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Colocalization of atriopeptin-like immunoreactivity with choline acetyltransferase-and substance P-like immunoreactivity in the pedunculopontine and laterodorsal tegmental nuclei in the rat
Brain Research, 382 (1986) 163-168
163
Elsevier BRE 21762
Colocalization of atriopeptin-like immunoreactivity with choline acetyltransferaseand substance P-like immunoreactivity in the pedunculopontine and laterodorsal tegmental nuclei in the rat DAVID G. STANDAERT 1'2, CLIFFORD B. S A P E R 2-4'6, DAVID B. R Y E 3'5'6 and BRUCE H. W A I N E R 3'5'6
Departments of 1Pharmacology, 2Neurology and Anatomy, and Neurobiology, Washington University School of Medicine, Saint Louis, MO 63110 and Departments of 3Pharmacological and Physiological Sciences, 4Neurology, 5pathology, and 6The Brain Research Institute, University of Chicago, Chicago, IL 60637 ( U. S. A.) (Accepted May 13th, 1986)
Key words: atrial natriuretic peptide - - electrolyte homeostasis - - cardiovascular system - - interpeduncular nucleus
Atriopeptin, the atrial natriuretic peptide, is a circulating hormone that plays an important role in the regulation of fluid and electrolyte balance. Immunohistochemical studies have shown that large, multipolar atriopeptin-like immunoreactive (APir) neurons are present in areas of the midbrain corresponding to the large neurons of the pedunculopontine tegmental (PPT) and lateral dorsal tegmental (TLD) nuclei, all of which can be stained immunohistochemically for choline acetyltransferase-like immunoreactivity (ChATir). A subpopulation of these cholinergic PPT and TLD neurons are also known to contain substance P-like immunoreactivity (SPir). Using an immunofluorescent technique that allows simultaneous localization of two antigens, we have studied the relationship between APir, SPir and ChATir in the pontine tegmentum of the rat. We have found that (i) the large multipolar APir neurons of the pontine tegmentum are identical to the ChATir neurons of the PT and TLD nuclei and (ii) a subpopulation of the APir neurons in PPT and TLD neurons are also SPir. A t r i o p e p t i n , the atrial natriuretic p e p t i d e , is a circulating p e p t i d e h o r m o n e that plays a central role in the regulation of fluid and electrolyte homeostasis. Synthesized and stored in the atria of the heart, atriopeptin is released into the circulation in response to increases in intravascular v o l u m e and causes natriuresis, diuresis, and vasorelaxation (see ref. 15 for review). Several recent studies have shown that neurons containing A P i r are present within the brain of the rat. The m a j o r i t y of these A P i r neurons are found within two regions, the h y p o t h a l a m u s and the pontine t e g m e n t u m 1°,22,24,27. M a n y of the A P i r neurons in the h y p o t h a l a m u s are l o c a t e d within structures implicated in regulation of the cardiovascular system 22'26. In contrast, relatively little is k n o w n about the organization and function of the A P i r neurons in the pontine t e g m e n t a l region. Two clusters of A P i r n e u r o n s are found in the pontine t e g m e n t u m of the rat 22,24,27. Both are c o m p o s e d of large neurons with a distinctive multipolar mor-
phology. T h e m o r e rostral of the two groups begins at the caudal end of the substantia nigra and extends caudally in association with the fibers of the superior cerebellar peduncle. T h e m o r p h o l o g y and distribution of these A P i r neurons is strikingly similar to the neurons constituting the PPT, as defined cytoarchitectonically in both h u m a n s 9A6 and the rat 18. The m o r e caudal group of A P i r neurons is located in the ventrolateral part of the central gray m a t t e r , at the level of the opening of the cerebral aqueduct into the fourth ventricle. These a p p e a r to c o r r e s p o n d to the magnocellular part of the T L D , as described by T o h y a m a et al. 28. A l l of the neurons in both P P T and the magnocellular part of T L D can be stained immunohistochemically using antibodies to C h A T 1'13'1s. A subpopulation of these neurons is known to contain SPir as well as C h A T i r 29. In o r d e r to examine the relationship between C h A T i r , SPir and A P i r neurons in the pontine tegmental region, we have used an immuno-
Correspondence: C.B. Saper, Department of Pharmacological and Physiological Sciences, University of Chicago, 947 East 58 Street, Chicago, IL 60637, U.S.A. 0006-8993/86/$03.50 (~) 1986 Elsevier Science Publishers B .V. (Biomedical Division)
164 fluorescent staining technique that allows the simultaneous localization of two antigenic markers with two colored fiuorochromes. Nine male S p r a g u e - D a w l e y rats were p r e t r e a t e d with colchicine, 150 ~g in 10 ¢tl, injected stereotaxically into the lateral cerebral ventricle u n d e r chloral hydrate anesthesia. Two days later, the rats were reanesthetized with chloral hydrate and perfused through the heart with 300 ml of normal saline, followed by 300 ml of 4 % p a r a f o r m a l d e h y d e in saline buffered with 100 m M sodium p h o s p h a t e (PBS), p H 6.5, and then with 300 ml of 4 % p a r a f o r m a l d e h y d e in PBS, p H 8.5 (ref. 2). Brains were r e m o v e d immediately, m o u n t e d on the chuck of a freezing m i c r o t o m e , and 50-gm coronal sections were cut from the level of
the cerebral aqueduct to the genu of the facial nerve. These sections were washed for 1 h each in PBS, PBS with 0.25% Triton X-100, and PBS with 3% normal goat serum (PBS-G). The sections were then incubated overnight at 4 °C in P B S - G containing antiserum A P 11 (1:500) and a mouse monoclonal antibody to either substance P (Sera-lab, 1:2000) or C h A T (AB8, 1:5(1 dilution of an a m m o n i u m sulfate precipitate of h y b r i d o m a supernatant) j. The next day, the sections were rinsed in PBS, and incubated in PBS-G containing fluorescein isothiocyanate-conjugated goat anti-rabbit serum (Miles, 1:50), and tetramethylrhodamine isothiocyanate-conjugated goat antirat serum (Cappel, 1:50). A f t e r a final wash in PBS, the sections were m o u n t e d on gelatin coated slides,
Fig. 1. Photomicrographs of immunohistochemically stained neurons in the PPT. A: PPT neuron stained for APir is indicated by curved arrow, B: same section as A, stained for SP. Curved arrow indicates same neuron as in A; two additional SPir neurons which are not APir are also seen (open arrows). C: PPT neurons stained for APir; one is seen clearly (arrow), two others lie out of the plane of focus (arrowheads). D: same section as C, stained for ChATir. Arrow indicates neuron seen in C, while the two other neurons lie out of the plane of focus (arrowheads).
165 coverslipped with glycerin containing 10% 1 M Tris buffer, pH 8.5, and 2 mg/ml paraphenylene diamine 17. Staining was observed using a Leitz microscope with Ploem optics and the Leitz 12 and N2 filter systems. The location of labelled neurons was recorded using an X - Y plotter coupled to the stage of the microscope. AP 11 was raised in rabbits using a 79-amino acid cyanogen bromide fragment of the high-molecularweight precursor of atriopeptin as an antigen, as described previously 22. The production and specificity of the substance P antibody 4 and AB8 (ref. 11) have been described. Absorption experiments were performed to demonstrate the specificity of these antisera in this system. When AP 11 was combined with the AB8 preabsorption with the purified cyanogen bromide fragment of the atriopeptin precursor (5 pg/ml) 6 blocked APir staining without affecting ChATir staining. Omission of the ChAT antibody prevented ChATir staining, but did not affect APir staining. .Similarly, when the AP 11 was combined with the substance P antibody, preabsorption with either synthetic substance P (Sigma, 50 pg/ml) or the purified cyanogen bromide fragment, blocked all staining of the corresponding antigen and did not affect the staining of the other. In sections stained for ChATir and APir (Fig. 1) we observed that the large, multipolar APir neurons of the dorsolateral tegmentum corresponded exactly to the ChATir neurons of the PPT and TLD nuclei. The neurons of the PPT and T L D nuclei observed with this method were similar in morphology, number and distribution to previous descriptions 1'13,18. Rostrally, the large, multipolar perikarya of the PPT nucleus, labelled for both ChATir and APir, were observed just dorsal to the caudal end of the substantia nigra. Caudally, additional similarly labelled neurons were found more dorsally and medially, following the superior cerebellar peduncle up to the medial parabrachial nucleus. As the cerebral aqueduct opened out into the fourth ventricle, a second cluster of large, ChATir and APir neurons comprising the magnocellular part of TLD could be seen within the central gray matter. Although all of the neurons in the PP and TLD nuclei were stained for both APir and ChATir, other areas contained neurons labelled for only one of these substances. At the level of the locus coeruleus,
a distinct group of much smaller, more closely packed APir perikarya that were not ChATir were found in the central gray matter between the lateral edge of T L D and the medial edge of the locus coeruleus. These probably correspond to Barrington's nucleus 2s which is thought to be involved in reflex control of micturition 12'23. In contrast, ChATir perikarya that were not APir were observed in the brainstem within the motor nuclei of the cranial nerves. Material stained for APir and SPit revealed that some but not all of the APir perikarya in the PPT and TLD nuclei were also SPir. The relationship between SPir and APir was more complex than that of ChATir and APir and will be described by reference to a series of drawings (Fig. 2). At the level at which the cerebral aqueduct opens into the fourth ventricle (Fig. 2A), the neurons of both the PPT and TLD nuclei may be identified. At this level, a small minority of the APir neurons in both PPT and TLD are also SPir. Neurons which are SPir but not APir are scarse; a small cluster is present in the central gray matter at the dorsolateral tip of TLD. More caudally, at the caudal end of the inferior colliculus (Fig. 2B, C), PPT shifts medially and ventrally, so that its medial border merges with TLD. At this level, the majority of the APir neurons in PPT and T L D are also SPir. A few neurons that are SPir only are present; most of these are located ventral to the APir neurons. At the level of the caudal part of the parabrachial nucleus (Fig. 2D), SPir perikarya are rare. An additional cluster of smaller neurons that were APir and not SPir were observed in B A R 28. Our results demonstrate the identity of the large, multipolar APir neurons of the dorsolateral pontine tegmentum with the previously described ChATir neurons of the PPT and T L D nuclei. In agreement with a report of the colocalization of SPir and ChATir in the PPT and TLD nuclei 29we have found that a subpopulation of these neurons also contain SPir. Studies employing immunohistochemistry in combination with retrograde tracing 13'25 and lesions 19'21 have identified ChATir and SPir projections from neurons in the PPT and T L D nuclei to the thalamus and cortex. In addition, PPT has descending projections to the medullary reticular formation 2°. However, no APir fibers that are likely to represent projections of these neurons have been observed in these a r e a s 10,22,24,27. Thus it appears that although the peri-
166
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\ Fig. 2. Diagrams of coronal sections showing the distribution of APir, SPir, and ChATir neurons in the PPT and TLD. Open circles indicate neurons that were APir only, open triangles indicate SPir neurons, and filled circles indicate neurons stained for both APir and SPir.
karya of neurons in the PPT and T L D nuclei are APir in colchicine-treated animals, the A P immunoreactive material is not present in the thalamic, cortical or reticular terminals of these neurons in either normal or colchicine-treated animals in quantities that can be detected by present methods. The ChATir neurons of PPT and T L D also project to the interpeduncular nucleus 3,14,3°. Immunohistochemical staining for APir reveals a dense bundle of fibers emerging from the medial edge of the T L D nucleus and sweeping between the medial longitudinal
fasciculi, to form prominent APir terminal fields in the dorsal and lateral subnuclei of the interpeduncular nucleus 1°,22,24,27. These same subnuclei contain both SPir and C h A T terminal fields and stain intensely for acetylcholinesterase 5,7,8,19,3°. In a preliminary series of experiments, we have made injections of fluorescent retrograde tracers into the interpeduncular nucleus and observed retrograde labelling of APir neurons in both the PPT and T L D nuclei. We think it is likely that the primary APir projection of many of the neurons of the PPT and T L D nuclei is to the interpe-
167 duncular nucleus, and that these same n e u r o n s also
McKnight Scholar Award. This work was supported
supply C h A T i r and probably SPir i n n e r v a t i o n to this
by U S P H S Grants NS18669, NS22835, NS17661,
structure.
HD0453 and NS00631, N I G M S Grants GM07200, GM07281 and GM07805, a G r a n t - i n - A i d from the
The authors would like to thank Miss Q u a n H u e Ha and Mr. Joseph Hayes for technical and photographic assistance. C.B.S.
AB8 AP AP 11 BAR ChAT CUN DR DT IC icp ir LL mV mcp me5 mlf o
American Heart Association 850894, and the Monsanto Co.
is the recipient of a
ChAT antibody atriopeptin (-like) rabbit antiserum to AP Barrington's nucleus choline acetyltransferase cuneiform nucleus dorsal raphe nucleus dorsal tegmental nucleus inferior colliculus inferior cerebellar peduncle immunoreactivity lateral lemniscus motor nucleus of the trigeminal nerve middle cerebellar peduncle mesencephalic nucleus of the trigeminal nerve medial longitudinal fasciculus nucleus 'o'
1 Armstrong, D.M., Saper, C.B., Levey, A.I., Wainer, B.H. and Terry, R.D., Distribution of cholinergic neurons in the rat brain: demonstrated by the immunocytochemical localization of choline acetyltransferase, J. Comp. Neurol., 216 (1983) 53-68. 2 Berod, A., Hartman, B.K. and Pujol, J.F., Importance of fixation in immunocytochemistry: use of formaldehyde solutions at variable pH for the localization of tyrosine hydroxylase, J. Histochem. Cytochem., 29 (1981)844-850. 3 Contestabile, A. and Flumerfelt, B.A., Afferent connections of the interpeduncular nucleus and topographic organization of the habenulo-interpeduncularpathway: an HRP study in the rat, J. Comp. Neurol., 196 (1981) 253-270. 4 Cuello, A.C., Detection of substance P in the central nervous system by a monoclonal antibody, Proc. Natl. Acad. Sci. U.S.A., 76 (1979) 3532-3536. 5 Fass, B. and Hamill, G.S., The subnuclear organization of acetylcholinesterase-containing neurons in the interpeduncular nucleus of the rat, Brain Res. Bull. 13 (1984) 503-508. 6 Geller, D.M., Currie, M.G., Seigel, N.R., Fok, K.F., Adams, S.P. and Needleman, P., The sequence of an atriopeptigen: a precursor of the bioactive atrial peptide, Biochem. Biophys. Res. Commun., 121 (1984) 802-807. 7 Hamill, G.S., Olschowka, J.A., Lenn, N.J. and Jacobowitz, D.M., The subnuclear distribution of substance P, cholecystokinin, vasoactive intestinal peptide, somatostatin, leu-enkephalin, dopamine beta hydroxylase, and serotonin in the rat interpeduncular nucleus, J. Comp. Neurol., 226 (1984) 580-596. 8 Hemmingdinger, L.M. and Moore, R.Y., Interpeduncular nucleus organization in the rat: cytoarchitecture and histo-
PBI
PBm PBS PBS-G PPT,PP scp SNV SP TLD V
lateral part of the parabrachial nucleus c - - central subnucleus d - - dorsal subnucleus el - - extreme lateral subnucleus i - - internal subnucleus k f - - Kolliker-Fuse nucleus v - - ventral subnucleus medial part of the parabrachial nucleus phosphate-buffered saline PBS with 3% goat serum pedunculopontine tegmental nucleus superior cerebellar peduncle spinal nucleus of the trigeminal nerve substance P laterodorsal tegmental nucleus trigeminal nerve
chemical analysis, Brain Res. Bull., 13 (1984) 163-179. 9 Jacobsohn, L., [fiber die Kerne des Menschlichen Hirnstamms: (Medulla Oblongata, Pons, und Pedunculus Cerebri). K6nigliche Akademie von Wissenschaften, Berlin, 1909. 10 Kawata, M., Nakao, K., Morri, N., Kiso, Y., Yamashita, H., Imura, H. and Sano, Y., Atrial natriuretic polypeptide: topographical distribution in the rat brain by radioimmunoassay and immunohistochemistry, Neuroscience, 16 (1985) 521-546. 11 Levey, A.I., Armstrong, D.M., Atweh, S.F., Terry, R.D. and Wainer, B.H., Monoclonal antibodies to choline acetyltransferase: production, specificity, and immunohistochemistry, J. Neurosci., 3 (1983) 1-9. 12 Loewy, A.D., Saper, C.B. and Baker, R.P., Descending projections from the pontine micturition center, Brain Research, 172 (1979) 533-538. 13 Mesulam, M.M., Mufson, E.J., Wainer, B.H. and Levey, A.I., Central cholinergic pathways in the rat: an overview based on an alternative nomenclature (Chl-Ch6), Neuroscience, 10 (1983) 1185-1201. 14 Marchand, E.R., Riley, J.N. and Moore, R.Y., Interpeduncular nucleus afferents in the rat, Brain Research, 193 (1980) 339-352. 15 Needleman, P., Adams, S.P., Cole, B.R., Currie, M.G., GeUer, D.M., Michner, M.L., Saper, C.B., Schwartz, D. and Standaert, D.G., Atriopeptins: potential cardiac hormones, Hypertension, 7 (1985) 469-482. 16 Olszewski, J. and Baxter, D., Cytoarchitecture of the Human Brainstem, Karger, Basel, 1954. 17 Renfroe, J.B., Cbronister, R.B., Haycock, J.W. and Waymire, J.C., The localization of tyrosine hydroxylase-like
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18
19
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22
23
immunoreactivity in the central nervous system: methodological considerations, Brain Res. Bull., 13 (1984) 109-126. Rye, D.B., Saper, C.B. and Wainer, B.H., Cytoarchitectonic/cytochemical features of the nucleus tegmenti pedunculo-pontis (NTPP) in the rat are distinct from "extrapyramidal' projections to the mesopontine tegmentum, Soc. Neurosci. Abstr., 11 (1985) 1024. Rotter, A. and Jacobowitz, D.M., Neurochemical identification of cholinergic forebrain projection sites of the nucleus tegmentalis dorsalis lateralis, Brain Res. Bull.. 6 (1981) 525-529. Rye, D.B., Lee, H., Ronnett, B. and Wainer, B.H., Descending cholinergic projections from the mesopontine tegmentum, Soc. Neurosci. Abstr., 10 (1984) 900. Sakanaka, M., Shiosaka, S., Takatsuki, K. and Tohyama, M., Evidence for the existence of a substance P-containing pathway from the nucleus laterodorsalis tegmenti (Castaldi) to the medial frontal cortex in the rat, Brain Research, 259 (1983) 123-126. Saper, C.B., Standaert, D.G., Currie, M.G., Geller, D.M. and Needleman, P., Atriopeptin immunoreactive neurons in the brain: presence in cardiovascular regulatory areas, Science, 227 (1985) 1047-1049. Satoh, K., Shimizu, N., Tohyama, M. and Maeda, T., Localization of the micturition reflex center at the dorsolateral pontine tegmentum of the rat, Neurosci. Len., 8 (1978) 27-33.
24 Skofitsch, G., Jacobowitz, D.M., Eskay, R.L. and Zamir, N., Distribution of atrial natriuretic factor-like immunoreactive neurons in the rat brain, Neuroscience. 16 (1985) 917-948. 25 Sonfroniew, M.V., Priestly, J.V., Consolazione, A., Eckenstein, F. and Cuello, A.C., Cholinergic projections from the midbrain and pons to the thalamus in the rat, identified by combined retrograde tracing and choline acetyltransferase immunocytochemistry, Brain Research, 329 (1985) 213-223. 26 Standaert, D.G., Saper, C.B. and Needleman. P., Atriopeptins: potent hormones and potential neuromediators, Trends Neurosci., 8 (1985) 509-511. 27 Standaert, D.G., Needleman, P. and Saper, C.B., Organization of atriopeptin immunoreactive neurons in the central nervous system of the rat, J. Comp. Neurol., in press. 28 Tohyama, M., Satoh, K., Sakumoto, T., Kimoto, Y., Takahashi, Y., Yamamoto, K. and Itakura, T., Organization and projections of the neurons in the dorsal tegmental area of the rat, J. Hirnforsch., 19 (1978) 165-176. 29 Vincent, S.R., Satoh, K., Armstrong, D.M. and Fibiger, H.C., Substance P in the ascending cholinergic reticular system, Nature (London), 306 (1983) 688-691. 30 Woolf, N.L. and Butcher, L.L., Cholinergic systems in the rat brain. II. Projections to the interpeduncular nucleus, Brain Res. Bull., 14 (1985) 63-68.
163
Elsevier BRE 21762
Colocalization of atriopeptin-like immunoreactivity with choline acetyltransferaseand substance P-like immunoreactivity in the pedunculopontine and laterodorsal tegmental nuclei in the rat DAVID G. STANDAERT 1'2, CLIFFORD B. S A P E R 2-4'6, DAVID B. R Y E 3'5'6 and BRUCE H. W A I N E R 3'5'6
Departments of 1Pharmacology, 2Neurology and Anatomy, and Neurobiology, Washington University School of Medicine, Saint Louis, MO 63110 and Departments of 3Pharmacological and Physiological Sciences, 4Neurology, 5pathology, and 6The Brain Research Institute, University of Chicago, Chicago, IL 60637 ( U. S. A.) (Accepted May 13th, 1986)
Key words: atrial natriuretic peptide - - electrolyte homeostasis - - cardiovascular system - - interpeduncular nucleus
Atriopeptin, the atrial natriuretic peptide, is a circulating hormone that plays an important role in the regulation of fluid and electrolyte balance. Immunohistochemical studies have shown that large, multipolar atriopeptin-like immunoreactive (APir) neurons are present in areas of the midbrain corresponding to the large neurons of the pedunculopontine tegmental (PPT) and lateral dorsal tegmental (TLD) nuclei, all of which can be stained immunohistochemically for choline acetyltransferase-like immunoreactivity (ChATir). A subpopulation of these cholinergic PPT and TLD neurons are also known to contain substance P-like immunoreactivity (SPir). Using an immunofluorescent technique that allows simultaneous localization of two antigens, we have studied the relationship between APir, SPir and ChATir in the pontine tegmentum of the rat. We have found that (i) the large multipolar APir neurons of the pontine tegmentum are identical to the ChATir neurons of the PT and TLD nuclei and (ii) a subpopulation of the APir neurons in PPT and TLD neurons are also SPir. A t r i o p e p t i n , the atrial natriuretic p e p t i d e , is a circulating p e p t i d e h o r m o n e that plays a central role in the regulation of fluid and electrolyte homeostasis. Synthesized and stored in the atria of the heart, atriopeptin is released into the circulation in response to increases in intravascular v o l u m e and causes natriuresis, diuresis, and vasorelaxation (see ref. 15 for review). Several recent studies have shown that neurons containing A P i r are present within the brain of the rat. The m a j o r i t y of these A P i r neurons are found within two regions, the h y p o t h a l a m u s and the pontine t e g m e n t u m 1°,22,24,27. M a n y of the A P i r neurons in the h y p o t h a l a m u s are l o c a t e d within structures implicated in regulation of the cardiovascular system 22'26. In contrast, relatively little is k n o w n about the organization and function of the A P i r neurons in the pontine t e g m e n t a l region. Two clusters of A P i r n e u r o n s are found in the pontine t e g m e n t u m of the rat 22,24,27. Both are c o m p o s e d of large neurons with a distinctive multipolar mor-
phology. T h e m o r e rostral of the two groups begins at the caudal end of the substantia nigra and extends caudally in association with the fibers of the superior cerebellar peduncle. T h e m o r p h o l o g y and distribution of these A P i r neurons is strikingly similar to the neurons constituting the PPT, as defined cytoarchitectonically in both h u m a n s 9A6 and the rat 18. The m o r e caudal group of A P i r neurons is located in the ventrolateral part of the central gray m a t t e r , at the level of the opening of the cerebral aqueduct into the fourth ventricle. These a p p e a r to c o r r e s p o n d to the magnocellular part of the T L D , as described by T o h y a m a et al. 28. A l l of the neurons in both P P T and the magnocellular part of T L D can be stained immunohistochemically using antibodies to C h A T 1'13'1s. A subpopulation of these neurons is known to contain SPir as well as C h A T i r 29. In o r d e r to examine the relationship between C h A T i r , SPir and A P i r neurons in the pontine tegmental region, we have used an immuno-
Correspondence: C.B. Saper, Department of Pharmacological and Physiological Sciences, University of Chicago, 947 East 58 Street, Chicago, IL 60637, U.S.A. 0006-8993/86/$03.50 (~) 1986 Elsevier Science Publishers B .V. (Biomedical Division)
164 fluorescent staining technique that allows the simultaneous localization of two antigenic markers with two colored fiuorochromes. Nine male S p r a g u e - D a w l e y rats were p r e t r e a t e d with colchicine, 150 ~g in 10 ¢tl, injected stereotaxically into the lateral cerebral ventricle u n d e r chloral hydrate anesthesia. Two days later, the rats were reanesthetized with chloral hydrate and perfused through the heart with 300 ml of normal saline, followed by 300 ml of 4 % p a r a f o r m a l d e h y d e in saline buffered with 100 m M sodium p h o s p h a t e (PBS), p H 6.5, and then with 300 ml of 4 % p a r a f o r m a l d e h y d e in PBS, p H 8.5 (ref. 2). Brains were r e m o v e d immediately, m o u n t e d on the chuck of a freezing m i c r o t o m e , and 50-gm coronal sections were cut from the level of
the cerebral aqueduct to the genu of the facial nerve. These sections were washed for 1 h each in PBS, PBS with 0.25% Triton X-100, and PBS with 3% normal goat serum (PBS-G). The sections were then incubated overnight at 4 °C in P B S - G containing antiserum A P 11 (1:500) and a mouse monoclonal antibody to either substance P (Sera-lab, 1:2000) or C h A T (AB8, 1:5(1 dilution of an a m m o n i u m sulfate precipitate of h y b r i d o m a supernatant) j. The next day, the sections were rinsed in PBS, and incubated in PBS-G containing fluorescein isothiocyanate-conjugated goat anti-rabbit serum (Miles, 1:50), and tetramethylrhodamine isothiocyanate-conjugated goat antirat serum (Cappel, 1:50). A f t e r a final wash in PBS, the sections were m o u n t e d on gelatin coated slides,
Fig. 1. Photomicrographs of immunohistochemically stained neurons in the PPT. A: PPT neuron stained for APir is indicated by curved arrow, B: same section as A, stained for SP. Curved arrow indicates same neuron as in A; two additional SPir neurons which are not APir are also seen (open arrows). C: PPT neurons stained for APir; one is seen clearly (arrow), two others lie out of the plane of focus (arrowheads). D: same section as C, stained for ChATir. Arrow indicates neuron seen in C, while the two other neurons lie out of the plane of focus (arrowheads).
165 coverslipped with glycerin containing 10% 1 M Tris buffer, pH 8.5, and 2 mg/ml paraphenylene diamine 17. Staining was observed using a Leitz microscope with Ploem optics and the Leitz 12 and N2 filter systems. The location of labelled neurons was recorded using an X - Y plotter coupled to the stage of the microscope. AP 11 was raised in rabbits using a 79-amino acid cyanogen bromide fragment of the high-molecularweight precursor of atriopeptin as an antigen, as described previously 22. The production and specificity of the substance P antibody 4 and AB8 (ref. 11) have been described. Absorption experiments were performed to demonstrate the specificity of these antisera in this system. When AP 11 was combined with the AB8 preabsorption with the purified cyanogen bromide fragment of the atriopeptin precursor (5 pg/ml) 6 blocked APir staining without affecting ChATir staining. Omission of the ChAT antibody prevented ChATir staining, but did not affect APir staining. .Similarly, when the AP 11 was combined with the substance P antibody, preabsorption with either synthetic substance P (Sigma, 50 pg/ml) or the purified cyanogen bromide fragment, blocked all staining of the corresponding antigen and did not affect the staining of the other. In sections stained for ChATir and APir (Fig. 1) we observed that the large, multipolar APir neurons of the dorsolateral tegmentum corresponded exactly to the ChATir neurons of the PPT and TLD nuclei. The neurons of the PPT and T L D nuclei observed with this method were similar in morphology, number and distribution to previous descriptions 1'13,18. Rostrally, the large, multipolar perikarya of the PPT nucleus, labelled for both ChATir and APir, were observed just dorsal to the caudal end of the substantia nigra. Caudally, additional similarly labelled neurons were found more dorsally and medially, following the superior cerebellar peduncle up to the medial parabrachial nucleus. As the cerebral aqueduct opened out into the fourth ventricle, a second cluster of large, ChATir and APir neurons comprising the magnocellular part of TLD could be seen within the central gray matter. Although all of the neurons in the PP and TLD nuclei were stained for both APir and ChATir, other areas contained neurons labelled for only one of these substances. At the level of the locus coeruleus,
a distinct group of much smaller, more closely packed APir perikarya that were not ChATir were found in the central gray matter between the lateral edge of T L D and the medial edge of the locus coeruleus. These probably correspond to Barrington's nucleus 2s which is thought to be involved in reflex control of micturition 12'23. In contrast, ChATir perikarya that were not APir were observed in the brainstem within the motor nuclei of the cranial nerves. Material stained for APir and SPit revealed that some but not all of the APir perikarya in the PPT and TLD nuclei were also SPir. The relationship between SPir and APir was more complex than that of ChATir and APir and will be described by reference to a series of drawings (Fig. 2). At the level at which the cerebral aqueduct opens into the fourth ventricle (Fig. 2A), the neurons of both the PPT and TLD nuclei may be identified. At this level, a small minority of the APir neurons in both PPT and TLD are also SPir. Neurons which are SPir but not APir are scarse; a small cluster is present in the central gray matter at the dorsolateral tip of TLD. More caudally, at the caudal end of the inferior colliculus (Fig. 2B, C), PPT shifts medially and ventrally, so that its medial border merges with TLD. At this level, the majority of the APir neurons in PPT and T L D are also SPir. A few neurons that are SPir only are present; most of these are located ventral to the APir neurons. At the level of the caudal part of the parabrachial nucleus (Fig. 2D), SPir perikarya are rare. An additional cluster of smaller neurons that were APir and not SPir were observed in B A R 28. Our results demonstrate the identity of the large, multipolar APir neurons of the dorsolateral pontine tegmentum with the previously described ChATir neurons of the PPT and T L D nuclei. In agreement with a report of the colocalization of SPir and ChATir in the PPT and TLD nuclei 29we have found that a subpopulation of these neurons also contain SPir. Studies employing immunohistochemistry in combination with retrograde tracing 13'25 and lesions 19'21 have identified ChATir and SPir projections from neurons in the PPT and T L D nuclei to the thalamus and cortex. In addition, PPT has descending projections to the medullary reticular formation 2°. However, no APir fibers that are likely to represent projections of these neurons have been observed in these a r e a s 10,22,24,27. Thus it appears that although the peri-
166
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\ Fig. 2. Diagrams of coronal sections showing the distribution of APir, SPir, and ChATir neurons in the PPT and TLD. Open circles indicate neurons that were APir only, open triangles indicate SPir neurons, and filled circles indicate neurons stained for both APir and SPir.
karya of neurons in the PPT and T L D nuclei are APir in colchicine-treated animals, the A P immunoreactive material is not present in the thalamic, cortical or reticular terminals of these neurons in either normal or colchicine-treated animals in quantities that can be detected by present methods. The ChATir neurons of PPT and T L D also project to the interpeduncular nucleus 3,14,3°. Immunohistochemical staining for APir reveals a dense bundle of fibers emerging from the medial edge of the T L D nucleus and sweeping between the medial longitudinal
fasciculi, to form prominent APir terminal fields in the dorsal and lateral subnuclei of the interpeduncular nucleus 1°,22,24,27. These same subnuclei contain both SPir and C h A T terminal fields and stain intensely for acetylcholinesterase 5,7,8,19,3°. In a preliminary series of experiments, we have made injections of fluorescent retrograde tracers into the interpeduncular nucleus and observed retrograde labelling of APir neurons in both the PPT and T L D nuclei. We think it is likely that the primary APir projection of many of the neurons of the PPT and T L D nuclei is to the interpe-
167 duncular nucleus, and that these same n e u r o n s also
McKnight Scholar Award. This work was supported
supply C h A T i r and probably SPir i n n e r v a t i o n to this
by U S P H S Grants NS18669, NS22835, NS17661,
structure.
HD0453 and NS00631, N I G M S Grants GM07200, GM07281 and GM07805, a G r a n t - i n - A i d from the
The authors would like to thank Miss Q u a n H u e Ha and Mr. Joseph Hayes for technical and photographic assistance. C.B.S.
AB8 AP AP 11 BAR ChAT CUN DR DT IC icp ir LL mV mcp me5 mlf o
American Heart Association 850894, and the Monsanto Co.
is the recipient of a
ChAT antibody atriopeptin (-like) rabbit antiserum to AP Barrington's nucleus choline acetyltransferase cuneiform nucleus dorsal raphe nucleus dorsal tegmental nucleus inferior colliculus inferior cerebellar peduncle immunoreactivity lateral lemniscus motor nucleus of the trigeminal nerve middle cerebellar peduncle mesencephalic nucleus of the trigeminal nerve medial longitudinal fasciculus nucleus 'o'
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PBI
PBm PBS PBS-G PPT,PP scp SNV SP TLD V
lateral part of the parabrachial nucleus c - - central subnucleus d - - dorsal subnucleus el - - extreme lateral subnucleus i - - internal subnucleus k f - - Kolliker-Fuse nucleus v - - ventral subnucleus medial part of the parabrachial nucleus phosphate-buffered saline PBS with 3% goat serum pedunculopontine tegmental nucleus superior cerebellar peduncle spinal nucleus of the trigeminal nerve substance P laterodorsal tegmental nucleus trigeminal nerve
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