Brain Research, 581 (1992) 339-343 t~) 1992 Elsevier Science Publishers B.V. All fights reserved. 0006-8993/92/$05.00
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Localization and retention in vitro of fluorescently labeled aortic baroreceptor terminals on neurons from the nucleus tractus solitarius David Mendelowitz a, Mingyong Yang b, Michael C. Andresen b and Diana L. Kunze a ~Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX 77030 (USA) and bDepartment of Physiology and Biophysics, University of Texas Medical Branch Galveston, TX 77550 (USA) (Accepted 11 February 1992) Key words: Anterograde; Baroreflex; Cardiovascular; DiA; Dissociated; Horseradish peroxidase; Solitary tract; Tracer
The anterograde fluorescent tracer DiA was used to visualize baroreceptor fibers and synaptic terminals both in living and fixed tissue. Baroreceptor fibers labeled with DiA terminated as a dense synaptic field in the medial nucleus tractus solitarius (NTS), making synaptic contact on the soma, as well as processes of neurons that they innervated. A similar distribution and morphology was observed in baroreceptor fibers and terminals labeled with horseradish peroxidase. DiA also identified baroreceptor terminals and the neurons receiving these synaptic contacts in vitro. NTS neurons were dissociated from their surrounding tissue and identified by attached baroreceptor terminals that retained the fluorescent dye. These results will enable us to study the electrophysiological properties of dispersed neurons that receive identified baroreceptor synaptic terminals. In contrast to the vast body knowledge concerning baroreceptors, little is known of the distribution, morphology and electrophysiology of second order neurons in the medulla that receive baroreceptor synaptic activity. Fibers within the vagus and carotid sinus nerves terminate in the dorsal medulla, with the densest concentrations of terminals at the nucleus tractus solitarius (NTS) 4-6'15-18'30'33. However, these nerves contain gastrointestinal and/or respiratory sensory neurons in addition to baroreceptor fibers 3°. Surprisingly, only 5 anatomical studies 5,6,17,18,33 have examined the terminations of the aortic nerve, and in only 2 of these studies 5'33 did the aortic nerve exclusively contain baroreceptor fibers. Attempts to examine the electrophysiology of neurons that receive identified baroreceptor synaptic activity have met with limited success. In vivo electrophysiological experiments of neurons within the NTS are very difficult due to their small size and the mechanical instability of the dorsal medulla. In addition, monosynaptic activation from an isolated and identified population of baroreceptor fibers would be necessary to unequivocally establish their identity. To obtain greater stability and access, in more easily manipulated environments, neurons from the NTS have been studied using in vitro slice preparations or as dissociated n e u r o n s 1,3,7-9,19,24-26. However, the functional identity of neurons in these in vitro preparations is lost. The goal of this study was to identify the synaptic ter-
minals of aortic baroreceptors, and the neurons that receive baroreceptor synaptic contacts, with a technique that is compatible with electrophysiological studies. A new family of lipophilic, fluorescent anatomical tracers have been developed that can be used to identify and examine the morphology of living neurons 12. These nontoxic dyes are readily absorbed into the cell membrane of intact nerves, diffuse both in an anterograde and retrograde direction, do not leak into surrounding tissue, and can identify neurons for up to 2 years 12. In the present study, we used both horseradish peroxidase (HRP) and a fluorescent dye, D i A (4-(4-dihexadecylaminostyryl)-N-methylpyridinium iodine, Molecular Probes), to label the aortic depressor nerve of the rat. This nerve is unique in rats and rabbits since it contains only baroreceptor fibers 27'28. In the first part of this study baroreceptor terminals labeled with either H R P or D i A were found primarily in the medial NTS. In the second part of this study we demonstrate that neurons from this region can be dissociated from their surrounding tissue and identified by retention of attached fluorescent baroreceptor terminals. Therefore, unlike HRP, which can only be visualized in fixed and processed tissue, the fluorescent dye allows visual identification of living neurons that receive synaptic contact from aortic baroreceptors prior to electrophysiological experiments. To visualize baroreceptor terminals, both in fixed tissue and as synaptic boutons attached to dissociated NTS
Correspondence: D. Mendelowitz, Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX 77030, USA. Fax: (1) (713) 798-3475.
340 neurons, a two-stage procedure was used. In the first procedure, rats were anesthetized with pentobarbital (30-40 mg/kg) and the aortic nerve was identified where it joins the superior laryngeal nerve prior to entering the nodose ganglia 2'5'11'2°. The aortic nerve was freed from surrounding tissue for 3-4 mm and placed on a section of parafilm. After the aortic nerve was adequately isolated from other tissue, either the nerve was cut and a 20-40% solution of H R P was applied, or the nerve was left intact and crystals of DiA were placed in contact with the nerve. When H R P was used, the central end of the aortic depressor nerve was encased inside a piece of tubing that was plugged at the distal end. To insulate the aortic nerve and tracer the area was imbedded with fast hardening SilGel (Wacker-Chemie GMBH, Munich, Germany). After the insulating compound hardened visual examination indicated that the dye remained affixed to the nerve and did not inadvertently make contact with other tissue. The surgical area was closed and the animal was allowed to recover for 1-6 weeks. To determine the distribution and morphology of baroreceptor terminals in the medulla, animals were anesthetized with pentobarbital, sacrificed, and perfused with 4% paraformaldehyde. The medulla was removed and cut into 40-#m-thick horizontal sections with a vibratome. The tissue from animals labeled with DiA could be viewed without any additional processing using a microscope with appropriate fluorescent filters (Nikon filter B-2A). To visualize H R P the sections of tissue were processed with tetramethylbenzimide (TMB) as the substrate for H R P 23. Neurons from the NTS were dissociated with methods similar to that described previously7'8'19. In brief, animals were anesthetized with ether and sacrificed by cervical dislocation. The brains were quickly removed, placed in cold (5°C) buffer (in mM) 110 NaC1, 10 sodium succinate, 5 KC1, 0.2 CaC12, 5 MgC12, 15 glucose, 15 HEPES, p H 7.4, equilibrated with 100% 02) and mounted on a vibratome. Five hundred/~m horizontal sections, that included the NTS, were excised and placed in 10 ml of HEPES buffer (same as above) at 35°C containing trypsin (Sigma type XI, 4-5 mg) and DL a-aminoadipate (Sigma, 1 mg) for 50-70 min, after which they were removed and placed in HEPES buffer without enzyme. All solutions were agitated and oxygenated by bubbling with 100% 02. Neurons were dissociated by gently triturating the tissue with serially smaller pipettes until individual cells were freed from surrounding tissue. As described previously7, if the concentration of calcium in the bath solutions is kept at 0.2 raM, neurons are dissociated with attached and functional synaptic boutons. Electromicroscopic (EM) examination of neurons with this preparation demonstrate that the contacts that sur-
J
ST
\ I Iobeled~ \~) terminals mNTS C
Fig. 1. Camera lucida drawing of a horizontal section of the medulla showing HRP-labeled aortic baroreceptor fibers in the solitary tract. Terminal arborizations were densely localized to the medial region of the NTS, between 0.5 mm rostral and 2 mm caudal to the obex. ST, solitary tract; mNTS, medial region of the nucleus tractus solitarius; v, spinal tract of the trigeminal nerve; AP, area postrema; c, cuneate nucleus; g, gracile nucleus.
round these neurons are separated from the neurons by synaptic clefts and contain synaptic vesicles7. Dissociated neurons from the NTS with attached fluorescent baroreceptor terminals were visualized using a microscope with appropriate fluorescent filters. Aortic baroreceptor fibers that were labeled with H R P traveled within the solitary tract and terminated in the NTS. The densest labeling was found in the medial subnucleus between 0.5 mm rostral and 2 mm caudal to the obex, see Fig. 1. Some additional and sparser labeling was observed modestly (up to 200/~m) ventral to the tract, and in the interstitial regions. No fibers or baroreceptor terminals were found elsewhere in the medulla. No cell bodies were filled, confirming previous reports that the aortic depressor nerve in rats is comprised solely of afferent fibers. Unfortunately, only approximately 50% of the rats treated with H R P possessed labeled fibers and terminals. H R P labeling was undetectable in the remaining animals. The reason for this variability is unknown. Baroreceptor fibers that were labeled with DiA were also found in the solitary tract and terminated in the NTS, see Fig. 2A and B. Unlike the results with HRP, the labeling of baroreceptor fibers with DiA was successful in all animals. The arborization of fibers and terminal field of baroreceptor endings were localized to the
341
A
B
C
F Fig. 2. A series of photographs showing baroreceptor fibers and terminals labeled with the fluorescent tracer DiA. As illustrated in A and B, aortic baroreceptor fibers in the solitary tract terminate as a dense synaptic field in the medial region of the NTS, between 0.5 mm rostral and 2 mm caudal to the obex. With higher magnification, shown in C and D, the fluorescent label can be observed to form partial circles or rings that surround the soma of neurons. Identified synaptic contacts are retained when the neurons are dissociated from their surrounding tissue, as illustrated in the paired photographs in panels E and E The labeled terminals, identified with fluorescent exposure, contact the neurons that are shown with phase contrast illumination (below). Bar -- 200/zm in panels A and B, 100 pm in panel C, 20 pm in panel D, and 50 #m in panels E and E
342 medial region of the NTS. Examination of this area with greater magnification demonstrated that the label formed partial circles, or rings, that appeared to surround the cell bodies of neurons in the medial NTS, as shown in Fig. 2C and D. Another set of experiments were performed to examine whether DiA-labeled baroreceptor terminals could remain attached and be used to identify electrophysiologically viable neurons dissociated from the NTS. Neurons from the medial region of the NTS were dissociated from their surrounding tissue. As shown in Fig. 2E and F, the fluorescent baroreceptor terminals remained attached during the dissociation procedure. Neurons with attached baroreceptor terminals were bipolar, extending approximately 15-30 /~m in length and 10-15 ~m in width, typically with two processes extending from either end. Identified neurons closely resemble the medial NTS neurons used in previous studies in which synapticaUy evoked currents 7, voltage-dependent calcium 19, and excitatory amino acid evoked currents 7'8'26 were characterized. The results from this study demonstrate that aortic baroreceptor fibers terminate as a dense synaptic field localized to the medial region of the NTS with sparser labeling in the interstitial region. The distribution of baroreceptor terminals found in this study is in general agreement with results from the only other study of aortic baroreceptor terminals in the rat. Ciriello 5 found the densest labeling of aortic baroreceptor terminals were at the level of the obex in the medial and dorsolateral NTS, as well as the interstitial nucleus. In the rabbit, an animal in which the aortic nerve is also comprised only of baroreceptor fibers, aortic nerve terminals were localized medial and dorsal to the solitary tract 33. In species such as the cat 6'17 and pigeon 18 in which the aortic nerve contains chemoreceptor as well as baroreceptor fibers, aortic nerve terminals were mostly found in the medial, dorsal, and dorsolateral regions of the NTS. There are two novel and interesting main findings from this study concerning the labeling and distribution of baroreceptor terminals. (1) D i A was found to be more reliable and consistent than HRP, labeling fibers and terminals in all animals examined. Additionally, D i A labeled the arborization of
1 Andresen, M.C. and M. Yang. Non-NMDA receptors mediate sensory afferent synaptic transmission in medial nucleus tractus solitarius, Am. J. Physiol., 259 (1990) H1307-H1311. 2 Andrew, B.L., A laryngeal pathway for aortic baroreceptor impulses, J. Physiol., 125 (1954) 352-360. 3 Barnes, K.L., Knowles, W.D. and Ferrario, C.M., Angiotensin II and Angiotensin (1-7) excite neurons in the canine medulla in vitro, Brain Res. Bull., 24 (1990) 275-280. 4 Berger, A.J., Distribution of carotid sinus nerve afferent fibers
baroreceptor terminals more effectively than HRP. The reasons for this difference is unknown. One potentially significant factor is that the nerve remained intact in DiA-treated animals, whereas the nerve was sectioned when H R P was used. It-is possible that section of the nerve in the HRP-treated animals decreased intra-axonal transport, and/or changed the synaptic field of baroreceptor terminals in the NTS. Another possibility is that the intensity of the lipophilic DiA, but not HRP, may be greatest where there is turnover of the membrane by endocytosis, such as at synaptic terminals 12. (2) The results from this study demonstrate that baroreceptors make dense synaptic contacts on the soma, as well as on the processes, of the neurons that they innervate. A similar synaptic morphology has been observed in pulmonary slowly adapting stretch receptors that synaptically innervate the soma and dendrites of neurons in the ventral, ventrolateral and intermediate regions of the N T S 15'16. The major advantage of using fluorescent tracers such as D i A to identify neurons is that neurons can be subsequently studied in vitro using microelectrode or patch clamp electrophysiological techniques without loss of identification. The combination of in vitro electrophysiology and fluorescent tracing techniques has been used to study the ionic currents in the soma of identified aortic baroreceptors 1°'22, parasympathetic cardiac motorneurons 21, and neurons projecting to the spinal c o r d TM 14,29, visual cortex3~and tongue 32. In conclusion, this is the first study to~ demonstrate that the synaptic terminals of sensory neurons, and the cells that they innervate, can be identified with the fluorescent tracer DiA. Baroreceptor fibers terminate mainly as a dense synaptic field in the medial NTS. Neurons that receive baroreceptor synapses are bipolar neurons 15-30/~m in length and 10-15/~m in width. These neurons can be dissociated from their surrounding tissue and identified by attached and fluorescently labeled baroreceptor terminals.
This work was supported by grants from the National Heart, Lung and Blood Institute to D.L. Kunze (HL-36840) and M.C. Andresen (HL-41119). D. Mendelowitz is a recipient of a National Research Service Award (HL 08375).
to solitary tract nuclei of the cat using transganglionic transport of horseradish peroxidase, Neurosci. Lett., 14 (1979) 153-158. 5 Ciriello, J., Brainstem projections of aortic baroreceptor afferent fibers in the rat, Neurosci. Lett., 36 (1983) 37-42. 6 Ciriello, J., Hrycyshyn, A.W. and Calaresu, F.R., Horseradish peroxidase study of brain stem projections of carotid sinus and aortic depressor nerves in the cat, J. Auton. Nerv. Syst., 4 (1981) 43-61. 7 Drewe, J.A., Childs, G.V. and Kunze, D.L., Synaptic trans-
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mission between dissociated adult mammalian neurons and attached synaptic boutons, Science, 241 (1988) 1810-1813. Drewe, J.A., Miles, R. and Kunze, D.L., Excitatory amino acid receptors of guinea pig medial nucleus tractus solitarius neurons, Am. J. Physiol., 259 (1990) H 1389-H1395. Feldman, P.D. and Felder, R.B., Alpha-adrenergic influences on neuronal responses to visceral afferent input in the nucleus tractus solitarius, Neuropharrnaeology, 28 (1989) 1081-1087. Hajduczok, G. and Abboud, F.M., Identification and isolation of baroreceptor neurons in culture (abstract), Physiologist, 34 (1991) 240. Heymans, C. and Neil, E., Reflexogenic areas of the cardiovascular system, Churchill, London, 1958. Honig, M.G. and Hume R.I., Fluorescent carbocyanine dyes allow living neurons of identified origin to be studied in longterm cultures, J. Cell Biol., 103 (1986) 171-187. Huang, L.-Y.M., Electrical properties of acutely isolated identified rat spinal dorsal horn projection neurons, Neurosci. Lea., 82 (1987) 267-272. Huang, L.-Y.M., Calcium channels in isolated rat dorsal horn neurons including labelled spinothalamic and trigeminothalamic cells, J. Physiol., 411 (1989) 161-177. Kalia, M. and Richter, D., Morphology of physiologically identified slowly adapting lung stretch receptor afferents stained with intra-axonal horseradish peroxidase in the nucleus of the tractus solitarius of the cat. I. A light microscopic analysis, J. Comp. Neurol., 241 (1985) 503-520. Kalia, M. and Richter, D., Morphology of physiologically identified slowly adapting lung stretch receptor afferents stained with intra-axonal horseradish peroxidase in the nucleus of the tractus solitarius of the cat. II. An ultrastructure analysis, J. Comp. Neurol., 241 (1985) 521-535. Kalia, M. and Welles, R.V., Brain stem projections of the aortic nerve in the cat: a study using tetramethyl benzidine as the substrate for horseradish peroxidase, Brain Res., 188 (1980) 2332. Katz, D.M. and Karten, H.J., The discrete anatomical localization of vagal aortic afferents within a catecholamine-containing cell group in the nucleus solitarius, Brain Res., 171 (1979) 187195. Kunze, D.L., Calcium currents of cardiovascular neurons isolated from adult guinea pigs, Am. J. Physiol., 252 (1987) H867H871. McCubbin, J.W., Masson, G.M.C. and Page, I.H., Aortic depressor nerves of the rat, Arch. Int. Pharmacodyn., 154 (1958)
303-306. 21 Mendelowitz, D. and Kunze, D.L., Identification and dissociation of cardiovascular neurons from the medulla for patch clamp analysis, Neurosci. Lett., 132 (1991) 217-221. 22 Mendelowitz, D. and Kunze D.L., Characterization of calcium currents in aortic baroreceptor neurons, J. Neurophysiol., in press. 23 Mesulam, M.-M., Tetramethyl benzidine for horseradish peroxidase neurohistochemistry: a non-carcinogenic blue reaction product with superior sensitivity for visualizing neuronal afferents and efferents, J. Neurochem. Cytochem., 223 (1978) 106117. 24 Miles, R., Frequency dependence of synaptic transmission in nucleus of the solitary tract in vitro, J. Neurophysiol. 55 (1986) 1076-1090. 25 Miller, B.D. and Felder R.B., Excitatory amino acid receptors intrinsic to synaptic transmission in nucleus tractus solitarii, Brain Res., 456 (1988) 333-343. 26 Nakagawa, T., Shirasaki, T., Tateishi, M., Murase, K. and Akaike, N., Effects of Antagonists on N-methyl-o-aspartate response in acutely isolated nucleus tractus solitarii neurons of the rat, Neurosci. Lett., 113 (1990) 169-174. 27 Sapru, H.N and Krieger, A.J., Carotid and aortic chemoreceptor function in the rat, J. Appl. Physiol., 42 (1977) 344-348. 28 Sapru, H.N., Gonzalez, E. and Krieger, A.J. Aortic nerve stimulation in the rat: cardiovascular and respiratory responses, Brain Res. Bull., 6 (1981) 393-398. 29 Sun, M.-K., Young, B.S,, Kackett, J.H. and Guyenet, P.G., Reticulospinal pacemaker neurons of the rat dorsal ventrolateral medulla with putative sympathoexcitatory function: an intracellular study in vitro, Brain Res., 442 (1988) 229-239. 30 Spyer, K.M., Neural organization and control of the baroreceptor reflex, Rev. Physiol. Biochem. Pharmacol., 88 (1981) 23124. 31 Tanelian, D.L. and Maclver, M.B., Simultaneous visualization and electrophysiology of corneal A-delta and C fiber afferents, J. Neurosci. Methods, 32 (1990) 213-222. 32 Viana, E, Gibbs, L. and Berger A.J., Double- and triple-labeling of functionally characterized central neurons projecting to peripheral targets studied in vitro, Neuroscience, 38 (1990) 829841. 33 Wallach, J.H. and Loewy, A.D., Projections of the aortic nerve to the nucleus tractus solitarius in the rabbit, Brain Res., 188 (1980) 247-251.
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Localization and retention in vitro of fluorescently labeled aortic baroreceptor terminals on neurons from the nucleus tractus solitarius David Mendelowitz a, Mingyong Yang b, Michael C. Andresen b and Diana L. Kunze a ~Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX 77030 (USA) and bDepartment of Physiology and Biophysics, University of Texas Medical Branch Galveston, TX 77550 (USA) (Accepted 11 February 1992) Key words: Anterograde; Baroreflex; Cardiovascular; DiA; Dissociated; Horseradish peroxidase; Solitary tract; Tracer
The anterograde fluorescent tracer DiA was used to visualize baroreceptor fibers and synaptic terminals both in living and fixed tissue. Baroreceptor fibers labeled with DiA terminated as a dense synaptic field in the medial nucleus tractus solitarius (NTS), making synaptic contact on the soma, as well as processes of neurons that they innervated. A similar distribution and morphology was observed in baroreceptor fibers and terminals labeled with horseradish peroxidase. DiA also identified baroreceptor terminals and the neurons receiving these synaptic contacts in vitro. NTS neurons were dissociated from their surrounding tissue and identified by attached baroreceptor terminals that retained the fluorescent dye. These results will enable us to study the electrophysiological properties of dispersed neurons that receive identified baroreceptor synaptic terminals. In contrast to the vast body knowledge concerning baroreceptors, little is known of the distribution, morphology and electrophysiology of second order neurons in the medulla that receive baroreceptor synaptic activity. Fibers within the vagus and carotid sinus nerves terminate in the dorsal medulla, with the densest concentrations of terminals at the nucleus tractus solitarius (NTS) 4-6'15-18'30'33. However, these nerves contain gastrointestinal and/or respiratory sensory neurons in addition to baroreceptor fibers 3°. Surprisingly, only 5 anatomical studies 5,6,17,18,33 have examined the terminations of the aortic nerve, and in only 2 of these studies 5'33 did the aortic nerve exclusively contain baroreceptor fibers. Attempts to examine the electrophysiology of neurons that receive identified baroreceptor synaptic activity have met with limited success. In vivo electrophysiological experiments of neurons within the NTS are very difficult due to their small size and the mechanical instability of the dorsal medulla. In addition, monosynaptic activation from an isolated and identified population of baroreceptor fibers would be necessary to unequivocally establish their identity. To obtain greater stability and access, in more easily manipulated environments, neurons from the NTS have been studied using in vitro slice preparations or as dissociated n e u r o n s 1,3,7-9,19,24-26. However, the functional identity of neurons in these in vitro preparations is lost. The goal of this study was to identify the synaptic ter-
minals of aortic baroreceptors, and the neurons that receive baroreceptor synaptic contacts, with a technique that is compatible with electrophysiological studies. A new family of lipophilic, fluorescent anatomical tracers have been developed that can be used to identify and examine the morphology of living neurons 12. These nontoxic dyes are readily absorbed into the cell membrane of intact nerves, diffuse both in an anterograde and retrograde direction, do not leak into surrounding tissue, and can identify neurons for up to 2 years 12. In the present study, we used both horseradish peroxidase (HRP) and a fluorescent dye, D i A (4-(4-dihexadecylaminostyryl)-N-methylpyridinium iodine, Molecular Probes), to label the aortic depressor nerve of the rat. This nerve is unique in rats and rabbits since it contains only baroreceptor fibers 27'28. In the first part of this study baroreceptor terminals labeled with either H R P or D i A were found primarily in the medial NTS. In the second part of this study we demonstrate that neurons from this region can be dissociated from their surrounding tissue and identified by retention of attached fluorescent baroreceptor terminals. Therefore, unlike HRP, which can only be visualized in fixed and processed tissue, the fluorescent dye allows visual identification of living neurons that receive synaptic contact from aortic baroreceptors prior to electrophysiological experiments. To visualize baroreceptor terminals, both in fixed tissue and as synaptic boutons attached to dissociated NTS
Correspondence: D. Mendelowitz, Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX 77030, USA. Fax: (1) (713) 798-3475.
340 neurons, a two-stage procedure was used. In the first procedure, rats were anesthetized with pentobarbital (30-40 mg/kg) and the aortic nerve was identified where it joins the superior laryngeal nerve prior to entering the nodose ganglia 2'5'11'2°. The aortic nerve was freed from surrounding tissue for 3-4 mm and placed on a section of parafilm. After the aortic nerve was adequately isolated from other tissue, either the nerve was cut and a 20-40% solution of H R P was applied, or the nerve was left intact and crystals of DiA were placed in contact with the nerve. When H R P was used, the central end of the aortic depressor nerve was encased inside a piece of tubing that was plugged at the distal end. To insulate the aortic nerve and tracer the area was imbedded with fast hardening SilGel (Wacker-Chemie GMBH, Munich, Germany). After the insulating compound hardened visual examination indicated that the dye remained affixed to the nerve and did not inadvertently make contact with other tissue. The surgical area was closed and the animal was allowed to recover for 1-6 weeks. To determine the distribution and morphology of baroreceptor terminals in the medulla, animals were anesthetized with pentobarbital, sacrificed, and perfused with 4% paraformaldehyde. The medulla was removed and cut into 40-#m-thick horizontal sections with a vibratome. The tissue from animals labeled with DiA could be viewed without any additional processing using a microscope with appropriate fluorescent filters (Nikon filter B-2A). To visualize H R P the sections of tissue were processed with tetramethylbenzimide (TMB) as the substrate for H R P 23. Neurons from the NTS were dissociated with methods similar to that described previously7'8'19. In brief, animals were anesthetized with ether and sacrificed by cervical dislocation. The brains were quickly removed, placed in cold (5°C) buffer (in mM) 110 NaC1, 10 sodium succinate, 5 KC1, 0.2 CaC12, 5 MgC12, 15 glucose, 15 HEPES, p H 7.4, equilibrated with 100% 02) and mounted on a vibratome. Five hundred/~m horizontal sections, that included the NTS, were excised and placed in 10 ml of HEPES buffer (same as above) at 35°C containing trypsin (Sigma type XI, 4-5 mg) and DL a-aminoadipate (Sigma, 1 mg) for 50-70 min, after which they were removed and placed in HEPES buffer without enzyme. All solutions were agitated and oxygenated by bubbling with 100% 02. Neurons were dissociated by gently triturating the tissue with serially smaller pipettes until individual cells were freed from surrounding tissue. As described previously7, if the concentration of calcium in the bath solutions is kept at 0.2 raM, neurons are dissociated with attached and functional synaptic boutons. Electromicroscopic (EM) examination of neurons with this preparation demonstrate that the contacts that sur-
J
ST
\ I Iobeled~ \~) terminals mNTS C
Fig. 1. Camera lucida drawing of a horizontal section of the medulla showing HRP-labeled aortic baroreceptor fibers in the solitary tract. Terminal arborizations were densely localized to the medial region of the NTS, between 0.5 mm rostral and 2 mm caudal to the obex. ST, solitary tract; mNTS, medial region of the nucleus tractus solitarius; v, spinal tract of the trigeminal nerve; AP, area postrema; c, cuneate nucleus; g, gracile nucleus.
round these neurons are separated from the neurons by synaptic clefts and contain synaptic vesicles7. Dissociated neurons from the NTS with attached fluorescent baroreceptor terminals were visualized using a microscope with appropriate fluorescent filters. Aortic baroreceptor fibers that were labeled with H R P traveled within the solitary tract and terminated in the NTS. The densest labeling was found in the medial subnucleus between 0.5 mm rostral and 2 mm caudal to the obex, see Fig. 1. Some additional and sparser labeling was observed modestly (up to 200/~m) ventral to the tract, and in the interstitial regions. No fibers or baroreceptor terminals were found elsewhere in the medulla. No cell bodies were filled, confirming previous reports that the aortic depressor nerve in rats is comprised solely of afferent fibers. Unfortunately, only approximately 50% of the rats treated with H R P possessed labeled fibers and terminals. H R P labeling was undetectable in the remaining animals. The reason for this variability is unknown. Baroreceptor fibers that were labeled with DiA were also found in the solitary tract and terminated in the NTS, see Fig. 2A and B. Unlike the results with HRP, the labeling of baroreceptor fibers with DiA was successful in all animals. The arborization of fibers and terminal field of baroreceptor endings were localized to the
341
A
B
C
F Fig. 2. A series of photographs showing baroreceptor fibers and terminals labeled with the fluorescent tracer DiA. As illustrated in A and B, aortic baroreceptor fibers in the solitary tract terminate as a dense synaptic field in the medial region of the NTS, between 0.5 mm rostral and 2 mm caudal to the obex. With higher magnification, shown in C and D, the fluorescent label can be observed to form partial circles or rings that surround the soma of neurons. Identified synaptic contacts are retained when the neurons are dissociated from their surrounding tissue, as illustrated in the paired photographs in panels E and E The labeled terminals, identified with fluorescent exposure, contact the neurons that are shown with phase contrast illumination (below). Bar -- 200/zm in panels A and B, 100 pm in panel C, 20 pm in panel D, and 50 #m in panels E and E
342 medial region of the NTS. Examination of this area with greater magnification demonstrated that the label formed partial circles, or rings, that appeared to surround the cell bodies of neurons in the medial NTS, as shown in Fig. 2C and D. Another set of experiments were performed to examine whether DiA-labeled baroreceptor terminals could remain attached and be used to identify electrophysiologically viable neurons dissociated from the NTS. Neurons from the medial region of the NTS were dissociated from their surrounding tissue. As shown in Fig. 2E and F, the fluorescent baroreceptor terminals remained attached during the dissociation procedure. Neurons with attached baroreceptor terminals were bipolar, extending approximately 15-30 /~m in length and 10-15 ~m in width, typically with two processes extending from either end. Identified neurons closely resemble the medial NTS neurons used in previous studies in which synapticaUy evoked currents 7, voltage-dependent calcium 19, and excitatory amino acid evoked currents 7'8'26 were characterized. The results from this study demonstrate that aortic baroreceptor fibers terminate as a dense synaptic field localized to the medial region of the NTS with sparser labeling in the interstitial region. The distribution of baroreceptor terminals found in this study is in general agreement with results from the only other study of aortic baroreceptor terminals in the rat. Ciriello 5 found the densest labeling of aortic baroreceptor terminals were at the level of the obex in the medial and dorsolateral NTS, as well as the interstitial nucleus. In the rabbit, an animal in which the aortic nerve is also comprised only of baroreceptor fibers, aortic nerve terminals were localized medial and dorsal to the solitary tract 33. In species such as the cat 6'17 and pigeon 18 in which the aortic nerve contains chemoreceptor as well as baroreceptor fibers, aortic nerve terminals were mostly found in the medial, dorsal, and dorsolateral regions of the NTS. There are two novel and interesting main findings from this study concerning the labeling and distribution of baroreceptor terminals. (1) D i A was found to be more reliable and consistent than HRP, labeling fibers and terminals in all animals examined. Additionally, D i A labeled the arborization of
1 Andresen, M.C. and M. Yang. Non-NMDA receptors mediate sensory afferent synaptic transmission in medial nucleus tractus solitarius, Am. J. Physiol., 259 (1990) H1307-H1311. 2 Andrew, B.L., A laryngeal pathway for aortic baroreceptor impulses, J. Physiol., 125 (1954) 352-360. 3 Barnes, K.L., Knowles, W.D. and Ferrario, C.M., Angiotensin II and Angiotensin (1-7) excite neurons in the canine medulla in vitro, Brain Res. Bull., 24 (1990) 275-280. 4 Berger, A.J., Distribution of carotid sinus nerve afferent fibers
baroreceptor terminals more effectively than HRP. The reasons for this difference is unknown. One potentially significant factor is that the nerve remained intact in DiA-treated animals, whereas the nerve was sectioned when H R P was used. It-is possible that section of the nerve in the HRP-treated animals decreased intra-axonal transport, and/or changed the synaptic field of baroreceptor terminals in the NTS. Another possibility is that the intensity of the lipophilic DiA, but not HRP, may be greatest where there is turnover of the membrane by endocytosis, such as at synaptic terminals 12. (2) The results from this study demonstrate that baroreceptors make dense synaptic contacts on the soma, as well as on the processes, of the neurons that they innervate. A similar synaptic morphology has been observed in pulmonary slowly adapting stretch receptors that synaptically innervate the soma and dendrites of neurons in the ventral, ventrolateral and intermediate regions of the N T S 15'16. The major advantage of using fluorescent tracers such as D i A to identify neurons is that neurons can be subsequently studied in vitro using microelectrode or patch clamp electrophysiological techniques without loss of identification. The combination of in vitro electrophysiology and fluorescent tracing techniques has been used to study the ionic currents in the soma of identified aortic baroreceptors 1°'22, parasympathetic cardiac motorneurons 21, and neurons projecting to the spinal c o r d TM 14,29, visual cortex3~and tongue 32. In conclusion, this is the first study to~ demonstrate that the synaptic terminals of sensory neurons, and the cells that they innervate, can be identified with the fluorescent tracer DiA. Baroreceptor fibers terminate mainly as a dense synaptic field in the medial NTS. Neurons that receive baroreceptor synapses are bipolar neurons 15-30/~m in length and 10-15/~m in width. These neurons can be dissociated from their surrounding tissue and identified by attached and fluorescently labeled baroreceptor terminals.
This work was supported by grants from the National Heart, Lung and Blood Institute to D.L. Kunze (HL-36840) and M.C. Andresen (HL-41119). D. Mendelowitz is a recipient of a National Research Service Award (HL 08375).
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