Tachykinin neurokinin-1 and neurokinin-3 receptor-mediated responses in guinea-pig substantia nigra: an in vitro electrophysiological study

Pergamon

PII:

Neuroscience Vol. 78, No. 3, pp. 745–757, 1997 Copyright ? 1997 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/97 $17.00+0.00 S0306-4522(96)00625-2

TACHYKININ NEUROKININ-1 AND NEUROKININ-3 RECEPTOR-MEDIATED RESPONSES IN GUINEA-PIG SUBSTANTIA NIGRA: AN IN VITRO ELECTROPHYSIOLOGICAL STUDY E. NALIVAIKO,* J.-C. MICHAUD,* P. SOUBRIE u ,* G. LE FUR* and P. FELTZ† *Sanofi Recherche, 371 rue du Prof. Blayac, 34184 Montpellier, France †Laboratoire de Physiologie Ge´ne´rale, Universite´ Louis Pasteur, 21 avenue Rene´ Descartes, 67084 Strasbourg, France Abstract––The effects of tachykinin receptor agonists and antagonists were investigated using intra- and extracellular recordings on spontaneously firing nigral neurons in guinea-pig brain slices. In 70 of 76 electrophysiologically identified dopaminergic neurons, a concentration-dependent increase in firing rate was induced by the selective neurokinin-3 tachykinin agonist senktide and by the natural tachykinin agonists neurokinin B and substance P, with 50 values of 14.7, 31.2 and 12200 nM respectively. These responses were inhibited in a concentration- and time-dependent manner by the selective non-peptide neurokinin-3 receptor antagonist SR 142801 (1–100 nM; n=23), but neither by its S-enantiomer SR 142806 (100 nM; n=4) nor by selective antagonists of neurokinin-1 (SR 140333) or neurokinin-2 (SR 48968) receptors (both at 100 nM; n=3). The selective neurokinin-1 agonist [Sar9,Met(O2)11]substance P (30–100 nM; n=23) and the selective neurokinin-2 agonist [Nle10]neurokinin A(4–10) (30–100 nM; n=13) were without any effect on dopaminergic cells. In 13 of 21 electrophysiologically identified, presumably GABAergic neurons located in the pars compacta of the substantia nigra, excitatory responses were evoked concentration dependently by substance P and [Sar9,Met(O2)11]substance P, with 50 values of 18.6 and 41.9 nM respectively. These responses were inhibited by SR 140333 (100 nM; n=3), but neither by its R-enantiomer SR 140603 nor by SR 142801 (both at 100 nM; n=3). Senktide and [Nle10]neurokinin A(4–10) (both at 30–100 nM; n=10) were without effect on these presumed GABAergic neurons. A small population (12%) of pars compacta neurons was insensitive to any of the three selective tachykinin agonists. In the nigral pars reticulata, 12 neurons were recorded which had an electrophysiological profile similar to that of presumed GABAergic neurons in the pars compacta. Of these 12 cells, seven did not respond to any of the selective tachykinin agonists tested, while five were excited by senktide in a concentration-dependent manner (50=98.5 nM). Although this value was significantly higher than that found for dopaminergic neurons in the pars compacta, senktide-evoked responses were inhibited by SR 142801 (100 nM; n=3). We conclude that, in the guinea-pig substantia nigra, tachykinins evoke excitatory responses in both dopaminergic and non-dopaminergic neurons; however, the sensitivity to tachykinin agonists (neurokinin-1 versus neurokinin-3) depends on both neuronal type and localization. ? 1997 IBRO. Published by Elsevier Science Ltd. Key words: brain slices, tachykinin receptors, tachykinin antagonists, dopaminergic neurons, GABAergic neurons, firing rate.

Substance P (SP), neurokinin A and neurokinin B are endogenous preferential agonists of tachykinin neurokinin-1, neurokinin-2 and neurokinin-3 receptors respectively.18,27,40 The recent development of more selective agonists and selective non-peptide antagonists10–12,17,26 for each tachykinin receptor Abbreviations: ACSF, artificial cerebrospinal fluid; AHP, afterhyperpolarization; AP, action potential; DA, dopaminergic; SN, substantia nigra; SNc, pars compacta of the substantia nigra; SNr, pars reticulata of the substantia nigra; SP, substance P; SR 48968, selective neurokinin-2 receptor antagonist; SR 140333, selective neurokinin-1 receptor antagonist; SR 140603, inactive enantiomer of SR 140333; SR 142801, selective neurokinin-3 receptor antagonist; SR 142806, inactive enantiomer of SR 142801; TH, tyrosine hydroxylase.

type has allowed substantial progress in the field of tachykinin pharmacology. Although tachykinins and tachykinin receptors are abundantly distributed in the mammalian CNS,7,13,17,26,27,53 direct action of tachykinin agonists on neuronal activity has been described only in a limited number of brain structures.7,22,34,36,46,47 Among them, of special interest is the substantia nigra (SN), where tachykinins could potentially modulate the input from the striatum, and a co-localization of GABA and SP has been demonstrated in striatonigral synaptic terminals.41 However, data concerning the action of tachykinins in the SN are contradictory: whereas dopaminergic (DA) nigral neurons responded to neurokinin-1, neurokinin-2 and neurokinin-3 agonists when examined in vivo,36 experiments performed in rat brain

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slices revealed that DA cells responded exclusively to the selective neurokinin-3 agonist senktide,22 and no functional neurokinin-1 receptor-mediated response has been reported so far in identified neurons of the SN. Another mismatch consists of the fact that the SN contains the highest concentration of SP (which is a preferential neurokinin-1 agonist) in the brain,3,32 and apparently very few neurokinin-1 receptors.28,48,53 The neuronal composition of the SN is presently well established: the majority (85–95%) of its pars compacta (SNc) is represented by DA cells which project mostly to the striatum. These neurons display a specific electrophysiological profile, especially in vitro: slow and highly regular firing rate, longduration action potentials (APs) and the presence of inward rectification.15,19,24,29,31,54 Their DA nature has been demonstrated by the detection of tyrosine hydroxylase (TH) in the cytoplasm.16,54 Another, less numerous, population of the SNc (5–15%) possesses different electrophysiological characteristics: highfrequency firing rate (10 Hz) and short-duration APs.15,19,24,29,31,54 Neurons of this type were also reported in the pars reticulata of the SN (SNr). Although their transmitter phenotype is still to be identified, evidence exists for its GABAergic nature, since intranigral localization of these neurons paralleled the distribution of cells positive for the immunoreactivity of glutamate decarboxylase (a marker enzyme for GABAergic neurons), and these neurons were TH negative.54 In addition, two other neuron types were electrophysiologically identified in the rostral part of the SNc, one of which originates from the neighbouring subthalamic nucleus,37 while the other probably represents a second type of DA nigral neurons.31 Nigral neurons belonging to different populations in the SNc also differ in the spectrum of synaptic receptors which they express: DA neurons respond to dopamine D2 agonists but not to µ-opioid agonists, while the pharmacological sensitivity of presumably GABAergic neurons is reversed.24 Recent evidence suggests a further pharmacological difference between different types of nigral neurons, and it has been suggested that neurokinin-3 receptors may be selectively expressed in DA neurons, whereas neurokinin-1 receptors are located in GABAergic neurons.5,49,53 In order to examine further the modulatory role of tachykinins in different types of nigral neurons, we have investigated the effects of tachykinin agonists and antagonists on the spontaneous activity of electrophysiologically identified cells in the SN. Species differences in tachykinin pharmacology have been reported, particularly for non-peptide receptor ligands.7,13,14 Since the binding affinity of SR 142801, the selective neurokinin-3 receptor antagonist, for neurokinin-3 receptors in guinea-pig brain is considerably higher than in rat brain, and is close to that for human neurokinin-3 receptors expressed in Chinese hamster ovary cells,10 our experiments

were performed in guinea-pig mesencephalic slices. Although the electrophysiology of guinea-pig nigral neurons is well described,19,25,29,31 there are as yet no data available concerning the action of tachykinins in these cells. EXPERIMENTAL PROCEDURES

Slice preparation and solutions used Mesencephalic slices containing the SN were obtained from male Hartley guinea-pigs (Charles River, France; 150–180 g), as described elsewhere.31 Briefly, under ketamine anaesthesia (200 mg/kg, i.p.), animals were decapitated; the brain was removed and placed in an ice-cold artificial cerebrospinal fluid (ACSF), where sodium chloride (120 mM) was totally replaced by sucrose (240 mM).1 Procedures were approved by the ‘‘Comite´ d’Expe´rimentation Animale’’ (Animal Care and Use Committee) of Sanofi Recherche and were carried out in accordance with the French legislation which implemented the European Community Council Directive 86/609/ECC. Coronal sections of 400 µm thickness were cut using a Vibratome tissue slicer (Campden Instruments, U.K.). Slices placed between two nylon meshes in the experimental chamber were completely submerged and continuously perfused at a flow rate of 2 ml/min by an ACSF composed as follows (in mM): NaCl, 120; KCl, 2.3; KH2PO4, 1.2; CaCl2, 2.5; MgSO4, 1.2; NaHCO3, 26; glucose, 10; and saturated by a gas mixture of 95% O2/5% CO2. Electrophysiological recordings and data analysis Experiments were performed on neurons located in the SN. This brain structure was detected visually as an area located dorsolaterally from the accessory optic tract. Subtypes of nigral neurons were identified electrophysiologically. Intracellular recordings were performed by means of glass microelectrodes filled with 3 M potassium acetate (90–130 MÙ) in current-clamp mode, using an Axoclamp 2B amplifier and pCLAMP software (Axon Instruments, U.S.A.). Some neurons were recorded extracellularly, in which case microelectrodes filled with the ACSF had a resistance of 30–40 MÙ. Experiments were started at least 2 h after dissection. Upon impalement, a hyperpolarizing current was applied during a 5- to 10-min recovery period; subsequent recording of spontaneous electrical activity was made at zero holding current. Data were continuously recorded with a digital paper recorder. Rate-meter histograms were generated on-line using an event detector and a second computer running MRATE software (CED Ltd., U.K.). All drugs were applied by bath perfusion. Perfusion time for all agonists was 1 min. Experiments were carried out at 33–34)C. Excitatory effects evoked by tachykinin receptor agonists were quantified by counting the number of action potentials for 60 s at the maximum of agonist action and subsequent subtraction of the number of spikes during the 60-s control period. Theoretical equations were fitted to experimental data using the least squares method (SigmaPlot software, Jandel Scientific, U.S.A.). Concentration–response curves of the effects of SP and [Sar9,Met(O2)11]SP were analysed using the equation R=Rmax/(1+(50/[agonist])n), where Rmax is the maximum response observed, 50 is the value of agonist concentration which evokes 50% of the Rmax and n is a factor describing the steepness of the curve. Since concentration–response curves for senktide and neurokinin B appeared to be bell shaped, a logistic equation according to Ariens et al.4 was used: R=(a/(1+50/[agonist]))#(1+(a1/ (1+50"1/[agonist]))) where, in addition, 50"1 is the value of agonist concentration which reduces maximal response by 50%, and a and a1 are factors describing the steepness of the ascending and descending parts of the curve

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Fig. 1. Three types of neurons in the SNc. Intracellular recordings of spontaneous activity from cells of type I (row A), type II (row B) and type III (row C) are shown. No holding current was applied. The difference in AP amplitude is due to a digitization artifact. Note the differences in firing frequency and AP shape and duration. Dotted lines correspond to 0 mV. Scale bars: voltage, 40 mV for all panels; time, 1 s (Aa, Ba, Ca); 150 ms (Ab, Bb); 100 ms (Cb); 1.5 ms (Ac, Bc, Cc).

respectively. Finally, analysis of the concentrationdependent effect of SR 142801 was performed using the equation R=1/(1+ [antagonist]/50), where 50 is the value of antagonist concentration which reduced the agonistevoked response by 50%. All data in the text and figures are expressed as mean&S.E.M. Differences were considered statistically significant when P<0.05 in Student’s t-test. Materials SR 140333 (neurokinin-1 receptor antagonist), SR 140603 (R-enantiomer of SR 140333), SR 48968 (neurokinin-2 receptor antagonist), SR 142801 (neurokinin-3 receptor antagonist) and SR 142806 (S-enantiomer of SR 142801) were synthesized at Sanofi Recherche (Montpellier, France). [Sar9,Met(O2)11]SP, [Nle10]neurokinin A(4–10) and neurokinin B were obtained from Neosystem (Strasbourg, France); senktide, septide and SP were obtained from Cambridge Research Biochemicals (Cambridge, U.K.); quinpirole was obtained from RBI (Natick, MA, U.S.A.). All drugs were diluted in ACSF from 1 mM stock solutions (in distilled water for peptides and quinpirole, and in dimethyl sulphoxide for SR compounds). RESULTS

Electrophysiological identification of nigral neurons On the basis of several criteria, three different cell types were clearly distinguishable in the guinea-pig SNc. The majority of recorded neurons (119 of 152 cells) possessed a uniform electrophysiological profile: they fired spontaneously at a low rate (1.7&0.2 Hz); this firing pattern was highly regular (Fig. 1Aa) and, once stabilized after impalement, did

not change under control conditions for the whole period of recording. APs were of long duration (2.1&0.5 ms, measured at the threshold level), had a notch at the repolarization phase and were followed by a prominent slow afterhyperpolarization (AHP), which usually exceeded by 3–5 mV the fast AHP observed immediately upon spike termination (Fig. 1Ab, c). Spontaneous firing could be suppressed by injecting hyperpolarizing holding current; in this case, hyper- and depolarizing current pulses evoked active membrane responses (Fig. 2A). If the current step caused a membrane hyperpolarization below "100 mV, a time-dependent inward rectification became clearly distinguishable in all neurons. After the hyperpolarizing current step, an ‘‘off’’ response could be observed, usually as a positively directed slow oscillating potential (not shown). Suprathreshold depolarizing current pulses (0.1 nA, 250 ms) evoked single APs, followed by fast and slow AHPs (similar to those observed during spontaneous firing). No burst activity was observed in this population of nigral cells. The above-described neurons will be referred to in the present work as type I or as identified DA neurons, since their electrophysiological profile corresponded to the one described for DA cells.54 A second SNc neuronal population consisted of neurons with irregular firing rate, ranging from 0.2 to 5.3 Hz. These neurons were far lower (19 of 152 cells) in number than the DA ones. Recordings of their

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Fig. 2. Responses of the three types of SNc neurons to hyper- and depolarizing current pulses of 250-ms duration. Same cells as in Fig. 1. Spontaneous activity was suppressed by hyperpolarizing holding current. Note the differences in post-spike AHP and in the degree of inward rectification. Dotted lines correspond to 0 mV. Scale bars: 40 mV, 50 ms.

spontaneous activity are presented in Fig. 1B. APs of these neurons were also of long duration (1.9&0.4 ms); however, when compared to type I cells, the ‘‘notch’’ was less consistently present at the repolarization phase, and the slow AHP was either indistinguishable from the ascending phase after the fast AHP or, if present, did not exceed the amplitude of the fast AHP (Fig. 1Bb, c; also compare the shape of APs on upper traces in Fig. 2A and B). Inward rectification was significantly less prominent with respect to type I neurons. Suprathreshold depolarizing current pulses (0.1 nA, 250 ms) evoked either single APs (n=8), or bursts of two to three spikes (n=11). This cellular population will be denoted as type II neurons. Finally, a small group (14 of 152) of neurons in the SNc (type III cells) possessed electrophysiological characteristics which differed markedly from the other two populations. They discharged regularly at a high frequency (10 Hz), had short-duration APs (1.3&0.2 ms) with a straight phase of repolarization, and only a fast AHP could be observed upon spike termination (Fig. 1C). Electrical stimulation performed after the suppression of spontaneous activity

by hyperpolarization evoked responses different from those of type I or type II neurons: no inward rectification was apparent even at current pulses producing hyperpolarization below "120 mV; the ‘‘off ’’ response was usually observed as a burst of two to three spikes; suprathreshold depolarizing current steps (0.1 nA, 125 ms) evoked bursts of three to six APs without any sign of accommodation (Fig. 2C). For the pharmacological study, 10 more neurons of this type were recorded extracellularly; they could be attributed to type III on the basis of their fast firing rate and short APs (see Table 1). The input resistances were found to be 112.5&5.2, 120.9&5.6 and 139.0&9.3 MÙ in type I, type II and type III neurons respectively; the differences in these values were not significant. Five type I, three type II and one type III neurons were silent; nevertheless, they could easily be attributed to the corresponding neuronal populations using parameters of evoked electrical responses. These cells were excluded from the subsequent study. In addition, 12 neurons were recorded in the SNr. Since the density of neurons is much lower in the SNr than in the SNc, recordings from only two of these 12 cells were performed by means of intracellular microelectrodes; the others were recorded extracellularly. All of them had a high-frequency firing rate (10 Hz) and short-duration (1.2&0.2 ms) APs. The electrophysiological profile of the two intracellularly recorded cells did not differ from that of fast-firing neurons in the SNc. Effect of quinpirole on identified nigral neurons Quinpirole, a selective agonist for dopamine D2 receptors, was tested on neurons from all three groups in the SNc. When applied through bath perfusion at concentrations of 1–3 µM to type I neurons, quinpirole caused a strong and reversible inhibition of the spontaneous firing rate, accompanied by a hyperpolarization of 3–5 mV. Spontaneous activity was abolished in nine of 12 cells tested, and the frequency was reduced to less than 15% of control values in the three others. On the contrary, no effect of the agonist was observed in any of five type III neurons tested. As for the type II cells, in three of five neurons tested, quinpirole did not produce any effect at all, while in the other two cells a weak inhibitory effect was observed. No effect of quinpirole was found in the SNr neurons (n=4). Tachykinin responses in identified dopaminergic nigral neurons The effects of neurokinin-1 ([Sar9,Met(O2)11]SP), neurokinin-2 [[Nle10]neurokinin A(4–10)] and neurokinin-3 (senktide) tachykinin receptor agonists were tested on a total of 76 spontaneously active neurons of type I. While [Sar9,Met(O2)11]SP (n=23) or [Nle10]neurokinin A(4–10) (n=13) were without

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Table 1. Summary of some electrophysiological and pharmacological properties of guinea-pig nigral neurons Electrophysiological properties Freq. (Hz) Type I, n=119 Type II, n=19 Type III, n=14 SNr, n=12

Regular, 1.7&0.2 Irregular, 0.2&5.3 Regular, 12.8&4.1 Regular, 14.6&3.9

Pharmacological sensitivity

TAP (ms)

Late AHP

Inward rectif.

Burst activity

QP

SP

SarMetSP

Nle-NKA

NKB

Senktide

2.1&0.5

Present Weak or absent

No

15/15

9/10*

0/23

0/13

8/8*

70/76*

1.9&0.4

Present Weak or absent

No/yes

2/5

0/3

0/11

0/11

—

2/11

1.3&0.2

Absent

Absent

Yes

0/5

4/4†

13/21†

0/10

—

2/10

1.2&0.2

Absent

Absent

Yes

0/4

—

0/6

0/4

—

5/12*

Abbreviations: Frequency, spontaneous firing frequency; TAP, action potential duration; QP, quinpirole; SP, substance P; SarMet-SP, [Sar9,Met(O2)11]substance P; Nle-neurokinin A, [Nle10]neurokinin A(4–10). Note that in type I neurons, SP was tested at micromolar concentrations. *Responses were antagonized by the selective neurokinin-3 antagonist SR 142801. †Responses were antagonized by the selective neurokinin-1 antagonist SR 140333.

Fig. 3. Responses of the three types of SNc neurons to different tachykinin receptor agonists. A, B and C represent rate-meter histograms recorded in type I, II and III neurons respectively. Bin width, 5 s. Arrowheads indicate the beginning of the 1-min perfusion period with the agonist indicated above. Abbreviations: SarMet-SP, [Sar9,Met(O2)11]SP; Nle-NKA, [Nle10]neurokinin A(4–10). SP was applied at a concentration of 1 µM, all other drugs at a concentration of 100 nM.

any effect or caused a hardly detectable increase in firing frequency when applied at concentrations up to 100 nM, senktide (1–100 nM) potently increased firing rate in 70 of 76 cells tested (Fig. 3A). Frequency changes could be detected 1–1.5 min after switching to the agonist-containing solution, reached their maximum within the next 2–3 min and then slowly returned to the basal level over 7–12 min (depending on the concentration of the agonist). In all DA neurons sensitive to senktide, enhancement of firing

frequency was accompanied by a significant decrease in the late AHP amplitude. Such responses were present in 92% of all identified nigral DA neurons; they were concentration dependent, reversible and reproducible for several hours of recording provided that intervals between drug applications were of at least 15 min. Consecutive applications of senktide at increasing concentrations (1–100 nM) in six neurons allowed us to establish a concentration–response curve (Fig. 4) and to calculate the 50, which was

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Fig. 4. Tachykinin-mediated responses in SNc type I/DA neurons. (A) Rate-meter histogram from a representative experiment showing responses to senktide. The agonist was applied for 1 min (arrowheads) at increasing concentrations (10, 30, 100 and 300 nM). Bin width, 5 s. (B) Concentration–response curves of senktide (-), neurokinin B (5) and SP (/). Results are expressed as percentage of maximal response. Values are the means&S.E.M. (n=4–6). The calculated 50 values for senktide, neurokinin B and SP are 14.7 nM, 31.2 nM and 12.2 µM respectively.

found to be 14.7 nM. The minimal effective concentration was 1 nM, the maximal response being observed at 100 nM. In three cells for which senktide was also applied at 300 nM, responses were smaller than those obtained at 100 nM. The average increase of firing frequency in response to the saturating concentration of senktide (100 nM) was 212% (from 1.7&0.2 Hz before agonist application to 3.6&0.3 Hz at the maximum of the response). In addition, in three cells we observed a depolarization block caused by the agonist (30– 100 nM); in such a case, after an initial increase in firing frequency, a silent period appeared. If during this period the membrane potential was returned to the initial value by passing hyperpolarizing current, rhythmic activity was immediately restored. In order to verify whether the effect of senktide was mediated through pre- or postsynaptic receptors, the agonist was also tested in a low-calcium (0.3 mM), highmagnesium (3.7 mM) medium (n=4). In these conditions, senktide evoked excitatory responses similar to those observed in control. Addition of the selective neurokinin-3 receptor antagonist SR 142801 to the bath perfusion medium caused a time-dependent inhibition of senktideevoked responses. The time-course of block was rather slow: no sign of antagonism could be observed within the first 15–20 min, and a steady level of

inhibition was reached only after 90 min of perfusion (Fig. 5A). The concentration–effect relationship of SR 142801 was studied in 19 neurons to which senktide was applied repetitively at 15-min intervals at a concentration of 30 nM; because of the slow action of the antagonist, data were collected after a 90-min incubation period. The minimal effective concentration of SR 142801 was 1 nM, while at 100 nM it caused a significant decrease of the senktide-evoked response (by 89&4%). The 50 value was found to be 11.1 nM when senktide was used at a concentration of 30 nM (Fig. 5B). No recovery of senktide-evoked responses was observed 2 h after washout of SR 142801 applied at 100 nM (n=3). In another set of experiments, the concentration– response curve of senktide was determined after a 90-min perfusion period with SR 142801 at a concentration of 30 nM (n=4). In this case, a rightward parallel shift was observed, and the 50 for the agonist increased from 14.7 nM in control to 71.5 nM after SR 142801. In the latter case the concentration–response curve was also bell shaped (Fig. 5C). SR 142806, the S-enantiomer of SR 142801, applied at 100 nM in the same experimental protocol as shown in Fig. 5A, exhibited a much lower activity, since it decreased senktide-evoked responses (30 nM) by only 9.6&2.4% (n=4). Excitatory responses to senktide were affected neither by the selective neurokinin-1 antagonist SR 140333, nor by the selective neurokinin-2 antagonist SR 48968 (both at 100 nM; n=3). A concentration-dependent increase of the spontaneous firing frequency in identified nigral DA neurons was also evoked by the natural tachykinin agonists neurokinin B (n=8; 50=31.2 nM) and SP (n=9); however, in the latter case the agonist efficacy was markedly lower, since the 50 was found to be 12.2 µM (Figs 3A, 4B). Responses to both agonists were antagonized by SR 142801: at 100 nM, this compound significantly reduced responses to neurokinin B (100 nM) by 82&8% (n=3) and responses to SP (10 µM) by 91&5% (n=4). Tachykinin responses in identified non-dopaminergic neurons in the pars compacta of the substantia nigra Tachykinin receptor agonists were applied to a total of 21 type III neurons, 10 of which were recorded extracellularly. In 13 cells, [Sar9,Met(O2)11]SP (1–300 nM) produced an increase in the firing frequency (Fig. 3C, Fig. 6A); this effect was reversible and concentration dependent (Fig. 6B; n=4). The minimal effective agonist concentration was 10 nM, and the maximal response (22.1&4.7 Hz with respect to the basal level of 12.8&4.1 Hz) was evoked at the concentration of 300 nM. The 50 of [Sar9,Met(O2)11]SP was found to be 41.9 nM (n=4). SP (1–100 nM) was tested in four type III neurons which were also sensitive to [Sar9,Met(O2)11]SP; in

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Fig. 5. Inhibition by SR 142801 of senktide-induced excitation in SNc type I/DA neurons. (A) Rate-meter histogram showing the excitatory effect of senktide and its time-dependent suppression by SR 142801. Senktide was applied at a concentration of 30 nM for 1 min at 15-min intervals, as shown by arrowheads. The black bar indicates the period of slice perfusion with SR 142801 at a concentration of 100 nM. Bin width, 5 s. (B) Concentration–effect relationship of SR 142801. Results are expressed as percentage of the response obtained before antagonist application. Values are means&S.E.M. (n=4). The calculated 50 value for SR 142801 (for 30 nM senktide) was 11.1 nM. (C) Rightward shift in concentration–response curve of senktide after a 90-min perfusion period with SR 142801 at a concentration of 30 nM (/). Control curve (-) is the same as in Fig. 4B.

Fig. 6. SP- and [Sar9,Met(O2)11]SP-mediated responses in SNc type III/presumably GABAergic neurons. (A) Rate-meter histogram from a representative experiment showing responses to [Sar9,Met(O2)11]SP applied for 1 min (arrowheads) at increasing concentrations (30, 100 and 300 nM). Bin width, 5 s. (B) Concentration–response curve of SP (-) and [Sar9,Met(O2)11]SP (/). Results are expressed as percentage of maximal response. Values are mean&S.E.M. (n=3–4). The calculated 50 values for SP and [Sar9,Met(O2)11]SP were 10.1 and 41.9 nM respectively.

all of them, SP evoked excitatory responses in a concentration-dependent manner, with an 50 of 10.1 nM (Fig. 6B). No changes in the spontaneous firing rate were observed in the type III neurons in response to [Nle10]neurokinin A(4–10) tested at a concentration of 100 nM (n=10). In addition, senktide (100 nM) evoked excitatory responses in only two of the 10 type III neurons tested; however, these

two cells did not respond to [Sar9,Met(O2)11]SP (100 nM). Since these two neurons were recorded in the area where the SNc and SNr could overlap, they probably belonged to the SNr neuronal population (see below). Excitatory responses to [Sar9,Met(O2)11]SP and SP were reduced by the selective neurokinin-1 receptor antagonist SR 140333. The kinetics of the antagonist

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Fig. 7. Inhibition by SR 140333 of [Sar9,Met(O2)11]SPinduced excitation in SNc type III neurons. Rate-meter histogram showing the excitatory effect of [Sar9, Met(O2)11]SP and its time-dependent suppression by SR 140333. [Sar9,Met(O2)11]SP was applied at a concentration of 100 nM for 1 min (arrowheads). The black bar indicates the period of slice perfusion with SR 140333 at a concentration of 100 nM. Bin width, 5 s.

shaped, as observed in DA neurons from the SNc, the 50 found in the present case was considerably higher (91.3 nM). In order to test whether the effect of senktide in SNr neurons was mediated through a separate type of tachykinin receptor (termed ‘‘septide-sensitive’’14a), septide (100 nM) was applied to three SNr cells which were sensitive to senktide; in none of them did septide evoke any detectable response. Senktide-evoked responses (30–100 nM) in SNr neurons were reduced by the selective neurokinin-3 receptor antagonist SR 142801 (100 nM) to 87&3% of their initial value. The main electrophysiological and pharmacological properties of guinea-pig nigral neurons recorded in our study are summarized in Table 1.

DISCUSSION

Different neuron types in the substantia nigra action were slow, both in onset and in development of the maximal effect (Fig. 7), and appeared similar to those of SR 142801. Since type III neurons were less numerous than DA cells, we were unable to examine the concentration-dependent effect of the antagonist. Therefore, SR 140333 was tested at a single concentration of 100 nM: in these conditions, after a 90-min perfusion period with the antagonist, [Sar9,Met(O2)11]SP-induced responses (100 nM) were decreased by 93&6% (n=3) and SP-induced responses by 89&5% (n=3). The excitatory action of [Sar9,Met(O2)11]SP in type III neurons was affected neither by SR 140603 (the R-enantiomer of SR 140333) nor by the selective neurokinin-3 receptor antagonist SR 142801 (both at 100 nM; n=3). None of the three selective tachykinin agonists applied at 100 nM affected the spontaneous firing frequency in nine of 11 type II cells tested (Fig. 3B); in the other two neurons, senktide evoked weak excitatory responses. Applied alone, none of the selective tachykinin receptor antagonists produced any detectable change in the basal firing rate or in the membrane properties in all neurons tested. Tachykinin responses in neurons in the pars reticulata of the substantia nigra Tachykinin agonists were tested in 12 fast-firing neurons in the SNr. While [Sar9, Met(O2)11]SP (n=6) and [Nle10]neurokinin A(4–10) (n=4) were without any effect when applied at a concentration of 100 nM, senktide evoked concentration-dependent excitatory responses in five of 12 cells (Fig. 8). The maximal increase in spontaneous firing frequency (19.6&5.2 Hz with respect to the basal level of 14.6&3.9 Hz) occurred when the agonist was perfused at 300 nM. Although the concentration– response curve for the action of senktide was bell

This study is a comprehensive description of tachykinin pharmacology in the guinea-pig SNc. According to electrophysiological and pharmacological criteria, we were able to identify three different neuronal populations resembling those already described in this brain area in rat15,24 and guineapig.19,29,31,54 Type I cells, due to both their sensitivity to the dopamine D2 agonist quinpirole and their typical combination of slow regular firing rate, long-duration APs, late AHP and prominent inward rectification, fit with the description of ‘‘nonbursting’’,54 ‘‘rhythmic’’31 or ‘‘principal’’24 nigral cells and could therefore be considered as DA neurons. In agreement with previous reports, we found that such cells represented the large majority (78%) of the SNc neuronal population. Type III cells, in addition to their lack of sensitivity to quinpirole, exhibited a distinct electrophysiological profile (fast firing rate, short-duration APs and absence of both late AHP and inward rectification), suggesting that they are non-DA, presumably GABAergic cells corresponding to ‘‘bursting’’54 or ‘‘secondary’’24 neurons. These cells may account for 9% of the total SNc neuronal population. Type II cells (13% of SNc neurons in our study) resembled the ‘‘phasic’’ neurons described by Nedergaard and Greenfield31 in the guinea-pig SNc. They differed from type I/DA neurons by their irregular firing rate and less prominent late AHP and/or inward rectification, and from type III/GABAergic neurons by lower firing rates, longer APs and the presence of a slow AHP. Finally, neurons originating from the neighbouring subthalamic nucleus with different pharmacological and electrophysiological characteristics, described by Overton et al.37 in guinea-pig SNc, were never observed in our study. This could be explained by the use of younger animals in our experiments, assuming that the subthalamic nucleus and the SNc do not overlap at this age.

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Fig. 8. Senktide-mediated responses in neurons located in the SNr. (A) Rate-meter histogram from a representative experiment showing responses to senktide applied for 1 min (arrowheads) at increasing concentrations (30, 100, 300 and 1000 nM). Bin width, 5 s. (B) Concentration–response curve of senktide. Results are expressed as percentage of maximal response. Values are mean&S.E.M. (n=3). The calculated 50 value for senktide was 91.3 nM.

Selective responses of dopaminergic neurons to neurokinin-3 receptor stimulation Similar to the majority of observations performed in other brain areas and in other species,7,22,34,46,47 responses to the tachykinin receptor agonists in the guinea-pig SN were excitatory. Our study also confirms the global insensitivity of guinea-pig nigral cells to the neurokinin-2 receptor agonist [Nle10]neurokinin A(4–10), as already reported for another neurokinin-2 agonist in rat SNc22 and ventral tegmental area46 in vitro. We suggest that neurokinin-2 agonist-mediated effects observed in some studies were either due to species differences or to non-specific activation of neurokinin-1 or neurokinin-3 receptors. Interestingly, experiments in which an inhibitory action of tachykinin agonists has been detected in some nigral neurons were performed in vivo either by pressure ejection or by microiontophoresis,8,36 and thus the actual agonist concentration at the receptor site was unknown. Since we have observed a depolarization block provoked by the direct action of senktide or SP at a high concentration, it is possible that the inhibitory action of the tachykinin agonists in the cited studies was due to this phenomenon. One of the most significant findings of the present study is that while both type I/DA and type III/ GABAergic neurons were excited by tachykinin receptor agonists, these cells exhibited a radically different sensitivity to neurokinin-3 versus neurokinin-1 receptor stimulation. Several observations converge to indicate that activation of neurokinin-3 receptors preferentially, if not exclusively, affects type I/DA neurons in guinea-pig SNc. Applied in the nanomolar range, only selective (senktide) or preferential (neurokinin B) neurokinin-3 receptor agonists were found to increase spontaneous firing rate in these cells, while selective neurokinin-1 and neurokinin-2 agonists were without effect. The activity and the rank order of potency of senktide and neurokinin B revealed in the present study agree with those reported by others on rat DA cells of the A9/A10 complex.22,46 The action of senktide was

shifted or blocked by the selective non-peptide neurokinin-3 receptor antagonist SR 142801 in a concentration-dependent manner, with an 50 value in the nanomolar range (11 nM), in full agreement with its affinity for brain neurokinin-3 receptors10 and its activity on functional neurokinin-3 receptordependent experimental models.2,10,20,35,38 The onset and the development of SR 142801 action were slow, since complete block of the senktide-evoked response required 60–90 min of drug application. A delayed, slowly reversible in vitro activity, as distinct from that observed in vivo, seems to be a typical feature of compounds of this chemical series (see Jung et al.20). As expected from the binding data,10 SR 142806, the S-enantiomer of SR 142801, demonstrated a significantly lower potency, confirming a stereoselective action of the antagonist. Neither neuronal firing rate nor the shape of the active membrane responses were affected by SR 142801 application, thus indicating that, at the concentrations used, none of the voltagegated ion conductances were disturbed (also see Emonds-Alt et al.10). Finally, clearly distinct from the effects observed with SR 142801, neither SR 140333 nor SR 48698, selective non-peptide antagonists of neurokinin-1 and neurokinin-2 receptors respectively,11,12 were able to affect the rate-increasing effects of senktide on type I/DA cells. The mechanism of action of neurokinin-3 receptor agonists requires a separate study; nevertheless, since senktide- and neurokinin B-evoked excitatory responses were always accompanied by a reduction of late AHP amplitude, it can be speculated that they may be mediated, at least in part, by the modulation of calcium-dependent potassium channels.44,45 Type I neurons in the SNc were also sensitive to SP, the preferential neurokinin-1 receptor agonist; however, its potency was found to be about three orders of magnitude lower than that of senktide. This difference corresponds to the relative affinity of SP versus senktide for neurokinin-3 binding sites,9,47 and since SP-evoked responses were suppressed by SR 142801, we suggest that SP action in DA cells was mediated through neurokinin-3 receptor activation.

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These receptors may therefore represent one of the targets for the SP liberated from the striatonigral synaptic terminals; such a ‘‘non-specific’’ action of endogenous SP was recently demonstrated in guineapig gall bladder neurons.30 Thus, co-liberation of GABA and SP from the same synaptic terminal could probably underlie the complex postsynaptic response observed in nigral neurons following striatal52 or cortical23 stimulation. In addition, these receptors could be activated by another natural tachykinin agonist, neurokinin B, which is also present in the SN21 and which might be released from the afferents originating in the nucleus accumbens.28a Due to the persistence of the senktide-induced response in low-calcium medium, our electrophysiological results suggest that, in the guinea-pig SNc, neurokinin-3 receptors are located on type I/DA nigral cells. This conclusion is consistent with two lines of evidence. First, in cultured mesencephalic cells from gerbils, neurokinin-3 but not neurokinin-1 receptor activation enhanced [3H]dopamine release and intracellular Ca2+ mobilization, both biochemical events being likely to occur almost exclusively in DA neurons.2 Secondly, within the rat A9/A10 region, in situ hybridization studies revealed that mRNA encoding for neurokinin-3 receptors is preferentially associated with TH-immunoreactive cells,53 while an almost complete suppression of neurokinin-3 nigral binding was observed after local 6-hydroxydopamine lesion.50 In contrast, levels of nigral mRNA for neurokinin-1 receptors were not affected after destruction of DA neurons with 6-hydroxydopamine.49 Interestingly, Keegan et al.22 reported that 75% of DA nigral neurons were excited by senktide, and a slightly lower (70%) proportion of senktide-sensitive DA cells were estimated by Seabrook et al.47 in the rat ventral tegmental area. The high proportion of senktide-sensitive type I/DA neurons found in our study (92%) would indicate that, in the SNc of guinea-pig, almost all DA cells express neurokinin-3 receptors, the activation of which causes an increase in spontaneous neuronal firing rate. Differential sensitivity of non-dopaminergic nigral neurons to tachykinin agonists Another important aspect of this study is the report for the first time of a selective response of nigral cells to neurokinin-1 receptor stimulation in vitro. The efficacy of [Sar9,Met(O2)11]SP observed in our experiments was close to that reported in rat medial habenula neurons in vitro.34 This response was found exclusively in neurons identified as type III/non-DA (presumably GABAergic), located in the pars compacta. Several observations indicate that the rate-enhancing effect of [Sar9,Met(O2)11]SP was mediated through neurokinin-1 receptor activation. First, selective neurokinin-2 and neurokinin-3 receptor agonists were without any effect in these neurons.

Secondly, responses to [Sar9, Met(O2)11]SP of type III neurons were suppressed by the selective neurokinin-1 receptor antagonist SR 140333, whereas these responses were affected neither by SR 140603 (the R-enantiomer of SR 140333 with low affinity for neurokinin-1 receptors) nor by the selective neurokinin-3 receptor antagonist SR 142801. As expected, type III neurons in the SNc were also sensitive to SP, the natural preferential neurokinin-1 receptor agonist. In agreement with the data obtained on central34 or peripheral9 neurokinin-1 receptor models, this peptide appeared to be a slightly more potent agonist than [Sar9,Met(O2)11]SP. Our electrophysiological data indicating that, in the guinea-pig SNc, presumed GABAergic neurons and not DA neurons are sensitive to neurokinin-1 receptor stimulation are consistent with the observation that the level of neurokinin-1 receptor mRNA was unaffected after destruction of nigral DA neurons,50 and suggest that neurokinin-1 receptors might be expressed in GABAergic cells. According to previous in vivo18,55 and in vitro42,43,54 observations, fast-firing neurons located in the SNr have membrane properties similar to presumably GABAergic cells in the SNc, and therefore cannot be distinguished electrophysiologically. Our present data demonstrate pharmacological differences between these neurons, so that in the SNc they are sensitive to neurokinin-1 agonists, while in the SNr excitation was provoked exclusively by the selective neurokinin-3 receptor agonist senktide. Similarly to DA neurons, responses to senktide were likely to be mediated through neurokinin-3 receptors, since selective neurokinin-1 and neurokinin-2 agonists were without effect in these cells, and since senktide-evoked responses were inhibited by the selective neurokinin-3 receptor antagonist SR 142801. However, the significantly lower efficacy of senktide in SNr cells (50=91.3 nM compared to 14.7 nM in nigral DA neurons) does not exclude the existence of a subtype of neurokinin-3 receptor with a lower affinity for senktide. Alternatively, tonic autoinhibition through the recurrent collaterals could be another reason for the lower potency of senktide in these cells. This neuronal population might correspond to neurokinin-3 receptor-positive but THnegative (i.e. non-DA) neurons found occasionally in rat SN.53 Interestingly, nigral cells possessing reverse properties (i.e. TH positive, but neurokinin-3 receptor negative) were reported in the same study. Although our tachykinin-insensitive type II neurons still require further identification concerning their neurotransmitter phenotype, it is possible that they belong to this second minor DA neuronal population. Functional implications Contrary to the rather homogeneous population of DA neurons in the SNc, multiple electrophysiological

Tachykinin responses in guinea-pig substantia nigra

data indicate that fast-firing nigral neurons belong to different functional groups, composed of nigral interneurons (including those located in the SNr, which represent an important inhibitory input for DA cells in the SNc33) and output neurons projecting from both SNc and SNr to the striatum, the thalamus and other brain areas.18 Since we have never observed inhibitory effects of tachykinin receptor agonists, it is likely that putative nigral interneurons are not sensitive to neurokinins (otherwise their activation would result in a decrease of spontaneous firing rate in DA cells). We can further speculate that neurokinin-1 receptor agonist-sensitive neurons in the SNc constitute either a second, fast nigrostriatal inhibitory output,17 or the initial stage in the nigrothalamocortico-striatal loop, which has been demonstrated to facilitate presynaptically dopamine release from nigrostriatal terminals.6 The latter suggestion could explain the complex biphasic effect of the SP injection in the SN on the striatal dopamine release:19a while low doses of SP excited non-DA neurons through their neurokinin-1 receptors, applied at high doses it could evoke excitation of DA neurons through activation of their neurokinin-3 receptors.

755

CONCLUSION

The main conclusion which can be made on the basis of our results is that, in the guinea-pig SN, tachykinin agonists selectively affect different neuronal populations, thus contributing to the complex tachykinin–dopamine interaction. Taken together with the data concerning tachykinin receptor mRNA localization in the SN, our data provide an argument in favour of the proposal that the behavioural difference in the action of neurokinin-1 versus neurokinin-3 agonists39,51 is, at least in part, due to the selective activation of nigral output neurons (DA by neurokinin-3 agonists and non-DA by neurokinin-1 agonists).

Acknowledgements—This article is dedicated to the memory of Professor Paul Feltz, who died on 24th January 1996, in whose laboratory this work was initiated. We would like to thank Dr X. Emonds-Alt and Dr I. A. Lefevre for helpful comments on the manuscript.

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