Proc. Natl Acad. Sci. USA Vol. 79, pp. 6717-6721, November 1982 Neurobiology Synthesizing enzymes for four neuroactive substances in motor neurons and neuromuscular junctions: Light and electron microscopic immunocytochemistry (transmitters/choline acetyltransferase/tyrosine hydroxylase/cysteine sulfinic acid decarboxylase/glutamic acid decarboxylase) VICTORIA CHAN-PALAY*, ANDREW G. ENGELt, SANFORD L. PALAYt, AND JANG-YEN WU§ Departments of *Neurobiology and tAnatomy, Harvard Medical School, Boston, Massachusetts 02115; tNeuromuscular Research Laboratory, Mayo Clinic, Rochester, Minnesota 55905; and §Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030 Contributed by Sanford L. Palay, July 30, 1982 ABSTRACT Immunocytochemical evidence is presented for ninergic, whereas others are peptidergic and still others are the existence of choline acetyltransferase (ChoAcTase), cysteine both (9-11). Recently, it has been shown that cerebellar Pur- sulfinic acid decarboxylase (CSADCase), tyrosine hydroxylase kinje cells are heterogeneous chemically as well as morpholog- (TyrOHase), and glutamic acid decarboxylase (GluDCase) in large ically, some beingpeptidergic, some containing 'y-aminobutyric motor neurons ofthe hypoglossal nucleus and the spinal cord and acid (GABA), some containing taurine, and others containing in nerve terminals ofmotor end plates in tongue and skeletal mus- various combinations of peptides, taurine, and GABA (12-15). cle of five mammalian species, including man. These enzymes, Therefore, it is important to inquire whether the motor neuron which are responsible for the synthesis of acetylcholine (AcCho), and its axonal terminals at the neuromuscular junction might taurine, dopamine, and y-aminobutyrate (GABA), respectively, be more diversified than has been thought. were detected by immunocytochemical studies with monoclonal also or polyclonal antibodies raised against the enzymes. Electron mi- The present paper reports evidence from an immunocyto- croscopy of the neuromuscular junctions showed that the immu- chemical study of five mammalian species (including man) to noreactivity in each case was confined to the cytoplasmic matrix the effect that motor neurons and nerve terminals at the neu- of presynaptic nerve terminals. Immunoreactivity obtained for romuscular junction contain not only the enzyme ChoAcTase each enzyme antibody varied with the species. It was highest in but also other transmitter synthesizing enzymes: cysteine sul- fresh, unfixed muscle and lowest in aldehyde-fixed specimens. finic acid decarboxylase (CSADCase; EC 4.1.1.29), which cata- Negative controls were obtained with preimmune sera and anti- lyzes the conversion of cysteine sulfinic acid or cysteic acid to sera preabsorbed with pure ChoAcTase, CSADCase, or Glu- taurine; glutamic acid decarboxylase (GluDCase; EC 4.1.1.15), DCase antigen. Double-labeling studies with ChoAcTase antibod- the rate-limiting enzyme in the synthesis of GABA; and the ies and acetylcholinesterase (AcChoEase) antibodies, AcChoEase catecholamine biosynthetic enzyme tyrosine 3-hydroxylase enzyme activity, or a-bungarotoxin binding indicated that (TyrOHase; EC 1.14.16.2). ChoAcTase, AcChoEase, and AcCho receptors were colocalized at the same end plates. MATERIALS AND METHODS The following antibodies and controls were used in this study: Acetylcholine (AcCho) occurs in motor nerve fibers along with monoclonal antibody and polyclonal antibody against Cho- the enzyme choline acetyltransferase (ChoAcTase; EC 2.3.1.6) AcTase (for specificity see ref. 16); polyclonal antibody against which catalyzes its synthesis. This enzyme has been localized CSADCase (17); polyclonal antibody against GluDCase (18, 19); by immunocytochemistry to the motor neurons in the spinal polyclonal antibody against TyrOHase (20); and monoclonal an- cord and brainstem (1). AcCho is released upon stimulation of tibody against AcChoEase (AE-3) (7). Control sera included the motor nerve and evokes a contraction in the innervated preimmune rabbit serum and anti-CSADCase, anti-GluDCase, muscle (2, 3). Furthermore, the postsynaptic potentials induced or anti-ChoAcTase preabsorbed with the corresponding puri- by the nerve impulse and the spontaneous miniature end plate fied antigen. The appropriate conjugated second antibodies potentials found at the neuromuscular junction can be mim- were used for the indirect peroxidase-labeled second antibody icked by the controlled application of AcCho to the surface of method and, in some instances, followed by the peroxidase-an- a muscle fiber close to the nerve ending (4). The specific hy- tiperoxidase (PAP) reaction. For the monoclonal antibodies, drolyzing enzyme acetylcholinesterase (AcChoEase; EC 3.1.1.7) treatment with peroxidase-labeled goat anti-mouse immuno- occurs both in the motor neuron and in the synaptic cleft ofthe globulin was followed by the application of mouse PAP or flu- neuromuscular junction as demonstrated by histochemical (5, orescein-labeled goat anti-mouse immunoglobulin. For the 6) and immunocytochemical methods (7). Finally, the postsyn- polyclonal antibodies, peroxidase-labeled goat anti-rabbit im- aptic effects of both the nerve impulse and applied AcCho can munoglobulin (PGARIg) was followed by rabbit PAP or fluo- be blocked by the same specific antagonists (8). Thus, the ex- rescein-labeled goat anti-rabbit immunoglobulin. The peroxi- clusive role of AcCho at the neuromuscular junction has been dase reactions were visualized with diaminobenzidine (DAB). and for the last half century. Central nervous system motor neurons in medullary brain- established universally accepted stem and cervical and thoracic spinal cord were examined in The apparent chemical uniformity of the somatic motor sys- Vibratome sections. Swiss albino mice of both sexes (body tem contrasts, however, with the heterogeneity that has been Tissues were fixed and Vibratome discovered in other parts of the nervous system. For example, weight, 6-20 g) were used. are whereas some neurons in sympathetic ganglia adrenergic Abbreviations: AcCho, acetylcholine; AcChoEase, acetylcholinester- others are cholinergic. Some cells in the raphe nuclei are seroto- ase; BTX, a-bungarotoxin; ChoAcTase, choline acetyltransferase; CSADCase, cysteine sulfinic acid decarboxylase; DAB, diaminoben- The publication costs ofthis article were defrayed in part by page charge zidine; GABA, y-aminobutyric acid; GluDCase, glutamic acid decar- payment. This article must therefore be hereby marked "advertise- boxylase; PAP, peroxidase-antiperoxidase; PGARIg, peroxidase-la- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. beled goat anti-rabbit immunoglobulin; TyrOHase, tyrosine hydroxylase. 6717 Downloaded by guest on October 1, 2021 6718 Neurobiology: Chan-Palay et al. Proc. Natl. Acad. Sci. USA 79 (1982) sections were processed for light microscopic immunocyto- RESULTS chemical study according to described procedures (15). Animals Large and medium-sized neurons in the hypoglossal nucleus were injected intraperitoneally with demecolcine (Sigma, 1 mg/ and spinal cord displayed immunoreactivity to the antibodies kg) or intrathecally with colchicine (1 mg/kg) 16 hr before trans- raised against the transmitter synthesizing enzymes (ChoAc- cardiac perfusion with 4% formaldehyde in 0.12 M phosphate Tase, TyrOHase, CSADCase, and GluDCase) as well as to an- buffer (first at pH 6.5 and then at pH 11.0) at 40C. The tissues tibodies against AcChoEase. Background staining or reactions were left overnight in the same fixative at pH 7.4. Loose 20-pum obtained with control sera (preimmune, preabsorbed CSAD- sections were incubated with antibodies and control sera at di- Case, preabsorbed GluDCase, preabsorbed ChoAcTase) were lutions of 1:1,000. Unfixed cryostat sections of mouse spinal cord negligible. The results are shown in Table 1 and Figs. 1 and 2. (injected with colchicine intrathecally) and human muscle Both monoclonal and polyclonal ChoAcTase antibodies re- were also examined after immunocytochemical processing. vealed immunoreactive cell bodies that were also reactive with Specimens from different muscles and species were sampled AE-3 in adjacent serial sections. ChoAcTase antibody consis- as follows: from mouse and rat-tongue, masseter, omohyoid, tently produced the most intense reactions in neuronal peri- sternothyroid, sternocleidomastoid, digastric, gastrocnemius, karya, primary dendrites, large- and small-caliber axons, and soleus, anterior tibial, hand and toe extensors, and interossei; terminals in the immediate area. The monoclonal antibodies from guinea pig-gastrocnemius; and from monkey (Macaca against ChoAcTase and AcChoEase revealed similar numbers mulatta)-biceps, intercostal, and dorsal interosseus. Diagnos- of neurons but with less intense reactivity against a very faint tic muscle biopsy specimens from six humans were used: deltoid background. These antibodies stained approximately40% ofthe (79-yr-old man), triceps (67-yr-old woman), deltoid (42-yr-old large hypoglossal, cervical, and thoracic motor neurons. CSAD- woman), and vastus lateralis (two 24-mo-old boys and one 18- Case antibodies also showed a sharp reaction in approximately mo-old girl). Serial cryostat sections (5 ,m) were cut in trans- 40% ofmotor neurons in both locations. This immunoreactivity verse, oblique, or longitudinal planes. Mouse tongue and skel- was not evident in material incubated in anti-CSADCase preab- etal muscle fixed by cardiac perfusion were also embedded