Quantitative light microscopic localization of [3H]naltrindole binding sites in the rat brain

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Brain Research, 602 (1993) 138-142 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00

BRES 25524

Quantitative light microscopic localization of [3H]naltrindole binding sites in the rat brain Edward J. Drower a, Clifford R. Dorn b Charles S. Markos b, James R. Unnerstall d, Michael F. Rafferty a and Patricia C. Contreras c Neurological Disease Research and b Department of Drug Metabolism, G.D. Searle and Company, Research and Development Division, Skokie, IL 60077 (USA), c Cephalon, West Chester, PA (USA) and a University of Illinois, Department of Anatomy and Cell Biology, Chicago, IL 60612 (USA) (Accepted 27 October 1992)

Key words: Naltrindole; Receptor binding; Autoradiography; fi-Opioid antagonist; Rat

The binding of radiolabeled naltrindole ([3H]NTI), a selective 6-opioid antagonist, was characterized using receptor autoradiography. Receptor binding properties were established in brain paste slices which demonstrated one site receptor occupancy with an apparent K a of 0.25_+ 0.08 nM (Bmax of 597.5 fmol/mg protein). Autoradiographic localization of [3H]NTI binding sites in the rat brain revealed high densities of these sites in the cortex (layers 1-3 and 6), caudate putamen, accumbens, claustrum, and internal plexiform layer of the olfactory bulb. Moderate to low levels of specific binding were observed in the hippocampus, thalamus, and substantia gelatinosa of the spinal cord.

Autoradiographic localization of the 3-opioid receptor has been refined throughout the years as more selective ligands for the 6-receptor have evolved. The receptor distribution patterns and densities of binding for such ligands as DPDPE, DSBULET and DADLE in the rat brain indicate marked similarities 2'8'12'1435. However, further characterization of the 6-receptor has been limited by the 6-antagonists available. Peptidergic antagonists such as ICI-174,864 possess high selectivity but relatively poor affinity for the 3-receptor 4,6,7. Recently, naltrindole (NTI), a rigid morphinoid derivative of naltrexone, has been shown to be a selective high-affinity ligand for the 6-opioid binding site 5,17,19. Moreover, the pharmacologic selectivity of NTI as well as the CNS accessibility of this drug following systemic administration has been confirmed ~0.~3 To further evaluate the selectivity of this compound for the 3-0pioid binding site, this study sought to examine the distribution of tritiated NTI ([3H]NTI) binding sites in the rat brain and spinal cord. [3H]NTI (spee. act. 40 Ci/mmole) was synthesized by the Radiochemistry Division at Searle 9. Conditions for radioligand binding to slide-mounted tissues were predetermined on a tissue paste matrix, prepared from pooled dissected cortex and hippocampus from male

Sprague-Dawley rats (250-300 g). The matrix was prepared over ice and hand homogenized. Aliquots of tissue were placed in plastic tubes and centrifuged (20,000 g, 15 min, 4°C) to remove air bubbles. The tissue was promptly frozen in a dry ice/acetone bath and stored at -80°C. Brain paste slices (20/zm) were cut in a refrigerated microtome (-17°C), thawmounted on gelatin-coated slides and stored at -80°C. Optimal binding conditions were predetermined using these slide-mounted brain paste sections. In general,conditions for binding of fi-selective ligands such as DPDPE and NTI are similar. However, it is important to note that the autoradiographic conditions used for labeling slices with [3H]NTI were temperature dependent. Adequate specific binding was only present when sections were incubated at 37°C. Frozen sections were warmed to room temperature prior to preincubation for 15 min in 50 mM Tris-HCl buffer (pH 7.4) containing 100 mM NaC1 at 25°C. Equilibrium binding of [3H]NTI was reached within 90 min of incubation in 50 mM Tris-HC1 (pH 7.4) containing 5 mM MgC12 and 0.2% BSA at 37°C (Fig. 1A). Sections were then washed (3 × 10 min) in 4°C baths containing 50 mM Tris-HCl, 5 mM MgCI 2 and 1% BSA at pH 7.4. Slices were wiped off with cotton swabs and placed into 10 ml

Correspondence: E.J. Drower, Research Associate, G.D. Searle and Company, Skokie, IL 60077, U.S.A. Fax: (1)(708)982-4701.

139 Aquassure scintillation cocktail for determination of radioactivity by scintillation spectrometry. Non-specific binding was determined in the presence of 10 /zM levorphanol. Protein content (4-8% protein) for the paste slices were determined from random slices of paste using the Bradford method 3. Specific binding in paste slices was determined to be as high as 70% of total binding under these conditions. Autoradiography was performed on coronal brain and spinal cord slices (20/zm) incubated with 0.4 nM [3H]NTI (representing 65% receptor occupancy) under conditions described above. After the final wash, slides were dried under a cold stream of air. The autoradiograms were imaged on [3H]Hyperfilm (Amersham) with exposure times of 6-8 weeks. Autoradiograms were analyzed on a RAS-1000 Image (Loats) and quantified following standarization using 3H-microscales (Amersham). Optical densities in specific regions of each slice was determined from 3-6 different sampled areas of 52 /Zm2 within each region (n = 2 brains). Specific binding was determined by subtraction of non-specific binding measured in adjacent images incubated in [3H]NTI in the presence of 10 /zM levorphanol. Anatomical localization was confirmed by apposing emulsion-coated coverslips (Kodac NTB3 undiluted) to some [3H]NTI labeled sections. Receptor binding experiments for [3H]NTI in brain paste sections revealed a single binding site using the computer program LIGAND (G.A. McPherson, BIOSOFT). Equilibrium was obtained within 90 min. An association rate constant (Kobs) based on a single concentration of ligand (0.4 nM) was determined to be 7.3 × 10 -3 min -1 using a pseudo-first order process previously described by Bennett and Yammaura 1. Upon reaching saturation (90 min) 85% of specific binding was obtained on the paste slices. An apparent K d value of 0.25 n M + 0.08 and Bmax of 597.5 fmol/mg protein was determined by saturation analysis of 3 separate experiments using the computer program LIGAND (BIOSOFT) (Fig. 1). Overall, [3H]NTI demonstrated a 10-20 fold greater affinity over DPDPE or DSBULET for rat brain 6-opioid receptors 8'12. Moreover, possible cross-reactivity with /z-selective agents has been ruled out as displacement of [3H]NTI with the /z-selective agonists PL-1719 and DAMPGO 5 and morphine 5 were in the low /ZM range in brain homogenate binding experiments. In addition, ancillary binding studies in brain paste mounted sections failed to demonstrated displacement of 0.4 nM [3H]NTI with either DAMPGO or morphine at doses exceeding 3 /ZM. The relative density of [3H]NTI binding sites was sampled in various regions of the brain following au-

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SH.Naltrindole (riM) Fig. 1. Representative saturation isotherm and Rosentha] plot (inset) of [3H-na]triondole in brain paste slices. [3H]NTI (0.05-3.2nM) incubated for 90 min in absence and presence of 10 uM levorphanol. Apparent K d 0.25nM; Brn~x of 59?.5 fmol/mg protein. ( • ) total, (151) non-specific and (o) specific binding.

toradiography of intact, labeled brain sections (Table I, Fig. 2A-E). Similar to the binding observed with other 6-selective ligands, somatosensory cortical areas possessed moderate to high binding densities (15-27 fmol/tissue equivalent) with consistently lower levels of binding observed in layer 4. Other areas that contained high densities of binding were the claustrum, caudate putamen, olfactory tubercle and plexiform layers of the olfactory bulb. The binding within these areas also corresponds to areas associated with the binding of DPDPE and DSBULET 8"~e. Regions that demonstrated moderate to low densities of [3H]NTI binding sites were ventral regions of the thalamus, frontal cortex, hippocampus, globus pallidus and hypothalamus. In the spinal cord, binding was low and homogeneous throughout the grey matter with the exception of the substantia gelatinosa that displayed low specific binding. The apparent binding observed in the hippocampal formation, particularly within the inner molecular layer of the dentate gyrus would appear to contrast with the the reports of other investigators who utilized the selective 6-0pioid ligand [3H]DPDPE 16'4. On the other hand, Blackburn et al. reported high levels of [3H]DPDPE binding in the adjacent area of the dentate gyrus. These discrepancies suggest subtle differences in the selectivity of these two opioid receptor ligands. Detailed comparisons between the binding of [3H]NTI and [3H]DPDPE are needed to resolve these potential differences. Differences in the density of [3H]NTI binding sites in distinct cortical regions as well as other subcortical forebrain structures such as the nucleus accumbens correlated well with the immunohistochemical, anatomical and functional compartmentalization of these

140 a r e a s 2'8'll'12As For example, the dorsal lateral, and caudal caudate has higher binding site densities in comparison to m o r e medial and ventral areas (Fig. 2C).

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On the other hand, the nucleus accumbens displayed differences in binding site densities with a decrease in binding caudally. This is visually suggested in a slightly

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Fig. 2. A-E: [3H]NTI receptor distribution of ~-opioid binding sites in the rat. The images shown represent total (left) and non-specific (right) binding from adjacent sections. Abbreviations: Fr, frontal cortex; AO, anterior olfactory nucleus; IGr, internal granular layer; EPL, external plcxiform layer; fmi, corpus callosum; CI, claustrum; Acb, nucleus accumbens; Tu, Olfactory tubercle; CTX, cortex; Cg, cingulate cortex; CPu, caudate putamen; GP, globus pallidus; Pit, piriform cortex; Rsg, retrosplenial granular cortex; Hip, hippocampus; Hyp, hypothalamus; DTh, dorsal thalamis; Ent, entorhinal cortex.

141

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Fig. 2 (continued).

rostral/caudal offset section (Fig. 2C, Table I). Further, it appears that no distinctions in 6-opioid binding site densities were observed between the striasome and matrix compartments. Thus, while the anatomical relationships are complex, 6 binding sites tend to be associated more with sensory motor as opposed to limbic portion of the basal forebrain. The binding within the claustrum also followed a rostral-caudal differentiation with an increase in binding in more caudal sections (Fig. 2B-D). In conclusion, these data further support the 6selectivity of [3H]NTI. The similarities in this 6-selecTABLE I

Relati~,e binding densities of [~H]NTI in the rat Brain area

Specific bound a (fmol / tissue equivalent)

Hippocampus (entire) Dentate gyrus (inner laminar) Rostral caudate (dorsal-lateral) Globus pallidus Thalamus Dorsal medial Ventral medial Hypothalamus Cortex Laminae 1-3 Laminae 4 Laminae 5-6 Retrosplenial granular cortex Internal capsule Nu. accumbens (rostral) Claustrum (rostral) Inferior olive Piriform cortex Olfactory bulb External plexiform layer Internal granular layer Anterior olfactory nucleus Olfactory tuberacle Frontal cortex Entorhinal cortex Spinal cord Substantia gelatinosa

5.64 7.63 34.67 6.00 3.97 24.50 5.67 27.83 15.25 26.83 8.00 6.75 43.50 14.17 8.50 7.17 45.58 21.00 11.58 30.72 19.00 5.17 9.00

~' Values represent fmol/tissue equivalents derived from 3H-microscale based calibrations. Each number represents 3-6 determinations within each area of two different brains.

tive antagonist when compared to other select 6agonists make it a useful tool for studying 6-opioid function. Subtle differences seen in the distribution of agonist and antagonist binding sites may also make it a useful tool in examining differential 6-opiate selectivity. We would like to thank Judith Balazs and Glyn McCord for their technical assistance. 1 Bennett, J.P. Jr. and Yamamura, H.I., Neurotransmitter, hormone or drug receptor binding methods. In H.I. Yamamura, S.H. Enna and M.J. Kuhar (Eds.), Neurotransmitters in Receptor Binding, Raven, New York, 1985. 2 Blackburn, T.P., Cross, A.J., Hille, C. and Slater, P., Autoradiographic localization of 8-opiate receptors in rat and human brain, Neuroscience, 27 (1988) 497. 3 Bradford, M.M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal Biochem., 72 (1976) 248. 4 Cohen, M.L., Shuman, R.T., Osborne, J.J. and Gesellchen, P.D., Opioid agonist activity of ICI 174,864 and its carboxypeptidase degradation product LY281217, J. Pharmacol. Exp. Ther., 238 (1986) 769. 5 Contreras, P.C., Tam, L., Drower, E.J. and Rafferty, M.F., [3H]naltrindole: a potent and selective ligand for labeling 6-opioid receptors, Brain Research, in press. 6 Cotton, R., Kosterlitz, H.W., Paterson, S.J., Rance, M.J. and Traynor J.R., The use of [3H]-[D-Pen2,D-PenS]enkephalin as a highly selective ligand for the g-binding site, Eur. J. Pharmacol., 128 (1985) 303. 7 Cowan, A., Zhu, X.Z. and Porreca, F., Studies in vivo with ICI 174,864 and [D-Pen2,D-PenS]enkephalin, Neuropeptides, 5 (1985) 311. 8 Delay-Goyet, P., Zajac, J.-M. and Roques, B.P., Improved quantitative radioautography of rat brain 6-opioid binding sites using [3H]DSTBULET, a new highly potent and selective linear enkephalin analogue, Neurochem. Int., 16 (1990) 341. 9 Dorn, C.R., Markos, C.S., Dappen, M.S. and Pitzele, B.S., Synthesis of [3H]naltrindole, J. Labeled Compounds Pharm., in press. 10 Drower, E.J., Stapelfeld, A., Rafferty, M.F., de Costa, B.R., Rice, K.C. and Hammond D.L., Selective antagonism by naltrindole of the antinociceptive effects of the 6-opioid agonist cyclic[D-penicillamine2-D-pencillamineS]enkephalin in the rat, J. Pharmacol. Exp. Ther., 259 (1991) 725. 11 Gerfen, C.R., The neostriatal mosaic: striatal patch-matrix organization is related to cortical lamination, Science, 246 (1989) 385. 12 Gulya, K., Gehlert, D.R., Wamsley, J.K., Mosberg, H., Hruby, V.J. and Yamamura, H.I., Light microscopic autoradiographic localization of g-opioid receptors in the rat brain using a highly selective bis-penicillamine cyclic enkephalin analog, J. Pharmacol. Exp. Ther., 238 (1986) 720.

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