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Autoradiographic Localization of Tryptamine Binding Sites in the Rat and Dog Central Nervous System

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Autoradiographic Localization of Tryptamine Binding Sites in the Rat and Dog Central Nervous System The Journal of Neuroscience January 1986, 6(l): !+I-101 Autoradiographic Localization of Tryptamine Binding Sites in the Rat and Dog Central Nervous System James K. McCormack, Alvin J. Beitz, and Alice A. Larson Department of Veterinary Biology, University of Minnesota, St. Paul, Minnesota 55108 Tryptamine, an endogenous trace amine, is currently postulated posing evidence for such a role has been reviewed by Jones to be a neuromodulator or neurotransmitter in the mammalian (1982). CNS. High-affinity binding sites have been described for trypt- More recently, additional supporting evidence that docu- amine in rat brain homogenate preparations. The present study ments a high-affinity binding site for H3-tryptamine in rat brain further characterizes tryptamine binding throughout the CNS homogenateshas been presented(Cascio and Kellar, 1982; Kel- and delineates its distribution using in viiro receptor binding in lar and Cascio, 1982). Since then, other investigators have also conjunction with autoradiographic techniques. Saturation stud- demonstrated that tryptamine binding sites are present in the ies on ZO-pm-thick brain sections suggest a single class of bind- CNS and are distinct from those of serotonin binding sites ing sites (Hill coefficient = 0.97 + 0.04) with a high affinity (Rommelspacher and Kaufmann, 1983; Wood et al., 1984). (K, = 4.79 f 1.55 nM). In competition studies, kynuramine and Tryptamine binding has been found to be strongly inhibited by tetrahydrobetacarboline significantly inhibited HI-tryptamine beta-carbolines(Cascio and Kellar, 1983; Rommelspacherand binding while serotonin, dopamine, and phenylethylamine failed Kaufmann, 1983; Wood et al., 1984), kynuramine (Charlton et to significantly inhibit it. The most potent inhibitor of H3-trypt- al., 1984), and derivatives of phenylethylamine (PEA) (Cascio amine binding was tryptamine (K, = 4.19 f 2.13 nM). In rat and Kellar, 1983; Martin et al., 1984). This suggeststhat several brain sections processed for in vitro autoradiography, highest endogenously found compounds are possible ligands for the binding occurred in the following limbic structures: the accum- tryptamine binding site. bensnucleus, the amygdalohippocampalarea, the lateral septal All the binding studiesdescribed above have been performed nucleus,the entorhinal cortex, and the anterior olfactory nucle- on brain homogenatepreparations. Although thesehomogenate us. At diencephalic levels, the highest binding was observedin studieshave provided data on tryptamine binding in large brain the reuniensthalamic nucleus,the paraventricular thalamic nu- regions, they fail to delineate the localization of tryptamine- cleus,the medial habenular nucleus,the central medial thalamic binding sites among brain nuclear groups. The in vitro autora- nucleus,and the arcuate hypothalamic nucleus.In the midbrain diographic technique developed by Young and Kuhar (1979) of the rat, binding was most notable in the interpeduncular nu- allows precise delineation of binding sitesamong brain nuclei. cleus, the superficial layer of the superior colliculus, the peri- Two preliminary reports have describedthe useofthis technique aqueductalgray, and the paranigral nucleus.In the lower brain to elucidate the localization of tryptamine binding sites in the stem of the dog, binding was evident in the external cuneate CNS (McCormack et al., 1983; Perry et al., 1982). The purpose nucleus,the spinal trigeminal nucleus, and in the region of the of the presentinvestigation is to employ in vitro autoradiograph- solitary nucleus. Binding was also present in both the ventral ic technique to map tryptamine binding sites in the rat and dog and dorsal horns of the canine spinal cord. Tryptamine binding CNS. sites appear widely distributed throughout the CNS, exhibiting the highest densitiesof binding in the more rostra1 portions of the brain. Materials and Methods Male Sprague-Dawley rats, weighing between 250 and 300 gm, were Tryptamine is present in all major brain regions in all species anesthetized with chloral hydrate and perfused transcardially with ice examined thus far (Marsden and Curzon, 1974; Martin et al., cold isotonic saline. Brains were immediately removed, slowly frozen 1972; Philips et al., 1974; Saavedra and Axelrod, 1972; Sloan to - 2o”C, and used for binding studies within 24 hr. Dogs of either sex, et al., 1975; Snodgrassand Horn, 1973). Mass spectrometric weighing between 10 and 15 kg, were premeditated with atropine and acetylpromazine and anesthetized with pentobarbital. Dogs were can- quantification reveals that the concentration of tryptamine in nulated in the carotid artery and perfused with ice cold isotonic saline the caudate(2.93 ng/gm) is almost six times that ofwhole brain, over a 30 min period. The spinal cord and lower brain stem were and concentrations in the hypothalamus are almost double that removed and slowly frozen to -20°C after gross dissection into the of whole brain (Philips et al., 1974). Tryptamine has been pre- following six regions: medulla, Cl to C5, C6 to T2, T3 to T13, Ll to viously postulated to be a neuromodulator or neurotransmitter L3, and L4 to S3. in the mammalian CNS (Frankhuijzen and Bonta, 1974; Quack To establish appropriate biochemical parameters for HI-tryptamine and Weick, 1978; Vane et al., 1961). Both supporting and op- binding, rat brains were first sectioned at 20 pm on an American Optical cryostat at -20°C. Serial sections from the rostra1 commissure through the caudal hippocampus were thaw-mounted onto clean glass slides and refrozen until used. Slide-mounted sections were then placed in prein- Received Apr. 2, 1985; revised June 17, 1985; accepted July 8, 1985. cubation baths for 5 and 2 min each, transferred to an incubation bath This work was supported by NIH Grants NS19208 and NS17407, NSF Grant for 60 min, and subsequently washed twice in trizma citrate buffer, as BNS83- I 12 14, as well as by a grant from the American Veterinary Medical As- described in Table 1. After the last bath, the sections were scraped off sociation. the slides using a 2.4-cm-diameter glass microfiber filter (Whatman) Correspondence should be sent to Dr. Alice A. Larson, Dept. of Veterinary and placed in 10 ml of Scintiverse E scintillation fluid. After a minimum Biology, 295 Animal Science/Veterinary Medicine Building, St. Paul, MN 55 108. of 12 hr, the sections were counted in a liquid scintillation counter Copyright 0 1986 Society for Neuroscience 0270-6474/86/010094-08$02.00/O (Beckman Model LS7000). The amount of protein in 20 pm tissue 94 The Journal of Neuroscience Autoradiography of Tryptamine Binding 95 Table 1. HWyptamine binding procedure - Bath Total binding 0 Preincubation I 0.1 M trizma citrate 5 min, 25°C pH 7.4 5 mM ascorbic acid Preincubation II 0.1 M trizma citrate 2 min, 25°C pH 7.4 5 mM ascorbic acid 20 PM pargyline Incubation bath 0.1 M trizma citrate For binding pH 1.4 60 mitt, 25°C 5 mM ascorbic acid For autoradio 20 PM pargyline ” YU 30 min, 25°C 2 nM HI-tryptamine time (min) Q Wash I 0.1 M trizma citrate 4 n 0 0 6.5 min, 4°C pH 1.4 IL 0 5 mM ascorbic acid 20 jt~ pargyline Wash II 0.1 M trizma citrate 6.25 min, 4°C pH 7.4 5 mM ascorbic acid ” . 20 PM pargyline 0 1; 3b 4; sb 7; $0 Nonspecific binding was measured in the presence of 2 pi unlabeled tryptamine. Time (min) Figure 1. Effect of varying incubation times on the total (Cl), specific sections was determined using the Coomassie Brilliant Blue method, as (O), and nonspecific (0) binding of H%yptamine to mounted tissue described by Macart and Gerbaut (1982). sections. See text for details. Each point represents the average of three For autoradiography, both canine and rodent tissue sections were cut or more experiments, except at 75 and 90 min, which are based on two at 20 pm and processed according to the protocol described in Table 1 and one experiments, respectively. The insertdepicts the association with the exception that tissues were incubated for only 30 min. This curve, based on linear regression of data in the main graph. The asso- shorter incubation time was used because it provided a higher specific/ ciation rate constant was calculated to be 0.059 nM-’ min-I. nonspecific binding ratio. Brains from rats were sectioned from the rostra1 pole of the forebrain to the caudal medulla. The canine caudal brain stem and spinal cord were also sectioned, incubated and processed ditions was determined to be 15.5 min while the dissociation for autoradiography. Autoradiography of tissue sections was carried out rate constant (Km,) was calculated to be 0.045 min-l (Fig. 2). If as described previously (Beitz et al., 1984). After the last bath, sections pargyline hydrochloride was omitted, specific H’-tryptamine were briefly dipped in distilled water, dried, and apposed to LKB Ul- binding was dramatically reduced becauseof the rapid oxidation trotilm. After an appropriate exposure period, the film was developed by MAO. in D- 19 developer and analyzed using a computerized photometer sys- tem. Since tritium standards were not used in this analysis, the density Saturation studies values obtained represent relative values for the nuclear groups studied. In addition, no attempt was made to account for the regional white- Slide-mounted tissuesections were incubated with different con- matter quenching, which varies according to the different ratios of gray centrations of H%-yptamine to determine total binding, and to white matter in the brain regions studied (Unnerstall and Kuhar, also in the presenceof excessunlabeled tryptamine (1OOO-fold 1985). Brain structures were identified according to the atlas of Paxinos excess)to determine nonspecific binding. All sectionswere in- and Watson (1982) for the rat and of Singer (1962) for the dog. cubated for 60 min to establish equilibrium. This longer incu- Tritiated tryptamine hydrochloride, with a specific activity ranging bation bath time was neededprimarily at the lower concentra- between 23.9 and 41.5 Ci/mmol, was purchased from New England tions of tritiated tryptamine.
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