Preprotachykinin-A mRNA Expressionin the Human and Monkey Brain:An In Situ Hybridization Study

YASMIN L. HURD,1* EVA KELLER,2 PETER SOTONYI,2 AND GORAN SEDVALL1

1Psychiatry Section, Department of Clinical Neuroscience, Karolinska Institute,S-171 76 Stockholm, Sweden

2Department of Forensic Medicine, Semmelweis University of Medicine,1091 Budapest, Hungary

ABSTRACTThe mRNA expression for preprotachykinin-A (PPT-A) was studied throughout the

human and cynomolgus monkey brain to assess the neuroanatomical expression pattern ofthe PPT-A gene in primates. In situ hybridization showed that the PPT-A mRNA is expressedhighly in specific regions of the postmortem human brain, including the striatum, islands ofCalleja, hypothalamus (posterior, premammillary, medial mammillary, and ventromedialnuclei), superior and inferior colliculi, periaqueductal gray, and oculomotor nuclear complex.PPT-A mRNA-expressing neurons also were present in the paranigralis (ventral tegmentalarea) and were scattered in the bed nucleus stria terminalis throughout the sublenticularsubstantia innominata region, including the diagonal band of Broca and the nucleus basalis ofMeynert. In the hippocampus, high PPT-A mRNA expression was localized predominantly tothe polymorphic layer of the dentate gyrus; no labeled cells were present in the granular layer.Positively labeled cells also were found scattered in the CA regions as well as in theamygdaloid complex. Neocortical expression of PPT-A mRNA was localized mainly to the deeplaminae (layers V/VI), except for the striate cortex (labeling was seen also in superficiallayers). The subiculum, thalamus, globus pallidus, ventral pallidum, substantia nigra parscompacta, red nucleus, pontine nuclei, and cerebellum were characterized by very weak toundetectable expression of PPT-A mRNA. An expression pattern was evident in the monkeyforebrain similar to that observed in the human, except for the absence of PPT mRNA-expressing cells in the medial mammillary nucleus despite intense expression in supramam-millary, lateral mammillary, and premammillary nuclei. Overall, more similarities thandifferences are apparent between primate species in the expression pattern of the PPT-A gene.J. Comp. Neurol. 411;56–72, 1999. r 1999 Wiley-Liss, Inc.

Indexing terms: substance P; primate; islands of Calleja; striatum; hippocampus; substantia

innominata

Considerable interest has been focused on the role ofneuropeptides in the central nervous system (CNS), be-cause these diverse neuroactive substances are present inhigh concentrations in the brain with a wide distributionand influence over various functions and behaviors. One ofthe best characterized neuropeptides is substance P. Sub-stance P belongs to the family of tachykinin neuropeptidesthat are generated from the preprotachykinin (PPT) gene.Specifically, substance P derives from the PPT-A (PPT-A)gene that also encodes neurokinin A (Bannon et al., 1992;Krause et al., 1987). Animal studies have provided a largebody of evidence showing that substance P is involved inmotor behavior, neuroendocrine function, and nociception(for reviews, see Maggio, 1988; Pernow, 1983). Human

postmortem studies also have revealed an associationbetween tachykinin-containing neurons and specific neuro-logical disorders. For example, a depletion of substance Pimmunoreactivity has been observed in cortical neurons ofpatients with Alzheimer’s disease (Bouras et al., 1990;

Grant sponsor: The Swedish Medical Research Council; Grant numbers:11252, 10591, and 03560; Grant sponsor: the National Institute of DrugAbuse; Grant number: DA08914; Grant sponsor: the Harald and GretaJeansson Stiftelse; Grant sponsor: the Sigurd and Elsa Goljes Minne Fund.

*Correspondence to: Dr. Yasmin Hurd, Psychiatry Section, Departmentof Clinical Neuroscience, Karolinska Institute, S-171 76 Stockholm, Swe-den. E-mail: yasmin.hurd@neuro.ks.se

Received 26 April 1998; Revised 16 February 1999; Accepted 12 March1999

THE JOURNAL OF COMPARATIVE NEUROLOGY 411:56–72 (1999)

r 1999 WILEY-LISS, INC.

Quigley and Kowall, 1991) and in the basal ganglia ofsubjects with Parkinson’s disease (Fernandez et al., 1992;Mauborgne et al., 1983; Sakamoto et al., 1992). In Hunting-ton’s chorea, the levels of substance P are reduced (Emsonet al., 1980; Ferrante et al., 1986; Goto et al., 1989; Grafe etal., 1985; Kowall et al., 1993; Marshall et al., 1983; Reineret al., 1988; Sapp et al., 1995), and the PPT-A mRNA also isdecreased in the striatum of Huntington’s subjects in agrade-related manner (Richfield et al., 1995). Postmortemstudies of the tachykinin system in psychiatric disordersare not as well investigated, but cerebrospinal fluid levelsof substance P have been shown to be elevated in schizo-phrenic subjects (Terenius et al., 1976).

In the human brain, substance P immunoreactivity islocalized mainly to discrete populations of cells and nerveterminal fibers in the cerebral cortex as well as basalganglia structures, including the striatum, internal globuspallidus, and substantia nigra (Bouras et al., 1984; DelFiacco et al., 1984, 1987; Goto et al., 1989; Haber andWatson, 1985; Mai et al., 1986; Nomura et al., 1987;Sakamoto et al., 1987). The distribution of the PPT-AmRNA expression has been well characterized in thehuman striatum (Chesselet and Robbins, 1989; Chesseletand Affolter, 1987; Hurd and Herkenham, 1995; Nisbet etal., 1995). Although much attention has been focused onthe striatal expression of the PPT-A gene in normal andclinical subjects, only partial information is availableregarding the mRNA expression in other brain regions. Inlight of the limited information available about the ana-tomic organization of the PPT-A mRNA expression in thehuman brain, a more widespread study of the human brainwas thought necessary. Such studies are important be-cause it has become more evident that a better understand-ing of the neurochemical organization of the human brainis needed to interpret the vast amounts of data accruedfrom experimental animals regarding the role of tachyki-

nins in brain function. In the present study, in situhybridization histochemistry was performed on cryosec-tions at different levels of the postmortem human andmonkey brain to map the expression pattern of the PPT-Agene in primates.

MATERIALS AND METHODS

Tissue

Normal control human brains were obtained at autopsyfrom the Forensic Medicine Department at the KarolinskaInstitute under guidelines approved by the ethics commit-tee and the Swedish Board of Health and Social Welfare.The subjects were three males and one female, ages 36years, 53 years, 58 years, and 59 years, respectively, with apostmortem delay time between 13 hours and 22 hours. Allsubjects died from acute cardiorespiratory failure. Thetoxicological reports showed no presence of neuroactivedrugs, including alcohol, antidepressants, antipsychotics,or minor tranquilizers. None of the brains showed anyevidence of macroscopic pathology. In addition, there wereno indices of substance abuse, or psychiatric or neurologicdisease in any of the subjects, as assessed from examina-tion of medical records gathered from hospitals in theStockholm region. The brains were divided along themidsagittal line, and the whole hemispheres were frozen(285°C) on glass plates with the sagittal plane facingdown, as described previously (Brene et al., 1994; Hall etal., 1993). In preparation for cryosectioning, the brainhemisphere, oriented so that the anterior and posteriorcommissures were positioned on the same horizontal line,was embedded in a metal frame mounted on a cooled stagewith a carboxymethylcellulose semiliquid gel and frozen at285°C. Subsequently, the hemisphere was placed in a cold

Abbreviations

ac anterior commissureaCg anterior cingulate gyrusAlv alveusAmy amygdalaBNST bed nucleus stria terminalis, lateralCA cornu ammonisCb cerebellumCl claustrumCN caudate nucleusCNt caudate nucleus, tailDBB diagonal band of BrocaDg dentate gyrusEc entorhinal cortexFg fusiform gyrusGL granular layerGPe globus pallidus, externalHi hippocampusHy hypothalamusI insulaIC inferior colliculusic internal capsuleIsC islands of CallejaiTg inferior temporal gyrusLG lateral geniculate bodyLg lingual gyrusLM lateral mammillary nucleusMM medial mammillary nucleusmPf medial prefrontal cortexmTg medial temporal gyrusNAc nucleus accumbensOcc occipital cortexOg orbital gyrus

ot optic tractPAG periaqueductal grayPBP parabrachial pigmented nucleusPc piriform cortexpCg posterior cingulate gyrusPH posterior hypothalamusPHg parahippocampal gyrusPL polymorphic layerPM premammillary bodyPn paranigralisPN pontine nucleiPu putamenRg rectal gyrusRN red nucleusS subiculumSC superior colliculusSCg subcallosal gyrussF superior frontal cortexSI substantia innominataSLEA sublenticular extended amygdalaSM supramammillary hypothalamic nucleusSo stratum oriensSp hippocampal pyramidal cell layersp spleniumSr stratum radiatumSt stria terminalissTg superior temporal gyrusT temporal cortexTh thalamusTu olfactory tubercleUg uncal gyrusVMH ventromedial hypothalamus

PPT-A mRNA EXPRESSION IN HUMAN AND MONKEY BRAIN 57

(220°C) cryomicrotome (PMV400 LKB2250; LKB, Stock-holm, Sweden), and, after equilibration to the cryostattemperature, 100-µm-thick sections were taken through-

out the entire brain hemisphere in the horizontal plane.The sections were placed onto poly-l-lysine-treated glassplates and stored frozen at 230°C until later use.

Fig. 1. Distribution of preprotachykinin-A (PPT-A) mRNA expres-sion in whole human hemisphere horizontal cryosections. The low-resolution, high-contrast images show the PPT-A hybridization signal

at five dorsoventral levels throughout the human brain. The levels areapproximately 71 mm (A), 81 mm (B), 86 mm (C), 91 mm (D), and 103mm (E) below vertex. For abbreviations, see list. Scale bar 5 20 mm.

58 Y.L. HURD ET AL.

Normal human brain specimens (three males and onefemale; postmortem interval 4–10 hours; 46–73 years ofage) also were obtained at autopsy at the Forensic Medi-cine Department of Semmelweis University under theguidelines approved by the Semmelweis University Hu-man Ethical Committee. These brains immediately werecut into 1.5-cm-thick coronal slabs, frozen in dry ice-cooledisopentane, and stored at 240°C. The slabs subsequentlywere cut into coronal blocks of tissue containing specificbrain regions (striatum, hippocampus, and neocortex).Twenty-micron-thick cryosections were taken from theseblocks by using a Jung-Frigocut 2800E cryostat (Leica,Heidelberg, Germany), dried onto poly-l-lysine-treatedglass slides, and stored frozen at 230°C until later use.

The forebrains of two cynomolgus monkeys were ob-tained from the National Bacteriological Laboratory (Stock-holm, Sweden). The monkeys had been used as control,untreated subjects in an unrelated experiment and re-ceived a lethal dose of pentobarbital. The animals weretreated in accordance with protocols approved by theAnimal Ethical Committee of Stockholm. The brains hadbeen rapidly frozen in dry ice-cooled isopentane and keptfrozen at 270°C until the time of cryosectioning. Twenty-micron-thick coronal cryosections were taken throughoutthe rostral-to-caudal extent of the right hemisphere, andsagittal cryosections were taken throughout the dorsoven-

tral extension of the left hemisphere. The cryosectionswere dried onto poly-l-lysine-treated glass slides and storeddesiccated at 230°C.

In situ hybridization histochemistry

The tissues were prefixed in preparation for in situhybridization, as described previously (Hurd, 1996), whichwas carried out under conditions to prevent contaminationby RNases. Briefly, for in situ hybridization histochemis-try, the glass-mounted tissue sections were brought toroom temperature and fixed by immersion in 4% formalde-hyde in phosphate-buffered saline (PBS), pH 7.4, for fiveminutes at 25°C; rinsed twice in PBS; treated with 0.25%acetic anhydride in 0.1 M triethanolamine/0.9% saline, pH8.0, for 10 minutes; dehydrated in 70%, 80%, 95%, and100% ethanol; delipidated in chloroform for five minutes;rinsed in 100% and 95% ethanol; and air dried. Allsolutions were made with autoclaved 0.1% diethylpyrocar-bonate-treated water.

RNA probes were synthesized from 1.0-kb human cDNA(directed against bases 16–1003 of the human PPT-A gene;Genbank accession no. M28019) inserted into pSP65 vec-tor (kindly provided by Drs. T.I. Bonner and H.-U. Af-folter). The plasmid containing the PPT-A cDNA insert waslinearized with HindIII, and transcription was initiatedfrom the SP6 promoter. In vitro transcription was carried

Figure 1 (Continued)

PPT-A mRNA EXPRESSION IN HUMAN AND MONKEY BRAIN 59

out by using [35S]uridine 58-[a-thio]triphosphate (NewEngland Nuclear, MA) to radiolabel the probes, and hybrid-ization was carried out with 20 3 104 cpm-labeled probeper mm2. The sections were covered with 2,000 µl (wholehemisphere sections) and 200 µl (small coronal blockedsections) of heat-denatured hybridization solution [4 3standard sodium citrate (SSC; 1 3 SSC 5 3 M sodiumchloride, 0.3 M sodium citrate, pH 7.0); 10% (weight/volume) dextran sulfate; 1 3 Denhardt’s (0.02% Ficoll,0.2% polyvinylpyrrolidone, 0.2 mg/ml bovine serum albu-min); 0.5 mg/ml sheared, single-stranded salmon spermDNA; 250 µg/ml yeast tRNA; 200 mM dithiothreitol (DTT);and 50% formamide; the chemicals used in the hybridiza-tion procedure were purchased mainly from Sigma, St.Louis, MO]. The tissue sections were coverslipped andplaced in an incubation oven (55°C) overnight with 4 3SSC in a humidified environment. The coverslips wereremoved, and the tissue sections were placed in 2 3 SSCdiluted in 1 mM DTT solution and were put through thefollowing washes: 10 minutes in RNase A buffer (5 M NaCl;1 M Tris, pH 8.0; 0.5 M ethylenediamine tetraacetic acid,pH 8.0) at 37°C; 30 minutes in RNase A (20 µg/ml) plusRNase A buffer at 37°C; 5–10 minute series of washes indecreasing concentrations of SSC diluted in 1 mM DTT atroom temperature; two 30-minute rinses in 0.1 3 SSC/1mM DTT at 53°C; one-minute rinse in 0.1 3 SSC/1 mMDTT at room temperature. These steps were followed bydehydration of the tissue in ascending one-minute ethanolrinses containing 300 mM ammonium acetate and 100%ethanol, and then the tissue was dried. Subsequently, theglass-mounted sections were apposed to film (Hyperfilmb-Max, Amersham, Buckinghamshire, United Kingdom)in x-ray cassettes for 5–28 days with 14C standards, anddeveloped (D19; Eastman-Kodak, Rochester, NY) for fiveminutes at 20°C. The same sections that were used in thehybridization experiments or near adjacent sections laterwere stained with cresyl violet for gross histologic examina-tion of Nissl substances. Acetylcholinesterase activity (seeHurd and Herkenham, 1995) also was performed on nearadjacent sections for histologic characterization.

Film autoradiograms were viewed by using a light box,MTI CCD72 high-resolution video camera with a CanonMacro FD/50 mm lens (Canon, Inc., Tokyo, Japan), andMacintosh-based image analysis software system (NIHImage; Wayne Rasband, NIMH, Bethesda, MD). Gain andblack level settings were standardized with the MTICCD72 control unit for the camera. Digitized images alsowere made from the autoradiograms of the whole hemi-sphere sections; the films were scanned (ScanMarker III;Microtek Electronics, DYsseldorf, Germany) at a resolu-tion of 300–500 dots per inch. Densitometric readings weretaken of structures based on histochemical landmarks inconjunction with information obtained from publishedsources (Parent, 1996; Paxinos, 1990) as well as theDeArmond et al. (1989) and Duvernoy (1991) human brainatlases and the Stephan et al. (1980) monkey atlas. In situhybridization also was carried out on near adjacent brainsections to localize tyrosine hydroxylase mRNA expres-sion, which was used to identify catecholamine popula-tions in the brainstem, and prodynorphin mRNA expres-sion, which was used to dissociate the patch (highprodynorphin mRNA expression; Hurd and Herkenham,1995) versus matrix (low prodynorphin mRNA expression)compartments in the striatum. In the whole hemispherebrain sections, measurements were taken throughout the

rostral-to-caudal extent of each structure of interest and inthe dorsal and ventral subregions of any structure thatwas visible at different levels of the brain sections studied.Measurements obtained for each brain region were aver-aged. Light transmittance values were converted to disin-tegrations per minute/milligram (dpm/mg) values by using14C standards. Background labeling in the surroundingwhite matter was subtracted from dpm/mg values ob-tained in gray matter structures. Emulsion-dipped slideswere examined by the use of an Optiphot-2 microscope(Nikon, Tokyo, Japan) equipped with episcopic illumina-tion from a mercury light source and a band-pass filter(450–490 nm). Only silver grains, and not the Nisslsubstances, reflect light under the episcopic illuminationconditions. Positively labeled neurons were defined as cellswith light cresyl violet staining with overlying silvergrains (at least four times the number of scattered grainsin the immediate surrounding neuropil) in contrast to glialcells, which appeared small and were stained darkly withan absence of silver grains. Figures were prepared digi-tally by using NIH Image and the PowerPoint (version 4.0;Microsoft, Inc., Redmond, WA) or PageMaker (version 6.5;Adobe Systems, Mountain View, CA) software programsand contrast was adjusted for some images.

RESULTS

PPT-A mRNA expression in the human brain

A heterogeneous hybridization pattern was apparent inthe postmortem human brain using the 35S-labeled ribo-probe against the PPT-A gene (Fig. 1). Although there weredifferences between subjects in terms of the magnitude ofPPT-A mRNA expression, all subjects showed the samegeneral anatomic pattern of expression. The total disinte-grations per minute/milligram (dpm/mg) values measuredthroughout different structures in the whole human hemi-sphere cryosections is presented in Figure 2. The distribu-tion pattern generated by the PPT-A riboprobe was foundto be quite distinct from hybridization patterns obtainedwith other riboprobes (e.g., the nontachykinin neuropep-tides, prodynorphin, and proenkephalin; see Hurd, 1996)that had been studied in near adjacent brain sections ofthe same specimens. The specificity of the PPT hybridiza-tion signal also appeared to represent specific RNA label-ing, because RNase pretreatment abolished the PPT-Asignals (data not shown).

Forebrain. PPT-A mRNA expression was relativelylow in the human cerebral cortex, except for the olfactorypaleocortical area, where intense hybridization signalswere found in the olfactory tubercle generally in clusters ofcells consistent with the anterior olfactory nucleus andislands of Calleja (Figs. 1–3). In the neocortex, the samelaminar distribution pattern was apparent in most re-gions, with the highest PPT-A mRNA expression localizedto the deep cortical layers; the lamination most distinct inthe superior temporal and insular cortices (Fig. 1). Al-though they were not abundant, high-power microscopicexamination of emulsion-dipped sections of the cerebralcortex showed that the PPT-A mRNA-expressing cells withdense clusters of silver grains were small and medium-sized and were localized primarily to layers V and VI (Fig.4). Moderate and weakly labeled nerve cells also werefound scattered throughout other layers, and positivelylabeled neurons were detected as well in the subcorticalwhite matter (Fig. 4). The striate cortex, however, showeda different laminar pattern, with the most abundant

60 Y.L. HURD ET AL.

expression in both deep (layers IVc and VI) and superficial(layers II/III) layers (data not shown). A distinct laminarorganization of PPT-A mRNA expression was lacking inthe entorhinal cortex (Fig. 1E), with labeled cells foundscattered throughout this cortical area.

Abundant PPT-A mRNA expression was present in thestriatum (caudate nucleus, putamen, and ventral stria-tum; Fig. 1A–C). Consistent with previous results (Chesse-let and Robbins, 1989; Hurd and Herkenham, 1995),clusters of cells with the highest PPT-A mRNA expressionwere evident throughout the striatal area, most often inregister with high prodynorphin mRNA-expressing zones(data not shown), which delineates the patch (striosome)compartment in the human caudate nucleus and putamen(Hurd and Herkenham, 1993, 1995). PPT-A mRNA expres-sion also displayed a moderate medial-to-lateral gradientwithin the caudate nucleus, with the highest levels foundin the medial regions. All subjects but one showed higher

expression levels of PPT-A mRNA in the dorsal comparedwith the ventral striatal (nucleus accumbens) area. Figure2 shows the summary data of all subjects. Exclusion of theone subject with higher PPT-A mRNA expression in theventral striatum versus the dorsal striatum showed thatthe levels in the striatum were as follows: nucleus accum-bens, 132.01 6 23.26 (mean 6 S.D.); caudate nucleus,174.08 6 17.26; and putamen, 167.09 6 21.41. IntensePPT-A mRNA hybridization signals were apparent alongthe medial edge of the nucleus accumbens, consistent withthe islands of Calleja, as well as in some neurons of the bednucleus of stria terminalis. PPT-A mRNA-expressing cellsalso were scattered throughout the substantia innominata(sublenticular extended amygdala), including the area ofthe diagonal band of Broca and the nucleus basalis ofMeynert (Figs. 1B,C, 5). Silver grains were seen predomi-nantly over the medium-sized and small neurons, and notover the large neurons, within this area (Fig. 6). No

Fig. 2. Bar graph representing disintegrations per minute/milligram (dpm/mg) values (subtracted from background white matterlevels) measured within the total specified brain area in whole humanhemisphere sections from four subjects. Numbers in parenthesis

correspond to Brodmann cerebral cortex nomenclature. d, dorsalportion of the specified brain region; v, ventral portion of the specifiedbrain region. For abbreviations, see list.

PPT-A mRNA EXPRESSION IN HUMAN AND MONKEY BRAIN 61

positive PPT-A hybridization signals were detected in theventral pallidum (Fig. 5) or the globus pallidus (Figs. 1A,5), except in a few subjects under very long exposure time;even then, the signal was barely higher than background.The dorsal portion of the claustrum showed low-to-moderate PPT-A mRNA expression (Fig. 1A).

PPT-A mRNA was expressed in discrete cell populationsof the hippocampal formation (Figs. 1A–D, 7). The highesthybridization signals were measured in the region of thedentate gyrus (Fig. 2). High-magnification microscopicexamination showed that the hybridization signal arosepredominantly from intense PPT-A mRNA-expressing cellswithin the hilar (polymorphic layer) area of the dentategyrus; no labeled cells were found in the granular layer(Fig. 7). Lower mRNA expression levels were found in theCA regions and uncal gyrus. Cells showing dense silvergrains in the CA regions were located predominantly in thestratum oriens (Fig. 8). A few PPT-A mRNA-positive cellsalso were found scattered in the pyramidal layer of the CAregion, but generally with a lower abundance of overlyingsilver grains. Very low to undetectable PPT-A mRNAhybridization signals were apparent in the subicular com-plex (Fig. 1B–D). The amygdaloid complex was character-ized by very weak hybridization signals in the low-resolution, whole hemisphere brain sections (Fig. 1D).However, microscopic examination of emulsion-dipped sec-tions from the amygdala showed the presence of PPT-AmRNA-positive cells scattered throughout the amygdala

nuclei (data not shown). No positive PPT-A hybridizationsignals were detected in any thalamic nuclei examined,including the geniculate bodies (Fig. 1A).

Very high levels of PPT-A mRNA expression were seenwithin the hypothalamus, primarily in the posteriornucleus, premammillary nucleus, medial mammillarynucleus (mammillary body), and caudal tuberal area (Fig.1B–D). One subject, however, showed only moderate expres-sion in the mammillary body despite similar intenseexpression in the premammillary nucleus, a finding thatcould not be explained by the documented clinical history.Low-to-moderate PPT-A mRNA expression was observedin other hypothalamic nuclei, including the dorsomedialand lateral nuclei. The anterior hypothalamic region wascharacterized by very low PPT-A hybridization signals.

Midbrain and cerebellum. Intense hybridization sig-nals were visible throughout the mesencephalic tegmen-tum, in areas such as the periaqueductal gray (Fig. 1C)and the oculomotor nuclear complex (Fig. 9), as well as thesuperior colliculus (in particular, the external gray layer)and the inferior colliculus (Fig. 1B–D). At this mesence-phalic brain level, low-to-moderate PPT-A mRNA expres-sion was observed in the raphe (caudal linear nucleus;data not shown) and in paranigral and parabrachialpigmented nuclei (dorsal to the substantia nigra), whichcorrespond to the rat A10 ventral tegmental area (Bogerts,1981; Fig. 1C). In the substantia nigra pars compacta, nopositively labeled cells were present in medially localized

Fig. 3. PPT-A mRNA-expressing cells in the islands of Calleja (IsC)within the olfactory tubercle area of the human brain. The coronalsection was visualized under brightfield (A) and episcopic (B) illumina-tion. The white grain clusters (episcopic illumination) identify the

positively labeled cells. Note the overlap of the PPT-A hybridizationsignal with the dense cluster of small cells that characterizes the IsCregion. Scale bar 5 100 µm.

62 Y.L. HURD ET AL.

neuronal populations (Fig. 10A,B); however, in more cen-tral and lateral areas, some (mainly nonpigmented) neu-rons with weak-to-moderate silver grain density wereobserved after long exposure times. A few labeled cells(large, nonpigmented) with dense overlying silver grainsalso were found scattered in ventral areas of the substan-tia nigra (data not shown). Microscopic examination of theneuronal populations immediately medial and dorsal tothe substantia nigra, which constitutes the ventral tegmen-tal area, showed that the intensely labeled cells werenonpigmented neurons (Fig. 10C,D). No labeling was

present in the red nucleus or in the pontine nuclei (Fig.1C–E). The cerebellar cortex and dentate nucleus showedno positive PPT-A mRNA hybridization signals (Fig. 1D–E).

PPT-A mRNA expression in the monkey brain

A heterogeneous pattern of PPT-A mRNA expressionalso was found throughout the cynomolgus monkey fore-brain (Figs. 11, 12). Low to high hybridization signals weredetected in the cortical mantle, with the olfactory, piri-form, and temporal cortices showing the highest expres-

Fig. 4. PPT-A mRNA-expressing cells in the human insular cortex.The coronal section was visualized under brightfield (A,C,E) andepiscopic (B,D,F) illumination. The white grain clusters (episcopicillumination) identify the positively labeled cells. Arrows point to

intensely labeled cells in layer III (A,B), in deep layer V (C,D), and inthe white matter (WM; E,F). Arrowheads point to unlabeled cells.Scale bar 5 50 µm.

PPT-A mRNA EXPRESSION IN HUMAN AND MONKEY BRAIN 63

sion levels. In most areas of the neocortex, intenselylabeled cells showing positive expression of the PPT mRNAwere found mainly within the deep laminae (particularlyin layer V; Fig, 13), but the striate cortex showed PPTmRNA expression in both superficial and deep laminae(data not shown). The only cortical area without a discretelaminar organization was the entorhinal cortex, wheremoderate and high numbers of PPT-A expressing cellswere found scattered in all layers (Fig. 11).

Intense PPT-A mRNA expression levels were present inthe caudate nucleus and putamen (Figs. 11, 12). Clustersof the highest levels of PPT-A hybridization were visiblemainly in the rostral areas, because the hybridizationsignal increased in the caudal area in particular withinregions surrounding the patches so that the caudal regionshowed a more homogenous expression pattern. There alsowas a dorsal-to-ventral striatal gradient with lower hybrid-ization signals in the main accumbens area. Intensehybridization signals were evident in the olfactory tu-bercle and the islands of Calleja (Fig. 11A,B). Low and afew moderately labeled cells were found scattered through-out the substantia innominata and bed nucleus of the striaterminalis. No positive PPT-A mRNA expression wasdetected in the pallidum (Fig. 11D).

PPT-A mRNA was expressed weakly in the monkeyhippocampus (Fig. 11D). Similar to the human, micro-scopic examination showed that the hippocampal PPT-AmRNA-expressing cells were localized preferentially to thepolymorphic layer of the dentate gyrus, with no labelingdetected in the granule cells. Scattered moderate hybridiza-tion signals were present in the CA region, and thesubicular region showed very low expression of PPT-A

mRNA (data not shown). Positively labeled (low-to-moderate overlying silver grains) PPT-A mRNA cells werefound scattered throughout the amygdaloid complex, withthe lowest abundance in the lateral nuclei, which contrib-uted to a generally weak hybridization signal present inthis region on low-resolution film autoradiographic exami-nation (Fig. 11C,D).

At the hypothalamic levels studied (the infundibularstalk and a few other medial nuclei could not be examinedin the primate brain specimens obtained), the highesthybridization signals were apparent in the ventromedial,supramammillary, lateral mammillary, and premammil-lary nuclei as well as throughout the posterior hypothala-mus (Fig. 11C,D). Low and moderate PPT-A mRNA-expressing cells were also found scattered in portions ofthe dorsomedial and lateral hypothalamus. However, noPPT-A mRNA-expressing cells were detected in the mam-millary body (Fig. 11D). No positive hybridization signalswere observed in the main body of the thalamus (Fig. 12).

DISCUSSION

Although the distribution of PPT-A mRNA expressionhad been characterized previously in the rat centralnervous system (Harlan et al., 1989; Warden and Young,1988), no complementary information existed with regardto the overall tachykinin gene expression in the primatebrain. In the current study, we provide for the first time ageneral map of the PPT-A mRNA expression in the humanand monkey brain. The results show that, in addition tothe very well established information known about thehuman striatum, high PPT-A mRNA-expressing regions inthe human brain include the olfactory tubercle, hypothala-mus, hippocampus, periaqueductal gray, and superiorcolliculus.

The highest PPT-A mRNA expression found in thehuman hippocampal formation was localized preferen-tially to cells within the polymorphic layer of the dentategyrus. This finding is consistent with the pattern ofdistribution of substance P-immunoreactive neurons de-scribed previously in the human and monkey hippocam-pus, in which abundant substance P-positive cells wererestricted to the polymorphic area of the dentate gyrus,and low numbers were present in the strata oriens andpyramidal CA area (Del Fiacco et al., 1987; Iritani et al.,1989; Pioro et al., 1990; Seress and Leranth, 1996). Basedon the anatomic distribution of PPT-A-expressing cells inthe hippocampus, it has been suggested that these cellpopulations are not regulated directly by the main excita-tory input to the hippocampus through the perforantpathway but, instead, are regulated by the granule cellswithin of the dentate gyrus (Seress and Leranth, 1996).The hippocampal formation is involved in epileptic sei-zures, and, as part of the local intrinsic circuitry of thehippocampal formation, PPT-A mRNA-expressing cellsmay play a role in epilepsy. In fact, the numbers ofhippocampal substance P-immunoreactive neurons havebeen found to be reduced in epileptic patients (de Lanerolleet al., 1992; Sloviter, 1987, 1994). PPT-A cells located in thepolymorphic zone normally decrease the excitation ofcortical input onto the granule cells (Sloviter, 1994). Thus,the reduction of substance P neurons in epilepsy would beconsistent with a diminished ability to reduce the excitabil-ity of hippocampal cells.

Fig. 5. Distribution of PPT-A mRNA expression in a coronal sectionof the human basal forebrain at the level of the sublenticular extendedamygdala. Midline is to the right. For abbreviations, see list. Scalebar 5 5 mm.

64 Y.L. HURD ET AL.

The most notable feature of PPT-A mRNA expressionwithin the human hypothalamus was the high abundancewithin the premammillary nucleus and mammillary body.These nuclei situated in the medial region of the hypothala-mus are thought to play a role in goal-oriented behavior forsurvival of the individual (Canteras and Swanson, 1992).The mammillary body is an intricate component of the‘‘Papez circuit’’ (Papez, 1937), which is hypothesized to beimportant for limbic function relevant for emotional expres-sion, learning, and memory. Intense prodynorphin mRNAexpression has been found within the human premammil-lary nuclei; however, unlike the PPT-A mRNA pattern, theprodynorphin gene is not expressed in the mammillarybody (Hurd, 1996; Sukhov et al., 1995). Different roles in

limbic function are expected for the premammillary nucleusand the mammillary body due to their distinct anatomicconnectivity. In contrast to the mammillary body, whichreceives direct input from the hippocampal formation, thepremammillary nuclei receive their primary innervationfrom the anterior hypothalamus, which is innervateddirectly by the prefrontal cortex, amygdala, and hippocam-pus (Canteras and Swanson, 1992). Both the premammil-lary nuclei and the mammillary body, however, receiveinput from the ventral tegmental area (Shibata, 1987), thelimbic-related area of the mesencephalon involved in drugreinforcement and associated motor behaviors (Koob, 1992).The PPT-A mRNA cell populations may be involved invarious limbic function through regulation of hormonal

Fig. 6. A–D:High-magnification images of the diagonal band ofBroca (DBB) region in the human brain taken under brightfield (A,C)and episcopic (B,D) illumination. White grain clusters (episcopic

illumination) identify the PPT-A mRNA-expressing cells. Note the lackof positive labeling over the large-sized cells (A,C) but the highlabeling over smaller sized neurons (B,D). Scale bar 5 100 µm.

Fig. 7. High-magnification images of the human dentate gyrustaken under brightfield (A) and episcopic (B) illumination. Whitegrain clusters (episcopic illumination) identify the PPT-A mRNA-expressing cells. Note that intensely labeled PPT-A mRNA-expressing

cells are localized primarily to the polymorphic layer (PL) region closeto the granule cell layer (GL), where no labeled cells are present. Scalebar 5 100 µm.

PPT-A mRNA EXPRESSION IN HUMAN AND MONKEY BRAIN 65

and motor response within limbic-related brain regions.Although abundant numbers of substance P-immunoreac-tive neurons also have been detected in the hypothalamictuberal area, a finding consistent with the high mRNAexpression we observed in the caudal portion of this area,no substance P-immunoreactive neurons were found in thehuman mammillary body (Pioro et al., 1990). Furtherexamination of a larger number of human subjects mighthelp to reveal the nature for this discrepancy and whether

PPT-A mRNA in the mammillary body is expressed differ-entially in different populations of subjects. However, inagreement with the current results, a recent microscopicexamination of the human hypothalamus also reportedintense PPT-A mRNA expression in the mammillary body(Chawla et al., 1997).

The limbic anatomical association of the PPT-A mRNApattern also was evident due to the intense expression ofthe tachykinin gene within the islands of Calleja and

Fig. 8. High-magnification images of the human hippocampal CAregion taken under brightfield (A) and episcopic (B) illumination.White grain clusters (episcopic illumination) identify the PPT-AmRNA-expressing cells. Moderate to intensely labeled PPT-A mRNA-expressing cells are present along the stratum oriens (So), with some

scattered cells (lower intensity) evident in the pyramidal cell layer(Sp). Arrows point to positively labeled cells in So and Sp. No labeledcells are present in the alveus (Alv) or the stratum radiatum (Sr).Scale bar 5 100 µm.

66 Y.L. HURD ET AL.

specific neuronal populations within the rostral and sublen-ticular substantia innominata. This is consistent with thedistribution in the rat brain, in which high substance PmRNA expression characterizes the Calleja granule cells(Harlan et al., 1987; Warden and Young, 1988). Althoughthe function of the granule cells of the islands of Calleja isstill unclear, in addition to PPT-A, these neurons also arecharacterized by high expression of dopamine D1 mRNA(Le Moine and Bloch, 1996) and D3 mRNA (Bouthenet etal., 1991; Diaz et al., 1995; Landwehrmeyer et al., 1993; LeMoine and Bloch, 1996; Suzuki et al., 1998). Because cellsin the islands of Calleja complex that do express PPT-AmRNA also appear to express the D1 and D3 receptor genes(Le Moine and Bloch, 1996), and because this complex doesreceive dopamine innervation from the ventral tegmentalarea (Fallon et al., 1983), it is possible that PPT-A mRNA-expressing cells within the islands of Calleja could beaffected by dopamine and also play a role in psychiatric(e.g., schizophrenia) and drug-abuse disorders. The atypi-cal antipsychotic agent, clozapine, but not haloperidol,induces long-term induction of c-fos activity in the islandsof Calleja (Guo et al., 1995; Hurley et al., 1996; MacGibbonet al., 1994). Activation of the mesolimbic neurons in theventral tegmental area also induces c-fos activation in theislands of Calleja (Chergui et al., 1996; Cornish and vanden Buuse, 1996). Thus, alteration of Fos protein transcrip-tion factors, which regulate the control of gene expression,could affect PPT-A mRNA expression in these neuronalpopulations, especially considering that the PPT-A genecontains the consensus sequence for AP-1 factors such, asc-fos (Carter and Krause, 1990). The finding that thePPT-A phenotype defines the specificity of cocaine-inducedc-fos activation within striatal neurons (Kosofsky et al.,

1995; Moratalla et al., 1993) also might apply for theislands of Calleja.

Although the limbic relevance of the PPT-A mRNA-expressing cell populations was evident throughout manycircuits of the human brain, the amygdaloid complex wasnotable in its very low abundance of PPT-A mRNA expres-sion. The weak levels of mRNA expression are consistentwith the low numbers of substance P-immunoreactiveneurons previously observed in the human amygdala(Pioro et al., 1990). Although PPT-A cell populations mightnot contribute directly to behaviors mediated by theamygdala, such as fear, anxiety, social behavior, andstimulus-response (see Aggleton, 1992), their role cannotbe discounted completely, because PPT-A mRNA-express-ing cells were found scattered throughout most amygdalanuclei. It remains to be determined whether the normallow expression of the PPT-A gene in the human amygdalais altered in specific neuropsychiatric disorders.

The present results confirm the very high PPT-A mRNAexpression within the limbic-related patch compartment ofthe striatum (Chesselet and Robbins, 1989; Hurd andHerkenham, 1995) and the very low to undetectableexpression in the globus pallidus (Bannon et al., 1992;Nisbet et al., 1995). Based on the expression pattern ofPPT-A mRNA in different human brain structures, itwould appear that the major source of substance P andneurokinin A found in the human dorsal striatum (Beachand McGeer, 1984; Mai et al., 1986) does not originate fromthe thalamus, because there were no detectable PPT-AmRNA signals apparent in this structure. Although theventral tegmental area (which showed abundant expres-sion of PPT-A mRNA) does project to the striatum, thoseterminal inputs are localized mainly to the nucleus accum-bens region (Dahlstrom and Fuxe, 1964), and the dopamin-ergic (neuromelanin-pigmented) substantia nigra pars com-pacta neuronal populations, which innervate the caudatenucleus and putamen (Carpenter and Peter, 1972), dis-played no or weak PPT-A mRNA expression. Striatalsubstance P immunoreactivity may arise from the cerebralcortex, but this contribution might be low considering therelative weak abundance of PPT-A mRNA expression inthe human neocortex and the weak association with thelarge (pyramidal) cells. Instead, a major source of sub-stance P and neurokinin A present in the caudate nucleusand the putamen in all probability arises from axoncollaterals of the striatal medium spiny projection neu-rons, which have intense expression of the PPT-A gene.

A strong involvement of tachykinin neurons in humansomatosensory function is supported by the high expres-sion of the PPT-A gene in the superior colliculus, a regioninvolved in influencing head and eye movement in re-sponse to visual, auditory, and somatic stimuli, and in theinferior colliculus, which is the major brainstem auditoryrelay center receiving information from the medial genicu-late body and transmitting impulses to the primary audi-tory cortex. The current findings agree with immunohisto-chemical studies showing substance P-immunoreactivecell bodies in the collicular region in the human brain(Bouras et al., 1986; Mai et al., 1986; Pioro et al., 1990). Animportant role of the PPT-A gene in oculomotor functionalso is strengthened by the abundance of PPT-A-express-ing cells in the mesencephalic oculomotor nuclear complex.High expression of the PPT-A mRNA in the human periaq-ueductal gray also is very much in line with the well-known involvement of substance P in nociception and

Fig. 9. Low magnification image of PPT-A mRNA expression in themidline dorsal mesencephalic tegmentum (coronal section) of thehuman brain. Note the intense PPT-A hybridization signals within theoculomotor complex and periaqueductal gray. Aq, cerebral aqueduct;CLi, caudal linear raphe nucleus; OMn, oculomotor nuclei; PAG,periaqueductal gray. Scale bar 5 2 µm.

PPT-A mRNA EXPRESSION IN HUMAN AND MONKEY BRAIN 67

analgesia (see Maggio, 1988; Meyer and Frenk, 1988;Pernow, 1983).

In addition to the above-mentioned structures, such asthe thalamus and the substantia nigra, there was anotable absence of PPT-A mRNA expression in the humancerebellum. High-magnification microscopic examinationof these areas might have allowed the ability to detectscattered PPT-A mRNA-expressing cells in this brainregion. However, the absence of PPT-A mRNA expressioncurrently observed in the cerebellar cortex and dentatenucleus is consistent with previous immunohistochemicalstudies (Beal et al., 1988; Del Fiacco et al., 1988). Althoughsubstance P immunoreactivity has been documented inthe human cerebellum, the levels are most abundant innewborns, with only faint levels present in the adult brain(Del Fiacco et al., 1988). The rare occurrence of substanceP-immunoreactive neurons in the cerebellum suggest anextracerebellar origin of the substance P found in thisbrain region (Inagaki et al., 1982). Dysfunction of cerebel-lar substance P afferents, rather than abnormal intrinsicPPT-A cerebellar neuronal populations, might contributeto the increased levels of substance P present in thecerebellar dentate nucleus of Huntington’s disease sub-jects (Beal et al., 1988).

Species comparisons

PPT-AmRNAexpression in the monkey forebrain showedthe same general pattern of distribution that was observedin the human: highest cortical expression mainly to thedeep laminae in most neocortical areas, intense expressionin the islands of Calleja and olfactory tubercle area,patches of intense PPT-A mRNA expression in the caudateand putamen, lower PPT-A expression in the main portionof the nucleus accumbens, high to intense expression inthe hypothalamic tuberal and mammillary area, scatteredexpression in the amygdala, highest hippocampal expres-sion localized to the polymorphic layer and an absence ofexpression in the granule layer, and no detectable expres-sion in the thalamus or the pallidum. Two in situ hybridiza-tion studies have characterized the anatomic organizationof substance P mRNA expression in the rat brain (Harlanet al., 1989; Warden and Young, 1988); however, somedifferences were observed between these investigations.Aside from the species differences discussed below, theanatomic pattern of the PPT-A mRNA expression weobserved in the human brain was most similar to the ratsubstance P mRNA distribution pattern described byWarden and Young (1988). In the rat brain, regions show-ing high levels of substance P mRNA expression included

Fig. 10. High-magnification images of the human substantia nigrapars compacta (SNc; A,B) and paranigralis (Pn; C,D) taken underbrightfield (A,C) and episcopic (B,D) illumination. White grain clus-ters (episcopic illumination) identify the PPT-A mRNA-expressing

cells. Note that the paranigral neurons show expression of the PPT-AmRNA but that the neuromelanin-pigmented neurons (dark cells inbrightfield) in the medial SNc do not. Scale bar 5 100 µm.

68 Y.L. HURD ET AL.

Fig. 11. A–D:Distribution of PPT-A mRNA expression in whole-hemisphere coronal sections of the cynomolgus monkey brain. Thelow-resolution, high-contrast images show the PPT-A hybridization

signal at four rostrocaudal levels. The most rostral level is representedin A, and the most caudal level is represented in D. Midline is to theright. For abbreviations, see list. Scale bar 5 0.5 cm.

PPT-A mRNA EXPRESSION IN HUMAN AND MONKEY BRAIN 69

the striatum, islands of Calleja, olfactory tubercle, deepneocortical layers, hypothalamus (premammillary, supra-mammillary, and ventromedial hypothalamus), inferiorand superior colliculus, oculomotor nucleus, and periaque-ductal gray.

The most marked species difference we observed was inthe hypothalamus. No PPT-A mRNA was identified in themonkey mammillary body, a region that showed intenseexpression in the human brain (Chawla et al., 1997;current results), even though the premammillary nucleiexpressed very high PPT-A mRNA in both species. Consis-tent with the expression pattern observed in the monkey,substance P mRNA in the rat also is undetectable in themedial mammillary nucleus (Harlan et al., 1989). Thus, itappears that, with regard to the hypothalamic expressionof the PPT-A gene, there may be a closer similaritybetween monkeys and rats compared with humans. Al-though common anatomic features of PPT-A mRNA expres-sion are evident between the human, monkey, and ratbrain, it must be considered that experimental animals dohave a more limited exposure to the diverse pharmacologicand environmental conditions than that experienced byhumans, which might have led to the high expression ofthe PPT-A gene observed in the mammillary body. How-ever, such variables most likely would be expected toinfluence a wider number of neuronal populations ratherthan one specific brain area, such as the mammillary body.

Very limited information currently exists regarding thePPT system in the monkey; however, it had been reportedpreviously that no substance P-immunoreactive neuronsare present in the ventromedial hypothalamus (Ronnek-leiv et al., 1984). That finding is in contrast to the very highexpression of the PPT-A mRNA-expressing cells found inthis hypothalamic region in both the primate (Chawla etal., 1997; current results) and the rat (Harlan et al., 1989;Warden and Young, 1988). The reason for this discrepancyis unclear; however, the monkeys examined in one immu-nohistochemical study (Ronnekleiv et al., 1984) had been

gonadectomized, which might have altered the intensity ofthe substance P cells in the tuberal hypothalamic area.Further immunohistochemical and in situ hybridizationstudies clearly are needed to verify the anatomic differ-ences noted thus far in the monkey as well as investiga-tions into the relevance of the PPT-A cells in the humanmammillary body.

Although it was not as marked as the hypothalamicexpression pattern, there appeared to be a slight speciesdifference in the hippocampal expression of the tachykiningene. Although PPT-A mRNA cells barely are expressed inthe rat hippocampus (Harlan et al., 1989; Warden andYoung, 1988), they were detected more easily in themonkey hippocampus and, to an even greater extent, inthe human hippocampus. This might suggest an increasedPPT-A involvement in hippocampal function with increas-ing phylogenic complexity. Whether these differences re-late to the broader array of experiences (e.g., learning andmemory) incurred by humans than by rats and monkeysneeds further investigation.

Fig. 12. Distribution of PPT-A mRNA expression in a horizontalsection of the cynomolgus monkey brain at the level of the main body ofthe dorsal thalamus. Midline is oriented toward the bottom. Forabbreviations, see list. Scale bar 5 0.5 cm.

Fig. 13. PPT-A mRNA-expressing cells in deep layers of themonkey superior temporal cortex visualized with episcopic illumina-tion. Arrows point to positively labeled cells (white grain clusters) thatwere found predominantly in layer V. Scale bar 5 100 µm.

70 Y.L. HURD ET AL.

CONCLUSIONS

The role of the tachykinin system in human brainfunctions related to neuropsychiatric diseases is far fromclear. Advances in postmortem and in vivo methodologicapproaches to study the human brain should help to gainbetter insight into the possible role of the tachykininpeptides in psychiatric and neurologic disorders. ThePPT-A gene was found in this study to be highly expressedin a number of limbic-related (islands of Calleja, patchcompartment of striatum, hippocampus), sensory-related(colliculi, periaqueductal gray, oculomotor complex), motor-related (caudate nucleus, putamen), and neuroendocrine-related (hypothalamus) areas of the human brain. Overall,there appeared to be a comparable anatomic organizationof the PPT-A gene expression between primates and ro-dents. Although some caution should be taken whengeneralizing from rodents and monkeys to human, espe-cially in relation to hypothalamic (mammillary body)function, it appears to be possible to extrapolate certaintachykinin-related behaviors characterized in experimen-tal animals to humans.

ACKNOWLEDGMENTS

We thank Mrs. Barbro Berthelsson and Mrs. Siv Eriks-son for their skilled technical support and Ms. Pia Eriks-son for her valuable help with preparations of the figures.

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