In situ hybridization analysis of the distribution of neurokinin-3 mRNA in the rat central nervous system

THE JOURNAL OF COMPARATIVE NEUROLOGY 372:395-414 (1996)

In Situ Hybridization Analysis of the Distribution of Neurokinin-3 mRNA

in the Rat Central Nervous System

PAUL J. SHUGHRUE, MALCOLM V. LANE, AND ISTVAN MERCHENTHALER The Women’s Health Research Institute, Wyeth-Ayerst Research,

Radnor, Pennsylvania 19087

ABSTRACT The tachykinin family of neuropeptides, which includes substance P, neurokinin A, and

neurokinin B, have three distinct receptors; NK-1, NK-2, and NK-3. With the cloning of the rat NK-3 cDNA, it is now possible to evaluate the distribution of NK-3 mRNA in the rat brain. Female rat brains were sectioned and hybridized with a riboprobe complimentary to NK-3 mRNA. The results of these studies revealed an extensive distribution of NK-3 mRNA throughout the rostral-caudal extent of the brain, spinal cord, and retina. In agreement with previous binding studies, we observed NK-3 mRNA in the cortex, the amygdala, the hippocampus, the medial habenula, the zona incerta, the paraventricular and supraoptic nuclei of the hypothalamus, the substantia nigra, the ventral tegmental area, the interpeduncular nucleus, the raphe nuclei, the dorsal tegmental nucleus, and the nucleus of the solitary tract. In contrast with binding data, only a few NK-3 mRNA cells were detected in the striatum. In addition, the present study detected NK-3 mRNA in the olfactory bulb, the dentate gyrus and subiculum, the medial septum, the diagonal band of Broca, the ventral pallidum, the globus pallidus, the bed nucleus of the stria terminalis, the arcuate, the premammillary and mammillary nuclei, the dorsal and lateral regions of the posterior hypothalamus, the central gray, the cerebellum, the parabrachial nuclei, the nucleus of the spinal trigeminal tract, the dorsal horn of the spinal cord, and the retina. The results of these in situ hybridization histochemical studies have provided detailed and novel information about the distribution of NK-3 mRNA and have elucidated the putative sites of neurokinin B action in the rat central nervous system.

Indexing terms: tachykinin, neuromedin K, NKB, neurokinin B, retina

D 1996 Wiley-Liss, Inc.

The tachykinins, substance P, neurokinin A, and neurokinin B, are a family of neuropeptides that appear to have three distinct receptors; NK-1, NK-2, and NK-3 (Nakanishi, 1991). In vitro binding assays have attempted to determine the topography of tachykinin receptors in the rodent brain with a variety of endogenous (Beaujouan et al., 1986; Buck et al., 1986; Danks et al., 1986; Bergstrom et al., 1987; Saffroy et al., 1988; Mantyh et al., 1989) and synthetic (Beaujouan et al., 1986; Danks et al., 1986; Bergstrom et al., 1987; Mantyh et al., 1989; Dam et al., 1990; Stoessl and Hill, 1990) agonists. Unfortunately, the affinity of the tachykinins for multiple tachykinin receptors (Buck and Burcher, 1986) and the questionable specificity of synthetic agonists (Buck and Burcher, 1986; Quirion and Dam, 1988; Regoli et al., 1987) have made it difficult to ascertain the topography of the three tachykinin receptors in the rat brain.

In the late 1980s, the genes of rat NK-1 (Yokota et al., 1989; Hershey and Krause, 1990), NK-2 (Sasai and Nakani- shi, 19891, and NK-3 (Shigemoto et al., 1990) were cloned.

A comparison of the three tachykinin receptors revealed a high degree of homology (ranging from 53.7 to 66.3%) in the seven transmembrane domains as well as the cytoplas- mic tail (Shigemoto et al., 1990). RNase protection analysis of the regional distribution of NK-1, NK-2, and NK-3 mRNA in the rat indicated that the receptors are differen- tially expressed in the brain and peripheral tissues (Tsu- chida et al., 1990). In the brain, elevated levels of the substance P receptor (NK-1) mRNA were detected in the olfactory bulb, striatum, hypothalamus, and spinal cord, whereas the neurokinin B receptor (NK-3) mRNA levels were highest in the hypothalamus, cortex, and cerebellum (Tsuchida et al., 1990). The expression of NK-2 appears to be restricted to peripheral tissues (Tsuchida et al., 1990).

Accepted March 11, 1996. Address reprint requests to Dr. Pan1 J. Shughrue, Department of Func-

tional Morphology, Wyeth-Ayerst Research, 145 King of Prussia Road, Radnor, PA 19087. E-mail: [email protected]

o 1996 WILEY-LISS, INC.

396 P.J. SHUGHRUE ET AL.

Abbreviations

10 12 1-6 2n 3 v 4v I n AAD AAV ac Acb ACo AHiA Am AN AOB AOE AOL AOM AOP AOV AP Al’ir B BL BLA BLP BLV BM BMP BST BSTIA CA1-CA3

CC Ce CeC CG c1 CLi CnF CP CPU c u DA DBB DCIC DEn DG Dk DMN DMSp5 DMTg DPGi DpMe DTg ECIC Ent f fmi Fr2 Gi GI GP Gr Hab HDB HiF ic ICj IG IGr IP1 IPN IRt I-x LC LGN LH

cc

dorsal motor nucleus of the vagus nerve hypoglossal nucleus cortical laminae optic nerve third ventricle fourth ventricle nucleus of the facial nerve anterior amygdaloid area, dorsal division anterior amygdaloid area, ventral division anterior commissure nucleus accumbens anterior cortical amygdaloid nucleus amygdalohippocampal area amygdala arcuate nucleus accessory olfactory bulb anterior olfactory nucleus, external division anterior olfactory nucleus, lateral division anterior olfactory nucleus, medial division anterior olfactory nucleus, posterior division anterior olfactory nucleus, ventral division area postrema amygdalopiriform area basal nucleus of Meynert basolateral amygdaloid nucleus basolateral amygdaloid nucleus, anterior division basolateral amygdaloid nucleus, posterior part basolateral amygdaloid nucleus, ventral part basomedial amygdaloid nucleus basomedial amygdaloid nucleus, posterior division bed nucleus of the stria terminalis bed nucleus of the stria terminalis, intraamygdaloid division CA1-CAB fields of Ammon’s horn corpus callosum central canal central amygdaloid nucleus central cervical nucleus central gray claustrum caudal linear raphe nucleus cuneiform nucleus cerebral peduncle caudate putamen cuneate nucleus dorsal hypothalamic area diagonal band of Broca dorsal cortex, inferior colliculus dorsal endopiriform nucleus dentate gyms nucleus of Darkschewitsch dorsomedial hypothalamic nucleus spinal trigeminal nucleus, dorsomedial dorsomedial tegmental area dorsal paragigantocellular nucleus deep mesencephalic nucleus dorsal tegmental nucleus external cortex, inferior colliculus entorhinal cortex fornix forceps minor, corpus callosum frontal cortex, area 2 gigantocellular reticular nucleus glomerular layer, olfactory bulb globus pallidus gracile nucleus habenula horizontal limb of the diagonal band hippocampal fissure internal capsule islands of Calleja indusium griseum internal granular layer, olfactory bulb internal plexiform layer, olfactory bulb interpeduncular nucleus intermediate reticular nucleus laminae of the spinal cord locus ceruleus lateral geniculate nucleus lateral hypothalamic area

LHb lo LPB LPOA LS LSI LSO LSV LV MCLH MCPO MdD MdV Me MGN MHb Mi ml mlf MMN MnPO M05 MPA MPB MPON MS mt MTu MVe NTS OB ON OP ot

PC PCRt PDTg Pe Pir Pit PLCo PMCo PMD PMV Pn PnC POlN PON Pr PVN PY P3Tx RCh RD RF R U RM

Rob RPn Rt S SCP SFO sg SHY SI sm smt SN SNC SNR SON SP5 SP5 SpVe st STh

ox

RMg

lateral habenular nucleus lateral olfactory tract lateral parabrachial nucleus lateral preoptic area lateral septum lateral septal nucleus, intermediate division lateral superior olive lateral septal nucleus, ventral division lateral ventricle magnocellular lateral hypothalamic nucleus magnocellular preoptic nucleus medullary reticular nucleus, dorsal medullary reticular nucleus, ventral medial amygdaloid nucleus medial geniculate nucleus medial habenular nucleus mitral cell layer medial lemniscus medial longitudinal fasciculus medial mammillary nucleus, medial division median preoptic nucleus motor nucleus of the trigeminal nerve medial preoptic area medial parabrachial nucleus medial preoptic nucleus medial septal nucleus mammillothalamic tract medial tuberal nucleus medial vestibular nucleus nucleus tractus solitarii olfactory bulb olfactory nerve optic nerve layer of the superior colliculus optic tract optic chiasm posterior commissure parvocellular reticular nucleus posterodorsal tegmental nucleus periventricular hypothalamic nucleus piriform cortex anterior pituitary posterolateral cortical amygdaloid nucleus posteromedial cortical amygdaloid nucleus premammillary nucleus, dorsal division premammillary nucleus, ventral division pontine nuclei pontine reticular nucleus periolivary nuclei periolivary nucleus principal nucleus of trigeminal nerve paraventricular hypothalamic nucleus pyramidal tract pyramidal decussation retrochiasmatic area nucleus raphe dorsalis rhinal fissure rostra1 linear raphe nucleus nucleus raphe magnus nucleus raphe magnus nucleus raphe obscurus nucleus raphe pontis reticular thalamic nucleus subiculum superior cerebellar peduncle subfornical organ stratum granulosum, cerebellum septohypothalamic nucleus substantia innominata stratum moleculare, cerebellum stria medullaris thalami substantia nigra substantia nigra, pars compacta substantia nigra, pars reticulata supraoptic nucleus spinal tract of the trigeminal nerve spinal nucleus of the trigeminal nerve spinal vestibular nucleus stria terminalis subthalamic nucleus

NK-3 mRNA IN RAT BRAIN 397

The prTKR 3 plasmid was digested with Xcml, and the multiple cloning site with EcoRV to excise a large fragment of the cDNA (bases 553-2,159). The plasmid and remaining cDNA fragment were then treated with endonuclease and ligated together. The resulting pBluescript plasmid (NK3- 553) contained a 553 bp fragment (bases 1-553) of the NK-3 cDNA, with little sequence homology with NK-1 and

The NK3-553 plasmid was linearized and used to generate a S35-UTP-labeled probe that was complimentary to a portion of the rat NK-3 mRNA. Processed section-mounted slides were hybridized with 100-150 ~1 of an antisense or sense (control) riboprobe (4.7 x lo6 disintegration per minute (DPM) /slide) - 50% formamide hybridization mix (described in Miller et al., 1989; except with 5% dextran sulfate and 200 mM dithiothreitol (DTT) and were incu- bated overnight at 55°C in a chamber humidified with 50% formamide/600 mM NaCl. In the morning, the slides were placed in metal racks and immersed in 2 x SSC (0.3 M NaC1, 0.03 M sodium citrate, pH 7.0)/10 mM DTT to remove the excess hybridization mixture. The racks were then transferred to a large container (no. 3863; Rubber- maid, Winchester, VA) filled with 2 x SSC/10 mM DTT. When all of the slides were in the large container, the racks were transferred again to a new container and washed in 2 x SSCilO mM DTT for 15 minutes at RT with gentle agitation. The slides were then washed in RNase buffer at 37°C for 30 minutes, treated with RNase A (20 Fg/ml) for 30 minutes at 37"C, and washed for 15 minutes in RT 1 x SSC. Subsequently, the slides were washed (twice for 30 minutes) at 65°C in 0.1 x SSC to remove nonspecific label, rinsed at RT with 0.1 x SSC for 15 minutes, and dehy- drated with a graded series of alcoho1:ammonium acetate (70%, 95%, and 100%). Air-dried slides were opposed to x-ray film (Amersham, Arlington Heights, IL) for 9 days and then dipped in NTB2 nuclear emulsion (Eastman Kodak; diluted 1:l with 600 mM ammonium acetate). The slides were exposed for 24-64 days in light-tight, black desiccated boxes, photographically processed, stained in cresyl violet, and coverslipped. The slides from all animals were hybridized, washed, exposed, and photographically processed together to eliminate differences due to interas- say variation in conditions. Details concerning the preparation of tissue and riboprobes and the in situ hybridization method have been reported previously (Miller et al., 19891, with the modifications to this methods noted herein.

NK-2.

Sub1 SUM suo SuVe Tec Th TST TT tz TZ VDB vhc VL VMN w VTA ZI

Abbreviations (continued)

subincertal nucleus supramammillary nucleus superior olive superior vestibular nucleus tectum thalamus tractus solitarius tenia tecta trapezoid body nucleus of the trapezoid body vertical limb of the diagonal band ventral hippocampal area ventrolateral thalamic nucleus ventromedial hypothalamic nucleus ventral pallidum ventral tegmental area zona incerta

Recently, the distribution of NK-1 (Shigemoto et al., 1993) and NK-1 mRNA-containing cells (Elde et al., 1990; Gerfen, 1991; Maeno et al., 1993) have been elucidated in the striatum and throughout the rat brain with immunocy- tochemistry and in situ hybridization histochemistry. The results of these studies have clearly demonstrated the presence or absence of NK-1 mRNA in certain regions of the rat brain and have resolved several discrepancies emanating from in vitro binding assays. Although the distribution of the NK-1 receptor mRNA has been elucidated, little is known about the topography of NK-3 mRNA in the rodent brain. The purpose of the present study was to ascertain the topography of NK-3 mRNA in the female rat central nervous system (CNS), spinal cord, and eye.

MATERIALS AND METHODS Animals and tissue preparation

Sixty-day-old female Sprauge-Dawley rats (Taconic, Ger- mantown, NY) were housed in the Wyeth-Ayerst animal care facility (AAALAC certified) with a l%hours-light, 12-hours- dark photoperiod and with free access to tap water and rodent chow. After acclamation, the animals were exposed to a lethal dose of COz. The brains, spinal cords, and eyes were removed, frozen on dry ice, and stored at -80°C until sectioning. The studies described in this paper were re- viewed and approved by the Radnor Animal Care and Use Committee (RACUC) at Wyeth-Ayerst Research.

Twenty micron coronal cryostat sections were cut at - 14°C and collected on Silane-coated microscope slides (Histology Control Systems, Glen Head, NY). The section- mounted slides were dried on a slide warmer maintained at 42°C (Lab-Line Instruments, Melrose Park, IL) and then stored in desiccated (Dricap; Ted Pella, Redding, CA) slide boxes at -80°C. Prior to processing, the desiccated slide boxes were slowly warmed to room temperature [-20°C for 12-16 hours; 4°C for 2 hours; room temperature (RT) for 1 hour] to eliminate the formation of condensation on slides and, thus, minimize tissue and RNA degradation. The dry slides were loaded into metal racks, postfixed in 4% parafor- maldehyde, pH 9.0, for 5 minutes, and processed as previously described (Miller et al., 1989).

In situ hybridization A pBluescript plasmid (prTKR 3; Shigemoto et al., 1990)

containing the rat NK-3 cDNA was obtained from Dr. Shigetada Nakanishi (Kyoto University, Kyoto, Japan).

Evaluation Section-mounted slides, which were used to generate film

autoradiographic images, were scanned at low magnification with a light microscope to determine the regional distribution of silver grains in the brain, spinal cord, and eye. Adjacent sections, which were exposed for shorter periods of time, were then viewed with high magnification to determine the cellular distribution of hybridization signal and to verify that silver grains concentrated over cells. Cells with a concentration (> ~5 radioactivity in neuropil) of silver grains were considered labeled and mapped onto computer scans of the rat brain (Paxinos and Watson, 1982); their size and distribution represented the number of NK-3 mRNA-containing cells in a particular brain region.

Film autoradiograms were also used to assist in generat- ing a map of NK-3 mRNA in the female rat brain. The autoradiograms were digitized with a computer-assisted

P.J. SHUGHRUE ET AL. 398

image analysis system (C-Imaging Inc., Pittsburgh, PA), processed for contrast enhancement, imported into the Canvas (Deneba Systems, Miami, FL) illustrator program, and the image was excised from the background and arranged into plates.

RESULTS General considerations

The following description of the localization of NK-3 mRNA-containing cells is based on data obtained from 20 km coronal sections through the female rat brain and spinal cord. The schematic representation of this information (Fig. 1A-C) has been simplified, so that the distribution of labeled cells is depicted by dots that represent both the number and the distribution of labeled cells. The majority of the dots are of one size (five labeled cells), but, in a few circumstances (paraventricular, supraoptic, and medial habenular nuclei), a large dot ( > 30 labeled cells) was used because of space limitations.

Specificity of hybridization signal The NK3-553 plasmid, which contains the first 553 bases

of the NK-3 cDNA, was selected, because this portion of the receptor has little sequence homology with the cDNAs of NK-1 and NK-2 (see Nakanishi, 1991). A comparison of the distribution of NK-3 mRNA (Fig. 9A-C) with NK-1 mRNA (Maeno et al., 1993) clearly illustrates that our probe is specific for NK-3 mRNA. In addition, some section- mounted slides were hybridized with a sense probe (com- pare Fig. 8D and Fig. 8H) or were pretreated with RNase A prior to hybridization with an antisense probe. In all cases, the results of these control studies demonstrated that the signal detected in our assay was specific signal and was not an aberration of our methodology. Moreover, a melting study revealed a positive correlation between an increase in the temperature of the hot washes and a reduction in specific hybridization signal. Together, the results of the control experiments demonstrate that the hybridization signal detected in the present study was specific for the rat NK-3 mRNA. The changes in the previously published (Miller et al., 1989) in situ hybridization protocol (i.e., increasing the concentration of DTT, reducing the concentration of dextran sulfate in the hybridization mixture, and using an open-air humidification chamber) reduced the level of nonspecific signal and the inconsistent background that are commonly seen with other in situ methods, resulting in a greater signal-to-noise ratio.

Although the methodology utilized in the present study does not enable one to differentiate between neurons and glia, the size, morphology, and distribution of the NK-3 mRNA-containing cells suggests that these cells are primarily neurons. Future colocalization studies using specific neuronal and glial markers are required to resolve this question.

Distribution of NK-3 mRNA Telencephalon. NK-3 mRNA-containing perikarya were

distributed throughout the rostral-caudal extent of the telencephalon. In the olfactory bulb, the hybridization signal was localized predominantly in the internal granular, plexiform, and glomerular layers, although a few labeled cells were also seen in the anterior olfactory nucleus and the mitral cell layer (Fig. 2B). The cerebral cortex was

labeled extensively at all levels investigated. Analysis of the distribution of NK-3 mRNA in the cortex revealed that the majority of the labeled cells were concentrated in laminae IV-V, with fewer cells present in laminae VIb (adjacent to the corpus callosum) and only a few scattered cells localized in laminae 1-111 (Fig. 2A,C). NK-3 mRNA was also detected in cells of the piriform cortex and claustrum. In the hippocampus, NK-3 mRNA was seen throughout the dorsal to ventral extent of Ammon’s horn (CA1-CAS), dentate gyrus, and subiculum, although most of the labeled perikarya were concentrated in the more ventral aspects of the hippocampus. Hybridization signal was also detected in the septum, diagonal band, and associated areas (Fig. 2D,E). Scattered cells with NK-3 mRNA were seen in the basolat- era1 subdivision of the septum, just lateral to the vertical limb of the diagonal band, and extending from the dorsal portion of the lateral septum into the medial septum (Fig. 2D). The transitional region between the vertical and horizontal limbs of the diagonal band, just above the ventral surface of the brain and in front of the organum vasculosum of the lamina terminalis, also contained labeled cells (Fig. 2D). Within the basal ganglia, the majority of the hybridization signal was localized in the ventral pallidum, the horizontal limb of the diagonal band of Broca (Fig. 3A), the zona incerta (Fig. 4 0 , and the subthalamic nuclei. In contrast, the globus pallidus and the mediobasal extent of the caudate putamen contained only a few, if any, labeled cells (Fig. 8G). NK-3 mRNA was detected in numerous nuclei of the amygdala (Figs. 3B, 4C), with the highest degree of labeling localized in the basal (medial and lateral subdivisions) nuclei and fewer cells found in the central and cortical nuclei. In the bed nucleus of the stria terminalis, labeled cells were scattered throughout the anterior and posterior subdivisions of this nuclei (Fig. 3A).

The distribution of NK-3 mRNA-containing perikarya in diencephalon was rather striking, because the majority of the labeled cells were localized in the hypothalamus. There were, however, a few exceptions. In the thalamus, several cells containing NK-3 mRNA were detected in the posterior portion of the reticular nucleus and in close proximity to the periventricular fiber system in the posterior thalamus. In addition, a dense aggregation of labeled perikarya was seen in the medial habenula (Figs. 4B, 8D), and fewer cells were present in the lateral division of the habenula. A large number of nuclei in the hypothalamus contained NK-3 mRNA-accumulating cells, with the number of labeled cells increasing from the rostral to caudal levels. In the anterior hypothalamus, a well-demarcated population of NK-3 mRNA cells was localized to the median preoptic nucleus (Fig. 3A). In addition, scattered labeled cells were seen in the lateral preoptic area (Fig. 3A), the parvicellular paraventricular nucleus (Fig. 4A) and the lateral hypothalamus (Fig. 4C), and the dorsomedial nucleus. Large labeled cells were concentrated in the supraoptic nucleus (Fig. 4C) and magnocellular subdivisions of the paraventricular nucleus (Figs. 4A, 8C) as well as the lateral hypothalamus, just medial to the cerebral peduncle (Fig. 4C). In the arcuate nucleus, small cells with NK-3 mRNA were concentrated in the basolateral subdivision throughout its rostral- caudal extent, with only a few cells inthedorsomedial portion (Figs. 4C, 5A). Labeled cells were also seen in the medial mammillary (Fig. 5B) and dorsal premammillary nuclei.

In the ventral midbrain, a dense accumulation of labeled cells formed a complex that extended from the interpeduncular nucleus to the ventral tegmental

Diencephalon.

Mesencephalon.


BSTlA

F

Fig. 1. A schematic representation of coronal sections (A-C) depict- ing the distribution of neurokinin-3 (NK-3) mRNA in the female rat brain and spinal cord. The information shown in these schematics has been simplified, so that the distribution of labeled cells is depicted by

area and terminated in the pars compacta of the substantia nigra (Figs. 5C, 6A,B). Additional NK-3 mRNA-containing cells were also seen in the midbrain central gray (Fig. 7A), the superior colliculus, the deep mesencephalic nuclei, the

dots that represent both the number and distribution of labeled cells. Small dots, five labeled cells; large dots, 230 labeled cells. The schematics were modified from the atlas of Paxinos and Watson (1982). For abbreviations. see list.

medial geniculate nucleus, and the rostral linear raphe nucleus.

In the pons, numerous labeled cells were seen in the pontine nuclei (Fig. 6C), the central gray,

Metencephalon.


Figure 1 (Continued.)

the posterodorsal tegmental nucleus, the parabrachial nucleus, and the sensory nuclei of the trigeminal nerve, whereas only a few NK-3 mRNA cells were seen scattered throughout the locus ceruleus, the raphe magnus, and the periolivary nucleus. In the cerebellum, the majority of the cells in the granular layer contained NK-3 mRNA (Fig.

8A,B), although this was difficult to resolve because of the density of cells in this layer.

The vast majority of labeled cells in the medulla were present in the nucleus of the solitary tact and the dorsal nucleus of the vagus (Fig. 7D). In addition, labeled cells were also seen in the nucleus of the spinal

Myelencephalon.


' PY

DMSp5

P5


trigeminal tract and scattered throughout the reticular formation and the raphe magnus and obscurus (Fig. 7B,C).

Spinal cord. A dense localization of NK-3 mRNA- containing cells was present in the superficial layers (11-111) of the dorsal horn (Fig. 7E), whereas additional cells were scattered throughout the remaining layers of the spinal cord (Fig. 7F), with the exception of lamina IX, which did not contain NK-3 mRNA-expressing cells.

In the eye, a moderate level of NK-3 mRNA was seen in the internal and external granular layers of the retina (Fig. 8E,F).

Retina.

DISCUSSION Comparison of NK-3 mRNA

with NK-3 binding sites The overall distribution of NK-3 mRNA-containing peri-

karya in the female rat CNS reported herein is similar to the distribution of binding for radiolabeled NK-3 agonists (Beaujouan et al., 1986; Buck et al., 1986; Danks et al., 1986; Bergstrom et al., 1987; Saffroy et al., 1988; Mantyh et al., 1989; Dam et al., 1990; Stoessl and Hill, 1990). In contrast to in vitro binding studies with putative NK-3-


Fig. 2. Representative autoradiograms of NK-3 mRNA in the telencephalon of the female rat by in situ hybridization. A: Parietal cortex. B: Anterior olfactory nucleus. C: Cingulate cortex. D: Medial septum and horizontal limb of the diagonal band of Broca. Note the concentration of NK-3 mRNA-containing perikarya in layers IV-V (stars) of the cortex, the sparse labeling in the remainingcortical layers,

and the absence of labeling in the corpus callosum. Small arrows demarcate the surface of the cortex (A), and large arrows indicate NK-3 mRNA-containing perikarya in layers 2-3 (A). The small asterisks in C indicate the interhemispherical fissure, and the large asterisks in D indicate the optic nerves. The arrow in D indicates the midline. For abbreviations, see list. Magnification: X 100 in A,B, x50 in C,D.

selective agonists, the present study was unable to detect NK-3 mRNA in the caudate putamen and the suprachiasmatic nucleus. Moreover, the present study found NK-3 mRNA-containing cells, which are not detected with most in vitro binding studies, in the anterior olfactory nucleus, the mitral cell layer of the olfactory bulb, the deep layer of

the cortex (VIb), the medial septum, the nucleus accumbens, the median preoptic nucleus, the parvicellular subdivisions of the paraventricular nucleus, the arcuate nucleus, the medial mammillary nuclei, the raphe nuclei, the lateral parabrachial nucleus, the cerebellum, the dorsal tegmental nucleus, the pontine nuclei, the spinal trigeminal nucleus,


Fig. 3. Representative autoradiograms of NK-3 mRNA in the stria terminalis, and the ventral pallidum (A). In the amygdala (B), many nuclei contained NK-3 mRNA-accumulating perikarya, including the anterior and posterior basolateral nuclei and central nucleus. Asterisks indicate the third ventricle. For abbreviations, see list. Magnification: x 100 in A,B.

female rat brain by in situ hybridization. Note the dense accumulation of NK-3 mRNA-containing cells in the median preoptic nucleus (A). Labeled perikarya are also present in the lateral preoptic area, the horizontal limb of the diagonal band of Broca, the bed nucleus of the


Fig. 5. NK-3 mRNA-containing perikarya in the female rat brain by in situ hybridization. Note the localization of NK-3 mRNA in the arcuate nucleus, the magnocellular lateral hypothalamus, and the dorsal hypothalamic area of the posterior hypothalamus (A); in the mammillary body (B); and in the interpeduncular nucleus, the ventral

tegmental area, and the substantia nigra and the pars compacta of the rostra1 mesencephalon (C). The third ventricle is indicated by the asterisks in A. Stars in B indicate the midline. For abbreviations, see list. Magnification: x 100 in A,B,C.

layers 11-VIII and X of the spinal cord, and the retina. In addition to these differences in the location of NK-3 mRNA and NK-3 binding sites in the CNS, there were several

Fig. 4. NK-3 mRNA-containing perikarya in the female rat hypothalamus by in situ hybridization. In the paraventricular nucleus (A), the labeled cells are present in the magnocellular subdivisions (arrows), although scattered perikarya can be seen in the parvicellular subdivisions, medial to the magnocellular subgroups (arrowheads). A dense accumulation of labeled cells is also seen in the medial habenular nucleus (B). In the hypothalamus, labeled cells are present in the arcuate nucleus, the lateral hypothalamus, the supraoptic nucleus, and the zona incerta (C). Within the amygdala, labeled cells are concentrated in medial and lateral basal nuclei and central nucleus (C). The third ventricle is indicated by 3V in B and by the asterisks in A and C. For abbreviations, see list. Magnification: x 100 in A,B, x 50 in C.

discrepancies in the number of NK-3 cells and the intensity of signal. For example, the results of binding studies indicated the presence of only a few scattered NK-3 neurons in the olfactory bulb, the medial habenula, the central gray matter of the mesencephalon and pons, the superior colliculi, and the ventral tegmental area (Beaujouan et al., 1986; Bucket al., 1986; Danks et al., 1986; Bergstrom et al., 1987; Saffroy et al., 1988; Mantyh et al., 1989; Dam et al., 1990; Stoessl and Hill, 1990). Interestingly, the present report detected a large numbers of NK-3 mRNA-containing cells in these regions of the brain. There are several possible explanations for the differences between NK-3 mRNA distribution and binding of NK-3 agonists in the rat brain. The most plausible reason for this discrepancy is the affinity of both natural and synthetic NK-3 agonists for multiple tachykinin receptors (Buck and Burcher; 1986;


Fig. 6. NK-3 mRNA-containingperikaryain thefemalemesencepha- Ion and pons by in situ hybridization. Note the localization of NK-3 mRNA in the interpeduncular nucleus (large arrow; A,B), the ventral tegmental area (A), the substantia nigra, pars compacta (A, small

arrow), and in the pontine nucleus (C). The asterisk in C indicates the midline. For abbreviations, see list. Magnification: X 5 0 in A, X 100 in B,C.

Regoli et al., 1987; Quirion and Dam, 1988). The potential for ligands to bind to other specific or nonspecific sites is readily apparent when one compares the distribution of various NK-3 agonists (Beaujouan et al., 1986; Buck et al., 1986; Danks et al., 1986; Bergstrom et al., 1987; Saffroy et al., 1988; Mantyh et al., 1989; Dam et al., 1990; Stoessl and Hill, 1990) and sees the marked differences in binding. Moreover, it is important to note that some of the binding studies employed peptides labeled with [12511 and conju- gated to the Bolton-Hunter group (Beaujouan et al., 1986; Bucket al., 1986; Danks et al., 1986; Bergstrom et al., 1987; Saffroy et al., 1988; Mantyh et al., 1989). The large size of this molecule, modified hydrophobicity, and charge may

have impaired receptor specificity and sensitivity when compared to the native ligand. Indeed, it has been shown that the addition of a Bolton-Hunter group can affect the receptor specificity of certain aliphatic tachykinins (Lee et al., 1986; Bergstrom et al., 1987). It is also possible that the ligand binding seen in brain regions that lack NK-3 mRNA may be due to the transportation and localization of NK-3 in certain nerve terminals. The absence of binding in regions of the brain that express NK-3 mRNA is probably due to a difference in sensitivity and resolution of tech- niques. Alternatively, NK-3 mRNA may not be translated into a functional receptor in every region of the brain. The possibility of this option is unlikely, but it is plausible,

Fig. 7. NK-3 mRNA-containing perikarya in the female rat lower brainstem by in situ hybridization. Scattered labeled cells are seen in the central gray of the pons (A). Additional labeled cells are seen in the posterodorsal tegmental nucleus and raphe pontis nucleus (B). A dense accumulation of labeled cells is found in the spinal trigeminal nucleus (C) and in the nucleus of the solitary tract (D). NK-3 mRNA is present in perikarya of the substantia gelatinosa of the dorsal horn of the spinal

cord (E). Scattered labeled cells are seen in the ventral horn of the spinal cord (F). The asterisks in A and D indicate the central canal, and the midline is indicated by stars in A and by arrows in B and D. Dashed lines indicate the border of the central canal in A and the border of the dorsal horn in E. For abbreviations, see list. Magnification: x50 in A,B,D, x 100 in C, ~ 2 0 0 in E,F.


Fig. 8. Brightfield photographs showing NK-3 mRNA-containing perikarya in the brain and retina by in situ hybridization. Note the localization of NK-3 mRNA in the granular layer of the cerebellum (A,B), whereas the molecular layer is unlabeled. A dense accumulation of silver grains is also seen in the paraventricular nucleus (C) and in the medial habenula (D). In the retina (E,F), NK-3 mRNA-containing cells are present in the external (IV) and internal granular layers. The

caudate putamen (G) is free of NK-3 mRNA-containing cells. H is a representative autoradiogram of the habenula when tissue is hybridized with a sense probe. Note the lack of signal compared with the habenula hybridized with an antisense probe (D). The habenula is demarcated with a broken line (H). For abbreviations, see list. Magnifi- cation: x 600 in A-D,F,G, ~ 4 0 0 in E, ~ 2 0 0 in H.


Fig. 9. Autoradiograms of NK-3 mRNA in the female rat brain and spinal cord by in situ hybridization (A-Y). The coronal rat brain sections are arranged in rostral-caudal order, and major regions and structures are indicated on the right side of each autoradiogram. For abbreviations, see list.

because several examples of nontranslated neuropeptide mRNAs (e.a.. cholecvstokinin in the thalamus) have been

Maggio, 1991; Marksteiner et al., 1992; Merchenthaler et al.. 1992). Within the brain. the densest accumulation of

documentei (Voigt and Uhl, 1988). the ligand and the receptor is present in the olfactory bulb, the cerebral cortex, the amygdala, the hypothalamus, the interpeduncular nucleus, the periaqueductal gray, the parabrachial nucleus, the nucleus of the solitarv iract. and the

Of NK-3 mRNA with neurokinin B-immunoreactive terminals

The overall distribution of NK-3 mRNA-expressing cells and neurokinin B-immunoreactive (neurokinin B-i) fibers/ nerve terminals in the CNS is similar (Table 1; Too and

dorsal vagal nucleus. On the other hand, t iere are several discrepancies between the location of neurokinin B and NK-3. In certain areas of the CNS, NK-3 mRNA is not

410


P.J. SHUGHRUE ET AL.

associated with the neurokinin B immunoreactivity that is present in nerve fibers and terminals. These include the internal granular and glomerular layers of the olfactory bulb, lamina VI of the cerebral cortex, and the cortical nucleus of the amygdala of the telencephalon; the dorsomedial, premammillary, and medial mammillary nuclei; the parvicellular subdivisions of the paraventricular nucleus of the hypothalamus; the inferior colliculus and geniculate nucleus of the mesencephalon; the cerebellum; the dorsal

tegmental, laterodorsal tegmental, and pontine nuclei and locus ceruleus of the pons; the dorsal motor nucleus of the vagus; the hypoglossal nucleus; most of the raphe nuclei; and the paraolivary and spinal trigeminal nuclei.

Within the spinal cord, the majority of NK-3 mRNA- containing perikarya and the densest accumulation of neurokinin B-i fibersiterminals were seen in the substantia gelatinosa (laminae 1-111). Only scattered NK-3 mRNA- accumulating perikarya and neurokinin B-i fibers/ termi-

NK-3 mRNA IN RAT BRAIN

Q

R

411

U

V

W

S

X

T Y Figure 9 (Continued.)

nals are present in other laminae (Too and Maggio, 1991). Lamina X, however, contains only neurokinin B-i fibers1 terminals and not NK-3 mRNA-expressing cells. The origin of neurokinin B-i fibers in the substantia gelatinosa is not known precisely. Although the majority of fibers in the substantia gelatinosa arise from dorsal root ganglia (Ruda et al., 1986), neurokinin B (Too and Maggio, 1991) and NK-3 mRNA (present study) are not present in dorsal root ganglia neurons. Moreover, deafferentation or cordotomy

depletes substance P immunoreactivity, but not neurokinin B immunoreactivity, from the substantia gelatinosa (Ogawa et al., 19851, providing further support for the idea that neurokinin B in this area is of intrinsic origin. Indeed, the presence of neurokinin B mRNA-containing cell bodies predominantly in the superficial laminae has been reported (Warden and Young, 1988). Similar to substance P, neurokinin B-i fibers in the anterior column may originate from local neurons or from neurons with descending processes in


TABLE 1. Comparison of Neurokinin-B Terminals and NK-3 mRNA- Containing Perikarya in the Rat CNS, Spinal Cord, and Retina’ TABLE 1. (continued)

Tissue Neurokinin B NK-3 mRNA Tissue Neurokinin B NK-3 mRNA

Telencephalon Olfactory bulb

External plexiform layer Internal granular layer Internal plexiform layer Mitral cell layer Glomerular layer Anterior olfactory nucleus

Cerebral cortex Layers 11-111 Layers N-V Layer VIb

Cingulate cortex Entorhinal cortex Piriform cortex Claustrum Amygdala

Anterior cortical nucleus Baal nuclei Central nucleus Cortical nucleus Lateral nucleus Medial nucleus

Amygdalohippocampal area Bed nucleus of the stria terminalis Caudate putamen Glohus pallidus Hippocampus

CA1-CAB fields of Ammon’s horn Dentate gyrus Subiculum

Nucleus accumbens Nucleus of the diagonal hand (Brocaj

Horizontal limh Vertical limb

Medial Lateral

Septum

Substantia innominata Ventral pallidum Diencephalon Thalamus

Paraventricular nucleus Reticular nucleus

Zona incerta Subthalamic nucleus Hypothalamus

Arcuate nucleus Dorsomedial nucleus Lateral hypothalamic nucleus Median preoptic nucleus Lateral prenptic nucleus Medial mammillary nucleus Paraventricular nucleus (magnocellularj Paraventricular nucleus (parvicellular) Periventricular nucleus Premamillary nucleus Suprachiasmatic nucleus Supraoptic nucleus

Medial hahenula Lateral habenula

Mesencephalon Dorsal raphe nucleus Geniculate nucleus Inferior colliculus Interpeduncular nucleus Periaqueductal gray Substantia nigra

Pars compacta Pars reticularis

Superior cnlliculus Superficial layer Intermediate layer Deep layer

Ventral tegmental area Metencephalon: Cerebellum Granular layer of cerebellum Metencephalon: Pons Dorsal tegmental nucleus Lateral dorsal tegmental nucleus Locus ceruleus Parabrachial nucleus

Epithalamus

Medial Lateral

Pontine nucleus Myelencephalon. Medulla oblongata Dorsal motor nucleus of the vagus Hypoglossal nucleus

+++ - - ++

+++ ++ + ++ - ++ + + + + + ++++ - ++ + ++++ + + + + + +

~ -~

Nucleus of the solitary tract Parabigeminal nucleus Paraolivary nucleus Raphe nuclei

Magnus Obscurus

Spinal trigeminal nucleus Spinal cord

11-111 IV-VIII IX X

Dorsal root ganglia Retina

External granular layer Internal manular laver

- -

+ +++ ++ ++

+ + + + + + +

+++ ++ + + + + ++ + ++ + ++++ + +

-

-

++ ++ ++ + ++ + ++ + + + + ++

+ +/ - + + + +++ + ++

++ ++ - + + +++

++ ++ + + + ++++ +

- +++ +

++

- -

- + + +++

+++ +++ + + + + / -

-

- + + -

+++ +++ ++ ++ + +++ + + + +++ + +++ + ++ + ++++ - +++

++ ++ ++ ++ - +++ - ++++ - +

+ +

++ +

++ ++

? ?

+++i ++ ++ + +

++ +++ -

++

++ ++

~ ~ ~~

‘Data from Too and Maggio (1991), Marksteiner et al. (1992), and Merchenthaler et al. (1992). -, No detectable immunoreactivity or mRNA, +/-, very low levels (less than five cells per section); +, low levels; + + , moderateimedium levels; + + + , high levels; + + + +, very high levels of immunoreadivity or mRNA.

the brainstem (Wessendorph and Elde, 1987). The substantia gelatinosa is an important site for the modulation of incoming somatosensory information, including nociception (Besson and Chaouch, 1987). The presence of neurokinin B binding sites (Ninkovich et al., 1985; Yashpal et al., 1990) and NK-3 mRNA-expressing cells in the superficial layers of the spinal cord strongly indicate that neurokinin B, as with several other neuropeptides, participates in the processing of nociceptive information. NK-3 mRNA and neurokinin B have not been detected in the dorsal root ganglia.

Comparison of NK-3 and NK-1 mRNA distribution in the brain

Among the three tachykinin receptors, NK-3 and NK-1 are the most abundant in the rat CNS (Tsuchida et al., 1990). To date, the presence of NK-2 in the rat CNS is still controversial (Saffroy et al., 19881, although several pharmacological studies have indicated the involvement of NKA in the central regulation of certain reproductive endocrine functions (Debeljuk et al., 1990; Kalra et al., 1992). There- fore, our discussion of the tachykinin receptor mRNAs in the brain will be restricted to NK-3 and NK-1.

The comparison of NK-3 mRNA described in the present report and NK-1 mRNA (Maeno et al., 1993) revealed a comparable distribution in the rat CNS, although there were several striking differences among these tachykinin receptor mRNAs. For example, NK-3 mRNA was abundant in the internal granular layer of the olfactory bulb, the anterior olfactory nucleus, the cerebral cortex, the supraoptic nucleus, the lateral hypothalamus, the zona incerta, the medial mammillary nucleus, the pontine nucleus, the ventral tegmental area, the substantia nigra, and the interpeduncular nucleus, whereas NK-1 mRNA (Maeno et al., 1993) was sparse or absent. In contrast, NK-1 mRNA (Maeno et al., 1993) was abundant in the striatum as well as the motor neurons of cranial and spinal nerves. In addition to these dramatic differences in the localization of NK-3 and NK-1 mRNA (Maeno et al., 19931, there appear to be several instances where both receptors were present but where the apparent levels of expression were markedly different. The areas of the brain that seem to have more NK-1 mRNA (Maeno et al., 1993) were the piriform cortex, the nucleus of the diagonal band of Broca, the amygdalohippocampal area, the locus ceruleus, the parabrachial nucleus, and the inferior olive. Alternatively, the distribution of NK-3 mRNA appears to be greater than NK-1 mRNA

NK-3 mRNA IN RAT BRAIN

(Maeno et al., 1993) in the arcuate nucleus, the parvicellular divisions of the paraventricular nucleus, and the nucleus of the spinal trigeminal tract. Moreover, the present study detected a concentration of NK-3 mRNA in the granular layer of the cerebellum, an area that was described as containing “ambiguous expression of NK- 1 mRNA” (Maeno et al., 1993). Although the above differences in NK-3 and NK-1 mRNAs most likely represent a genuine difference in receptor expression, it should be noted that these mRNAs were not evaluated in the same gender.

413

The results of these in situ hybridization histochemical studies have provided detailed and novel information about the distribution of NK-3 mRNA in the rat CNS and associated tissues. These morphological data have also elucidated the putative sites of neurokinin B action in the CNS, which is an important step toward understanding the neuronal systems that are modulated by this neuropeptide.

Functional considerations The present study provides a detailed description of the

topography of NK-3 receptor mRNA-containing perikarya in the CNS; therefore, it may serve as the foundation for further physiological, pharmacological, and morphological studies aimed at exploring the function of neurokinin B in the CNS. Evaluation of the sites of NK-3 mRNA expression in the CNS suggests that neurokinin B action is associated primarily with processing sensory information. This con- cept is supported by the observation that NK-3 mRNA is abundant in the main sensory processing areas. These areas include the nuclei of the limbic system, such as the olfactory bulb, the amygdala, the hippocampus, the habenula, and the interpeduncular nucleus; areas associated with visual processing, such as the granular layers of the retina, the superior colliculi, and the visual cortex; somatic and autonomic sensory nuclei, including the superficial layers of the spinal cord, the nucleus of the solitary tract, and the dorsal nucleus of the vagus; and nuclei associated with pain transmission, including the superficial layers of the spinal cord, the nucleus of the spinal trigeminal tract, the parabrachial nuclei, the central gray matter of the mesencephalon, the pons, and the cerebral cortex. Interest- ingly, the thalamus, which is a major relay between the cortex and the brainstem, has very few NK-3 mRNA cells. Intense hybridization signal was also detected in areas of the brain that are not associated with sensory processing, such as the supraoptic and paraventricular nuclei of the hypothalamus. Because the primary role of these hypothalamic nuclei is osmoregulation, neurokinin B may be in- volved in this process.

Previous immunocytochemical studies have shown that neurokinin B perikarya are concentrated in a region of the arcuate nucleus that projects to the external zone of the median eminence (Merchenthaler et al., 1992). This observation and the presence of neurokinin B terminals in the median eminence (Merchenthaler et al., 1992) suggest that neurokinin B may be released into the portal circulation, thereby regulating anterior pituitary function. In addition, the presence of NK-3 mRNA in the arcuate nucleus could provide a mechanism by which neurokinin B, released into the portal circulation, could feed back to the arcuate nucleus (an ultrashort feedback loop) and regulate neurokinin B synthesis and/or release.

It is intriguing that NK-3 was also abundant in the zona incerta, the ventral tegmental area, and the substantia nigra, areas of the brain that are rich in dopaminergic neurons that project to the basal ganglia. In contrast, NK-3 mRNA was absent in the dopaminergic neurons of the arcuate nucleus, which project to the median eminence and not to the basal ganglia. Thus, neurokinin B may selectively regulate the function of certain dopaminergic neuronal populations that are important in the regulation of specific dopaminergic pathways.

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In situ hybridization analysis of the distribution of neurokinin-3 mRNA in the rat central nervous system