Ultrastructural immunolocalization of adenosine deaminase in histaminergic neurons of the tuberomammillary nucleus of rat

Brain Research, 527 (1990) 335-341 Elsevier

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Ultrastructural immunolocalization of adenosine deaminase in histaminergic neurons of the tuberomammillary nucleus of rat T. Yamamoto, J. Ochi*, P.E. Daddona** and J.I. Nagy Department of Physiology, University of Manitoba, Winnipeg, Manitoba R3E OW3 (Canada) (Accepted 29 May 1990) Key words: Adenosine deaminase; Tuberomammillary nucleus; Electron microscopy; Immunohistochemistry; Purine; Histamine

Neurons in the tuberomammillary nucleus (TM) of the rat hypothalamus were immunolabelled for the enzyme adenosine deaminase (ADA) and investigated by electron microscopic immunohistochemical techniques. ADA-immunoreactivity was distributed throughout the somal and dendritic cytoplasm of TM neurons and in the karyoplasm of most, but not all of these neurons. Immunoreactive axons were rarely observed within the tightly packed cell clusters of the TM subdivisions examined. Dense deposition of immunoreaction product together with reasonable preservation of morphological detail facilitated identification of immunoreactive profiles and allowed characterization of the ultrastructural features of labelled neurons and the relationships of these with each other and with surrounding unlabeUed neuronal and glial elements. Immunoiocalization of ADA therefore represents a reliable and convenient method for the identification of TM neurons in EM studies of their ultrastructure and synaptic interactions. The tuberomammillary nucleus (TM) is located in the posterior basal hypothalamus and consists of a relatively broad distribution of neurons some of which are densely packed in a few discrete clusters, while others are more widely dispersed. The complex organization of these neurons has been revealed by histochemical or immunohistochemical localization of a variety of enzymes and substances they express 3'10'11'15'21'23'24'27. Markers of TM that allow visualization of the entire nucleus and are absent in other hypothalamic neurons have been particularly useful in studies of its constituent neurons, which are now known to have widespread projections throughout the brain 8'10'13'16'23-25'28. These include the enzymes histidine decarboxylase (HDC) 28 and adenosine deaminase ( A D A ) 13"21'24. The presence of H D C and its product histamine in TM neurons has led to the designation of TM as a histaminergic nucleus. The presence of high levels of A D A in TM and in several other CNS systems suggests augmented degradative capacity of adenosine or related purines 5,14. In contrast to the considerable body of information now available on the anatomical and cytochemical characteristics of TM at the LM level, only a few ultrastructural studies of this nucleus have been conducted 7'11'3°. In view of the dispersed nature of TM, it appears that such studies will require continued use of appropriate markers to aid identification of TM elements. Here we demonstrate some ultrastructural fea-

tures of TM as shown by immunohistochemical localization of A D A . Immunohistochemistry for A D A was conducted essentially as previously described 33 with some minor modifications. Male Sprague-Dawley (250-300 g) rats deeply anesthetized with chloral hydrate were perfused transcardiaUy with 70 ml of 0.1 M sodium phosphate buffer (pH 7.4) (PB) and then with 400 ml of fixative (4 °C) containing 4% paraformaldehyde, 0.2% picric acid, 0.2% glutaraldehyde and 0.1 M PB. Brains were removed, postfixed for 2 h in the same fixative without glutaraldehyde, and sectioned at a thickness of 20-30/~m on a vibratome. The sections were washed for 6-18 h in PB containing 0.9% saline (PBS) and then incubated for 48 h at 4 °C with rabbit anti-ADA antibody diluted 1:1000 in PBS containing 0.1% Photo-Flo and 1% bovine serum albumin (BSA). They were then washed in PBS for 45 min, incubated for 2 h at 22 °C with goat anti-rabbit IgG (Sternberger-Meyer) diluted 1:20 in PBS containing 1% BSA, washed for 45 min in PBS, incubated for 2 h at 22 °C with rabbit peroxidase-antiperoxidase complex (Stemberger-Meyer) diluted 1:100 in PBS containing 1% BSA, and washed for 20 min in PBS and then for a further 20 min in Tris-HCl buffer, pH 7.4. The sections were then reacted with 3,3"-diaminobenzidine, rinsed in Tris-HCl buffer, postfixed in 0.1 M PB containing 2% osmium tetroxide, dehydrated in ethanol and flat-em-

* Permanent address: Dept. of Anatomy, Shiga University of Medical Science, Seta, Otsu City, 520-21, Japan. ** Permanent address: Centocor, Malvern, PA 19355 U.S.A. Correspondence: J.l. Nagy, Department of Physiology, Faculty of Medicine, University of Manitoba, 770 Bannatyne Avenue, Winnipeg, Man., R3E 0W3 Canada. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

336 bedded in Epon-812. Desired areas of TM were trimmed from the section and mounted onto resin blocks. Semithin sections were photographed by LM and adjacent ultrathin sections were counterstained with uranyl acetate and lead citrate. The specificity of the affinity-purified anti-ADA antibody employed has been demonstrated by preabsorption and other control procedures as previously described 2A3. Adenosine deaminase-immunoreactive (ADA-IR) neurons were examined in what we have previously termed the ventral and lateral subdivisions of TM 24. By LM, immunoreactive neurons in these subdivisions were closely packed (Fig. 1A, inset) and, by EM, had prominent and densely distributed cytoplasmic organelles. Immunolabelled somata were generally round or oval and had round, centrally located nuclei with one or two nucleoli (Fig. 1A). They contained many small, slender mitochondria distributed uniformly throughout the perikaryal cytoplasm, well-developed Golgi lamellae (3-7. layers) near the nucleus (Fig. 1B), numerous vacuoles of various sizes and many multivesicular bodies of fairly constant size (Fig. 1G). In the vast majority of cells, immunoreaction product was localized in most areas of the cytoplasm and karyoplasm, but was absent within cytoplasmic organelles and the non-granular portion of nucleoli (Fig. 1B). Immunolabelling was usually most concentrated on rough endoplasmic reticulum and associated polysomes (Fig, 1C). A few instances were found ( < 5 % ) where the nucleus of immunoreactive neurons was devoid of immunostaining (Fig. 1B, inset). Immunoreactive somata exhibited several small cytoplasmic protrusions or somatic spines per semi-thin section (Fig. 1A,B). These had narrow stems protruding into surrounding neuropil and occasionally bulbous endings surrounded by non-labelled terminals, the whole of which were sometimes totally or partially encapsulated by glial processes (Fig. 1C,D). Pinocytotic elements (Fig. 1E,F), which were occasionally coated (Fig. IF), were observed along the cytoplasmic membrane of ADA-IR cell bodies. On rare occasions, a cilium emerged from a soma and was pressed against the plasma membrane (Fig. 1G) or was seen protruding into the neuropil. Immunoreaction product in dendrites was seen on

microtubules, outer membranes of mitochondria, and ribosomes, but was absent in vacuoles and multivesicular bodies (Fig. 2A-G). Dendrites tended to have irregular contours with occasional varicosities (Fig. 2D) and were often apposed to one another (Fig. 2 A , B ) o r to ADA-IR somata (Fig. 2C). Labelled elements commonly exhibited swellings or indentations at such appositions (Fig. 2B). Two types of ADA-IR dendrites were distinguished on the basis of their contents. Type I contained many microtubules, only a few mitochondria, and often formed puncta adherens with ADA-IR somata (Fig. 2C) or type II dendrites (Fig. 2F). Type II dendrites had scanty cytoplasm, many mitochondria, a relatively small number of microtubules, and formed puncta adherens with labelled as well as non-labelled dendrites (Fig. 2E). Immunolabelled axon terminals were not observed in the TM subdivisions examined. Five types of non-labelled terminals formed synaptic contacts with ADA-IR somata and dendrites. The first type, only rarely observed, was located on ADA-IR somata and type II dendrites where they formed asymmetrical synapses. They were distinguished from other types by the presence of presynaptic dense bodies (PDB) and hence were designated PDB boutons. These dense bodies were located beneath the Synaptic thickening and separated from it by a space (27 nm) containing electron dense material. The bodies were composed of several linearly arranged agranular synaptic vesicles (Fig. 2G). The second type were the most often encountered and tended to exhibit an electron-dense matrix. These terminals, designated DPA (densely packed, asymmetric) boutons, were distributed on ADA-IR dendrites, contained many, densely packed, round, clear synaptic vesicles about 50 nm in diameter and very few (0-2) granular vesicles, (Fig. 3A), and formed asymmetrical synaptic contacts. The third type, designated LPA (loosely packed, asymmetric) boutons, was similar to the second, but displayed electron-lucent cytoplasm and tended to contain loosely packed, round, clear synaptic vesicles (Fig. 3C). The fourth type, designated DCV (dense core vesicle) boutons, was characterized by the presence of 5-20 dense-core synaptic vesicles 100 to 130 nm in diameter and round, clear vesicles 30-50 nm in

Fig. 1. Electron micrographs showing ADA-IR somata in the ventral subdivision (Vs) of TM. A: low magnification of 4 immunopositive neurons indicated by arrows in the photomicrograph of a semi-thin section shown in the inset. B: higher magnification showing ADA-immunoreactivity throughout neuronal cytoplasm and nuclei (N). Inset shows an example where the nucleus (N) of a positive neuron (arrow) is unlabelled. GA, Golgi apparatus; RER, rough endoplasmic reticulum; SP, somatic spine. C: higher magnificationof a bulbous spine (arrow) protruding from an immunopositive soma (S). The density of immunolabelling appears to be higher in the spine than the soma. D: a somatic spine (large arrow) surrounded by several axon terminals (small arrowheads) one of which forms a synaptic contact with the spine (small arrow). The spine-terminal complex appears to be entirely ensheathed by several glial processes (large arrowheads). E,F: two immunoreactive somata (S) showing pinocytotic profiles (arrows) of the plasma membrane adjacent to a glial process (E) and to an immunoreactive dendrite (F). G: a cilium (arrow) emanating from and lying adjacent to an immnnoreactive soma (S). Note the multivesicular bodies (MVB) in the soma and in the adjacent immunolabelled process. Magnifications: A, ×2100, inset x350; B, x7200, inset x 1900; C,D, x14,500; E,F, x23,200; G, x8400.

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339 diameter (Fig. 3B). These also formed asymmetric synaptic contacts. The fifth type, found on ADA-IR somata and large calibre dendrites, contained many round, clear synaptic vesicles 30-50 nm in diameter, formed symmetric synapses and were termed DPS (densely packed, symmetric) boutons (Fig. 3D). These tended to be flattened terminals that produced indentations at their appositions on somata. Occasionally several dendrites together with several synaptically related nonlabelled terminals of different types were encapsulated by glial sheaths in glomerulus-like structures (Fig. 3E). The ultrastructural features of A D A - I R neurons in the posterior basal hypothalamus were similar to those of hypothalamic neurons that were previously identified as belonging to TM on the basis of EM immunolabelling for H D C 7"3°. Moreover, ADA-negative neuronal somata in the vicinity of immunopositive cells exhibited the same general ultrastructural characteristics as those reported for HDC-negative neurons distributed among HDC-IR somata. These results are consistent with previous LM observations of the coexistence of HDC and A D A in TM neurons 1s'21 and indicate that at the EM level immunolabelling for A D A is restricted to these neurons in the posterior hypothalamus. Given the relatively good balance between preservation of antigenicity and morphological detail provided by immunohistochemistry for A D A together with the production of antibodies against A D A in a number of laboratories 1"9'12,17,2°,26, it appears that A D A is a convenient marker for further ultrastructural studies of TM neurons. Some properties displayed by TM neurons include somatic spines, cilia and endocytotic profiles, numerous somal and dendritic multivesicular bodies, and varicose dendrites. While none of these are unique to neurons of TM, they are nevertheless quite distinctive in these cells. Neuronal cilia have no known functional role, but may be involved in chemo- or mechano-reception or may represent vestigial remnants TM. Multivesicular bodies have been found in virtually all eukaryotic cells and there is now substantial evidence suggesting that they participate in receptor-ligand internalization through endocytosis, and thereby may contribute to both intracellular signalling mechanisms and lysosomal pathways 6,22,29. Their prevalence together with the occurrence of somal pinocy-

totic profiles in TM neurons might indicate that these neurons have particularly well developed internaiization mechanisms. This would be consistent with suggestions that the proximity of these cells to ventricular and brain surfaces may be indicative of communication functions with the local extracellnlar environment 11'2a. With regard to synaptic inputs, the present results represent only a preliminary account of the types of boutons making synaptic contact with TM neurons in only two subdivisions of the nucleus. A more systematic analysis of these as well as their arrangement in what sometimes appear as synaptic glomeruli will eventually allow correlation with various types to known 4'31'32 and yet to be determined sources of TM afferents. A separate point concerns possible relationships between A D A and adenosine or purinergic neuromodulatory mechanisms s,14. We had hoped that some aspects of the subcellular localization of this enzyme would provide some clues to specific functions related to its abundant expression in TM and certain other central neurons. However, A D A in both neuronal somata and dendrites exhibited a distribution pattern expected of a soluble cytoplasmic enzyme, at least within the limits of resolution allowed by EM immunohistochemistry based on visualization of somewhat diffusible DAB immunoreaction product. We are currently developing immunogold methods for detection of A D A in both perikarya and axons and these may be more revealing. Two observations are, nevertheless, worthy of comment. First, as previously noted only light ADA-immunoreactivity of glial cells was seen by LM even after application of immunoreaction intensification procedures14. Our failure to detect ADA-immunolabelling in glial somata and processes here further suggests that glial cells have much lower levels of A D A than TM neurons. And second, while the nuclei of all TM neurons appear to contain A D A as seen by LM 2~'23,24, a few neurons were found here to be totally devoid of nuclear immunolabelling. Since these neurons displayed intense cytoplasmic ADAimmunoreactivity, failure of antibody penetration to the nucleus is an unlikely possibility. Thus, TM neurons may have differential or state-dependent requirements for nuclear A D A and molecular control mechanisms may exist whereby A D A is incorporated into both the

Fig. 2. Electron micrographs showing ADA-IR dendritic profiles within the lateral subdivision (Ls) of TM. A: low magnification showing an unlabelled multivesicular body within a labelled dendrite (MVB, large arrow), axon terminals in association with immunolabelled dendrites (large arrowheads) and appositions (small arrows) and puncta adherens (small arrowheads) between positive dendrites. B: appositions between several immunolabelled dendrites (dr) one of which is surrounded by several axon terminals (arrowheads). C: apposition between a positive soma (S) and dendrite (arrow). D: an immunolabelled varicose dendrite (arrow). E,F: magnification of adherens junctions between an immunopositive and immunonegative dendrite (arrow in E) and between two positive dendrites (arrows in F). G: an immunolabelled dendrite (dr) in synaptic contact with an axon terminal (ax) which displays presynaptic dense bodies (arrow) beneath the synaptic thickening. Dendrites referred to as type I and type II in the text are labelled drI and drII, respectively,in A and F. Magnifications: A,C,D,E,G, x 17,800; B, x8200; F, x35,700.

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Fig. 3. Electron micrographs showing various types of axon terminals in synaptic contact (arrowheads) with ADA-IR dendrites and a soma. A: a densely packed asymmetric-type terminal (DPA) containing small, round, clear vesicles. B: a loosely packed asymmetric-type terminal (LPA) containing round, clear vesicles and a nearby DPA-type terminal each making synaptic contact with a different immunolabelled dendrite. C: a dense-core vesicle-type terminal (DCV), containing round, clear vesicles and large dense core vesicles. D: a densely packed symmetric-type terminal (DPS, arrow) making a symmetrical synaptic contact with an immunopositive soma. E: 3, immunolabelled dendrites (dr) in synaptic contact with a DPA-type terminal (DPA, arrow), a DVC-type terminal (DVC, arrow) and a LPA-type terminal (LPA, arrow). The dendrites and terminals appear to be ensheathed by glial processes in a synaptic glomerulus-like arrangement. Magnifications: A-C, x 17,400; D,E, x 13,200.

341 cytoplasmic and nuclear c o m p a r t m e n t s of most T M n e u r o n s and is excluded from the latter in a small percentage of these cells.

1 Chechik, B.E. and Sengupta, S., Detection of human, rat and mouse adenosine deaminase by immunochemical and immunomorphologic methods using antiserum to calf enzyme, J. lmmunol. Methods, 45 (1981) 165-176. 2 Daddona, P.E. and Kelley, W.N., Human adenosine deaminase. Purification and subunit structure, J. Biol. Chem., 252 (1977) 110-115. 3 Ericson, H., Watanabe, T. and Kthler, C., Morphological analysis of the tuberomammillary nucleus in the rat brain: Delineation of subgroups with antibody against L-histidine decarboxylase as a marker, J. Comp. Neurol., 263 (1987) 1-24. 4 Ericson, H., Blomqvist, A. and Kfhler, C., Brainstem afferents to the tuberomammillary nucleus in the rat brain with special reference to monoaminergic innervation, J. Comp. Neurol., 281 (1989) 169-192. 5 Geiger, J.D. and Nagy, J.I., Adenosine deaminase and 3Hnitrobenzylthioinosine as markers of adenosine metabolism and transport in central purinergic systems. In M. Williams (Ed.), Adenosine Receptors, Humana Press, NJ, 1990, pp. 225-288. 6 Goldstein, J.L., Brown, M.S., Anderson, R.G.W., Russell, D.W. and Schneider, W.J., Receptor-mediated endocytosis: concepts emerging from the LDL receptor system, Ann. Rev. Cell Biol., 1 (1985) 1-39. 7 Hayashi, H., Takagi, T., Takeda, N., Kubota, Y., Tohyama, M., Watanabe, T. and Wada, H., Fine structure of histaminergic neurons in the caudal magnocellular nucleus of the rat as demonstrated by immunocytochemistry using histidine decarboxylase as a marker, J. Comp. Neurol., 229 (1984) 233-241. 8 Inagaki, N., Yamatodani, A., Ando-Yamamoto, M., Tohyama, M., Watanabe, T. and Wada, H., Organization of histaminergic fibers in the rat brain, J. Comp. Neurol., 273 (1988) 283-300. 9 Ingolia, D.E., Yeung, C.Y., Orengo, I.E, Harrison, M.L., Frayne, E.G., Rudolph, EB. and KeUems, R.E., Purification and characterization of adenosine deaminase from a genetically enriched mouse cell line, J. Biol. Chem., 160 (1985) 13261-13267. 10 KOhler, C., Swanson, L.W., Haglund, L. and Wu, J.-Y., The cytoarchitecture, histochemistry and projections of the tuberomammillary nucleus in the rat, Neuroscience, 16 (1985) 85-110. 11 Maeda, T., Kimura, H., Nagai, T., Imai, H., Arai, R., Sakumoto, T., Sakai, K., Kitahama, K. and Jouvet, M., Histochemistry of the magnocellular neurons in the posterior hypothalamus, with special reference to MAO activity and ability of 5-HTP uptake and decarboxylation, Acta Histochem. Cytochem., 17 (1984) 179-193. 12 Miguel-Hidalgo, J.-J., Senba, E., Matsutani, S., Takatsuji, K., Fukui, H. and Tohyama, M., Laminar and segregated distribution of immunoreactivities for some neuropeptides and adenosine deaminase in the superior colliculus of the rat, J. Comp. Neurol., 280 (1989) 410-423. 13 Nagy, J.I., La Bella, L.A., Buss, M. and Daddona, P.E., Immunohistochemistry of adenosine deaminase: implications for adenosine neurotransmission, Science, 224 (1984) 166-168. 14 Nagy, J.I., Geiger, J.D. and Staines, W.A., Adenosine deaminase and purinergic neuroregulation, Neurochem. Int., 16 (1990) 211-221. 15 Panula, P., Yang, H.-Y.T. and Costa, E., Histamine-containing neurons in the rat hypothalamus, Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 2572-2576. 16 Panula, P., Pirvola, U., Auvinen, S. and Airaksinen, M.S., Histamine-immunoreactive nerve fibers in the rat brain, Neuroscience, 28 (1989) 585-610. 17 Patel, B.T. and Tudball, N., Localization of S-adenosylhomocysteine hydrolase and adenosine deaminase immunoreactivities in

The authors wish to thank Lyn Poison and Mike Sawchuk for excellent technical assistance and Lyn Poison for typing the manuscript. This work was supported by grants from the Medical Research Council of Canada (MRC) and the University of Manitoba Faculty Fund. J.l. Nagy is a recipient of a MRC Scientist Award.

the rat, Brain Research, 370 (1986) 250-264. 18 Patel, B.T., Tudball, N., Wada, H. and Watanabe, T., Adenosine deaminase and histidine decarboxylase coexist in certain neurons of the rat brain, Neurosci. Lett., 63 (1986) 185-189. 19 Peters, A., Palay, S.L. and Webster, H.F., The fine structure of the nervous system: the neurons and supporting cells, W.B. Saunders, Philadelphia, 1976. 20 Schrader, W.P. and Stacey, A.R., Purification and subunit structure of adenosine deaminase from human kidney, J. Biol. Chem., 252 (1977) 6409-6415. 21 Senba, E., Daddona, P.E., Watanabe, T., Wu, J.-Y. and Nagy, J.I., Coexistence of adenosine deaminase, histidine decarboxylase, and glutamate decarboxylase in hypothalamic neurons of the rat, J. Neurosci., 5 (1985) 3393-3402. 22 Stahl, P. and Schwartz, A., Receptor-mediated endocytosis, J. Clin. Invest., 77 (1986) 657-662. 23 Staines, W.A., Yamamoto, T., Daddona, P.E. and Nagy, J.I., Neuronal colocalization of adenosine deaminase, monoamine oxidase, galanin and 5-hydroxytryptophan uptake in the tuberomammillary nucleus of the rat, Brain Res. Bull., 17 (1986) 351-365. 24 Staines, W.A., Daddona, P.E. and Nagy, J.I., The organization and hypothalamic projections of the tuberomammillary nucleus in the rat: an immunohistochemical study of adenosine deaminase-positive neurons and fibers, Neuroscience, 23 (1987) 571596. 25 Staines, W.A., Yamamoto, T., Daddona, P.E. and Nagy, J.I., The hypothalamus receives major projections from the tuberomammillary nucleus in rat, Neurosci. Lett., 76 (1987) 257-262. 26 Vielh, P. and Castellazzi, M., Murine monoclonal antibodies which recognize adenosine deaminase from calf, mouse, rat and man, Immunol. Left., 10 (1985) 145-149. 27 Vincent, S.R., HOkfelt, T., SkirboU, L.R. and Wu, J.-Y., Hypothalamic ~,-aminobutyric acid neurons project to the neocortex, Science, 220 (1983) 1309-1311. 28 Watanabe, T., Taguchi, Y., Inagaki, S., Tanaka, J., Kubota, H., Terano, Y., Tohyama, M. and Wada, H., Disribution of the histaminergic neuron system in the central nervous system of rats; a fluorescent immunohistochemical analysis with histidine decarboxylase as a marker, Brain Research., 295 (1984) 13-25. 29 Williams, M.C., Vesicles within vesicles: what role do multivesicular bodies play in alveolar type II cells?, Am. Rev. Resp. D/s., 135 (1987) 744-746. 30 Wouterlood, EG., Sauren, Y.M.H.E and Steinbusch, H.W.M., Histaminergic neurons in the rat brain: correlative immunocytochemistry, Golgi impregnation and electron microscopy, J. Comp. Neurol., 252 (1986) 227-244. 31 Wouterlood, EG., Steinbusch, H.W.M., Luiten, P.G.M. and Bol, J.G.J.M., Projection from the prefrontal cortex to histaminergic cell groups in the posterior hypothalamic region of the rat. Anterograde tracing with Phaseolus vulgaris leucoagglutinin combined with immunocytochemistry of histidine decarboxylase, Brain Research., 406 (1987) 330-336. 32 Wouterlood, EG. and Gaykema, R.P.A., Innervation of histaminergic neurons in the posterior hypothalamic re#on by medial preoptic neurons. Anterograde tracing with Phaseolus vulgaris leucoagglutinin combined with immunocytochemistry of histidine decarboxylase in the rat, Brain Research, 455 (1988) 170-176. 33 Yamamoto, T., Shiosaka, S., Daddona, P. E. and Nagy, J.I., Further observations on the relationships between adenosine deaminase-containing axons and trigeminal mesencephalic neurons: an electron microscopic, immunohistochemical and anterograde tracing study, Neuroscience, 26 (1988) 669-680.