Autoradiographic localization of delta opiate receptors in rat and human brain

A’euroscience Vol. 27, No. 2, pp. 497-506, 1988 Printed in Great Britain

0306-4522/M $3.00 + 0.00

Pergamon Press plc 0 1988 IBRO

AUTORADIOGRAPHIC LOCALIZATION OF DELTA OPIATE RECEPTORS IN RAT AND HUMAN BRAIN T. P. BLACKBURN,* A. J. CRoss,t C. HILLEt and P. SLaTt?Rt *Bioscience Department II, ICI plc, Pharmaceuticals Division, Alderley Park, Macclesfield SKI0 4TG, U.K. and tDepartment of Physiological Sciences, University of Manchester, Manchester M 13 9PT, U.K. Abatrae-1n oitro quantitative receptor autoradiography was performed on frozen sections of rat and human brain to visualize delta opiate receptors using the specific ligand [3H][D-Pen2, n-Pens]enkephalin. For comparison, rat brain sections were also labelled with [3H]o-A1a2,o-I&u’-enkephalin. Compounds which block mu and kappa binding were included to make the [3H]~-AlaZ, D-LeuS-enkephalin binding more specific. The two ligands had similar, but not identical, distributions in rat forebrain sections. Sites labelled with [‘H][D-Pen*@Pen’jenkephalin were distributed heterogeneously within the layers of the frontal and parietal cerebral cortex, with high densities in the superficial and deep cortical layers. The claustrum and striatum had the most delta sites, whereas the globus pallidus had no delta binding. The distribution of [‘HID-Ala*,o-Leu’-enkephalin binding sites was similar to that of [‘H][D-Pen’,DPen5]enkephalin, except that there was less heterogeneity in the frontal cortex. In the human brain regions studied, the highest delta binding was in caudate, putamen, temporal cortex and amygdala. There was less heterogeneity in the binding of [D-Pen’,D-Pen’lenkephalin in the human cortex than in the rat. No delta binding was seen in the medial and lateral segments of the globus pallidus. In both species, a discrepancy between the high enkephalin content of the globus pallidus and the absence of delta binding was apparent

The chronic spinal dog preparation used by Martin et ~1.~’ in which the pharmacological effects of

ethylketocyclazocine and N-allylnormorphine, metazocine (SKF 10,047) were compared, led them to propose the existence of three classes of opiate receptors: mu, kappa and sigma. Subsequently, the existence of another receptor was suggested mainly on the basis that the enkephalins were selective for a novel site in the mouse vas deferens. This additional site was termed the delta receptor. Ligand binding studies have shown that delta sites are present in brain and that their distribution appears to be different from that of the mu receptor.4x’0,24 The identification of an endogenous ligand for each opiate receptor subtype has not been finally resolved. One hypothesis suggested is that the mu and delta receptors are the preferred sites for the endogenous enkephalin peptides [Met]- and [Leu]enkephalin respectively.‘5~38 Some areas of brain especially rich in enkephalin-containing neurons and nerve terminals were identified in both rat and human brain by immunohistochemical techniques.i4*i9 These areas ought to contain correspondingly high levels of opiate receptors. Many of the brain regions where opiate receptors have been identified correlate well BSA, bovine serum albumin; DADLE, o-Ala* ,o-L.eu’-enkephalin; DAGO, o-Ala2, MePheS , [D-Pen*,D-Pen’lenkephalin; ICI Gly(o1); DPDPE, 174,864, N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH; ICI 154,129, N,N-bisallyl-Tyr-Gly-+ -(CH, S)-Phe-Leu-OH; SKF 10,047, N-allylnormetazocine; U50,488H, trms3,4-dichloro-N-methyl-N[2-( 1-pyrrolidinyl)cyclohexyl]benzeneacetamide.

Abbreviations:

with the immunohistochemical localization of terminals containing enkephalins or endorphins.40 However, there are sometimes wide discrepancies between the distribution of an endogenous opioid and the appropriate receptor. One of the highest concentrations of enkephalin immunoreactivity is in the globus pallidus, but very few opioid receptors are present in this ,nucleus.8 Thus, in deciding on the possible functions of the [Let+enkephalin-delta receptor system, it is vital to know the distribution patterns of both enkephalin neurons and the delta receptors. Quantitative receptor autoradiography is the method of choice for examining opiate receptor distribution in brain.” Some progress has been made in determining the distribution of delta opiate receptors in rat brain. However, the majority of ligands used to date, including o-Ala2 ,o-Leu5 -enkephalin (DADLE), are not selective for delta receptors, labelling mu sites as well. In contrast, very little information is available concerning the concentrations and distribution of delta receptors in different parts of the human brain. Recently, autoradiographs were prepared from brain sections in which opiate receptors were labelled with [31-IJetorphine and selective displacing agents were used to show the presence of delta sites.* Human brain was found to contain relatively low levels of delta sites compared with mu and kappa receptors. In the present study, we have used quantitative, in vitro receptor autoradiography to investigate the distribution of delta receptors in some parts of rat and human brain. In the rat forebrain, delta receptors have been labelled with [‘HIDADLE, in the presence

497

of compounds kappa

sites,

which and

block

binding

the

specific

with

to mu and delta

ligand

[3H][~-Pen2,~-Pen5]enkephalin.4~29 The latter ligand was also used to investigate the distribution of delta receptors in the basal ganglia and temporal lobe of human brain. EXPERIMENTAL

PROCEDURES

Four female SpragueeDawley rats (18@200 g) were anaesthetized with pentobarbitone (60 mg/kg i.p.) and were perfused transcardially with 200 ml of 0.1% formalin in buffered saline followed by 200 ml of formalin-free saline. The brains were removed, frozen in isopentane at -45°C and were stored at -70’C. Human brains were removed at post-mortem examination from two subjects (one male, one female) with no history of neurological or psychiatric illness. Each had died from an acute illness. The brains were cut into coronal blocks (1 cm thick) through the basal ganglia an the temporal lobes and were frozen in isopentane at -45°C. The mean delay between death and freezing the brains was 15 h. Sections (20,hm) from the frozen rat and human brains were cut on a cryostat, thaw-mounted onto glass slides and stored at -70°C. Prior to labelling, the sections were thawed and washed for 15 min in Tris-HCl buffer (50 mM, pH 7.4) containing 5 mM MgCl,, 100 mM NaCl and 20 pg/ml bacitracin. For the labelling of receptors, sections were covered with Tris buffer solution containing 5 mM MgCl, , 20 p g/ml bacitracin and 5 nM [‘HI [D-Pen’ ,o-Pen’]enkephalin ([‘HIDPDPE). For determining non-specific binding, 100 PM of naloxone was added to the incubation solution. Sections were incubated with the ligand for 1 h at 20°C followed by three lo-min washes in fresh buffersolution containing MgCI, and bacitracin. The sections were then dipped briefly in cold distilled water and dried under a current of air at room temperature. Freeze-drying was used to complete the drying of the sections. Rat brain sections were also labelled with [‘HIDADLE. Sections were thawed and washed for 15 min in 50 mM Tris-HCl buffer (PH 7.4). Sections were then covered with buffer solution containing 2 nM [3H]DADLE, 20nM D-Ala2; MePhes, Gly(o1) (DAGO) to block mu receptor binding and 100 nM ethylketocyclazocine (to block kappa receptor binding). For determining non-specific binding, 100 nM of unlabelled DADLE was added to the incubation solution. After incubation for 30 min at room temperature, the sections were washed twice in fresh buffer (5 min), dipped briefly in cold distilled water, dried in a current of air and freeze-dried. Some brain sections were scraped from the slides into vials containing liquid scintillant and the radioactivity was

counted m order to determme the proportmn ,)I spccihc (displaceable) binding. The majority of the labelled sec~mn\ were used for autoradiography. Autoradiograph) wri\ performed by exposing the sections to tritium-serrsrtr\c lilm (Ultrofilm, LKB) in X-ray cassettes for 0 wcehs ([‘HIDPDPE) or 20weeks ([‘HIDADLE) at 4 ( fritlum standards, consisting of 20-p, sections of tritium Micra,scales (Amersham) mounted on glass slides. nerc it,exposed with the brain sections. The films were developed in Kodak Dl9 developer and density readings on the nnages were made with a microcomputer-based image analyscr. Samples of cerebral cortex for ligand binding and displacement studies were removed from the human brains. Cortical tissue was homogenized (Ultra-Turrax, 10 s) 111 Tris HCI buffer (50mM, pH 7.4) and was washed three times by centrifugation and resuspension. The pellet was suspended in 30~01. of Tris buffer containing 2 mg;ml bovine serum albumin (BSA), 5 mM MgCl.,, and 20 l(g,ml bacitracin. Aliquots (160~1) of the crude membrane preparation were incubated for 60 min with 50 nM [‘HIDPDPE in the presence and absence of displacing compounds. Bound ligand was isolated and washed free of unbound isotope using a cell harvester and glass-fibre filter.‘” Liquid scintillation counting was used to determine the amount 01‘ bound ligand. Labelled and non-labelled opioid compounds used were: [‘HIDADLE (D-Ala2, u-Leu’-[tyrosyl-3.5(n)-‘Hlenkephalin, 37.6 Ci/mmol. Cambridge Research Biochemicals), [‘HIDPDPE (D-Pen’, o-Per?-]tyrosyl-3,5(n)-‘H]enkephalin. 28 Ci Immol, Amersham). u-A-$ ,MePhes .Gly(ol) (DAGO), N.N-diallvl-Tvr-Aib-Aib-Phe-Leu-OH (ICI 174.864). N.Nbisallyl-Tyr-dly-$ -(CHzS)-Phe-Leu-OH (ICI I54.129), ethylketocyclazocine, naloxone hydrochloride, rrarr.v-3.4. dichloro-N-methyl-N[2-( 1-pyrrolidinyl)cyclohexyl]henzeneacetamide (U50.488H) (Upjohn).

RESULTS

Rat brain

Much of the binding of [3H]DPDPE to sections of rat brain was displaced by co-incubation of the sections with an excess of the delta agonist DADLE. The mean specific (displaceable) binding defined in this way was 73% of the total (mean of eight determinations). Figure I shows two autoradiographs, one prepared from a rat brain section labelled with 5 nM [3H]DPDPE and the other from an adjacent section that was treated with 1 PM DADLE plus [‘HIDPDPE. The non-displaceable

Fig. 1. Representative autoradiographs showing the total binding of [3H]DPDPE to adjacent coronal sections of rat brain in the absence (1) and the presence (2) of unlabelled DADLE (1 PM). In this and subsequent autoradiographs, the dark areas correspond to high levels of binding sites.

Delta receptor autoradiography

binding was insufficient to produce an image on the film. Two series of autoradiographs were prepared from rat forebrain sections. One set of sections was labelled with [3H]DPDPE, and the other with [3H]DADLE in

499

the presence of mu and kappa receptor blockers. Some of the autoradiographs, showing sections cut in approximately the same rostrocaudal plane for each ligand, are shown in Fig. 2. There was a very close agreement between the detailed distribution of radio-

A

-

4

Fig. 2. Autoradiographs showing the distribution of total binding sites for (A) [‘HJDPDPE and (B) [3H]DADLE in rat brain sections. Each pair of sections (l-5) was cut from two brains between anterior 2.1 mm and anterior 8.9 mm.

500

T P.

BLACKRUKN

r/ (I/.

Table 1. Total binding of [o-Pen’,D-Pen’lenkephalin in rat brain sections (the quantitative data were obtained from autoradiographs) Brain area Striatum (rostral) Striatum (middle) Nucleus accumbens Claus&urn Cerebral cortex-average Septum Globus pallidus Thalamus-ventral Thalamus-posterior Hippocampus-average Hippocampus-pyramidal cell layer Dentate gyrus Amygdala-average Cingulate cortex Lateral geniculate nucleus Periaqueductal gray

A

[‘HIDPDPE bound (fmol/mg wet wt) 59.6 + 3.4 47.8 + 2.2 40.1 k4.5 72.8 + 5.0 43.6 Ifr9.1 1.7 + 0.6 3.1 f 0.4 3.6 + 0.7 11.4+ 1.1 5.0 & 0.6 52.5 & 1.8 15.0 + 2.3 23.9 + 2.1 27.5 + 2.2 7.9 * 0.5 11.4+ 1.2

Each result is the mean f S.E.M. of five determinations.

activity in the two sets of autoradiographs. Both ligands were distributed unevenly in the cerebral cortex. The highest concentrations of [‘H]DPDPE binding sites were in the superficial (laminae I-III) and deep (laminae V-VI) cortical layers with low binding in the intermediate layer (lamina IV). The binding of [‘HIDADLE was distributed similarly in cortex, except for a more dense labelling of lamina IV in frontal cortex sections. Density measurements were made on the [3H]DPDPE autoradiographs and on the co-exposed tritium standards in order to obtain quantitative data on delta receptor densities. The data are summarized in Table 1. There were no areas of brain that had a receptor concentration in excess of 73 fmol/mg. However, because we cannot be certain that binding conditions in brain sections were optimal or reached saturation levels, the values may not represent the true levels of delta sites in brain. The greatest concentrations of delta sites were in the claustrum and striatum. The abundance of delta receptors in the striatum and nucleus accumbens was most apparent in autoradiographs prepared from brain sections cut

Fig. 4. The displacement of [‘HIDPDPE binding to human cerebral cortex membranes by opiate agonists and antagonists. The compounds, incubated for 40 min with 5 nM [’ HIDPDPE, were: (A) naloxone (ICY0.89 1 M, ()), DAGO (IC, 5.67 PM, A), DADLE (IC, 0.04 PM, m); (B) U50.488 H (IC 828pM, .), ICI 174,864 (ICY 843/1M, A), ICI 154,129 (ICY> 900 PM, n ). Each result is the mean of three separate determinations.

in the sagittal plane, and an example is shown in Fig. 3. The globus pallidus, in contrast to the striatum, had almost no delta sites (Fig. 2). Although delta receptors were distributed unevenly in the hippocampus, the overall concentration of receptors in this structure was relatively low. The pyramidal 1~11layer was clearly visible on the autoradiographs as a band of high [3H]DPDPE binding. Human brain

Fig. 3. Autoradiograph prepared from a sag&al section of rat brain showing the distribution of total binding sites for [‘HIDPDPE in the striatum (s) and nucleus accumbens (a).

The specificity of the binding of [SW]DPDPE to d&a receptors in human brain crude membranes (cerebral cortex) was investi@ed. Opioid compounds were tested for their ability to displace the bin&g of PIQWDPE (5 nM). The mu-delta agonist DADLE and the non-selective opiate receptor antagonist naloxone displaced the binding of [‘HIDPDPE. The IC, values are shown in Fig. 4. ICI 154,129 and ICI 174,864, which are selective delta antagonists, did not displace the binding of

Delta receptor autoradio~phy [‘HIDPDPE in the assay conditions that were used. The mu agonist DAGO and the highly specific kappa receptor agonist U50,488H were inactive. In separate experiments using coronal sections of human brain cut through the basal ganglia, a high proportion of [3H]DPDPE binding was displaced by co-incubation with DADLE (1 FM). The mean

501

specific binding was 81% of the total (mean of eight determinations). The detailed dist~bution of [3H]DPDPE binding in structures contained within the basal ganglia and the temporal lobe of human brain was obtained from autoradiographs. Figure 5 shows autoradiographs prepared from basal ganglia sections cut at different

--a

Fig. 5. Autoradio~p~ (A-D) prepared from coronal sections of human brain through the basal ganglia showing the total binding of [%]DPDPE in the head of the caudate nucleus (c), putamen (p), globus pallidus (g), claustrum (cl) and amygdala (a).

502

T. P. BLACKBURN CI td

levels in the brain. The most noticeable feature was the relatively high binding of [‘HIDPDPE in the caudate and putamen but a total absence of binding in both segments of the globus pallidus. Autoradiographs were also prepared from brain sections that included temporal lobe structures (Fig. 6). Delta receptors were visualized in the hippocampal formation, especially in the dentate gyrus and in the amygdaloid complex Quantitative data on the regional distribution of delta receptors in human basal ganglia and temporal

structures, obtained from autoradiographs, arc gtvcn in Table 2. Caudate and putamen had similar levels of sites. but the overall concentration of [‘H]DPDPE binding sites was probably lower than in the cquivalent areas of rat brain (Table 1). Other regions wtth high levels of delta receptors were the temporal cortex and the amygdaloid complex. The distribution of [‘H]DPDPE binding within the gray matter of the cerebral cortex was examined in more detail by making density measurements on the autoradiographs from both rat and human brain

Fig. 6. Autoradiographs prepared from coronal sections of human brain temporal lobe cut in the anterior-posterior direction (A-D) showing the total binding of PH]DPDPE in the amygdala (a) and the hippocampal formation including the dentate gyrus (d), molecular layer of dentate (l), para-hippocampal gyrus (p) and entorhinal cortex (e).

Delta receptor autoradiography

503

has been a major complicating factor when using them to investigate opioid receptors. The introduction of the conformationally restricted analogue [‘HIDPDPE bound of enkephalin [‘H]DPDPE as a high-affinity delta (fmol/mg wet wt) Brain area ligand with no significant activity at mu sites was 29.0 Cerebral cortex therefore a major step forward in opiate receptor 34.2 Caudate nucleus studies.29 32.8 Putamen Previous autoradiographic studies aimed at reGlobus pallidus-internal and vealing the distribution of opiate delta receptors in 0 external segments brain were made using [‘H]DADLE to label rat brain 15.0 Claustrum 49.0 Temporal cortex sections.‘5~28~30 These earlier studies showed a unique 17.9 Dentate gyrus pattern of receptor sites in which the cortex and 26.8 Hippocampus striatum were densely labelled, but the septum, Amygdaloid complex globus pallidus, preoptic area and hypothalamus Each result is the mean of four determinations on sections were only lightly labelled. This type of distribution in from two brains. rat brain was reproduced in the [3H]DADLE autoradiographs generated in the present study. The evidence for multiple opiate receptors in sections. In the rat brain the distribution of delta brain*’ has been strengthened by both ligand bindreceptors was heterogeneous within the cortex (Fig. ing and autoradiographic techniques. Autoradio7). [3H]DPDPE binding was lowest in the middle graphically, it has been shown that mu, delta and parts of the cortical gray (corresponding approximately to lamina IV) with distinct bands of high kappa sites in brain sections have clearly distinct binding in the outer and innermost layers (corre- distributions and each has unique features.‘2,‘5*3’,36*40 Mu receptor sites seem to account for much of sponding to laminae I-III and V-VI respectively). In the human cortex, the binding of [3H]DPDPE was the characteristic clusters of binding seen in the caudate-putamen,15 whereas delta receptors were more even. distributed much more evenly. It was suggested that mu and delta receptors in the caudate-putamen may DISCUSSION be interconvertible because their ligand selectivity [3H]DPDPE, a stable analogue of enkephalin, has appeared to alter with the incubation conditions.* been used as a selective delta receptor ligand.26,43 Kappa receptors appear to be distinct from delta sites in the rat brain, because delta sites were more However, at the concentrations of ligand needed for autoradiographic studies, [3H]DADLE will abundant in cortex and striatum, while kappa sites were found more in brainstem, hypothalamus and label other opioid sites, especially mu receptors. cerebellum.3 [3H]DADLE labels the putative mu, sites with an In the present study, the qualitative distribution of affinity similar to that for delta receptors.42 This lack delta sites in rat brain labelled with [3H]DADLE, in of specificity of some naturally occurring enkephalins

Table 2. Total binding of [D-Pen*@-Pen’lenkephalin in human brain sections (the quantitative data were obtained from autoradiographs)

A

8-

O-

I P

1

z

J

Coitex

depth

-mm

Fig. 7. Distribution of total [‘HIDPDPE binding through the depth of the cortical gray in human (A) and rat (B) brain. Optical density measurements (arbitrary units) were made on four autoradiographs from two to three brains starting at the outer surface. Vertical bars show S.E.M.

504

T. P.

BLACKBURN

the presence of compounds that prevent binding to mu and kappa receptors, was closely similar to that observed with [3HIDPDPE. In particular, several forebrain structures, including the nucleus accumbens, striatum and claustrum, had high levels of binding. Heterogeneity was especially pronounced in the cerebral cortex. The outer and inner layers of the cerebral cortex had the most binding sites, while the middle layers usually had a lower level. A similar distribution of [IH]DPDPE binding in rat and guinea-pig cortex was reported recently.34 This contrasts with an earlier report that in the cerebral cortex mu receptors occurred in laminae I and IV whereas delta receptors were found predominantly in laminae II, III and V.40 In rat brain sections labelled with [3H]DADLE but without any blockade of binding to mu sites, no laminar pattern of delta sites was seen in cortex. 25 Only very low levels of binding were seen in the globus pallidus, the area of brain that has the highest amount of enkephalin-like immunoreactivity.’ One of the earliest studies of opiate receptor distribution in human brain was that of Kuhar et uI.,~’ using the non-selective ligand [3Hldihydromorphine. The sites labelled by this compound showed wide regional variations. More recently, qualitative evidence for the occurrence of mu and delta sites in human brain was obtained using ligand binding In both cases, delta sites were techniques.‘,32 identified using [3H]DADLE as the ligand. Estimates were also made of the relative amounts of mu, delta and kappa sites in various brain areas by sequential displacement of [3Hldiprenorphine binding with mu and delta agonists. Human brain contained far fewer delta sites than either mu or kappa, and some areas such as the thalamus and substantia nigra had almost no delta receptors. A similar experimental approach, sequential displacement of a non-selective ligand, but combined with autoradiography, enabled us to make a preliminary examination of the three opioid receptor subtypes in human brain.* Once again, delta receptors were in a minority in human brain compared with mu and kappa receptors. A number of opioid compounds were tested for their ability to displace the binding of [‘HIDPDPE to human brain membranes. The findings suggest preferential binding by the ligand to delta sites. Mu and kappa agonists had very low activity, whereas DADLE, an agonist at both mu and delta receptors, was a potent displacer. The selective delta antagonists tested (ICI 154,129 and ICI 174,8647,“.33) did not displace the binding of [3H]DPDPE from human brain membranes. It is known that these compounds are not potent delta antagonists.6 However, ICI 174,864 was a weak displacer of [3H]DPDPE binding in guinea-pig and rat brain homogenates.(’ The binding medium used in the present study (Tris buffer) greatly reduces the delta antagonist action of these peptides, but in sodium-containing buffer they are much more potent delta antagonists (unpublished observations).

C’I ul.

Delta receptors labelled with [‘HIDPDPE were heterogeneously distributed in the human basal ganglia, cerebral cortex and temporal lobe. Other parts of the brain such as the thalamus had very low levels of delta sites (data not shown). A significant finding in the present study that requires explanation was the absence of delta sites in the rat globus pallidus and in both segments of the human pallidurn. This uas unexpected because in the rat brain there is a dense projection of leu-enkephahn-containing neurons to the globus pallidus.’ Similarly, both enkephalin- and dynorphin-like immunoreactivity are dense in the human external pallidal segment, but relatively weak in the internal segment.‘“,” Since enkephalins have been proposed as natural endogenous ligands for the delta receptor.4” there must be an explanation for the apparent discrepancy between transmitter and receptor. One possibility is that released enkephalin diffuses from the pallidum to reach target receptors, A similar mechanism was proposed for the striatum.” Alternatively, different subtypes of delta receptor may exist, only one of which is labelled by [3H]DPDPE. In this regard it is interesting to note that the potency of DADLE in [3H]DPDPE binding in human brain is considerably lower than that reported in rat brain.h There is some evidence obtained from w i;if:O studies that delta receptors are present in the globus pallidus. It is well established in experimental animals that opiate agonists have profound motor effects when injected directly into the pallidum.z2~35~37More specifically, intrapallidal administration of the delta receptor antagonist ICI 154,129 was active in an in vivo model of opiate delta receptor activation.‘h In general, the distribution of delta receptors in the human brain paralleled the distribution in rat brain. One difference between rat and human was the distribution through the depth of the cortical gray matter. In rat, delta receptors were localized to two distinct bands in layers I-III and V. In human cortex. these bands were much less pronounced, with delta receptors being more evently distributed throughout the cortex. This contrasts with kappa receptors, labelled with the specific ligand [3H]U69,583, which were concentrated in the deep cortical layers in human brain (unpublished observations). It would seem unlikely that delta receptors in human cerebral cortex are involved in specific functions which relate to their anatomical localization. CONCLUSION The present results demonstrate that delta opiate receptors are highly localized to specific regions of the rat and human brain. Delta receptors are concentrated particularly in the cerebral cortex and caudate-putamen. It is possible therefore that drugs active at delta opiate receptors may provide useful therapeutic agents for some human neuropsychiatric disorders.

Delta receptor autoradiography

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