LOCALIZATION OF RECEPTORS IN CENTRAL SYNAPSES

THOMAS L. LENTZ and JANICE CHESTER

From the Section of Cytology, Yale University School of Medicine, New Haven, Connecticut 065 l0

ABSTRACT The localization of cholinergic receptors in synaptosomes and in synapses of the midbrain reticular formation and hypothalamic preoptic nucleus has been demonstrated by means of a horseradish peroxidase-o~-bungarotoxin (HRP-ot- Btx) conjugate. Only a small proportion of the total number of synapses was reactive. terminals of reactive synapses contained primarily small clear vesicles, while synapses characterized by large numbers of dense core vesicles were unreactive. Toxin-binding sites were found to occur in a thickened zone of the postsynaptic surface. This procedure can be employed to study the regional distribution and localization of nicotinic sites in the central nervous system.

KEY WORDS acetylcholine receptors We have described recently a procedure in synapse synaptic membrane central which AChR are localized with t~-Btx conjugated nervous system cytoehemistry directly with horseradish peroxidase (HRP) (16). This procedure permits the localization of sites of The snake neurotoxin a-bungarotoxin (a-Btx) has toxin binding at high resolution with the electron proven useful in the localization of acetylcholine microscope. This technique has been applied to receptors (AChR) by virtue of its ability to bind the neuromuscular junction (16), and the present specifically and virtually irreversibly to nicotinic paper reports on its suitability for the localization receptor sites (15, 18). By using radioactively la- of AChR in synapses of the central nervous sys- beled a-Btx or markers attached to the toxin, it tem. has been possible to determine the location and number of these cholinergic receptor sites. For MATERIALS AND METHODS example, the toxin has been used to detect recep- tors in skeletal muscle (4, 6, 25), sympathetic Preparation of HRP-a-Btx Conjugate ganglia (8), and (35, 36). Bungarus multicinctus venom was obtained from the The brain contains nicotinic, muscarinic, and Sigma Chemical Co. (St. Louis, Mo.) or the Miami mixed cholinergic synapses, with the majority Serpentarium (Miami, Fla.). a-Btx was separated from the venom on a carboxymethylcelhilose cohman accord- being of the muscarinic type (3, 34). Several stud- ing to the method of Clark et al. (2). Conjugation of a- ies have demonstrated binding of ct-Btx to brain Btx to HRP was achieved utilizing the method of Nakane tissue or fractions indicating that a-Btx can be and Kawaoi (21). HRP (40 rag) and a,-Btx (8 mg) were used for the localization of nicotinic receptors in used in equimolar ratios in order to favor 1:1 conjugates. the central nervous system (1, 5, 12, 17, 19, 20, The products Of conjugation were separated by column 24, 28, 29, 31, 33). Toxin binding is highest in chromatography on Ultragel AcA44 (LKB Instruments, fractions containing synaptosomes (5, 12, 29). Inc., Hicksville, N. Y.) and fractions from an apparent

258 THE JOURNAL OF CELL BIOLOGY" VOLUME 75, 1977' pages 258-267 mol wt of 42,000 to 85,000 daltons were pooled and tion. Frontal slices (-2 mm thick) were made with a utilized for the cytochemical localization of AChR. This razor blade. Slices were employed to obtain maximum fraction was tested physiologically and produced a post- exposure and penetration of the conjugate into the tis- synaptic blockade at frog sartorius neuromuscular junc- sues, since a-Btx does not readily cross the blood-brain tion similar to that obtained with native a-Btx, except barrier (15). The unfixed slices were incubated for 2 h in that longer exposure to the conjugate was required. the conjugate (diluted 10-fold in Ringer's solution minus Miniature end plate potentials and muscle twitch were phosphate) with gentle agitation at room temperature blocked after 2-h exposure to the conjugate, and end and gassed with 95% 02, 5% CO2. Some slices were plate potentials were abolished after 3-4 h. Native a-Btx preincubated in native a-Btx (1 mg/6 ml Ringer's) for 2 (10 -e M) blocked the muscle twitch in 15 min, and end h and incubated in the conjugate or were incubated in plate potentials after 20 rain. These studies indicate that Ringer's solution alone. After incubation, the slices were the conjugate retains activity although its potency is rinsed in Ringer's solution (with phosphate) for 2 h with decreased, possibly because the coupling procedure uti- changes every 15 rain. Tissues were then fixed in 3% lizes some of the lysine residues of ot-Btx essential for its glutaraldehyde in 0.05 M cacodylate buffer (pH 7.2) for biologic activity (see reference 16). Vogel et al. (35) 1 h and rinsed in buffer with changes for -30 min. have analyzed the binding of HRP-a-Btx conjugate to The brain slices and synaptosome pellet blocks were solubilized nicotinic AChR from Torpedo electric organ. reacted for peroxidase activity by incubation in 3,3'- They found that the affinity of the conjugate for the diaminobenz/dine (DAB) (Sigma Chemical Co.) (50 rag/ receptor is lower than that of ]~SI-labeled a.-Btx, but that 100 ml Tris buffer, pH 7.2) and H202 (0,01%) for 60- it is capable of competing for receptor sites and protects 90 rain. Tissues and pellet blocks were rinsed in buffer a high proportion of sites against t2sI-iabeled a-Btx bind- and small blocks were excised from the brain slices. ing. Peroxidative activity of the conjugate was deter- Several regions of brain were surveyed and the midbrain mined by measuring the rate of decomposition of hydro- reticular formation and hypothalamic preoptic nucleus gen peroxide with o-dianisidine as hydrogen donor (38). were selected for study of synapses. Blocks were taken Conjugation was found not to produce a decrease in from the edges of the tissue slices. In the case of the peroxidative enzymatic activity. Further details of the preoptic nucleus, blocks were taken from the region conjugation procedure have been reported (16). adjacent to the third ventricle and included the periven- tricular zone of this nucleus. The blocks were dehy- Synaptosome Preparation and Incubation drated, infiltrated, and embedded in Epon 812. Thin Whole rat were homogenized with a glass ho- sections were viewed unstained with a Hitachi 8B or mogenizer in cold 0.32 M sucrose (10% wt/vol). A P2 HU11-ES electron microscope. fraction (containing myelin fragments, nerve ending par- RESULTS ticles, and mitochondria) was prepared according to the method of Gray and Whittaker (7). The pellet was Reactive synapses were observed in both synapto- resuspended in 3 ml of 0.32 M sucrose using a Vortex some preparations (Figs. 1 and 2) and tissue mixer (Scientific Industries, Inc,, Springfield, Mass.). blocks (Figs. 5-9). The reaction product for per- 12-15 vol of Ringer's solution were added slowly to the oxidase activity appeared as a dense, fine-textured suspension with mixing. The fraction was centrifuged at deposit, and occurred primarily on the postsyn- 9,000g for 5 min at 4~ and washed twice with Ringer's aptic portion of the synapses. Other membranes, solution in this manner. myelin, synaptic vesicles, and most mitochondria For localization of AChR, the pellet was resuspended were unreactive. Sites of reaction product occur- in 2 ml of Ringer's solution minus phosphate and con- ring in both HRP-a-Btx-treated tissues and con- taining 0.2 ml of the conjugate (produces a final conch of 10 mM phosphate). Controls consisted of preincubation trol preparations, and interpreted to represent en- for 1 h in 5 ml of Ringer's solution containing 0.4 mg of dogenous peroxidatic activity, are described be- native a-Btx followed by centrifugation and resuspen- low. sion in the conjugate or by suspension in Ringer's solu- Reactive synapses were similar in structure and tion alone. The preparations were incubated for 11/2 h at consisted of a presynaptic bouton and postsynaptic 4~ with occasional gentle mixing. After incubation, the element (Figs. 1, 2, 5-9). The axon terminals preparations were washed three times by suspension in contained numerous synaptic vesicles, 500 A in Ringer's solution and centrifugation at 9,000 g for 5 min. diam, with clear contents. Mitochondria and a few The pellets were fixed for 60-90 rain in 3% glutaralde- larger dense-core vesicles or granules usually were hyde. Small pieces were excised from the pellets and present as well. The synaptic cleft was about 150 processed as described below for tissue blocks. in width and contained moderately dense mate- Tissue Incubation and Cytochemistry rial. Finely filamentous, moderately dense mate- Young rats were anesthetized with ether and the rial was applied to the inner aspects of the postsyn- brains removed immediately and placed in Ringer's solu- aptic, and to a lesser extent, the presynaptic mem-

T. L. LENTZ AND J. CHESTER Localizationof Acetylcholine Receptors in Brain 259 260 THE JOURNAL OF CELL BIOLOGY" VOLUME 75, 1977 branes. The postsynaptic element sometimes con- Ringer's solution alone or were pretreated with tained a but generally contained native a-Btx to block specific cholinergic-binding few organized structures. In contrast, synapses in sites before incubation in the conjugate, did not which the axon terminal contained a large number show reaction product at the region of synaptic of larger dense-core vesicles were unreactive (Fig. contact. It should be noted though, that the finely 7). These terminals often contained some small, filamentous material applied to the inner aspects clear vesicles as well. of pre- and postsynaptic membranes varies in den- In reactive synapses, activity was primarily lo- sity and in some cases is moderately dense in calized to the postsynaptic membrane. The reac- control preparations (Figs. 10 and 11). In addi- tive region was located opposite the presynaptic tion, in controls, the pre-and postsynaptic mem- membrane and extended laterally a short distance branes in the region of synaptic contact are some- beyond the region of synaptic contact (Fig. 6). what darker than nonsynaptic membranes (Figs. Reaction product occurred in a uniform band 10 and 11). These inherent densities are to be which included the postsynaptic membrane (Figs. distinguished from the densities due to addition of 6, 8, and 9). The width of the reactive band (125 reaction product. The latter is extremely dense to A,) exceeded the width of the plasma membrane in opaque. In addition, in controls, the postsynaptic control preparations (75 A.) by about 50/~. membrane could be seen distinctly and was 75~ In intensely reacted specimens, reaction prod- in width (Figs. 10 and 11), while in reactive speci- uct extended into the postsynaptic density (Figs. 7 mens, postsynaptic reaction product occurred in a and 9). This appearance could be due to build-up band 125 A in width. of reaction product with trapping, and adherence While staining was not seen in the postsynaptic by the filamentous network forming the postsyn- membrane of control preparations, activity oc- aptic density. In addition, in a few instances, some curred in other regions in both control and experi- portions of the presynaptic axonal membrane mental preparations. Activity was most prominent were reactive. Reaction product was localized to in membrane-bounded granules and vacuoles the plasma membrane at the region of synaptic within and their processes, glia, and epen- contact (Fig. 9). Some staining also occurred in dymal cells. These structures may represent lyso- the membrane forming the sides of the terminal. somes or peroxisomes. Some mitochondria were Although the localization of reaction product in reactive and erythrocytes were increased in den- the axolemma was similar to that observed in the sity. Some processes contained dense particulate presynaptic axon terminals of the neuromuscular material of unknown nature, particularly in the a- junction (16), it was seen in only a few synapses in Btx controls. Sites of endogenous peroxidatic ac- heavily reacted preparations. Thus, no conclusions tivity in the brain have been described (11, 37). can be made at this time as to whether this staining Heme enzymes such as peroxidase, catalase, and is indicative of presynaptic receptors in central cytochromes are capable of catalyzing the oxida- synapses. tion of DAB. Another s~ource of activity is conju- Controls in which synaptosomes (Figs. 3 and 4) gate trapped within cellular processes and not and tissues (Figs. 10 and 11) were incubated in washed out during rinsing.

Fmu~ 1 Synaptosomes after incubation in HRP-a-Btx conjugate for 2 h and reaction for HRP. Reaction product is localized to the postsynaptic element (Po) upon which presynaptic profiles (Pr) terminate, x 56,000. FIrORE 2 Synaptosomes after incubation in the conjugate and reaction for HRP. Activity occurs postsynaptically (Po) at regions of synaptic contact. Pr, presynaptic element, x 65,000. Fmo~ 3 Synaptosome control preincubated in native a-Btx for 1 h, incubated in the conjugate for 2 h, and reacted for HRP. No activity occurs at the region of synaptic contact. Pr, presynaptic profile; Po, postsynaptic profile. • 55,000. FIrORE 4 Synaptosome control showing absence of reaction product at regions of synaptic contact (Sy). Moderately dense material is applied to the inner aspects of the pre- and postsynaptic membranes. • 54,000.

T. L. LENTZ AND J. CHESTm~ Localization of Acetylcholine Receptors in Brain 261 FIOURE 5 Midbrain reticular formation after procedure for localization of AChR. Activity occurs at a synapse and is localized to the postsynaptic membrane (Po). Other regions including the presynaptic terminal (Pr) and (Ax) are unreactive. • 45,000. FrauaE 6 Higher magnification of area enclosed in box in Fig. 5. Activity is restricted to the postsynaptic membrane opposite and extending a short distance beyond the region of synaptic contact. Note that the reactive region is thicker than unreactive membrane, x 120,000.

262 THE JOURNAL OF CELL BIOLOGY" VOLUME 75, 1977 Fmtn~ 7 Preoptic nucleus of hypothalam Js (neuropil of periventrieular zone) reacted for AChR. The postsynaptie region of the synapse on the left is intensely reactive. The axon terminal of this synapse contains predominantly small clear synaptic vesicles (SV). This synapse is considered to be chofinergic. The synapse on the right is unreactive and contains larger dense-core vesicles (DCV) along with smaller clear vesicles. • 54,500.

As in the case of muscle, staining of intact brain synaptic side of the synapses and in the presynap- tissue with this technique is variable, with some tic membrane as well. samples containing reactive synapses and others In the present study, reaction product was local- not. Furthermore, when present, reactive syn- ized primarily to a thickened region on the post- apses were located near the edges of the blocks synaptic surface. A similar thickened postsynaptic where tissue preservation was not always optimal zone of reaction product has been observed on the and cell relationships were disrupted. Such varia- upper portions of junctional folds of the neuro- bility may be due to the relatively large size of the muscular junction (16). Such a localization may be conjugate (mol wt 48,000 or larger) which could consistent with a transmembrane location of re- result in a low rate of diffusion and penetration ceptors projecting a short distance into the synap- into the tissues, and to the length of time neces- tic cleft, although caution is required in interpret- sary for incubation and washing of the unfixed ing the exact site of localization of reaction prod- tissues. uct relative to the membrane. The oxidized DAB may not lie precisely at the site of the receptor, DISCUSSION due to the size of the conjugate molecule bound to Nicotinic acetylcholine receptor sites have been the receptor and to accumulation, trapping, or identified in synapses of the reticular formation of adsorption of reaction product. However, at the the midbrain and preoptic nucleus of the hypothal- neuromuscular junction, the reactive zone coin- amus. Also employing an HRP-a-Btx conjugate, cides with the region where specializations sug- Jensen et al. (10) have reported binding to the gested to represent the morphological counterpart subsynaptic membrane in the optic rectum. Vogel of the AChR have been described (9, 23, 26, 27). et al. (35) found that 5-7% of the synapses in the Similarly, aggregations of particles have been de- inner plexiform layer of the chicken retina bound scribed in the outer leaflet of the postsynaptic the conjugate. They observed staining on the post- membranes of some central synapses (13, 14, 22,

T. L. LElcrz AND J. CrmSTER Localization of Acetylcholine Receptors in Brain 263 FIGURE 8 Reactive synapse from midbrain. Activity occurs in a thickened postsynaptic band along the region of synaptic contact, x 113,000. FXGURE 9 Intensely reactive synapse from midbrain. Activity occurs postsynaptically (Po) in an irregular layer that includes the postsynaptic membrane and postsynaptic density. In addition, reaction product is localized to the presynaptic membrane (Pr). • 111,000. Fmuw 10 Control preparation (midbrain) in which tissue was preincubated in native a-Btx before incubation in the conjugate and reaction for HRP. Reaction product is absent in the pre- and postsynaptic membranes and densities, x 112,000. Fmul~ 11 Control preparation (a-Btx preincubation, midbrain) showing absence of reaction product. Note that membranes in the region of synaptic contact are denser than membranes in other regions. The width of the postsynaptic membrane is less than the thickness of the postsynaptic band of reaction product in reactive synapses, x 103,000. 30). Since some central postsynaptic membranes diography after nsI-ot-bungarotoxin binding at have membrane granules and some bind conju- mouse neuromuscular junctions. J. Cell Biol. gated toxin, it is possible that, as in the case of the 69:144-158. neuromuscular junction, toxin binding takes place 7. GRAX, E. G., and V. P. Wrna'rAxEx. 1962. The isolation of nerve endings from brain: An electron- at regions occupied by membrane granules. microscopic study of cell fragments derived by ho- Previous studies have indicated that the reticu- mogenization and centrifugation. J. Anat. 96:79- lar system (32) and hypothalamic nuclei (33) con- 88. tain cholinergic neurons or fibers. This study dem- 8. Gv.r.Er~, L. A. 1976. Binding of a-bungarotoxin to onstrates the presence of cholinergic synapses in chick sympathetic ganglia: Properties of the recep- these regions. Reactive synapses were character- tor and its rate of appearance during development. ized by a predominance of small, clear vesicles Brain Res. 111:135-145. while synapses containing many dense-core vesi- 9. HEUSER,J. E., T. S. RE,E, and D. M. D. LANDXS. cles (possibly containing biogenic amines) were 1974. Functional changes in frog neuromuscular unreactive. It should be noted, however, that in junctions studied with freeze-fracture. J. Neurocy- tol. 3-109-131. both synaptosomes and reactive brain regions, 10. JEt, SEN, C., W. LtrrrN, J. FREEMAN, and R. only a small proportion of the total number of BRADY. 1975. Localization of ACh receptor synapses present were reactive, including those (AChR) in brain using horseradish peroxidase-con- containing clear vesicles. This finding may indicate jugated snake neurotoxin. J. Cell Biol. 67(2, Pt. a relatively small number of nicotinic cholinergic 2):191a (Abstr.). synapses in the central nervous system. Alterna- 11. KEE~R, D. A., and J. F. CHRIST. 1976. Distribu- tively, lack of staining could be due to a density of tion of endogenous diaminobenzidine-staining cells receptors below the limits of sensitivity of the in normal rat brain. Brain Res. 116:312-316. technique, or to insufficient penetration of the 12. KotrvEiAs, E. D., and L. A. GV,EEr~E. 1976. The conjugate. binding properties and regional ontogeny of recep- tors for a-bungarotoxin in chick brain. Brain Res. 113:111-126. This research was supported by a grant (BNS75-16434 13. LANDIS,D. M. D., and T. S. REESE. 1974. Differ- A01) from the National Science Foundation. ences in membrane structure between excitatory and inhibitory synapses in the cerebellar cortex. J. Received for publication 7 March 1977, and in revised Comp. Neurol. 155:93-126. form 17 June 1977. 14. LAr~DIS, D. M. D., T. S. 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T. L. L•NTZ AND J. CHESTER Localization of Acetylcholine Receptors in Brain 267