SUMMARY

1. In the present study, a comparison was made between thedistribution of tachykinin NK 1 and NK3 receptor immunoreac-tivity and the distribution of Fos-like immunoreactivity inducedby the tachykinin agonist substance P (SP) in the guinea-pigbrain.

2. In agreement with results from previous studies in ratbrain, NK 1 receptor-immunoreactive neurons were found to bewidely distributed throughout the brain in the striatum and indiencephalic and mesencephalic structures, while NK3 receptor-immunoreactive neurons were mainly in telencephalic struc-tures. Considerable overlap was observed between NK1 and NK3

receptor distributions.3. Substance P induced Fos-like immunoreactivity (Fos-LI)

in extensive areas of the guinea-pig brain. The induction ofFos-LI was markedly inhibited in many areas by pretreatmentwith the NK1 receptor antagonist SR 140333. The NK3 receptorantagonist SR 142801 reduced Fos-LI staining in fewer areas,although a reduction was observed in the cortex, striatum andhypothalamus.

4. In general, tachykinin receptors were located at sitescorresponding to areas of functional activation by SP, as shownby Fos-LI. These results extend previous studies by adding afunctional dimension to tachykinin receptor localization studies.

Key words: c-Fos, guinea-pig brain, immunohistochemistry,NK1 receptor, NK3 receptor, substance P, tachykinin.

INTRODUCTION

The tachykinins are a family of neuropeptides that exert their actionsvia NK1, NK2 and NK3 receptors. The recent discovery of the effec-tiveness of NK1 receptor antagonists in clinical depression inhumans1 has given a new impetus to studies on the functional rolesof central tachykinins.

It has been established in autoradiographic studies that NK1 andNK3 binding sites are present in higher densities in the centralnervous system (CNS) than NK2 sites.2,3 Recently, the availabilityof antibodies to specific amino acid sequences in tachykinin recep-tors has allowed the cellular localization of the receptors usingimmunohistochemical techniques and the distribution of NK1 andNK3 receptors in rat and human brain has been studied. However,the structure of tachykinin receptors varies between species, thereceptors in the human resembling those in the guinea-pig.3

Therefore, for functional studies, the guinea-pig is an ideal speciesin which to study tachykinin mechanisms. Because no systematicinvestigation of the distribution of NK1 and NK3 receptors in guinea-pig brain has been reported, the first aim of the present study wasto describe the localization of NK1 and NK3 receptors in the guinea-pig brain.

A useful method of obtaining a functional map of neuronsactivated by drugs is to localize Fos, the protein product of theimmediate-early gene transcription factor c-fos, which is inducedin the nuclei of activated neurons.4 The distribution of Fos-likeimmunoreactivity (Fos-LI) induced in the guinea-pig brain by theselective tachykinin NK1 receptor agonist [Sar9,Met(O2)11]-substanceP and the selective tachykinin NK3 receptor agonist senktide has beenreported previously.5,6 In the present study, a second aim was to com-pare the distributions of NK1 and NK3 receptors with the distribu-tion of Fos-LI neurons induced by intracerebroventricular (i.c.v.)injection of the most prevalent natural agonist substance P (SP) inthe presence and absence of specific antagonists for NK1 and NK3

receptors. Substance P was chosen because, although it acts pref-erentially on NK1 receptors, it has the potential to act on NK2 andNK3 receptors. This novel approach allowed comparison betweenthe distribution of receptors and of Fos-LI to ascertain whethertachykinin receptors were located at sites corresponding to those offunctional activation.

Proceedings of the Australian Physiological and Pharmacological SocietySymposium Tachykinins: The Challenge Continues

LOCALIZATION OF TACHYKININ RECEPTORS ANDFOS-LIKE IMMUNOREACTIVITY INDUCED BY SUBSTANCE P

IN GUINEA-PIG BRAIN

Jane Yip and Loris A Chahl

Experimental Pharmacology Unit, School of Biomedical Sciences, University of Newcastle, Newcastle,New South Wales, Australia

Correspondence: LA Chahl, Experimental Pharmacology Unit, School ofBiomedical Sciences, University of Newcastle, Newcastle, NSW 2308,Australia. Email: chahl@mail.newcastle.edu.au

Presented at the Australian Physiological and Pharmacological SocietySymposium Tachykinins: The Challenge Continues, September 1999. Thepapers in these proceedings have been peer reviewed.

Received 16 June 2000; accepted 26 June 2000.

Clinical and Experimental Pharmacology and Physiology (2000) 27, 943–946

944 J Yip and LAChahl

DISTRIBUTION OF NK 1 RECEPTORS

Adult guinea-pigs weighing 450–550g were used. Naïve animalswere given sodium pentobarbitone (80mg/kg) and were perfuse-fixed with 4% paraformaldehyde in phosphate buffer. Brains wereremoved, post-fixed for 2h and placed in 30% sucrose solution in0.5% paraformaldehyde for cryoprotection. Coronal brain sections(30mm) were cut and processed using the immunhistochemicalmethod described previously.5,6 The NK1-like immunoreactivity(NK1-LI) was detected using antibodies (1: 2000 dilution) raised inrabbit serum against a 15 residue synthetic peptide at the C-terminusof the rat NK1 receptor (NK1 393–407: KTMTESSSFYSNMLA)conjugated to bovine thyroglobulin (Novus Biologicals, Littleton,CO, USA). The NK1 receptor antibody pre-absorbed with the syn-thetic peptide for 24h was ineffective. Some sections were stainedfor glial fibrillary acidic protein (GFAP) using rabbit anti-cow GFAPantibody at a dilution of 1: 2000 (DAKO, Botany, NSW, Australia).Control sections were incubated without primary antibodies and/orsecondary antibodies. Sections were examined under a light micro-scope. Diagrammatic representation of the regional distribution ofreceptors was obtained by mapping onto templates of typical sec-tions using computer-assisted camera lucida drawing and the pro-gram Magellan Version 5.3 (PHalasz, Department of Anatomy,University of New South Wales, Sydney, NSW, Australia).

Tachykinin NK1 receptors were widely distributed throughout thebrain (see Table1). In the cortex, NK1-immunoreactive neurons wereseen in the more superficial layers. In the dorsal and medial caudateputamen and nucleus accumbens, large multipolar cells with dis-tinct fibre projections were observed. The NK1-immunoreactiveneurons were also found in the hippocampus (low numbers of faintlystained neurons in CA1, CA2, CA3 and dentate gyrus regions),thalamus, hypothalamus, amygdala, superior colliculus, ventral peri-aqueductal grey, interpeduncular nucleus, ventral tegmental area(VTA) and substantia nigra compacta, the nucleus tractus solitarius(NTS), raphe magnus, area postrema (AP; neurons and fibresobserved in the entire area), hypoglossal nuclei, the dorsal motornucleus of the vagus and the vestibular nuclei (magnocellular andparvocellular) and spinal trigeminal tract. Fibre staining was seenin the ventrolateral medulla (VLM).

The distribution of NK1-immunoreactive neurons in guinea-pigbrain was found to be similar to the distribution of NK1 binding sitespreviously found in autoradiographic studies.2,3The distribution wasalso similar to that found in rat brain in immunohistochemicalstudies.7,8 In agreement with studies in rat and human brain, theneurons that exhibited the most marked NK1 receptor immuno-reactivity were found in the dorsomedial caudate putamen (Fig.1).Many of these large multipolar NK1-immunoreactive neurons wereclearly outlined, indicating that the receptors were on the cell mem-brane and that they had long processes, similar to those describedpreviously in the human striatum.7

DISTRIBUTION OF NK 3 RECEPTORS

The NK3-LI was detected using antibodies raised in rabbit serumagainst a 15 residue synthetic peptide at the C-terminus of rat NK3

receptor (NK3 438–452: SSFISSPYTSVDEYS) conjugated tobovine thyroglobulin (Novus Biologicals). In agreement with pre-vious autoradiographic studies,2 NK3 receptors in guinea-pig brainwere most abundant in the cortex. The distribution of NK3 receptors

in the cortex differed from that of NK1 receptors in that NK3 recep-tors were distributed in both superficial and deep layers of the cor-tex. Beaded NK3-immunoreactive fibres perpendicular to thecortical surface arising from cells with the morphological character-istics of astrocytes were observed. Glial fibrillary acidic proteinstaining indicated that these NK3 receptors were in the region thatcorresponded to the glia limitans and many appeared to be locatedon glia. The distribution of NK3 receptors in the guinea-pig cortexwas similar to that in human cortex and differed to that in rat cortex,which lacks NK3 receptors in the glia limitans.7 This observation isimportant because it may indicate that not only do the tachykininreceptors differ in structure between species, but they may also differ in function. It is tempting to speculate that in some speciesNK3 receptors may play a role in maintenance of the blood–brainbarrier.

The distribution of NK3 receptors in other guinea-pig brain regions(see Table1) was similar to that described previously for rat andhuman brain.7 Fewer and less intensely stained cells were seen indiencephalic and mid- and hind-brain regions. In the hippocampus,extremely low levels of NK3-immunoreactive neurons wereobserved, although a number of intensely stained neurons wasevident in the caudal part of the dentate gyrus. The NK3-immuno-reactive neurons were also present in the medial habenula and severalother thalamic nuclei where fibres and small cells were evident andin the hypothalamus, amygdala, superior colliculus, periaqueductalgrey, VTA, substantia nigra compacta, interpeduncular nucleus, AP(marked staining), the NTS and the spinal trigeminal tract. Glial cellswere found in the AP and were prominent also in the area sur-rounding the spinal trigeminal nuclei. The NK3 receptor staining inthe APdiffered to that for NK1 receptors in that NK3 receptor neuronsformed more well-defined, densely clustered neuronal networkssurrounding the dorsal end of the AP and the area surrounding thecentral canal.

DISTRIBUTION OF FOS-LI INDUCED BY SP

Guinea-pigs used for Fos immunohistochemistry were anaes-thetized with ketamine hydrochloride (Ketalar; Parke-Davis Pty Ltd,Carringbah, NSW, Australia; 40mg/kg, s.c.) and xylazine (Rompun;Bayer Australia Ltd, Pymble, NSW, Australia; 4mg/kg, s.c.) and

Fig. 1 Neuron and processes in the dorsal striatum of the guinea-pig brainshowing NK1 receptor immunoreactivity. Bar, 20mm.

Tachykinin receptors and SP-induced Fos 945

intracerebroventricular (i.c.v.) bilateral guide cannulae were insertedas previously described.5 Three days later, SP(25nmol in 5mL eachside; total dose 50nmol, i.c.v.; Auspep, Melbourne, Victoria, Aus-tralia) or vehicle (sterile saline) was given (four animals per group).In addition, one guinea-pig was pretreated 40min before with oneof the following drugs (6mg in l0mL each side i.c.v.; total dose 12mg) prior to administration of SP: the NK1 receptor antagonistSR140333, its inactive enantiomer SR140603, the NK3 receptorantagonist SR142801, its inactive enantiomer SR142806 and a com-bination of the NK1 and NK3 receptor antagonists SR140333 andSR142801. One additional animal was given 5mL each side of

20mmol/L acetic acid (vehicle control). Another animal was nothandled or pretreated in any way and served as a naïve control. After90min, guinea-pigs were injected with sodium pentobarbitone(80mg/kg) and perfuse-fixed with 4% paraformaldehyde. For Fosimmunohistochemistry, sheep polyclonal antibody to Fos onco-protein (Cambridge Research Biochemicals, Northwich, Cheshire,UK; 1 : 1000 dilution) was used.

Substance Pinduced Fos-LI in extensive areas of the brain, inclu-ding superficial and deeper layers of the cortex, islands of Calleja,lateral and medial septal nuclei, caudate putamen, hippocampus andsubiculum, nucleus accumbens, thalamus, hypothalamus, amygdala,interpeduncular nucleus, periaqueductal grey, dorsal raphe nucleus,superior and inferior colliculus, AP, NTS, lateral parabrachial, locuscoeruleus, spinal trigeminal nucleus and cerebellar lobules. In con-trast, SPdid not induce significant Fos-LI in the substantia nigra orventral tegmental area. Control guinea-pigs exhibited very low levelsof Fos-LI.

EFFECT OF TACHYKININ ANTAGONISTS ONDISTRIBUTION OF FOS-LI INDUCED BY SP

The NK1 receptor antagonist SR140333, but not its less activeenantiomer SR140603, reduced Fos-LI induction by SPthrough-out the brain. In contrast, areas where the NK3 receptor antagonistreduced numbers of Fos-LI neurons were more circumscribed. TheNK3 receptor antagonist SR142801, but not its less active enanti-omer SR142806, reduced Fos-LI induced by SPin the piriform andcingulate cortex, dentate gyrus, lateral septum, caudate putamen, cor-tical amygdaloid nucleus, medial preoptic nucleus, paraventricularnucleus, anterior hypothalamus, interpeduncular nucleus and severalthalamic nuclei (paraventricular, central medial, laterodorsal,mediodorsal, ventrolateral and and rhomboid nuclei).

The reduction in SP-induced Fos-LI produced by the combinationof the NK1 and NK3 receptor antagonists SR140333 and SR142801was similar to that which may be expected from a combination of the two antagonists given independently. Thus, the level of Fos-LI induced by SPwas reduced towards control levels in mostregions by the combination of antagonists. The intense SP-inducedincrease in Fos-LI expression in the cortex was markedly reducedby the combination of NK1 and NK3 receptor antagonists, thussupporting the proposal of a functional involvement of NK1 and NK3

receptors in cortical processing. Similar profiles of inhibition havepreviously been found with the NK1 receptor antagonist SR140333against Fos-LI induced by the NK1 receptor-specific agonist[Sar9,Met(O)211]-SP6 and with the NK3 receptor antagonist SR142801 against Fos-LI induced by NK3 receptor-specific agonistsenktide.5

COMPARISON OF THE DISTRIBUTION OFRECEPTORS WITH THE DISTRIBUTION OF

FOS-LI INDUCED BY SP

The distribution of Fos-LI induced by SPwas similar to that expectedfrom the distribution NK1 receptors in most regions of the brain (seeTable1). However, in the cortex and thalamus, the distribution ofFos-LI was more extensive than would be expected from the distri-bution of NK1 receptors alone (data not shown). In these regions,the distribution of Fos-LI more closely matched the distribution ofa combination of NK1 and NK3 receptors.

Table1 A summary of the presence or absence of NK1 receptor immuno-reactivity, NK3 receptor immunoreactivity and Fos-like immunoreactivityinduced by substance Pin the guinea-pig brain

Brain area NK1 NK3 Fos-LI

CortexSuperficial layer 1 1 1

Deep layer – 1 1

HippocampusCA1 1 – 1

CA2 1 – 1

CA3 1 – 1

Dentate gyrus 1 1 1

StriatumCaudate putamen 1 – 1

Nucleus accumbens 1 – 1

AmygdalaMedial – – 1

Central – – 1

Cortical 1 1 1

Amygdalo-hippocampal 1 1 1

Basomedial 1 1 1

Basolateral 1 1 1

ThalamusParaventricular 1 1 1

Central median 1 1 1

Posteromedian 1 1 1

Posterior 1 1 1

Lateral posterior 1 1 1

Dorsal lateral 1 – 1

Lateral posterior – 1 1

Ventral posterior – 1 1

Medial geniculate 1 1 1

Medial and lateral habenula 1 1 1

HypothalamusVentromedial 1 – 1

Posterior 1 – –Lateral 1 – 1

Periventricular 1 1 1

Arcuate 1 1 1

Median eminence – 1 1

Mid- and hind-brainSuperior colliculus 1 1 1

Periaqueductal grey 1 1 1

Ventral tegmental area 1 1 –Substantia nigra compacta 1 1 –Interpeduncular nucleus 1 1 1

Area postrema 1 1 1

Nucleus tractus solitarius 1 1 1

Spinal trigeminal nucleus 1 1 1

1, immunoreactivity present; –, immunoreactivity absent. Fos-LI,Fos-like immunoreactivity.

946 J Yip and LAChahl

In previous studies, the substantia nigra has been found to containfew tachykinin binding sites.2 In the present study, both NK1 andNK3 immunoreactivity was found in the substantia nigra. This find-ing supports electrophysiological results showing that both NK1 andNK3 receptors are present in the guinea-pig substantia nigra.9

Nevertheless, despite the presence of both NK1 and NK3 receptors,SPfailed to induce Fos-LI in the substantia nigra. Similarly, therewas no increase in Fos-LI in response to SPin the VTA. PreviousFos immunohistochemical studies on the effects of [Sar9,Met(O2)11]-SPand senktide also showed little effect in this region.5,6 Theseunexpected results may be a reflection of the differential capacityof neurons to express Fos-LI in response to pharmacological stimuli.

One promising clinical application of NK1 receptor antagonistsis as anti-emetics in cancer chemotherapy.10 The presence of NK1 receptors and SP-induced Fos-LI in the NTS as well as the AP in the present study supports results from previous studies in other species showing that NK1 receptors play a role in emesis.However, the presence of NK3 receptors in the APindicates that thesereceptors may also play a role in emesis in the guinea-pig.

CONCLUSIONS

Tachykinin NK1 and NK3 receptor immunoreactivity was found tobe widely distributed throughout the guinea-pig brain. In severalregions, including the thalamus, hypothalamus, amygdala, peri-aqueductal grey and medulla, both types of receptor were found tobe present. The present study also revealed that tachykinin recep-tors were located at sites corresponding to areas of functional activ-ation by SPin the brain. In contrast, areas such as the substantianigra, which contained receptors but did not appear to be activated,may well contain neurons that do not readily express Fos under theconditions of these experiments.

ACKNOWLEDGEMENTS

This work was supported by a project grant from the National Healthand Medical Research Council of Australia. SR140333, SR140603,

SR142801 and SR142806 were kindly donated by Dr Emonds-Alt(SanofiRecherche, Montpellier, France).

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