GABAergic projection from the ventral tegmental area and substantia nigra to the periaqueductal gray region and the dorsal raphe nucleus

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Previous studies have shown that neurons in the ventral tegmental area (VTA) and substantia nigra (SN) project to the ventrolateral periaqueductal gray (PAGvl) and dorsal raphe nucleus (DR). Research has also shown that stimulation of neurons in the
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  GABAergic Projection from the VentralTegmental Area and Substantia Nigra tothe Periaqueductal Gray Region and theDorsal Raphe Nucleus GILBERT J. KIROUAC,* SA LI,  AND  GEHAN MABROUK Division of Basic Medical Sciences, Faculty of Medicine, Memorial University of Newfoundland, St. John’s, Newfoundland and Labrador, A1B 3V6, Canada ABSTRACTPrevious studies have shown that neurons in the ventral tegmental area (VTA) andsubstantia nigra (SN) project to the ventrolateral periaqueductal gray (PAGvl) and dorsalraphe nucleus (DR). Research has also shown that stimulation of neurons in the VTA/SNelicits cardiovascular depressor responses that are mediated by a projection to the PAGvl/DR. Anatomic and physiological experiments were done in the present study to determine theneurochemical identity of the VTA/SN projection to the PAGvl/DR. Experiments were done tocharacterize the srcin and chemical nature of this projection by combining cholera toxin Btracing with immunofluorescence for the 67K isoform of glutamic acid decarboxylase (GAD)and tyrosine hydroxylase. The PAGvl/DR region was found to receive a substantial inputfrom neurons in the VTA, SN, and deep mesencephalic nucleus. The DR was preferentiallyinnervated by neurons in the VTA, whereas the PAGvl was preferentially innervated byneurons in the SN. A proportion of neurons in the VTA and the reticular portion of the SNfound to project to the PAGvl/DR were GAD positive. In addition, experiments were done inurethane-anesthetized rats to determine whether injections of a  -aminobutyric acid (GABA)antagonist in the region of the PAGvl/DR attenuated the cardiovascular depressor responsesproduced by glutamate stimulation of the VTA/SN. Injections of the GABA-blocking agentpicrotoxin (2.5 nmol, 500 nl) into the PAGvl/DR eliminated the cardiovascular responses fromstimulation of the VTA/SN (0.01 M, 50 nl). The results of the present investigation provideevidence for a GABAergic projection from the VTA/SN to the PAGvl/DR. This projection maybe an important regulator of the PAGvl/DR, an area of the midbrain involved in the produc-tion of behavioral and physiological responses to pain and stress. J. Comp. Neurol. 469:170–184, 2004.  ©  2003 Wiley-Liss, Inc. Indexing terms: pain; cardiovascular regulation; dopamine; GABA; serotonin The periaqueductal gray matter (PAG) is well recog-nized for its role in the regulation of defensive behaviors,autonomic function, and nociception (Lovick, 1993; Ban-dler and Shipley, 1994; Behbehani, 1995). Bandler andcolleagues proposed that the PAG consists of functionallydistinct longitudinal columns that mediate specific phys-iological responses (see detailed reviews by Lovick, 1993;Bandler and Shipley, 1994; Behbehani, 1995; Bandler etal., 2000). For example, stimulation of the dorsolateralcolumn of the PAG with excitatory amino acids producesattack responses, hypertension, tachycardia, and an opi-oid independent antinociception. In contrast, stimulationof the ventrolateral column of the PAG (PAGvl) produceshypoactivity, hypotension, bradycardia, and an opioid de-pendent antinociception. The dorsal raphe nucleus (DR)embeddedinthecaudalportionofthePAGvlhasalsobeen Grant sponsor: Canadian Institute of Health Research; Grant number:42466.*Correspondence to: Gilbert J. Kirouac, Division of Basic Medical Sci-ences, Faculty of Medicine, Memorial University of Newfoundland, St.John’s, Newfoundland and Labrador, A1B 3V6, Canada.E-mail: gkirouac@mun.caReceived 17 March 2003; Revised 14 July 2003; Accepted 22 September2003DOI 10.1002/cne.11005Published online the week of December 15, 2003 in Wiley InterScience( THE JOURNAL OF COMPARATIVE NEUROLOGY 469:170–184 (2004) ©  2003 WILEY-LISS, INC.  implicated in antinociception and cardiovascular regula-tion (Robinson et al., 1986; Connor and Higgins, 1990;Lovick, 1993; Wang and Nakai, 1994) and is believed toplay an important role in regulating neural activity in thePAG (Lovick, 1993).The PAGvl receives afferents from several brain regionsimplicated in the regulation of the physiological responsesassociated with emotional reactions (Bandler and Shipley,1994; Behbehani, 1995). The brain regions with the mostprominent projections to the PAGvl and DR (PAGvl/DR)include the limbic cortices, hypothalamus, amygdala, dor-sal horn of the spinal cord, and a variety of brainstemregions associated with the regulation of the autonomic andsomatic nervous system (Sakai et al., 1977; Beitz, 1982;Marchand and Hagino, 1983; Peyron et al., 1995). Thesrcins of afferent projections to the PAGvl/DR are consis-tent with the hypothesis that this brain region serves tointegrate a variety of neural inputs to produce distinctivephysiological and behavioral responses involved in thesurvival of an organism (Lovick, 1993; Bandler and Ship-ley, 1994; Bandler et al., 2000). Previous tract tracing studies have demonstrated a projection from the ventraltegmental area (VTA) and the substantia nigra (SN) to thePAGvl/DR region (Sakai et al., 1977; Beckstead et al.,1979; Simon et al., 1979; Marchand and Hagino, 1983;Kale´n et al., 1988; Beitz, 1982; Peyron et al., 1995). In arecent study, Kirouac and Pittman (2000) demonstrated afunctional link between VTA and SN (VTA/SN) and thePAGvl/DR by showing that the cardiovascular depressorresponses elicited by stimulation of the VTA/SN were at-tenuated by injections of lidocaine and cobalt chloride intoPAGvl/DR region. In the same study, Kirouac and Pitt-man (2000) reported a large number of fiber terminals inthe PAGvl/DR after injections of biotinylated dextranamine into the VTA/SN. However, the location of the neu-rons in the VTA/SN that project to the PAGvl/DR couldnot be establish because injections of biotinylated dextranamine often involved several anatomically distinct regionsof the VTA/SN, including the reticular and compact por-tion of the substantia nigra (SNR and SNC, respectively)as well as the transitional region between the VTA andthe SNC (Kirouac and Pittman, 2000). An anatomic projection from the VTA/SN to thePAGvl/DR is of potential importance because of severalinvestigations reporting that the VTA and SN play a rolein modulating pain and arterial blood pressure, two func-tions that are also regulated by the PAGvl/DR (Lovick,1993; Bandler and Shipley, 1994; Behbehani, 1995; Bandleret al., 2000). For example, injections of    -aminobutyricacid (GABA) agonists or morphine into the SN have an-tinociceptive effects in anesthetized and conscious rats(Jurna et al., 1978; Barnes et al., 1979; Baumeister andFrye, 1986; Frye et al., 1986; Baumeister et al., 1987,1988, 1989, 1990, 1993; Baumeister, 1991; Hebert et al.,1990). Injections of morphine or substance P into the VTA also elicit antinociception (Altier and Stewart, 1993, 1996,1997, 1998; Morgan and Franklin, 1990). Several studieshave also implicated the VTA and SN in cardiovascularregulation. For example, stimulation of the VTA and theSN with excitatory amino acids produces cardiovasculardepressor responses in anesthetized rats (Kirouac andCiriello, 1997; Zhang et al., 1997; Kirouac and Pittman,2000), which appear to be mediated by a projection to thePAG (Kirouac and Pittman, 2000). It is also possible thatthe antinociception produced by stimulation of the VTA and SN may be mediated by a descending projection to thePAGvl, especially considering the prominent role that thePAGvl plays in the modulation of pain (Lovick, 1993;Bandler and Shipley, 1994; Behbehani, 1995; Bandler etal., 2000).Despite the potential significance of a connection be-tween these midbrain regions, the projection from the VTA/SN to the PAGvl/DR has not been the subject of detailed anatomic studies. The aim of the present studywas to describe the projections from the VTA/SN to thePAGvl/DR to determine the putative neurotransmitterusedbythisprojection.Theretrogradetracttracercholeratoxin B (CTb) was used to label VTA/SN neurons project-ing to the PAGvl/DR. Double-labeling experiments werealso done in an attempt to determine the neurochemicalidentity of the projection. In addition, physiological exper-iments were done to provide functional evidence for theexistence of this projection. MATERIALS AND METHODS The experimental protocols for this research were doneaccording to the guidelines set by the Canadian Council on Animal Care and were approved by Memorial Universityof Newfoundland. Injection of tracers Male Sprague-Dawley rats, each weighting 300 to 350 g,were used for the experiments. Rats were anesthetizedwith equithesin (0.3 ml/100 g, i.p.) and given supplemen-tary doses (0.15 ml/100 g) as needed during the surgicalprocedure. Rats were placed in a Kopf stereotaxic frame,with the incisor bar set at 3.3 mm below the interauralline (Paxinos and Watson, 1986). A burr hole of approxi-mately 1 mm in diameter was made over the PAG to injectCTb by using glass micropipettes (20–25   m tip diame-ters). The low-salt version of CTb was reconstituted withdistilled water to make a 0.5% CTb solution in 0.1 Msodium phosphate (List Biological Laboratory, Campbell,CA). Iontophoretic injections of CTb were done by apply-ing a 3- to 5-   A positive current (200 msec pulses at 2 Hzfor 10 to 15 minutes) through a chlorinated silver wireplaced in the pipette. The coordinates used for injecting CTb into the caudal PAGvl region were 1.5 to 2.0 mmanterior to the interaural line, 1.0 lateral to the midline,and 5.0 to 5.5 mm below the dorsal surface of the brain.Control injections were also done outside the PAGvl bymaking adjustments to the above coordinates. The scalpincision was sutured and rats were returned to their cagesfor recovery. Perfusion and tissue processing  After a survival period of 7 to 14 days, rats were deeplyanesthetized and perfused with 150 ml of saline followedby 500 ml of fixative composed of 4% paraformaldehyde in0.1 M phosphate buffer (pH 7.4). Brains were removed andpost-fixed at room temperature for 1 hour before being transferred to a 20% sucrose solution and stored at 4°Covernight. Coronal sections of the midbrain were cut at 30to 50  m by using a freezing microtome or a cryostat andcollected in PBS. All immunohistochemical reactions weredone on free-floating sections. Antibodies were diluted in0.1 M PBS containing normal donkey serum (5 to 10%),0.3% Triton X-100, and 0.1% sodium azide. After rinses inPBS (3    10 minutes), sections were incubated in goat 171GABAergic PROJECTION TO THE PAG  anti-CTb (1:10,000 to 40,000; List Biological Laboratories)at 4°C for 1 to 2 days. Sections were rinsed and thenincubated in biotinylated donkey anti-goat (1:500; Jack-son ImmunoResearch, West Grove, PA) for 2 hours fol-lowed by rinses and 1-hour incubation in an avidin-biotincomplex, according to kit directions (Elite ABC Kit, VectorLaboratories, Burlingame, CA). This step was followed bythree rinses, incubation in diaminobenzidine for 5 to 10minutes, according to kit directions (diaminobenzidineperoxidase substrate kit, Vector Laboratories), and sev-eral more rinses. The slide-mounted tissue was dried atroom temperature and coverslips were applied by using Fluka DPX mountant. For CTb and tyrosine hydroxylase(TH) double-labeling experiments, the tissue was reactedin a combination of goat anti-CTb (1:10,000–1:40,000; ListBiological Laboratories) and a monoclonal mouse anti-TH(1:40,000; Sigma). For the CTb and glutamic acid decar-boxylase (GAD) double-labeling experiments, we used acombination of goat anti-CTb and a monoclonal mouseanti-GAD (1:500; Chemicon International, Temecula, CA)at 4°C for 1 to 2 days. This GAD antibody recognizes the67K isoform of GAD, which is preferable for labeling cellbodies (Esclapez et al., 1994; Dirkx et al., 1995). After rinses, the brain sections were incubated in another mix-ture of antibodies containing Cy 2 -conjugated donkey anti-goat (1:500; Jackson ImmunoResearch) and Cy 3 -conjugated donkey anti-mouse for 2 hours (1:500; JacksonImmunoResearch). After a few more rinses, tissue sec-tions were mounted on slides and cover-slipped.Control experiments were done by removing the pri-mary antibody against CTb, TH, and GAD, which resultedin the absence of the labeling observed when the immu-nochemical reactions were done with the primary anti-body present. Triton X-100 was used at a concentration of 0.01% in the blocking buffer for experiments to show GAD,because this strategy resulted in more visible labeling of neurons (unpublished observations). To increase the de-tectable levels of GAD in the cell body of neurons, rats thathad previously received CTb injections in the PAGvl/DRwere treated with 100   g of colchicine (6   l, Sigma) in- jected into the lateral ventricles 24 hours before perfusionwith fixative.  Analysis of CTb, TH, and GADImmunohistochemistry  Coronal sections from the caudal region of the PAG tothe anterior region of the VTA/SN were examined using alight microscope. The location of the CTb injection site wasdefined, and the distribution of retrogradely labeled cellswas analyzed. In some representative cases, the locationof CTb-labeled neurons that resulted from injections of CTb in the different regions of the PAG, were drawn byusing a drawing tube. Double-labeled neurons were ana-lyzed with an Olympus fluorescent microscope (BX51)equipped with appropriate filter cubes for Cy 2  (U-MNB2,Olympus), Cy 3  (U-MNG2, Olympus) or combination nar-row green and blue filter (U-M51006ZZ, Olympus) com-bined with a blue/green excitation balancer (U-EXBABG,Olympus) to optimize the contrast under the single filtercube. We counted the number of CTb-labeled cells in the VTA/SN (CTb  ) that were also labeled for TH (TH  ) orGAD (GAD  ). Counts were done manually on four repre-sentative sections that represented the rostrocaudal ex-tent of the VTA and SN (approximate levels representedin Fig. 3B–E) in four rats that had CTb injections confinedthe PAGvl and wings of the DR. Counts were classifiedaccording to the area in which the CTb   neurons werefound, including the VTA, SNC, SNR and lateral portionof the SN (SNL). Photomicrographs were produced byusing a digital camera (Spot RT Slicer, Diagnostic Instru-ments, Sterling Heights, MI) and the images were im-ported in Adobe Photoshop 5.5 for contrast and color en-hancement. Physiological experiments Experiments were done on 10 male Sprague-Dawleyrats (each 300–450 g) that were anesthetized using ure-thane (1.6 g/kg, i.p.). Polyethylene catheters were insertedinto the femoral artery and vein for the recording of arte-rial pressure and the administration of drugs, respec-tively. Rectal temperature was monitored and maintainedat 35–37°C with a heating pad. Experiments were donewith rats placed in a stereotaxic frame with the nose baradjusted 3.3 mm below the interaural line (Paxinos andWatson, 1986). Arterial blood pressure was recorded by using a pres-sure transducer (MLT1050; ADInstruments, Mountain View, CA) connected to a bridge amplifier (ML110; ADIn-struments). The electronic signal for arterial pressure wasdigitized using a PowerLab 4SP device and software (AD-Instruments), and the data were captured and analyzedon a computer (Chart 4.0 data capture and analysis soft-ware; ADInstruments). Heart rate was calculated onlinefrom the pressure pulse, and the mean arterial pressure(MAP) was calculated as the diastolic pressure plus onethird of the pressure pulse. Glass micropipettes with tipdiameters of 30–50   m containing a 0.01 M glutamatesolution (sodium salt, Sigma) dissolved in 0.1 M PBS werelowered in the ventral midbrain on the right side (5.0–5.5mm caudal to bregma, 0.8–1.3 mm lateral to the midline,and 6.5–8.5 mm ventral to the dura), and 50 nl of theglutamate solution were microinjected by the applicationof pressurized nitrogen pulses controlled by a pneumaticpump (Medical Systems, Great Neck, NY). The injected volumes were measured by the direct observation of thefluid meniscus in the micropipettes with an engineering microscope fitted with a micrometer (Titan, Buffalo, NY).Regions of the VTA/SN were stimulated with glutamate tolocate a site that produced a depressor response of at least15 mm Hg. A minimal distance of 300  m separated eachinjection site. It was established previously that injectionof the vehicle in the VTA/SN does not produce changes inarterial pressure or heart rate (Kirouac and Ciriello, 1997;Zhang et al., 1997; Kirouac and Pittman, 2000). The car-diovascular depressor responses were retested after ad-ministration of the GABA-blocking substance picrotoxin(2.5 nmol/500 nl; Sigma) in the region of the PAGvl/DR.Glass micropipettes (50- to 75-  m tip) were used to micro-inject picrotoxin with pressure pulses (8.0 mm posterior tobregma; 0.5 mm lateral to the midline; 5.0 mm ventral tothe dura; angled 6 degrees in the posterior direction). Thefollowing protocol was used: (1) a site in the VTA/SN thatproduced a depressor response of a minimum of 15 mm Hg was identified; (2) 15 minutes later, picrotoxin was in- jected in the PAGvl/DR; (3) the VTA/SN site was restim-ulated at 5, 30, and 60 minutes after the picrotoxin injec-tion. Only one experiment was done in each animal, whichwas followed by transcardial perfusion of 100 ml of salinefollowed by 200 ml of 10% formalin solution. Sections of the brain were cut on a cryostat and stained with thionin 172 G.J. KIROUAC ET AL.  to verify the location of the microinjections. The effects of administration of picrotoxin in the PAGvl/DR on the de-pressor responses were analyzed by using two-way anal-ysis of variance (ANOVA) with repeated measures fortime combined with post hoc analysis by using Tukey’smultiple comparison test. A   P  value of   0.05 was taken toindicate a statistical difference. Values are expressed asmeans  standard error. RESULTSRetrograde tracing of the VTA/SNprojection to the PAGvl/DR Cholera toxin B injections were done in various portionsof the caudal half of the PAG and parts of the DR embed-ded in the PAGvl (Fig. 1A). The injections were subdividedinto different groups according to the extent of diffusion of the tracer. For description of the data, we will considerinjections that were largely confined to the PAGvl (n  8),DR (n    4), and injections involving the wings of the DRand parts of the PAGvl (n  3). Control injections of CTbwere also made in the dorsolateral region of the PAG (n  4) and in the region of the nucleus cuneiformis (n  4).Figures 2A, 3F, and 7A show the distribution of neuronslabeled in the midbrain in one rat after an injection of CTb, which was largely restricted to the PAGvl. Retro-gradely labeled neurons were found bilaterally in the ros-trocaudal extent of the PAG as well as areas around themedial lemniscus. A large number of retrogradely labeledneurons were found in the SN below the medial lemniscus(Figs. 2, 3, 7D–F) in both the SNC and SNR. A few CTb-labeled neurons were found scattered in various regions of the VTA (Figs. 3, 7B,E). A large number of neurons werealso retrogradely labeled in the SNL and the deep mesen-cephalic nucleus (DpMe) located dorsal to the SNL (Figs.2C, 3, 7C).Figure 4 shows a representative case of retrograde la-beling after an injection of CTb confined to the DR. Asbefore, the projection is bilateral in srcin with largernumbers of cells found in the VTA (Fig. 4B–E) than whatwas observed after injections of CTb in the PAGvl (Fig. 3).This midline to lateral arrangement in the VTA/SN wasconsistently seen in the injections that involved either theDR or PAGvl. Neurons were also labeled in regions aroundthe medial lemniscus, including in the region of the SNCand the dorsomedial aspect of the SNR. Injections of CTbinto the dorsolateral region of the PAG resulted in theretrograde labeling of a few neurons in the VTA or SN(Fig. 5). As with injections in the PAGvl and DR, CTbinjections in the dorsolateral PAG resulted in a largenumber of CTb-labeled neurons in the lateral region of theDpMe (Fig. 5). Control injections of CTb outside the PAGin the DpMe and the nucleus cuneiformis resulted inretrograde labeling in mostly the SNL and DpMe regionimmediately adjacent to the SNL (Fig. 6). Double-labeling experiments to determineneurotransmitter Double-labeling experiments were done to determinethe putative neurotransmitter contained in the projection(dopamine or GABA). Figure 1B illustrates the location of the CTb injections used for these double-labeling immu-nofluorescence experiments (n    8). The injections wererestricted to either the PAGvl or the DR, or involved acombination of these regions. Figure 7 shows the distribu-tion of CTb-labeled neurons in the VTA (Fig. 7B,E) and SN(Fig. 7D–F) after injections of CTb in the PAGvl/DR region(Fig. 7A) relative to TH-labeled neurons. CTb-labeled neu-rons found to project to the PAGvl/DR were found inter-spersed with TH-labeled neurons in the VTA and SN. Wefound a considerable overlap in the location of the CTb-labeled and TH-labeled neurons in the transition zonebetween the SNC and SNR (Fig. 7F). However, when weexamined the sections at higher magnification, we did notfind CTb-labeled neurons that were double labeled for TH(in four rats with CTb injections in the PAGvl/DR). Countsfor CTb and TH double-labeled cells are not reported here,because there was no evidence of double-labeling for thesemarkers.Tissue sections immunoreacted for both CTb and GADneurons showed a proportion of neurons double labeled forboth CTb and GAD (see Fig. 8 for examples). Double-labeled neurons (those that were retrogradely labeled byCTb and labeled for GAD) were counted in four represen-tative levels of the VTA/SN in four rats that had injectionslimited to the PAGvl/DR. The numbers of CTb   /GAD  neurons were 47/15 (32%) for the VTA; 198/75 (38%) forthe SNR; 78/7 (9%) for the SNC; 167/19 (11%) for theDpMe. It is possible that this finding represents an un-derestimation of the total number of CTb   /GAD   neu-rons making up the projection because several CTb   neu-rons were weakly stained for GAD and were not consideredas double-labeled neurons (see Fig. 8 for examples). Physiological experiments Injections of the GABA-blocking agent picrotoxin in thePAGvl/DR produced no immediate cardiovascular re-sponses. However, it did result in a trend toward an in-crease in MAP that did not reach significance level (prepi-crotoxin, 82.8    4.2 mm Hg; 5 minutes postpicrotoxin,99.7  6.5 mm Hg; 30 minutes postpicrotoxin, 94.2  7.1mm Hg; n    5), which was sometimes more apparent insome experiments (Fig. 9). In some cases, we noticed brief increases of approximately 1 minute in respiratory activ-ity after injections of picrotoxin, which occurred with theincreases in MAP. The ANOVA indicated that injections of picrotoxin attenuated the depressor responses elicited byactivation of the VTA/SN with glutamate (  F  3,19    3.238,  P    0.008; Fig. 9). The baseline response to VTA/SNstimulation was attenuated by 69% at 5 minutes (  P   0.05;n  5,Fig.9B,C)andby74%at30minutes(  P  0.05;n  5, Fig. 9B,C) postpicrotoxin administration. Restimu-lation of the same site in the VTA/SN at 60 minutespostpicrotoxin resulted in depressor response of similarmagnitude as the baseline response (Fig. 9B,C). Stimula-tion of the VTA/SN produced small and inconsistentchanges in HR responses that were unaffected by picro-toxin injections in the PAGvl/DR (data not shown). Thesites of the picrotoxin injections were verified on histologicsections to be located in the caudal portion of thePAGvl/DR region (Fig. 9A). Control injections of saline inthe PAGvl/DR had no effect on the cardiovascular depres-sor responses elicited from repeated stimulation of thesame site in the VTA/SN (n  5; Fig. 9B). DISCUSSION Four major observations can be made from results of the present study: (1) the PAGvl and DR receive a 173GABAergic PROJECTION TO THE PAG  substantial input from neurons in the VTA, SN, andDpMe; (2) this projection does not appear to use dopa-mine as a transmitter substance (TH negative); (3) aportion of VTA and SN neurons projecting to thePAGvl/DR likely use GABA as a transmitter substance(GAD positive); (4) the cardiovascular depressor re-sponses elicited by activation of neurons in the VTA/SNappear to be mediated by GABA release in the PAGvl/ DR, because picrotoxin injections in the PAGvl/DRblocked the responses. Fig. 1.  A,B:  Schematic diagram showing the location of cholera toxin B injections in the periaque-ductal gray region and the dorsal raphe nucleus for the immunohistochemical tract-tracing experiments(A) or the double-labeling immunofluorescence experiments (B). DR, dorsal raphe nucleus; PAGdl,dorsolateral periaqueductal gray; PAGvl, ventrolateral periaqueductal gray. 174 G.J. KIROUAC ET AL.
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