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Opioid binding sites in the nigrostriatal system of the guinea pig are not located on nigral dopaminergic neurons
Neuroscience Letters, 65 (1986) 341-345
341
Elsevier Scientific Publishers Ireland Ltd.
NSL 03876
OPIOID BINDING SITES IN THE NIGROSTRIATAL SYSTEM OF THE GUINEA PIG ARE NOT LOCATED ON NIGRAL DOPAMINERGIC NEURONS
R.W. FOOTE* and R. M A U R E R
Sandoz Ltd., Preclinical Research, CH-4002 Basel (Switzerland) (Received November 12th, 1985; Revised version received and accepted January 21 st, 1986)
Key words." [3HI( - )-bremazocine - x-binding site - #-binding site - glyoxylic acid fluorescence - quantitative autoradiography - guinea pig
Unilateral 6-hydroxydopamine injection into the compact zone of the guinea pig substantia nigra was used to destroy nigral dopamine neurons. The glyoxylic acid method for m o n o a m i n e fluorescence was employed to ascertain the loss of dopaminergic fibers in the striatum and substantia nigra. It could be shown that there was no change in the n u m b e r of [3H](-)-bremazocine binding sites in the substantia nigra or the striatum on the lesioned side of these guinea pigs.
Opioid binding sites were found early on in the rat striatum and substantia nigra [1, 15] and later characterized as being largely of the #-type, but 6-sites are also present [18]. Immunohistochemical studies of the endogenous opioid peptides in the rat have shown that the reticular zone of the substantia nigra contains only dynorphin; on the other hand peptides from both proenkephalin and prodynorphin were found in the striatum [7]. Although enkephalins were shown to be present in the guinea pig striatum several years ago [6], immunohistochemical investigations of opioid peptide distribution in the guinea pig are lacking. Quantitative autoradiographic studies in the guinea pig have revealed that in addition to high densities of opioid binding sites in the striatum dense binding occurs in the substantia nigra [5]. Lesion studies have demonstrated that a portion of the opioid binding sites in the rat striatum and substantia nigra are located on dopaminergic terminals, cell bodies or dendrites [10, 16, 17], but the majority in the striatum have been shown to be on intrinsic neurons [12]. It was the purpose of this study to investigate whether the opioid binding sites observed in the guinea pig substantia nigra and striatum are located on the neural processes which originate from the dopaminergic cells in the compact zone of the substantia nigra. The dopaminergic cells in the right compact zone of the substantia nigra were destroyed using the lesion technique developed by Ungerstedt [21, 22]. Eight male
*Author for correspondence. 0304-3940/86/$ 03.50 © 1986 Elsevier Scientific Publishers Ireland Ltd.
342
guinea pigs weighing 315-380 g were used. They were anesthetized with an i.p. injection of sodium pentobarbital (Nembutal; Abbott) and placed in a David K o p f stereotaxic instrument. 6-hydroxydopamine hydrobromide (Sigma) was dissolved in saline with ascorbic acid (0.1 mg/ml) added to prevent oxidation. A 10 #1 Hamilton syringe attached to a Sage Instruments infusion pump was used to administer 12.2 #g (base weight) of 6-hyroxydopamine hydrobromide in 4 #1 of solution over 5 min into the compact zone of the substantia nigra. After completion of the injection the cannula was left in place for two additional minutes and thereafter slowly withdrawn over 30 s. Control injections were performed on the left compact zone of the substantia nigra with the exception that 6-hydroxydopamine was omitted from the solution. The guinea pigs were decapitated 5 to 12 days after lesioning and their brains were immediately removed and frozen on dry-ice. Localization of dopaminergic perikarya and fibers was done by histochemical fluorescence using the sucrose-phosphate-glyoxylic acid method developed for labeling of monoamines by Lindvall et al. [8, 9] and refined by De la Torre and Surgeon [2, 3]. Briefly, cryostat sections (10/~m) of brains were warm-mounted on glass slides and immediately dipped 3 times in the sucrose-phosphate-glyoxylic acid solution (glyoxylic acid from Sigma) and quickly dried under a stream of air at room temperature. The dried sections were covered with paraffin oil and placed in an oven at 95°C for 2.5 min. Thereafter a coverslip was placed over the oil and the sections were examined under a fluorescent microscope. The autoradiographic experiments and quantification using tritium-sensitive Ultrofilm (LKB, Sweden) were performed as previously described [4, 19]. Coronal cryostat sections (10 #m thick) from guinea pig brains were incubated in a 2 nM solution of [3H](-)-bremazocine (32.2 Ci/mmol; New England Nuclear) for 40 min. The dried sections were apposed to the Ultrofilm in X-ray cassettes at 4°C for 56-101 days. Quantification was accomplished by projecting the image on a television screen and outlining the areas of interest with a light-sensitive pen. The optical densities were transformed to fmoles binding sites per milligram protein by a computer program using the method developed by Unnerstall et al. [23] and described by Palacios [13]. The background optical density of the film was taken for the zero value and the densities of areas within each film which had been exposed to known concentrations of tritium, mixed in a paste of homogenized brain tissue, were used for standardization. Non-specific binding was determined in adjacent sections which were incubated in a solution of the radioactive ligand mixed with 1000 nM of unlabeled tifluadom (Kali Chemie, F.R.G.) a benzodiazepine with good affinities to the x-, #and 6-opioid binding sites [20]. The values obtained were subtracted from all other measurements. The substantia nigra was sampled by taking coronal sections from one or more planes (therefore n is greater than 8 in Table I) the posterior, medial and anterior regions being represented where possible. Semiquantitative evaluation (by visual comparison of lesioned and intact sides) of the glyoxylic acid fluorescence for dopamine revealed that the striata on the lesioned side (right substantia nigra) of these guinea pigs had a reduction in fluorescence of well over 50% and in most cases there appeared to be less than a fourth of the fluores-
343 TABLE I EFFECT OF NIGRAL 6-HYDROXYDOPAMINE LESIONS ON THE NUMBERS OF [3HI(--)-BREMAZOCINE BINDING SITES IN THE GUINEA PIG Values represent fmol binding sites/rag prot. + S.E.M. Area
n
Left control
Right lesioned
Difference (paired)
Substantia nigra Striatum
17 8
401 ___20 472 _+41
434 ___18 482 _+42
33 ___11 10+ 18
cence remaining. In fact, 3 of the 8 guinea pigs had no discernible fluorescence in any of the 3 coronal planes of their right striata (anterior, middle and posterior regions) while their left striata exhibited the strong yellowish green fluorescence typical for dopamine. Moreover, a marked absence o f fluorescing cells was observed in the compact zone o f the right, but not the left substantiae nigrae. The lack of damage from control injections on the left side was evidenced by the strong fluorescence of perikarya immediately adjacent to the scar tissue left by the cannula. The numbers of [3H](-)-bremazocine binding sites in the control and lesioned substantiae nigrae and corresponding striata of these guinea pigs were approximately equal (Table I). The slightly higher values from the lesioned side lie within the errors of measurement. The absence of change in the number of [3H](-)-bremazocine binding sites measured in the substantia nigra indicates that the sites were neither located on the dendrites nor the perikarya of the dopaminergic cells found in that structure. It also follows that the binding sites in the striatum are not on the axonal arborizations arising from the dopaminergic cells in the compact zone of the substantia nigra. These findings in the guinea pig are in direct contrast to those from the rat in which 20-30% of the opiate receptors in the striatum and substantia nigra of the rat were on axonal and dendritic ramifications of the dopaminergic cells in the compact zone of the substantia nigra [10, 12, 16, 17]. It is likely that the striatal opioid sites which we have observed in the guinea pig are localized on intrinsic neurons as has been found by Murrin et al. in the rat [12]. It is interesting to note that in the rat the number o f opioid binding sites in the substantia nigra is only one fourth to one half of that found in the striatum [10, 1517], but it must not be forgotten that the density of opioid sites is very heterogeneous in the striatum [15]. Nevertheless, the differences between these two regions in the guinea pig are much less pronounced. The number of nigral sites amount to almost 90% of those in the striatum (Table I). The rat and guinea pig also have very different distributions and specificities of opioid sites within the substantia nigra. In the rat the opioid binding sites are mostly of the p-type with some ~ and they are most numerous in the central area of the compact zone [14, 15], while in the guinea pig the sites are of the x- and p-type [5] and here the central portion of the reticular zone contains the most prominent concentration. In the present study, using techniques similar to those previously employed [5],
344 we f o u n d that on the average 57% o f the o p i o i d sites in the s u b s t a n t i a nigra were o f the x-type (n = 11). T h e total specific b i n d i n g in the dense central a r e a o f the reticular zone on the intact side a m o u n t e d 0 f 4 8 7 fmol sites/mg prot. whereas the entire s u b s t a n t i a nigra h a d 401 fmoi sites/mg prot. (Table I). The a s s o c i a t i o n o f o p i o i d r e c e p t o r s with d o p a m i n e r g i c n e u r o n s in the rat is not surprising considering the d a t a from f u n c t i o n a l a n d b i o c h e m i c a l studies in this species which suggest an interaction between these two systems [17]. On the o t h e r h a n d the guinea pig is k n o w n to have a m u c h different o p i o i d - r e c e p t o r c o m p o s i t i o n [1 1] a n d d i s t r i b u t i o n [19], as well as lower sensitivity to d o p a m i n e r g i c drugs. Indeed, intact a n d lesioned guinea pigs into which we injected up to 10 m g / k g a p o m o r p h i n e i.p. showed only m i n o r b e h a v i o r a l changes whereas rats r e s p o n d e d with the typical s t e r e o t y p y a n d circling to one tenth this dose ( R . W . F . , u n p u b l i s h e d observations). O u r findings t h a t the guinea pig lacks o p i o i d b i n d i n g sites o n nigral d o p a m i n e r g i c n e u r o n s a n d has a dense p o p u l a t i o n o f nigral o p i o i d b i n d i n g sites, which is concent r a t e d in the reticular zone, further e m p h a s i z e s the i m p o r t a n c e o f differences between even relatively closely related species like these rodents. W e w o u l d like to t h a n k F. Hefti for his assistance with the fine p o i n t s o f neuroa n a t o m y a n d the glyoxylic acid m e t h o d a n d B.H. G/ihwiler a n d J.M. Palacios for their helpful criticism o f the m a n u s c r i p t . J.M. P a l a c i o s ' assistance with the c o m p u t e r q u a n t i f i c a t i o n is gratefully a c k n o w l e d g e d . 1 Atweh, S.F. and Kuhar, M.J., Autoradiographic localization of opiate receptors in rat brain. II. The brainstem, Brain Res., 129 (1977) 1-12. 2 De La Torre, J.C. and Surgeon, J.W., Histochemical fluorescence of tissue and brain monoamines: results in 18 minutes using the sucrose-phosphate-glyoxylic acid (SPG) method, Neuroscience, 1 (1976) 451~153. 3 De La Torre, J.C., An improved approach to histofluorescence using the SPG method for tissue monoamines, J. Neurosci. Meth., 3 (1980) I-5. 4 Foote, R.W. and Maurer, R., Autoradiographic localization of opiate x-receptors in the guinea-pig brain, Eur. J. Pharmacol., 85 (1982) 99-103. 5 Foote, R.W. and Maurer, R., Kappa opiate binding sites in the substantia nigra and bulbus olfactorius of the guinea pig as shown by in vitro autoradiography, Life Sci., 33, Suppl. 1 (1983) 243-246. 6 Henderson, G., Hughes, J. and Kosterlitz, H.W., In vitro release of Leu- and Met-enkephalin from the corpus striatum, Nature (London), 271 (1978) 677-679. 7 Khachaturian, H., Lewis, M.E., Sch/ifer, M. K.-H. and Watson, S.J., Anatomy of the CNS opioid system, TINS, 8 (1985) 111-119. 8 Lindvall, O., Bj6rklund, A., H6kfelt, T. and Ljungdahl, A., Application of the glyoxylic acid method to vibratome sections for improved visualization of central catecholamine neurons, Histochemie, 35 (1973) 31-38. 9 Lindvall, O. and Bj6rklund, A., The glyoxylic acid fluorescence histochemical method: a detailed account of the methodology for the visualization of central catecholamine neurons, Histochemistry, 39 (1974) 97-127. 10 Llorens-Cortes, C., Pollard, H. and Schwartz, J.C., Localization of opiate receptors in substantia nigra evidence by lesion studies, Neurosci. Lett., 12 (1979) 165-170. 11 Maurer, R., Multiplicity of opiate receptors in different species, Neurosci. Lett., 30 (1982) 303-307. 12 Murrin, L.C., Coyle, J.T. and Kuhar, M.J., Striatal opiate receptors: pre- and postsynaptic localization, Life Sci., 27 (1980) 1175-1183.
345 13 Palacios, J.M., Quantitative receptor autoradiography: application to the study of multiple serotonin receptors in rat cortex. In P. Mandel and F.V. DeFeudis (Eds.), CNS Receptors: From Molecular Pharmacology to Behaviour, Raven Press, New York, 1983, pp. 455--463. 14 Pearson, J., Brandeis, L., Simon, E. and Hiller, J., Radioautography of binding of tritiated diprenorphine to opiate receptors in the rat, Life Sci., 26 (1980) 1047-1052. 15 Pert, C., Kuhar, M.J. and Snyder, S.H., Opiate receptor: autoradiographic localization in rat brain, Proc. Natl. Acad. Sci. USA, 73 (1976) 3729-3733. 16 Pollard, H., Llorens-Cortex, C. and Schwartz, J.C., Enkephalin receptors on dopaminergic neurones in rat striatum, Nature (London), 268 (1977) 745-747. 17 Pollard, H., Llorens, C. and Schwartz, J.C., Gros, C. and Dray, F., Localization of opiate receptors and enkephalins in the rat striatum in relationship with the nigrostriatal dopaminergic system: lesion studies, Brain Res., 151 (1978) 393-398. 18 Quirion, R., Zajac, J.M., Morgat, J.L. and Roques, B.P., Autoradiographic distribution of mu and delta opiate receptors in rat brain using highly selective ligands, Life Sci., 33, Suppl. 1 (1983) 227-230. 19 Robson, L.E., Foote, R.W., Maurer, R. and Kosteditz, H.W., Opioid binding sites of the r-type in guinea-pig cerebellum, Neuroscience, 12 (1984) 621~627. 20 R6mer, D., Biischer, H.H., Hill, R.C., Maurer, R., Petcher, T.J., Zeugner, H., Benson, W., Finner, E., Milkowski, W. and Thies, P.W., An opiate benzodiazepine, Nature (London), 298 (1982) 759--760. 21 Ungerstedt, U., 6-Hydroxy-dopamine-induced degeneration of central monoamine neurons, Eur. J. Pharmacol., 5 (1968) 107-110. 22 Ungerstedt, U., Postsynaptic supersensitivity after 6-hydroxy-dopamine-induced degeneration of the nigro-striatal dopamine system, Acta Physiol. Scand., Suppl. 367 (1971) 69-93. 23 Unnerstall, J.R., Niehoff, D.L., Kuhar, M.J. and Palacios, J.M., Quantitative receptor autoradiography using [3H] Ultrofilm: application to multiple benzodiazepine receptors, J. Neurosci. Meth., 6 (1982) 59-73.
341
Elsevier Scientific Publishers Ireland Ltd.
NSL 03876
OPIOID BINDING SITES IN THE NIGROSTRIATAL SYSTEM OF THE GUINEA PIG ARE NOT LOCATED ON NIGRAL DOPAMINERGIC NEURONS
R.W. FOOTE* and R. M A U R E R
Sandoz Ltd., Preclinical Research, CH-4002 Basel (Switzerland) (Received November 12th, 1985; Revised version received and accepted January 21 st, 1986)
Key words." [3HI( - )-bremazocine - x-binding site - #-binding site - glyoxylic acid fluorescence - quantitative autoradiography - guinea pig
Unilateral 6-hydroxydopamine injection into the compact zone of the guinea pig substantia nigra was used to destroy nigral dopamine neurons. The glyoxylic acid method for m o n o a m i n e fluorescence was employed to ascertain the loss of dopaminergic fibers in the striatum and substantia nigra. It could be shown that there was no change in the n u m b e r of [3H](-)-bremazocine binding sites in the substantia nigra or the striatum on the lesioned side of these guinea pigs.
Opioid binding sites were found early on in the rat striatum and substantia nigra [1, 15] and later characterized as being largely of the #-type, but 6-sites are also present [18]. Immunohistochemical studies of the endogenous opioid peptides in the rat have shown that the reticular zone of the substantia nigra contains only dynorphin; on the other hand peptides from both proenkephalin and prodynorphin were found in the striatum [7]. Although enkephalins were shown to be present in the guinea pig striatum several years ago [6], immunohistochemical investigations of opioid peptide distribution in the guinea pig are lacking. Quantitative autoradiographic studies in the guinea pig have revealed that in addition to high densities of opioid binding sites in the striatum dense binding occurs in the substantia nigra [5]. Lesion studies have demonstrated that a portion of the opioid binding sites in the rat striatum and substantia nigra are located on dopaminergic terminals, cell bodies or dendrites [10, 16, 17], but the majority in the striatum have been shown to be on intrinsic neurons [12]. It was the purpose of this study to investigate whether the opioid binding sites observed in the guinea pig substantia nigra and striatum are located on the neural processes which originate from the dopaminergic cells in the compact zone of the substantia nigra. The dopaminergic cells in the right compact zone of the substantia nigra were destroyed using the lesion technique developed by Ungerstedt [21, 22]. Eight male
*Author for correspondence. 0304-3940/86/$ 03.50 © 1986 Elsevier Scientific Publishers Ireland Ltd.
342
guinea pigs weighing 315-380 g were used. They were anesthetized with an i.p. injection of sodium pentobarbital (Nembutal; Abbott) and placed in a David K o p f stereotaxic instrument. 6-hydroxydopamine hydrobromide (Sigma) was dissolved in saline with ascorbic acid (0.1 mg/ml) added to prevent oxidation. A 10 #1 Hamilton syringe attached to a Sage Instruments infusion pump was used to administer 12.2 #g (base weight) of 6-hyroxydopamine hydrobromide in 4 #1 of solution over 5 min into the compact zone of the substantia nigra. After completion of the injection the cannula was left in place for two additional minutes and thereafter slowly withdrawn over 30 s. Control injections were performed on the left compact zone of the substantia nigra with the exception that 6-hydroxydopamine was omitted from the solution. The guinea pigs were decapitated 5 to 12 days after lesioning and their brains were immediately removed and frozen on dry-ice. Localization of dopaminergic perikarya and fibers was done by histochemical fluorescence using the sucrose-phosphate-glyoxylic acid method developed for labeling of monoamines by Lindvall et al. [8, 9] and refined by De la Torre and Surgeon [2, 3]. Briefly, cryostat sections (10/~m) of brains were warm-mounted on glass slides and immediately dipped 3 times in the sucrose-phosphate-glyoxylic acid solution (glyoxylic acid from Sigma) and quickly dried under a stream of air at room temperature. The dried sections were covered with paraffin oil and placed in an oven at 95°C for 2.5 min. Thereafter a coverslip was placed over the oil and the sections were examined under a fluorescent microscope. The autoradiographic experiments and quantification using tritium-sensitive Ultrofilm (LKB, Sweden) were performed as previously described [4, 19]. Coronal cryostat sections (10 #m thick) from guinea pig brains were incubated in a 2 nM solution of [3H](-)-bremazocine (32.2 Ci/mmol; New England Nuclear) for 40 min. The dried sections were apposed to the Ultrofilm in X-ray cassettes at 4°C for 56-101 days. Quantification was accomplished by projecting the image on a television screen and outlining the areas of interest with a light-sensitive pen. The optical densities were transformed to fmoles binding sites per milligram protein by a computer program using the method developed by Unnerstall et al. [23] and described by Palacios [13]. The background optical density of the film was taken for the zero value and the densities of areas within each film which had been exposed to known concentrations of tritium, mixed in a paste of homogenized brain tissue, were used for standardization. Non-specific binding was determined in adjacent sections which were incubated in a solution of the radioactive ligand mixed with 1000 nM of unlabeled tifluadom (Kali Chemie, F.R.G.) a benzodiazepine with good affinities to the x-, #and 6-opioid binding sites [20]. The values obtained were subtracted from all other measurements. The substantia nigra was sampled by taking coronal sections from one or more planes (therefore n is greater than 8 in Table I) the posterior, medial and anterior regions being represented where possible. Semiquantitative evaluation (by visual comparison of lesioned and intact sides) of the glyoxylic acid fluorescence for dopamine revealed that the striata on the lesioned side (right substantia nigra) of these guinea pigs had a reduction in fluorescence of well over 50% and in most cases there appeared to be less than a fourth of the fluores-
343 TABLE I EFFECT OF NIGRAL 6-HYDROXYDOPAMINE LESIONS ON THE NUMBERS OF [3HI(--)-BREMAZOCINE BINDING SITES IN THE GUINEA PIG Values represent fmol binding sites/rag prot. + S.E.M. Area
n
Left control
Right lesioned
Difference (paired)
Substantia nigra Striatum
17 8
401 ___20 472 _+41
434 ___18 482 _+42
33 ___11 10+ 18
cence remaining. In fact, 3 of the 8 guinea pigs had no discernible fluorescence in any of the 3 coronal planes of their right striata (anterior, middle and posterior regions) while their left striata exhibited the strong yellowish green fluorescence typical for dopamine. Moreover, a marked absence o f fluorescing cells was observed in the compact zone o f the right, but not the left substantiae nigrae. The lack of damage from control injections on the left side was evidenced by the strong fluorescence of perikarya immediately adjacent to the scar tissue left by the cannula. The numbers of [3H](-)-bremazocine binding sites in the control and lesioned substantiae nigrae and corresponding striata of these guinea pigs were approximately equal (Table I). The slightly higher values from the lesioned side lie within the errors of measurement. The absence of change in the number of [3H](-)-bremazocine binding sites measured in the substantia nigra indicates that the sites were neither located on the dendrites nor the perikarya of the dopaminergic cells found in that structure. It also follows that the binding sites in the striatum are not on the axonal arborizations arising from the dopaminergic cells in the compact zone of the substantia nigra. These findings in the guinea pig are in direct contrast to those from the rat in which 20-30% of the opiate receptors in the striatum and substantia nigra of the rat were on axonal and dendritic ramifications of the dopaminergic cells in the compact zone of the substantia nigra [10, 12, 16, 17]. It is likely that the striatal opioid sites which we have observed in the guinea pig are localized on intrinsic neurons as has been found by Murrin et al. in the rat [12]. It is interesting to note that in the rat the number o f opioid binding sites in the substantia nigra is only one fourth to one half of that found in the striatum [10, 1517], but it must not be forgotten that the density of opioid sites is very heterogeneous in the striatum [15]. Nevertheless, the differences between these two regions in the guinea pig are much less pronounced. The number of nigral sites amount to almost 90% of those in the striatum (Table I). The rat and guinea pig also have very different distributions and specificities of opioid sites within the substantia nigra. In the rat the opioid binding sites are mostly of the p-type with some ~ and they are most numerous in the central area of the compact zone [14, 15], while in the guinea pig the sites are of the x- and p-type [5] and here the central portion of the reticular zone contains the most prominent concentration. In the present study, using techniques similar to those previously employed [5],
344 we f o u n d that on the average 57% o f the o p i o i d sites in the s u b s t a n t i a nigra were o f the x-type (n = 11). T h e total specific b i n d i n g in the dense central a r e a o f the reticular zone on the intact side a m o u n t e d 0 f 4 8 7 fmol sites/mg prot. whereas the entire s u b s t a n t i a nigra h a d 401 fmoi sites/mg prot. (Table I). The a s s o c i a t i o n o f o p i o i d r e c e p t o r s with d o p a m i n e r g i c n e u r o n s in the rat is not surprising considering the d a t a from f u n c t i o n a l a n d b i o c h e m i c a l studies in this species which suggest an interaction between these two systems [17]. On the o t h e r h a n d the guinea pig is k n o w n to have a m u c h different o p i o i d - r e c e p t o r c o m p o s i t i o n [1 1] a n d d i s t r i b u t i o n [19], as well as lower sensitivity to d o p a m i n e r g i c drugs. Indeed, intact a n d lesioned guinea pigs into which we injected up to 10 m g / k g a p o m o r p h i n e i.p. showed only m i n o r b e h a v i o r a l changes whereas rats r e s p o n d e d with the typical s t e r e o t y p y a n d circling to one tenth this dose ( R . W . F . , u n p u b l i s h e d observations). O u r findings t h a t the guinea pig lacks o p i o i d b i n d i n g sites o n nigral d o p a m i n e r g i c n e u r o n s a n d has a dense p o p u l a t i o n o f nigral o p i o i d b i n d i n g sites, which is concent r a t e d in the reticular zone, further e m p h a s i z e s the i m p o r t a n c e o f differences between even relatively closely related species like these rodents. W e w o u l d like to t h a n k F. Hefti for his assistance with the fine p o i n t s o f neuroa n a t o m y a n d the glyoxylic acid m e t h o d a n d B.H. G/ihwiler a n d J.M. Palacios for their helpful criticism o f the m a n u s c r i p t . J.M. P a l a c i o s ' assistance with the c o m p u t e r q u a n t i f i c a t i o n is gratefully a c k n o w l e d g e d . 1 Atweh, S.F. and Kuhar, M.J., Autoradiographic localization of opiate receptors in rat brain. II. The brainstem, Brain Res., 129 (1977) 1-12. 2 De La Torre, J.C. and Surgeon, J.W., Histochemical fluorescence of tissue and brain monoamines: results in 18 minutes using the sucrose-phosphate-glyoxylic acid (SPG) method, Neuroscience, 1 (1976) 451~153. 3 De La Torre, J.C., An improved approach to histofluorescence using the SPG method for tissue monoamines, J. Neurosci. Meth., 3 (1980) I-5. 4 Foote, R.W. and Maurer, R., Autoradiographic localization of opiate x-receptors in the guinea-pig brain, Eur. J. Pharmacol., 85 (1982) 99-103. 5 Foote, R.W. and Maurer, R., Kappa opiate binding sites in the substantia nigra and bulbus olfactorius of the guinea pig as shown by in vitro autoradiography, Life Sci., 33, Suppl. 1 (1983) 243-246. 6 Henderson, G., Hughes, J. and Kosterlitz, H.W., In vitro release of Leu- and Met-enkephalin from the corpus striatum, Nature (London), 271 (1978) 677-679. 7 Khachaturian, H., Lewis, M.E., Sch/ifer, M. K.-H. and Watson, S.J., Anatomy of the CNS opioid system, TINS, 8 (1985) 111-119. 8 Lindvall, O., Bj6rklund, A., H6kfelt, T. and Ljungdahl, A., Application of the glyoxylic acid method to vibratome sections for improved visualization of central catecholamine neurons, Histochemie, 35 (1973) 31-38. 9 Lindvall, O. and Bj6rklund, A., The glyoxylic acid fluorescence histochemical method: a detailed account of the methodology for the visualization of central catecholamine neurons, Histochemistry, 39 (1974) 97-127. 10 Llorens-Cortes, C., Pollard, H. and Schwartz, J.C., Localization of opiate receptors in substantia nigra evidence by lesion studies, Neurosci. Lett., 12 (1979) 165-170. 11 Maurer, R., Multiplicity of opiate receptors in different species, Neurosci. Lett., 30 (1982) 303-307. 12 Murrin, L.C., Coyle, J.T. and Kuhar, M.J., Striatal opiate receptors: pre- and postsynaptic localization, Life Sci., 27 (1980) 1175-1183.
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