22
Brain Research, 524 (1990) 22-30 Elsevier
BRES 15723
Inhibition of spontaneous and evoked unit activity in the rat medial prefrontal cortex by mesencephalic raphe nuclei J. Mantz, R. Godbout, J.-P. Tassin, J. Glowinski and A.-M. Thierry Chaire de Neuropharmacologie, 1.N.S.E.R.M. U. 114, CollOge de France, Paris (France) (Accepted 30 January 1990) Key words: Prefrontal cortex; Raphe nuclei; Serotonin; Noxious tail pinch; Mediodorsal thalamus
The rat medial prefrontal cortex (PFC) receives a serotoninergic (5-HT) innervation which originates from the mesencephalic raphe nuclei. In the present study we determined the influence of the 5-HT ascending systems on the spontaneous and evoked activity of PFC neurons in anesthetized rats. Stimulation of the dorsal (DRN) and of the median raphe (MRN) nuclei inhibited the spontaneous activity of 35.0% and 52.8% of the PFC cells tested (mean duration of the inhibition: 75.5 and 82.2 ms, respectively). These inhibitory responses are likely mediated by the 5-HT-containing neurons since they were decreased markedly following selective destruction of ascending 5-HT pathways induced by local injections of 5,7-dihydroxytryptamine. Moreover, the inhibitory effect of MRN stimulation could be blocked by systemic administration of the 5-HT2 receptor antagonists: ketanserin and ritanserin. The effects of MRN stimulation on two types of evoked responses were studied. The excitatory responses of PFC neurons induced by the stimulation of the mediodorsal nucleus of the thalamus (MD) were inhibited by MRN stimulation applied before that of MD. Similarly, the activation of PFC cells induced by a noxious tail pinch was suppressed by a concomitant stimulation of the MRN. These results indicate that 5-HT neurons exert an inhibitory control on spontaneous or evoked activity in the rat PFC. INTRODUCTION T h e rat medial prefrontal cortex (PFC) is one of the cortical areas innervated by the 3 aminergic ascending systems 1°'22. In a previous study, we have shown that the ascending d o p a m i n e r g i c ( D A ) and noradrenergic ( N A ) systems which originate respectively from the ventral t e g m e n t a l area ( V T A ) and the locus coeruleus (LC) m o d u l a t e m a r k e d l y neuronal activity in the P F C 23. H o w e v e r , m a j o r differences in the influence of D A and N A afferents on the spontaneous activity or e v o k e d responses of P F C cells were observed. I n d e e d , activation of the D A system induced a phasic inhibition of the spontaneous activity of P F C neurons and blocked the excitatory responses elicited by thalamic stimulation or by a noxious p e r i p h e r a l stimulus. In contrast, activation of the N A system p r o d u c e d a long-lasting inhibition of the basal firing of P F C neurons without blocking the e v o k e d responses e3. In o r d e r to compare the respective influences of the catecholaminergic and serotoninergic (5-HT) systems, we are now reporting on the control exerted by 5-HT neurons on the activity of PFC target cells. In the PFC, 5-HT fibers are distributed in all layers with a greater density in layer I, and originate from the dorsal ( D R N ) and median ( M R N ) raphe nuclei 4"5"8"12~34.
R a p h e nuclei stimulation has b e e n r e p o r t e d to induce mainly an inhibition of the activity of cortical cells r e c o r d e d in cingulate, parietal, and sensorimotor cortex 15'26"33. Neurons from various areas of the cerebral cortex are sensitive to m i c r o i o n t o p h o r e t i c application of 5-HT, but both excitatory and inhibitory responses have been o b s e r v e d 7'15'18'26"32"33"37. O n the o t h e r hand, radioligand binding techniques have r e v e a l e d the presence of several 5-HT r e c e p t o r subtypes in the central nervous system 14'3°. Quantitative a u t o r a d i o g r a p h i c m a p p i n g have shown that P F C is one of the regions containing primarily 5-HT 2 receptors 28. The aim of the present study was to analyze: (1) the effects of electrical stimulation of midbrain raphe nuclei on the activity of P F C neurons; (2) whether these effects are m e d i a t e d by 5 - H T neurons; and (3) the influence of M R N stimulation on the e v o k e d excitatory responses elicited either by stimulation of the M D or by noxious tail pinch. MATERIALS AND METHODS Experiments were performed on 51 male Sprague-Dawley rats (250-300 g body weight). Forty-eight rats were anesthetized with ketamine (80 mg/kg, i.p.), and additional injections (80 mg/kg, i.m.) were periodically delivered to maintain a stable level of anesthesia. Three other rats were anesthetized with halothane (0.8-1.0% in air)
Correspondence: A.-M. Thierry, I.N.S.E.R.M.U. 114, Coll6ge de France, Chaire de Neuropharmacologie, 11, Place Marcelin Berthelot, 75231 Paris Cedex 05, France. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
23
a
b
C~
w !
~
~ili~i~iii!!~~ i i ~ i ~ l l n ~ - ~ ii~i¸
~ ~ i ~ W
d
!
1trim Fig. 1. Photomicrographs of Nissl-stained brain sections. In a, the thick arrowhead points to a recording site in the PFC. In b, c and d, thick arrowheads point to stimulating sites in the MD, the VTA and the MRN, respectively.
delivered intratracheally by a continuous flow pump. Body temperature was monitored with a rectal probe connected to a thermostatequipped heating pad set at 37 °C. Animals were fixed in a stereotaxic head frame. Anatomical coordinates were determined relative to the interaurai line according to the atlas of Paxinos and Watson 27. Bipolar co-axial stimulation electrodes (300 gm tip to barrel, 200/~m diameter) were positioned in the DRN (A 1.36 ram, L 0.0 mm, H 3.4 mm), MRN (A 1.2 ram, L 0.0 mm, H 1.6 mm), MD (A 6.7 ram, L 0.8 ram, H 4.6 ram) and VTA (A 3.7 mm, L 0.5 ram, H 2.0 ram). Electrical stimuli consisted of square-wave pulses (0.5 ms duration, 50-150 # A intensity) delivered at a frequency of 1-10 Hz. Noxious (non-traumatic) tail pinch was applied for 10 s with surgical forceps. The activity of PFC neurons (A 11.7 to 13.2 mm, L 0.4 to 1.4 mm, 0.6 to 3.5 mm from the dura) was recorded with glass micropipettes filled with 4% Pontamine sky blue dissolved in a 0.4 M NaC1 solution (impedance 6-10 Mr2). Extracellular activity was amplified with a WPI DAM-5A differential preamplifier (filters: 100 Hz lowfrequency and 30 kHz high-frequency) and displayed on a memory oscilloscope. The action potentials were separated from the noise by means of a window discriminator and then fed to a digital computer (CED 1401 interface connected to an IBM PC) to generate on line rates or peristimulus time histograms. Antidromic spikes were identified by their fixed latency, collision with a spontaneous spike, and the ability to follow high frequency stimulation. PFC neurons normally exhibit a broad range of activity, from an almost silent pattern to 30 spikes/s. Inhibitory responses were analyzed on neurons which had a spontaneous activity higher than 0.5 spikes/s.
The responses induced by raphe nuclei or VTA stimulation were analyzed on peristimulus time histograms corresponding to 50 cumulative sweeps. Inhibitory periods were defined as the duration of complete cessation of firing after the stimulus. MRN stimulation was considered to inhibit excitatory responses evoked by MD stimulation or tail pinch when these were reduced by at least 50%. 5-HT denervation was achieved in 10 rats by means of bilateral microinjections of 5,7-dihydroxytryptamine (5,7-DHT, Sigma; 6/zg diluted in 1 gi of isotonic saline and delivered over 5 min) into the medial forebrain bundle (A 6.44 mm, L +1.8 mm, H 1.4 mm). Desmethylimipramine (Ciba Geigy; 25 mg/kg) and benztropine methane sulfate (Merck, Sharpe and Dohme; 30 mg/kg) were injected i.p. 30 min before 5,7-DHT microinjections in order to prevent the destruction of catecholaminergic neurons. Experimental recording sessions were performed 2 weeks after the microinjections. Following the recording sessions, lesioned and control (sham-operated) animals were sacrificed and their brains quickly removed and frozen for determination of 5-HT, D A and NA PFC content. This was performed by high-performance liquid chromatography (HPLC) with electrochemical detection 36. The effects of the 5-HT 2 receptor antagonists ketanserin (Janssen Pharmaceutica; 2.0 mg/kg, i.p.) and ritanserin (Janssen; 4.0 mg/kg, i.p.) on MRN- and VTA-induced inhibitions were analyzed in 11 and 5 rats, respectively. The effects of MRN and VTA stimulation were tested before and every 5 min following the drug administration. At the end of each experiment, recording sites were marked by the iontophoretic ejection of Pontamine sky blue (8/~A of cathoda! current for 20 min). Stimulation sites were marked by the electrical
24 deposit of iron at the electrode tip (10/~A of anodal current for 15 s) followed by a ferri-ferrocyanide reaction. The resulting blue points were observed on serial frozen sections (80 /~m) after conventional histological treatment (see Fig. 1).
Statistical analysis Results are expressed as means + S.E.M. and n refers to the number of neurons tested. Statistical differences between the effects of DRN, MRN and VTA stimulation in the control and 5, 7-DHT-treated groups were tested by the x2-method. The effect of noxious tail pinch and the influence of MRN stimulation on this effect were evaluated with the paired Student's t-test. RESULTS
Effects o f M R N and D R N stimulations on the spontaneous activity o f prefrontal cortical neurons M R N stimulation at a frequency of 1 Hz inhibited 52.9% of the PFC n e u r o n s recorded (n = 210, 9 rats) with an inhibition duration of 82.2 _+ 4.1 ms and a latency of 17.8 _+ 1.5 ms (Table I). Only 4 n e u r o n s responded by an excitation following M R N stimulation. The firing of the remaining cells tested was not affected by M R N stimulation. In 3 animals anesthetized with halothane, similar results were obtained. W h e n the stimulating electrode was placed outside the M R N (in the pontine reticular nucleus), the n u m b e r of inhibited cortical cells was only 16% (56 units tested). These results were excluded from the statistical analysis. D R N stimulation at 1 Hz was found to inhibit 35.0% of the PFC n e u r o n s recorded (n = 60, 4 rats) with a duration of inhibition of 75.5 _+ 5.0 ms and a latency of 17.5 + 2.5 ms (Table I). No excitatory response to D R N stimulation was observed. W h e n e v e r a PFC n e u r o n was inhibited by D R N stimulation, it was also inhibited by M R N stimulation, but the reverse was not true. A n t i d r o m i c responses in PFC cells were observed after stimulation of M R N (n = 39) and D R N (n = 5), with a m e a n latency of 12.2 _+ 1.3 and 16.0 + 2.3 ms, respectively. Based on these results, the m e a n conduction velocities of PFC cells projecting to M R N and D R N were estimated to be 1.3 and 0.9 m/s, respectively. Inhibitory responses were observed in 73.1% and 60.0% of these cells after M R N or D R N stimulation. Finally, the effects of raphe nuclei stimulation on the activity of PFC cells were compared to those of VTA stimulation in the same animals. The inhibitory responses induced by D R N or M R N stimulation were of shorter duration than those obtained following V T A stimulation (see Table I and Fig. 2). Effects o f M R N and D R N stimulation in 5, 7-DHT-treated rats Microinjection of 5 , 7 - D H T induced a 93.8 + 5.5% decrease of 5-HT levels in the PFC of treated animals relative to control values whereas D A and N A levels
TABLE I
Inhibitory responses of prefrontal cortical neurons induced by the stimulation of dorsal raphe nucleus, median raphe nucleus, and ventral tegmental area in control and in 5, 7-dihydroxytryptaminelesioned rats Durations and latencies of inhibitions are expressed as means + S.E.M. in ms.
MRN Controls 5,7-DHT DRN Controls 5,7-DHT VTA Controls 5,7-DHT
No. of cells tested
No. of cells inhibited
210 238
111 (52.9%) 21 (8.8%)*
82.2 + 4.1"* 84.3 + 6.8
17.8 + 1.5 17.4 + 4.4
60 145
21 (35.0%) 8 (5.5%)*
75.5 + 5.0** 78.8 + 14.8
17.5 + 2.5 20.5 + 4.5
128 230
94 (75.4%) 145 (63.0%)
Duration of inhibition
119.4+ 8.2 123.6+ 10.8
Latency of inhibition
16.4 + 2.1 16.2 + 2.3
*P < 0.05, x
Brain Research, 524 (1990) 22-30 Elsevier
BRES 15723
Inhibition of spontaneous and evoked unit activity in the rat medial prefrontal cortex by mesencephalic raphe nuclei J. Mantz, R. Godbout, J.-P. Tassin, J. Glowinski and A.-M. Thierry Chaire de Neuropharmacologie, 1.N.S.E.R.M. U. 114, CollOge de France, Paris (France) (Accepted 30 January 1990) Key words: Prefrontal cortex; Raphe nuclei; Serotonin; Noxious tail pinch; Mediodorsal thalamus
The rat medial prefrontal cortex (PFC) receives a serotoninergic (5-HT) innervation which originates from the mesencephalic raphe nuclei. In the present study we determined the influence of the 5-HT ascending systems on the spontaneous and evoked activity of PFC neurons in anesthetized rats. Stimulation of the dorsal (DRN) and of the median raphe (MRN) nuclei inhibited the spontaneous activity of 35.0% and 52.8% of the PFC cells tested (mean duration of the inhibition: 75.5 and 82.2 ms, respectively). These inhibitory responses are likely mediated by the 5-HT-containing neurons since they were decreased markedly following selective destruction of ascending 5-HT pathways induced by local injections of 5,7-dihydroxytryptamine. Moreover, the inhibitory effect of MRN stimulation could be blocked by systemic administration of the 5-HT2 receptor antagonists: ketanserin and ritanserin. The effects of MRN stimulation on two types of evoked responses were studied. The excitatory responses of PFC neurons induced by the stimulation of the mediodorsal nucleus of the thalamus (MD) were inhibited by MRN stimulation applied before that of MD. Similarly, the activation of PFC cells induced by a noxious tail pinch was suppressed by a concomitant stimulation of the MRN. These results indicate that 5-HT neurons exert an inhibitory control on spontaneous or evoked activity in the rat PFC. INTRODUCTION T h e rat medial prefrontal cortex (PFC) is one of the cortical areas innervated by the 3 aminergic ascending systems 1°'22. In a previous study, we have shown that the ascending d o p a m i n e r g i c ( D A ) and noradrenergic ( N A ) systems which originate respectively from the ventral t e g m e n t a l area ( V T A ) and the locus coeruleus (LC) m o d u l a t e m a r k e d l y neuronal activity in the P F C 23. H o w e v e r , m a j o r differences in the influence of D A and N A afferents on the spontaneous activity or e v o k e d responses of P F C cells were observed. I n d e e d , activation of the D A system induced a phasic inhibition of the spontaneous activity of P F C neurons and blocked the excitatory responses elicited by thalamic stimulation or by a noxious p e r i p h e r a l stimulus. In contrast, activation of the N A system p r o d u c e d a long-lasting inhibition of the basal firing of P F C neurons without blocking the e v o k e d responses e3. In o r d e r to compare the respective influences of the catecholaminergic and serotoninergic (5-HT) systems, we are now reporting on the control exerted by 5-HT neurons on the activity of PFC target cells. In the PFC, 5-HT fibers are distributed in all layers with a greater density in layer I, and originate from the dorsal ( D R N ) and median ( M R N ) raphe nuclei 4"5"8"12~34.
R a p h e nuclei stimulation has b e e n r e p o r t e d to induce mainly an inhibition of the activity of cortical cells r e c o r d e d in cingulate, parietal, and sensorimotor cortex 15'26"33. Neurons from various areas of the cerebral cortex are sensitive to m i c r o i o n t o p h o r e t i c application of 5-HT, but both excitatory and inhibitory responses have been o b s e r v e d 7'15'18'26"32"33"37. O n the o t h e r hand, radioligand binding techniques have r e v e a l e d the presence of several 5-HT r e c e p t o r subtypes in the central nervous system 14'3°. Quantitative a u t o r a d i o g r a p h i c m a p p i n g have shown that P F C is one of the regions containing primarily 5-HT 2 receptors 28. The aim of the present study was to analyze: (1) the effects of electrical stimulation of midbrain raphe nuclei on the activity of P F C neurons; (2) whether these effects are m e d i a t e d by 5 - H T neurons; and (3) the influence of M R N stimulation on the e v o k e d excitatory responses elicited either by stimulation of the M D or by noxious tail pinch. MATERIALS AND METHODS Experiments were performed on 51 male Sprague-Dawley rats (250-300 g body weight). Forty-eight rats were anesthetized with ketamine (80 mg/kg, i.p.), and additional injections (80 mg/kg, i.m.) were periodically delivered to maintain a stable level of anesthesia. Three other rats were anesthetized with halothane (0.8-1.0% in air)
Correspondence: A.-M. Thierry, I.N.S.E.R.M.U. 114, Coll6ge de France, Chaire de Neuropharmacologie, 11, Place Marcelin Berthelot, 75231 Paris Cedex 05, France. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
23
a
b
C~
w !
~
~ili~i~iii!!~~ i i ~ i ~ l l n ~ - ~ ii~i¸
~ ~ i ~ W
d
!
1trim Fig. 1. Photomicrographs of Nissl-stained brain sections. In a, the thick arrowhead points to a recording site in the PFC. In b, c and d, thick arrowheads point to stimulating sites in the MD, the VTA and the MRN, respectively.
delivered intratracheally by a continuous flow pump. Body temperature was monitored with a rectal probe connected to a thermostatequipped heating pad set at 37 °C. Animals were fixed in a stereotaxic head frame. Anatomical coordinates were determined relative to the interaurai line according to the atlas of Paxinos and Watson 27. Bipolar co-axial stimulation electrodes (300 gm tip to barrel, 200/~m diameter) were positioned in the DRN (A 1.36 ram, L 0.0 mm, H 3.4 mm), MRN (A 1.2 ram, L 0.0 mm, H 1.6 mm), MD (A 6.7 ram, L 0.8 ram, H 4.6 ram) and VTA (A 3.7 mm, L 0.5 ram, H 2.0 ram). Electrical stimuli consisted of square-wave pulses (0.5 ms duration, 50-150 # A intensity) delivered at a frequency of 1-10 Hz. Noxious (non-traumatic) tail pinch was applied for 10 s with surgical forceps. The activity of PFC neurons (A 11.7 to 13.2 mm, L 0.4 to 1.4 mm, 0.6 to 3.5 mm from the dura) was recorded with glass micropipettes filled with 4% Pontamine sky blue dissolved in a 0.4 M NaC1 solution (impedance 6-10 Mr2). Extracellular activity was amplified with a WPI DAM-5A differential preamplifier (filters: 100 Hz lowfrequency and 30 kHz high-frequency) and displayed on a memory oscilloscope. The action potentials were separated from the noise by means of a window discriminator and then fed to a digital computer (CED 1401 interface connected to an IBM PC) to generate on line rates or peristimulus time histograms. Antidromic spikes were identified by their fixed latency, collision with a spontaneous spike, and the ability to follow high frequency stimulation. PFC neurons normally exhibit a broad range of activity, from an almost silent pattern to 30 spikes/s. Inhibitory responses were analyzed on neurons which had a spontaneous activity higher than 0.5 spikes/s.
The responses induced by raphe nuclei or VTA stimulation were analyzed on peristimulus time histograms corresponding to 50 cumulative sweeps. Inhibitory periods were defined as the duration of complete cessation of firing after the stimulus. MRN stimulation was considered to inhibit excitatory responses evoked by MD stimulation or tail pinch when these were reduced by at least 50%. 5-HT denervation was achieved in 10 rats by means of bilateral microinjections of 5,7-dihydroxytryptamine (5,7-DHT, Sigma; 6/zg diluted in 1 gi of isotonic saline and delivered over 5 min) into the medial forebrain bundle (A 6.44 mm, L +1.8 mm, H 1.4 mm). Desmethylimipramine (Ciba Geigy; 25 mg/kg) and benztropine methane sulfate (Merck, Sharpe and Dohme; 30 mg/kg) were injected i.p. 30 min before 5,7-DHT microinjections in order to prevent the destruction of catecholaminergic neurons. Experimental recording sessions were performed 2 weeks after the microinjections. Following the recording sessions, lesioned and control (sham-operated) animals were sacrificed and their brains quickly removed and frozen for determination of 5-HT, D A and NA PFC content. This was performed by high-performance liquid chromatography (HPLC) with electrochemical detection 36. The effects of the 5-HT 2 receptor antagonists ketanserin (Janssen Pharmaceutica; 2.0 mg/kg, i.p.) and ritanserin (Janssen; 4.0 mg/kg, i.p.) on MRN- and VTA-induced inhibitions were analyzed in 11 and 5 rats, respectively. The effects of MRN and VTA stimulation were tested before and every 5 min following the drug administration. At the end of each experiment, recording sites were marked by the iontophoretic ejection of Pontamine sky blue (8/~A of cathoda! current for 20 min). Stimulation sites were marked by the electrical
24 deposit of iron at the electrode tip (10/~A of anodal current for 15 s) followed by a ferri-ferrocyanide reaction. The resulting blue points were observed on serial frozen sections (80 /~m) after conventional histological treatment (see Fig. 1).
Statistical analysis Results are expressed as means + S.E.M. and n refers to the number of neurons tested. Statistical differences between the effects of DRN, MRN and VTA stimulation in the control and 5, 7-DHT-treated groups were tested by the x2-method. The effect of noxious tail pinch and the influence of MRN stimulation on this effect were evaluated with the paired Student's t-test. RESULTS
Effects o f M R N and D R N stimulations on the spontaneous activity o f prefrontal cortical neurons M R N stimulation at a frequency of 1 Hz inhibited 52.9% of the PFC n e u r o n s recorded (n = 210, 9 rats) with an inhibition duration of 82.2 _+ 4.1 ms and a latency of 17.8 _+ 1.5 ms (Table I). Only 4 n e u r o n s responded by an excitation following M R N stimulation. The firing of the remaining cells tested was not affected by M R N stimulation. In 3 animals anesthetized with halothane, similar results were obtained. W h e n the stimulating electrode was placed outside the M R N (in the pontine reticular nucleus), the n u m b e r of inhibited cortical cells was only 16% (56 units tested). These results were excluded from the statistical analysis. D R N stimulation at 1 Hz was found to inhibit 35.0% of the PFC n e u r o n s recorded (n = 60, 4 rats) with a duration of inhibition of 75.5 _+ 5.0 ms and a latency of 17.5 + 2.5 ms (Table I). No excitatory response to D R N stimulation was observed. W h e n e v e r a PFC n e u r o n was inhibited by D R N stimulation, it was also inhibited by M R N stimulation, but the reverse was not true. A n t i d r o m i c responses in PFC cells were observed after stimulation of M R N (n = 39) and D R N (n = 5), with a m e a n latency of 12.2 _+ 1.3 and 16.0 + 2.3 ms, respectively. Based on these results, the m e a n conduction velocities of PFC cells projecting to M R N and D R N were estimated to be 1.3 and 0.9 m/s, respectively. Inhibitory responses were observed in 73.1% and 60.0% of these cells after M R N or D R N stimulation. Finally, the effects of raphe nuclei stimulation on the activity of PFC cells were compared to those of VTA stimulation in the same animals. The inhibitory responses induced by D R N or M R N stimulation were of shorter duration than those obtained following V T A stimulation (see Table I and Fig. 2). Effects o f M R N and D R N stimulation in 5, 7-DHT-treated rats Microinjection of 5 , 7 - D H T induced a 93.8 + 5.5% decrease of 5-HT levels in the PFC of treated animals relative to control values whereas D A and N A levels
TABLE I
Inhibitory responses of prefrontal cortical neurons induced by the stimulation of dorsal raphe nucleus, median raphe nucleus, and ventral tegmental area in control and in 5, 7-dihydroxytryptaminelesioned rats Durations and latencies of inhibitions are expressed as means + S.E.M. in ms.
MRN Controls 5,7-DHT DRN Controls 5,7-DHT VTA Controls 5,7-DHT
No. of cells tested
No. of cells inhibited
210 238
111 (52.9%) 21 (8.8%)*
82.2 + 4.1"* 84.3 + 6.8
17.8 + 1.5 17.4 + 4.4
60 145
21 (35.0%) 8 (5.5%)*
75.5 + 5.0** 78.8 + 14.8
17.5 + 2.5 20.5 + 4.5
128 230
94 (75.4%) 145 (63.0%)
Duration of inhibition
119.4+ 8.2 123.6+ 10.8
Latency of inhibition
16.4 + 2.1 16.2 + 2.3
*P < 0.05, x
