Proc. Nati. Acad. Sci. USAVol. 77, No. 9, pp. 5532-5536, September 1980Neurobiology

In vitro autoradiography of opiate receptors in rat brain suggestsloci of "opiatergic" pathways

(naloxone/neuropeptides/neurotransmitters/sensory systems/limbic system)

MILES HERKENHAM*t AND CANDACE B. PERTt*Laboratory of Neurophysiology and *Section on Biochemistry and Pharmacology, Biological Psychiatry Branch, National Institute of Mental Health,Bethesda, Maryland 20205

Communicated by Edward V. Evarts, June 4,1980

ABSTRACT Slide-mounted sections of unfixed frozen ratbrain can be labeled in vitro with [3Hlnaloxone to show the

-like ligand selectivity characterized in previous studies. Wehave developed an autoradiographic technique using hot par-aformaldehyde vapors to prevent diffusion of ligands with re-versible binding. Resolution at the light level is sufficient todetect concordance between receptor patterns and terminalfields of axonal projections marked-by tract-tracing techniques.The opiate receptor distribution suggests the existence ofwidespread intrinsic and several longer multisynaptic "opia-tergic" pathways within sensory and limbic circuits. One mul-tisynaptic pathway ma link olfactory structures with limbiccircuits in the amygdala and habenula. Another may lie inlimbic cortical structures. Opiate receptors are numerous alsoin sensory systems, and within primary sensory nuclei (visual,auditory, olfactory, somatic) they are found superficially inlaminated structures. Together, the opiate receptors are wellplaced to control incoming sensory and subsequent limbic in-formation processing.

Various histochemical techniques have revealed that subsetsof neurons secrete the same neurotransmitter and occupywell-defined positions in brain. Further advances towardmapping the chemical coding of tracts can be anticipated bythe concomitant use of two autoradiographic techniques tocompare (i) the terminal distributions of particular fiber tractswith (ii) receptor distribution patterns. Concordances may besufficiently close to allow one to surmise that the terminals arepresynaptic to the receptors. However, a major obstacle toroutine autoradiography of drug and neurotransmitter recep-tors is that often only reversible ligands are available for ra-diolabeling. These are prone to diffusion if conventional "wet"processing procedures are used (1). One method, wherebypredried emulsion-coated slides or coverslips are pressed ontoradiolabeled sections (2), has been applied to brain neuro-transmitter and drug receptors, not only in the earliest studiesafter injection of radiolabeled ligands into animals in vivo (3,4), but also in slide-mounted sections incubated in ligands "invitro" (5, 6). The advantages of the in vitro approach, includingits potential for studying human brain receptors, have beenemphasized (7). We now report how unfixed, slide-mountedsections can be radiolabeled in vitro, subsequently fixed withhot paraformaldehyde vapors and visualized by "wet" auto-radiography with its higher resolution.

MATERIALS AND METHODSMale Sprague-Dawley rats (approximately 200 g) were de-capitated, and their brains were rapidly immersed in isopentaneat -30'C, mounted on cryostat chucks, and cut into 25-,gm-thick coronal sections at -14'C. Sections were thaw-

mounted onto precleaned gelatin-coated slides and air driedat -14'C for 48 hr. Racks holding 30 slides were incubated in300 ml of the appropriate incubation medium. Preincubationfor 15 min at 250C (0.05 M Tris-HCl buffer, pH 7.4/100mMNaCl/2 ,uM GTP/bacitracin at 0.5 mg per ml/aprotinin at 100kallikrein inhibitory units per ml) was followed by incubationat 4VC for 30 min with a similar solution (lacking only GTP) towhich [3H]naloxone (50,000 cpm/ml, New England Nuclear,48 Ci/mmol; 1 Ci = 3.7 X 1010 becquerels) had been added.The slides were then transferred sequentially through six rinses(approximately 20 sec in each) of 0.05 M phosphate-bufferedsaline, pH 7.4, at 4VC, and then quickly frozen on dry ice. Thefrozen sections were lyophilized overnight, transferred to adesiccator jar containing paraformaldehyde powder, andheated for 2 hr at 800C under reduced pressure. Radioactivecontent of slides was monitored by scintillation spectrometryby adding 20 ml of detergent/fluor to the tissue-laden slidefragment (see legend, Fig. 1). To ascertain whether [3H]nal-oxone had been "fixed" to receptors, alternate sections werewashed in three sequential short (15-sec) dips in 40C water anddried. Washed and unwashed sections were indistinguishableafter visualization by autoradiography, and the wash watercontained no extracted radioactivity. Some sections were de-fatted in xylene and alcohol rinses without loss of labelingpatterns. Slides were dipped into Kodak NTB-2 emulsion asdescribed (1, 8). Coated slides were stored at -140C for 6 and12 weeks. After this exposure, the slides were developed andcounterstained with thionin, and coverslips were added. Thelocations of silver grains in the emulsion overlying [3H]nalox-one-labeled brain regions were microscopically examined underdark-field and bright-field illumination.

RESULTSFig. 1 shows the differential abilities of nine opiate ligands todisplace [3H]naloxone binding from brain slices on slides. Theirrank order of potency is identical to results from previous studiesof [3H]naloxone binding to rat brain membranes (9, 10) andguinea pig ileum (11), and it correlates extremely well (r > 0.9)with their ability to elicit analgesia in rodent pain tests (12) orto suppress smooth muscle contraction in the ileum (11).

General agreement with previously published autoradi-ographic distributions of opiate receptors (4, 5, 13-16) wasfound. However, a number of areas are intensely and discretelylabeled and show striking concordance with known patternsof tract terminations derived from neuroanatomical mappingstudies. This paper will focus on these sharply labeled areas.

Fig. 2 shows a section of rat brain at the level of the caudateand accumbens nuclei. The dense opiate receptor labeling,

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t To whom reprint requests should be addressed at: Laboratory ofNeurophysiology, Bldg. 36, Rm. 2D10, National Institute of MentalHealth, Bethesda, MD 20205.

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FIG. 1. Displacement of [3H]naloxone binding from unfixedslide-mounted sections at striatal levels. Tissue-laden slide fragmentswere fit into counting vials and incubated in a final volume of 3 mlwith four to seven different concentrations of various opiate ligands.The incubation was terminated by decanting the medium and washingtwice with 20 ml ofcold phosphate-buffered saline. Aquassure (NewEngland Nuclear) scintillation cocktail (20 ml) was added to eachwell-drained vial and radioactivity was measured at 35-40% efficiencyby liquid spectrophotometry. The mean 4 SEM of eight slides withno added drug (total binding) was 457 + 69 cpm and the "nonspecificbinding" obtained by maximal inhibition of drugs was 90 4 27cpm.

which appears white in the dark-field photomicrographs shownin Fig. 2, includes the patches and the subcallosal streak notedin previous studies (13, 16) as well as faint bands in layers I andIII of medial and dorsal walls of the cerebral hemispheres.Diffuse background striatal labeling can also be observed. Highmagnification of a striatal patch viewed in bright-field anddark-field illumination (Fig. 2 Lower) illustrates the intactmorphology. The distinct localization of grains over neuropiland not fiber fascicles militates against diffusion of ligand away

FIG. 2. (Upper) Low-power (X5.5) dark-field photomicrographof an autoradiogram (6-week exposure) of a [3H]naloxone-incubatedrat brain section at the level of the caudate and accumbens nuclei.Opiate receptor-rich areas appear as white patches and streaks. Morediffuse labeling appears as lighter shades of gray. (Lower) High-power(X108) bright-field (left) and dark-field (right) photomicrographsof a striatal section exposed to the emulsion for 6 days. In both high-power views, normal morphology ofneurons and glia is seen in relationto fiber fascicles that appear as clear areas surrounded by the neurons.In dark-field the silver grains appear as small white dots, situated overcells and neuropil, composing a discrete cluster in the center of thefield. No grains overlie the fiber fascicles.

from the receptor. When 0.1 uM levallorphan, a potent opiate,is included in the incubation (not shown) no labeling can bevisualized at low power. Inclusion of 0.1 MM dextrallorphan hadno effect on la1eling.

Proposal for a Five-Link Sequential "Opiatergic" Path-way. Early autoradiographic studies of opiate receptors (4, 13)revealed dense binding sites- in the lateral edge of the medialhabenula and the dorsal part of the interpeduncular nucleus.Herkenham and Nauta (8) noted these two opiate-rich areasare connected by habenulo-interpeduncular fibers. Fig. 3 showsthat opiate receptor distributions bear a striking resemblanceto tract terminations in the interpeduncular nucleus and medianraphe labeled by tritiated amino acids injected into the recep-tor-rich area of the habenula (bottom three pairs of photo-graphs) or in the habenula by amino acids injected into the bednucleus of the stria terminalis (top pair of photographs). Aparsimonious explanation of this concordance is that [3H]nal-oxone binding sites mark receptors postsynaptic to terminalsof "opiatergic" projections. Further putative opiatergic linksmay include the medial and posterior cortical nuclei of theamygdala, which project to the bed nucleus (Fig. 3B) (17).These amygdaloid nuclei are themselves rich in opiate receptors(Fig. 3 C and D), especially in their superficial portions thatreceive inputs from the accessory olfactory bulb (18), which isalso labeled (Fig. 3A). In summary, the concordances suggestsequential opiatergic paths originating in the vomeronasal organor, more likely, within intrinsic olfactory bulb neurons, andpassing to the amygdala and bed nucleus of the stria terminalisand then to the habenular border zone and, finally, relayed tothe interpeduncular nucleus and median raphe.

Opiate Receptors in Sensory Nuclei. As Figs. 3 and 4A il-lustrate, the points of termination of the primary and severalsubsequent neurons in the olfactory pathway are rich in opiatereceptors. The primary terminal zones of somatosensory inputswithin the spinal cord (Fig. 4I) and spinal trigeminal nucleus(Fig. 4H) are heavily marked with opiate receptors only in thesuperficial layers. Within the visual system, similar restrictionis noted: among the sites of termination of retinal ganglion cells,labeling is sparse in the dorsal lateral geniculate nucleus,moderate in the superficial gray stratum of the superior col-liculus (Fig. 4D), and heavy in the olivary pretectal and theaccessory optic nuclei (Fig. 4 B and C). Within the auditorysystem the dorsal portion of the dorsal cochlear nucleus, com-posing its molecular layer, is labeled (Fig. 4F). A noteworthyfeature of receptor localization in laminated structures is thepreferential distribution within superficial layers.

Visceral sensory fibers, conveyed by the vagus nerve to thenucleus of the solitary tract, terminate in a region rich in opiatereceptors (Fig. SG). The second synapse in this pathway is foundin the parabrachial and ambiguous nuclei (19), and both areasare labeled. Within the parabrachial nuclei, the lateral sectorsare the most densely labeled (Fig. 3E). Separate portions of thesolitary tract and parabrachial nuclei transmit gustatory af-ferents (19), and these are characterized by sparser labeling.Limbic Cortical Areas. Previous studies of opiate receptor

distribution in homogenized dissected tissue called attentionto opiate receptor-rich regions within the limbic system (20-22),but utilized ligands (etorphine and diprenorphine) or conditionsthat we now know label not only GTP-sensitive, alkaloid-pre-ferring, "type 1" receptors, but also "type 2" opiate receptors,which are GTP-resistant, peptide-preferring, and abundant inlimbic areas (23). In this study, using selective "type 1" condi-tions, we observed that the hypothalamus and much of theamygdala were not particularly well labeled. Conversely, allstriking opiate receptor-dense regions in cortex are withinstructures considered to be associated with the limbic system.

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FIG. 3. (A-D) Projection drawings (X10) of receptor-labeled sections at the levels of the frontal pole, showing olfactory structures (A),the posterior bed nucleus of the stria terminalis (B), and anterior (C) and posterior (D) levels of the amygdala. Stippling represents opiate receptordistributions in these sections. Arrows indicate neural pathways, derived from other studies (18), connecting the accessory olfactory bulb (aob)with the bed nucleus of the stria terminals (nst) and the medial (ma) and the posterior cortical (pca) nuclei of the amygdala. These amygdaloidnuclei project to the nst (17), which in turn projects to the border zone between the medial and lateral habenular nuclei (unpublished data, andE, left). (E-H) Paired photomicrographs (X60) of two kinds of autoradiography. In the left members of the pairs the transport of 3H-labeledamino acids has marked neural pathways from the nst to the habenula (hb), shown in E, and from this border zone to the interpeduncular nucleus(ip) and the median raphe (mr) as shown in F-H and also by figures 7 and 16 of Herkenham and Nauta (8). The identical corresponding levelsshown in the right members of the pairs illustrate opiate receptor distributions marked by [3H]naloxone. The striking concordances betweenthe terminal distribution of the nst-hb, hb-ip, and hb-mr paths and the receptor distributions suggests that these may be "opiatergic" tracts.Other abbreviations in this figure are: aon, anterior olfactory nuclei; bla, basolateral nucleus of amygdala; f, fornix; fc, frontal cortex; h, hippocampus;ic, internal capsule; mob, main olfactory bulb; mlf, medial longitudinal fasciculus; pc, piriform cortex; sm, stria medullaris; st, stria termi-nalis.

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FIG. 4. Dark-field photomicrographs (all X33) of [3H]naloxone binding in sensory nuclei. Sequence is from rostral to caudal. (A) Accessoryolfactory bulb (aob) and anterior olfactory nucleus (aon), molecular layer. (B-D) Primary sensory nuclei for vision. (B) Olivary pretectal nucleus(op) at level of pretectum and posterior commissure. (C) Medial terminal nucleus of accessory optic tract (mto) between mammillary nuclei(left) and substantia nigra (right). (D) Superficial gray stratum of the superior colliculus (sc). (E) Secondary visceral relay in the parabrachialnuclei (pb). (F) Dorsal cochlear nucleus (dc) of the auditory system. (G) Caudal portion of nucleus of the solitary tract (nts) at level of the areapostrema, which is not labeled. (H) Marginal and gelatinous layers of nucleus caudalis of spinal trigeminal complex (spV). (I) Marginal layerand substantia gelatinosa in dorsal horn (dh) of cervical spinal cord.

Moderate laminar labeling is found in medial frontal (Fig. 5A)and cingulate areas (Fig. 2). Labeling is strikingly dense in thepresubiculum (Fig. 5B). Sparse labeling characterizes the en-torhinal area and two strata of Ammon's horn: the lacuno-sum-moleculare and surrounding the pyramidale (Fig. 5C).While the underlying long- and short-axoned pathways whoseterminations correspond to these patterns are too numerous todescribe (see ref. 24 for a review), a tentative limbic opiatergiccircuit might involve the mediodorsal thalamic nucleus, itselfrich in opiate receptors, its projection field in the frontal cortex,the entorhinal area and presubiculum, which are peri-allo-cortical way stations, and the hippocampus proper. The labelingin the presubiculum (25) is the densest in the limbic corticalcircuit.

DISCUSSIONUsing the high-resolution method for visualizing brain receptorsat the light-microscopic level that we have developed, we haveobserved striking concordances between distribution patternsof opiate receptors and terminal fields demonstrated with

classical autoradiographic techniques. A wealth of biochemicaland neurophysiological evidence (12, 26) suggests that [3H]-naloxone binding sites, marked by the "type 1" incubationconditions used here (23), represent loci on the postsynapticmembrane where endogenous opiates alter sodium ion fluxafter secretion from the presynaptic element of "opiatergic"neurons. We have thus initiated a list of opiatergic pathwaysin rat brain which, while by no means complete, already makesup a remarkably extensive neurocircuitry. Confirmation maybe achieved by cell body destruction-induced depletion ofopiate receptor ligands in putative terminal areas, using asensitive selective radioreceptor assay (27).A particularly striking finding in this study is that sensory

input areas from nearly all modalities contain opiate receptorslocalized to corresponding loci-i.e., the molecular or super-ficial layers-wherever lamination is apparent. We cannotsurmise the origins of cell bodies innervating these sensory areasyet, but we emphasize that they must represent intrinsic orcentrifugal connections (not the primary afferents themselves),at least in the somatosensory system where dorsal root rhizotomycauses partial loss of opiate receptors (28) but no enkephalindepletion (29). A descending enkephalinergic projection from

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FIG. 5. Dark-field photomicrographs (all X33) of [3H]nalox-one-labeled limbic cortical structures. (A) Frontal cortex (fc), dor-somedial surface, showing receptors localized in the outer half of layerI and more indistinctly distributed in layer Ill. (B) Very dense labelingin the presubiculum (pres) bordering the caudal dentate gyrus dorsalto it and the parasubiculum ventral. (C) Sparse but laminated dis-tribution of receptors in the hippocampus. The bright stripes are thedensely packed pyramidal cells of Ammon's horn and granule cellsof the dentate gyrus.

the paragigantocellular nucleus to the dorsal horn has, in fact,been described recently (30).We wish to emphasize that in this paper we have neither

described nor considered the visualization of either "type 2"opiate receptors (23) or regions such as the periaqueductal graywhere "type 1" receptors are more sparsely distributed and lacksharp borders. However, we have already found that type 1 andtype 2 opiate receptors, which are somewhat analogous to ,u and6 receptors, respectively (31), have distinctly different patternsof brain distribution (32).The proposed pervasiveness of opiatergic circuitry is con-

sistent with the wide-ranging effects of exogenous and endog-enous opiates (33). Obviously, the setting of pain thresholds isbut a small component of this multi-origin cell body system:perhaps incoming sensory information from all modalities isplaced in an emotional context along a pleasure-pain contin-uum by the putative opiatergic pathways.

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