurons may be triggered by NMDA-mediated high-threshold Ca2+ spikes originating in distal dendrites,9,13,14 it could be suggested that the integrity of such a burst generator is lost in slices from adult rats. Therefore, a consequence of this study is that the age of the animal is an important variable not only for the occurrence of spontaneous bursts but also for NMDA-induced burst firing in DA neurons in slices. The fact that glutamate and kainate, unlike NMDA, increased the firing rate of DA neurons but failed to produce bursts suggests that the two phenomena may be differentially regulated.9,10 This possibility is supported by the lack of correlation between spontaneous firing rate and presence of bursting activity in DA neurons in both in vitro and in vivo preparations.5,9 However, one problem arises from the fact that glutamate failed to elicit bursting activity, even though it should activate both NMDAand non-NMDA receptors. It is possible that glutamate maximally stimulates non-NMDA receptors at concentrations insufficient to activate NMDA receptors, so that depolarization inactivation may ensue before bursting activity can be initiated. Accordingly, non-NMDA receptors have been shown to be more sensitive than NMDA receptors to glutamate.13,15

Spontaneous bursts in DA neurons CONCLUSIONS

This study indicates that DA neurons in slices offer a useful model for studying mechanisms controlling burst generation in these neurons. Our results suggest that NMDA receptors play an important role in controlling burst firing of DA neurons in slices and that this mechanism undergoes changes during development. These findings may have theoretical and practical consequences since bursting activity in DA neurons has been shown to be elicited in vivo by motivated movement6,7,24 and by different drugs, including drugs of misuse,10,17,19 and also to promote

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a much greater amount of DA release from nerve terminals than an equal number of action potentials delivered in a pacemaker mode.8,16 Therefore, the understanding of the mechanism involved in bursting pattern might indicate pharmacological manipulations of this phenomenon, thereby offering new strategies for treatment of the pathologies related to dopaminergic dysfunction.

Acknowledgement—We thank Stefano Aramo for his expertise and assistance.

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