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Bilateral projections of the pontine micturition center to the sacral parasympathetic nucleus in the rat
Brain Research 785 Ž1998. 185–194
Research report
Bilateral projections of the pontine micturition center to the sacral parasympathetic nucleus in the rat Sarah C. Nuding a , Irving Nadelhaft b
a,b,)
a Veterans Administration Medical Center, Bay Pines, FL 33744, USA Department of Pharmacology and Therapeutics, College of Medicine, UniÕersity of South Florida, Tampa, FL 33612, USA
Accepted 30 October 1997
Abstract Previous work has revealed that pontine micturition center ŽPMC. neurons send projections to the sacral parasympathetic nucleus ŽSPN. of the intermediolateral ŽIML. regions of L6-S1 spinal cord segments in rats. Although unilateral SPN injections will retrogradely label PMC neurons bilaterally, it is not known whether single PMC neurons project bilaterally to the SPN. There may be two different populations of PMC neurons on each side of the brainstem, with both groups independently connecting to the SPNs on opposite sides of the spinal cord. To verify one of these alternatives, a small injection of either rhodamine-labeled latex microspheres or a red fluorescent emulsion was made into the SPN on one side of the cord; a similar injection of either fluorescein-tagged microspheres or a green fluorescent emulsion was made into the other. After at least seven days, the rats were perfused. Inspection of 40 m m cord sections confirmed the similar placement of these injections along the rostrocaudal axis of the cord and that no tracer had spread across midline. Thirty-micron brain sections were examined for filled neurons. Red, green and double labeled neurons were found bilaterally in the PMC, subcoeruleus, and A5 regions. Although some red nucleus cells were also filled, they were only singly labeled and always located contralateral to the injection. Finally, immunohistochemical staining of dopamine-b-hydroxylase ŽDBH. containing cells confirmed that some labeled cells were also noradrenergic. We therefore conclude that some PMC, subcoeruleus, and A5 neurons send axons to the SPN on both sides of the lumbosacral cord. q 1998 Elsevier Science B.V. Keywords: Neuroanatomy; Spinal cord; Autonomic; Parasympathetic; Pontine micturition center
1. Introduction The micturition process involves complex pathways of afferent and efferent connections between the urinary bladder, urethral sphincter, lumbosacral cord, brainstem, and more rostral central nervous system ŽCNS. areas. One technique used in our laboratory to study this network of pathways has been to infect selected organs with pseudorabies virus ŽPRV. Žfor review, see Ref. w12x.. After injections of PRV into the bladder, the sequence of areas with infected cells outlines the temporal spread of the virus across functional synapses and delineates the neural pathways involving the bladder w21,22x. The virus is first transported from the site of infection in the bladder to postganglionic neurons in the major pelvic ganglia where it replicates. It is then exported to and transported within
) Corresponding author. Research and Development Service, VA Medical Center, Bay Pines, FL 33744, USA. Fax: q1-813-398-9467; E-mail: [email protected]
0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 1 3 4 7 - 4
the axons of the pelvic nerves to preganglionic neurons in the sacral parasympathetic nucleus ŽSPN. in the intermediolateral ŽIML. regions of L6-S1 spinal cord segments. The replicationrexport process is repeated in each infected neuron and, at longer postinfection times, the virus infects selected groups of neurons at progressively more rostral brain centers w22x. These include the nucleus of the solitary tract, raphe´ pallidus and obscurus, gigantocellular nucleus, locus coeruleus, subcoeruleus, periaquaductal gray, red nucleus, and especially, the pontine micturition center ŽPMC., originally described by Barrington w3x. Higher brain centers including the paraventricular region, the medial preoptic area and the cortex are also infected, but at times later than those of brainstem regions w22x. Following the injection of PRV into the bladder, all the areas exhibiting viral infected neurons are bilaterally symmetric. The bilateral nature of the PMC infection is maintained even if one pelvic nerve and both hypogastric nerves are transected before the initial PRV injection. This transection effectively restricts viral transfer to the CNS via the one remaining intact pelvic nerve w20x, as viral
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transmission to other areas via the blood stream has been ruled out ŽCard, personal unpublished communication.. This suggests that neurons in one SPN are contacted by neurons from both PMCs. Other experiments in our laboratory have established a monosynaptic connection between one SPN of the L6-S1 spinal cord segments and both PMCs by using retrograde transport of fluorescent microspheres w9,10x. After small Ž20–60 nl. unilateral injections into the SPN, equal numbers of neurons were labeled in the PMCs, suggesting that individual PMC neurons might have direct projections to both SPNs w19x. Electrophysiological stimulation of the PMC on one side of the brainstem induced firing of the contralateral, as well as the ipsilateral, bladder postganglionic nerves w23x, demonstrating a connection from the PMC to both sides of the bladder. Kuru and Yamamoto w11x also showed early evidence of a bilateral projection from Barrington’s nucleus when they noted bilateral degeneration of axons descending to the IML of the lumbosacral division after lesions in the dorsolateral pons of cats. More recently, after unilateral injections of biotinylated dextran amine ŽBDA. into the PMC and the adjacent ventral region, anterogradely labeled cells were found bilaterally in the SPN, suggesting that individual PMC neurons might have direct projections to both SPNs w5x. In addition, unilateral injections of the cholera toxin B subunit w5x into the IML and dorsal horn of rat L6-S1 cord segments retrogradely labeled cells bilaterally in the pontine tegmental area, either clustered in the area of the PMC or scattered ventral to it, again suggesting that the SPN receives input from this area on both sides of the pons. The results of all of these experiments can be explained by two possible hypotheses. There may be individual PMC neurons sending processes to both sides of the L6-S1 cord. Alternatively, there may be two different populations of PMC neurons on each side of the brainstem. Both groups might independently connect to the SPNs on opposite sides of the spinal cord, with no single cell projecting to both sides. The goal of the current experiments was to examine the connections between the PMC and the sacral parasympathetic nuclei ŽSPN. to determine if bilaterally projecting PMC neurons exist or if there are two groups of PMC neurons one of which projects to one SPN and the other to the contralateral SPN. To accomplish this aim, two different colors of either dye-labeled fluorescent latex microspheres w9,10x or fluorescent emulsions w6x were injected into the SPNs. A preliminary report of these results has been presented in abstract form w24x. 2. Materials and methods 2.1. Animal preparation The care and use of animals was approved by the Institutional Animal Care and Use Committee of the Bay
Pines Veterans Administration Medical Center. Adult Sprague–Dawley rats ŽHarlan. weighing between 230 and 300 g were anesthetized with pentobarbital Ž50 mgrkg i.p.. and body temperature was maintained at about 378C by an Aquamatic Heat Therapy unit ŽAmerican Pharmaseal.. A midline slit was made through the skin overlying the spinal column and muscle was detached from the vertebral processes until the L6-S1 vertebral interface was established. Using this bony landmark, the approximate location of the L6-S1 spinal cord segments was determined Ž; 2 mm rostral to the L1 and L2 vertebral boundary. and the surface of the cord exposed. After the dura was slit open, a small amount of 2% lidocaine was dripped on the surface to prohibit sudden movement during subsequent manipulations of the cord and spinal roots. Using a fire-polished glass pipette, any roots overlying the injection areas were gently pushed aside and then a 29-gauge needle was used to scratch superficial tears at the intended injection sites, about 0.5 mm lateral to midline. Without this last step, pipettes would not easily penetrate the cord surface. At this point, the animal was placed in a headholder and then suspended from a stereotaxic frame ŽKopf. by means of a clamp gripping the L2 and L3 spinous processes. In addition, the rat’s tail was draped over and then taped directly to a bar which connected both sides of the stereotaxic frame. This method of steadying the animal helped to reduce spinal movement due to breathing during the injection procedure. Lastly, the height and angle of the tail lift was adjusted so that the dorsal surface of the exposed cord was roughly parallel to the horizontal plane. 2.2. Injections A glass micropipette Žbumped to O.D.; 35 m m; I.D.; 14 m m. was coupled to both a micrometer syringe ŽGilmont. and an air pressure injection system Žmodified from Ref. w1x. via a two-way stopcock and polyvinylchloride tubing ŽTygon. and then clamped to the stereotaxic frame. The pipette was lowered into a 2-m l drop of a solution of either latex microspheres Žrhodamine-labeled or fluorescein-tagged, average diameter 0.050 m m and hereinafter referred to as beads, Lumafluor. or Fluoro-dye ŽFluoro-Red: excitation peak: 540 nm, emission peak: 585 nm; Fluoro-Green: excitation peak: 450 nm, emission peak: 505 nm; Tompow Pencil; see Ref. w6x.. Back suction applied via the syringe slowly filled the tip, shank, and some of the barrel of the micropipette. Once the pipette was sufficiently filled, the stopcock was connected to the injection system and the micropipette moved into position over one of the injection sites on the cord surface. The pipette penetrated the dorsal surface and advanced at an angle perpendicular to the plane defined by the stereotaxic frame and was driven into the cord to a depth of about 1.0 mm. After a short delay to settle the pipette in the tissue Ž1 min., the pipette was slowly withdrawn 0.3 mm. After
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another variable delay Ž1 to 5 min., the injection procedure began. The air pressure system, under stimulator control ŽGrass., delivered short duration pulses of nitrogen to the pipette, thereby effectively ejecting measured amounts of tracer w1x. ŽPrevious calibration of pressure, duration, and micropipette size parameters allowed for precise injections: w40 psi, 80 msx for 20 nl; w40 psi, 150 msx for 35 nl.. During each pulse, observation of the meniscus of fluid in
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the pipette barrel confirmed tracer ejection. If the meniscus did not move, even after repeated pulses, pipette blockage was assumed and a new micropipette was then filled and lowered into approximately the same location. After two or three successful pulses, final injection sizes were 40 to 90 nl in size. Once the injection was complete, the pipette was left in place for at least 30 min to lessen the amount of bead solution suctioned back up the electrode track during micropipette withdrawal. After slowly removing and dis-
Fig. 1. Examples of injections into L6-S1 cord segments. For these two experiments, fluorescein-tagged beads were injected on the left side, rhodamine-labeled beads on the right. A and C are fluorescent photomicrographs of two 40 m m sections showing injection locations for two experiments, with the solid white area demarcating the heaviest injection region. The filter set combination used allowed both injections to be visible simultaneously Žexcitation: BP 450–490; FT 510; emission: LP 520.. The plots of B and D show the extent of the injections over several contiguous sections for these experiments, with the center of each injection denoted by a region of solid color and cross-hatched areas outlining the full extent of the injection. Dorsal is to the top; plot scale bars are 500 m m. A and C: R1339. B and D: R1422.
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carding the micropipette following this injection into the SPN on one side of the cord, a similar injection was made into the other side, using the second fluorescent tracer. Upon completion of both injections, the wound was sutured closed and the skin over the tissue was stapled together. No gel foam or other material was left in place as this was observed to cause spinal tissue damage upon later dissection Žnote Fig. 1B.. 2.3. Histology Following at least a seven-day survival period, the deeply anesthetized animal Žpentobarbital w75 mgrkg i.p.x. was first injected through the heart with 100 units of Heparin, then perfused with a Krebs wash solution followed by a 4% paraformaldehyde fixative solution. The spinal cord Žsegments T13-S2. was then dissected and the L6-S1 region isolated and stored in fixative at 48C at least overnight, before being transferred to a solution of 25% sucrose at 48C. The brain was also removed and placed in fixative at 48C for a period of at least four days, before immersion in the 25% sucrose solution at 48C. Once the tissue had sunk, both samples were ready for sectioning. Forty-micron Žcord. or 30-m m Žbrain. transverse cryostat sections were collected in 0.1 M phosphate buffer and stored at 48C. For the cord, all tissue was mounted on gel-coated slides in order to identify the location and the extent of each injection. For the brain tissue, selected sections were mounted, dried, and then coverslipped with Krystalon ŽHarleco.. All slides were also stored at 48C to preserve bead or fluoro-dye longevity. Some sections were also processed by fluorescent immunohistochemistry to locate bead-filled cells which were also adrenergic by using an antiserum to dopamine-b-hydroxylase ŽDBH., the final enzyme used in noradrenergic production. The 30-mm sections were exposed for about 20 h at 48C to rabbit anti-DBH wEugene Tech Internationalx Ž1r1000 dilution in 1% normal donkey serum. followed by either donkey anti-rabbit fluorescein isothiocyanate ŽFITC. or tetramethyl rhodamine isothiocyanate ŽTRITC. at dilutions of 1r50 for 1 h at room temperature ŽJackson ImmunoResearch Laboratories.. Tissues were then mounted and coverslipped with Krystalon ŽHarleco. before storage at 48C. For each animal used in the analysis, inspection of 40
m m cord sections confirmed that the approximate center of each injection was in about the same rostrocaudal location; usually the core of each injection was offset by only a section or two Ž; 50 mm.. In addition, the injections had to include most, if not all, of the area occupied by the cells of the sacral parasympathetic nucleus within the intermediolateral cell column region. Finally, individual injections in each rat could not encroach upon the midline or the central canal. As individual preganglionic neurons within the rat IML do not extend their dendritic processes across the midline w18x, this stipulation prevented double labeling by inadvertent contralateral spread of the injection. Thirty-micron brain sections were examined under oil using a 40 = Planapochromat lens using a Zeiss standard microscope equipped for epifluorescence using Zeiss filter combinations to allow viewing either rhodaminerred emulsion- Žexcitation: BP 546r12; FT 580; emission: LP 590. andror fluoresceinrgreen emulsion- Žexcitation: BP 450–490; FT 510; emission: BP 520–560. filled neurons. Data was plotted using a computer-assisted mapping system ŽMDPlot, Minnesota Datametrics. andror photographed with either Kodak Tmax 400 Žfor black and white. or Ektachrome 400 = Žfor color. film. The general locations of filled cells were determined by comparing the tissue section with the compact rat brain atlas of Paxinos and Watson w26x.
3. Results Photographic examples and plots of sections illustrating successful fluorescent injections into the IMLs of both sides of the L6-S1 cord are shown in Fig. 1. The photograph of Fig. 1A shows an injection of fluorescein-tagged microspheres into the left side of the cord with the solid white area demarcating the heaviest injection region ŽR1339.. Fig. 1B is a depiction of the extent of this and the rhodamine-tagged microsphere injection over several contiguous sections Žleft: fluorescein; right: rhodamine.. For each injection, the center is denoted by a region of solid color; cross-hatched areas outline the full extent of the injection. Although the section shown in Fig. 1A and B is slightly elongated in the mediolateral dimension due to incidental dorsoventral compression of the cord, note that the IML regions were included in the injection areas of
Fig. 2. Photomicrographs of labeled cells located in different brainstem regions and a section redrawn from Paxinos and Watson w26x for reference. All paired photographs were taken from identical locations, only fluorescent filter set was changed. ŽA. Fluorescein-labeled bead-filled cell which was lying in the dorsal most extent of the PMC, medial to the locus coeruleus ŽLC.. Note the bright spheres of light which fill the cell and even some of its processes; single beads are clearly identifiable by their brilliance and individual diffraction patterns. ŽB. Two views of two adjacent PMC cells. ŽC. Cell located near the region containing A5 cells, among the streaming fibers of the rubrospinal tract, contained both rhodamine- and fluorescein-tagged microspheres. ŽD. Cell double-labeled with both of the fluorescent emulsions and located in the midline region in the raphe´ magnus ŽRMg.. ŽE and F. A small part of the PMC region after the injections outlined in Fig. 1D. Three double-labeled cells are marked by white arrows; several singly labeled filled cells are also apparent. ŽG. A pair of cells located in the ventral part of the subcoeruleus nucleus ŽsubCV.; note that although both cells were filled with fluorescein-tagged beads, only the more ventral of these cells is also labeled with rhodamine-tagged microspheres. All scale bars denote 25 m m. The brainstem section is adapted from Paxinos and Watson w26x; Bregma y9.68 mm.
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both sides and that there was no overlap between the injections. A photograph of a section and the corresponding plot showing tagged latex microsphere injections in another animal ŽR1422. are shown in Fig. 1C and D. The photographs of Fig. 1 were taken using a filter set combination which allowed both injections to be visible simultaneously Žexcitation: BP 450–490; FT 510; emission: LP 520.. Individually labeled cells, however, were
located using filter sets designed specifically for their absorption and emission spectra Žsee Section 2. so that rhodamine, fluorescein, and the individual fluorescent dye emissions would not overlap. Fig. 2 shows photographs of several filled cells located in several brainstem regions; a section redrawn from Paxinos and Watson w26x is included for reference ŽFig. 2F; Bregma y9.68 mm.. Fig. 2A is a close-up of a fluorescein-labeled bead-filled cell which
Fig. 3. Plots of a brainstem section and a section including the red nucleus showing labeled cells. Bead filled cells are denoted by open Žrhodamine. or filled Žfluorescein. circles; double-labeled cells are marked by asterisks. Filled squares are neurons from the mesencephalic trigeminal nucleus. ŽA. Solid-filled areas mark PMC regions; these areas were redrawn under higher magnification and are shown as insets. ŽB. Some neurons in the red nucleus were also filled, but they were always singly labeled by fluorescence and only from the contralateral injection. Dorsal is to the top; scale bars are 1 mm.
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was lying in the dorsal most extent of the PMC, medial to the locus coeruleus ŽLC.. Note the bright spheres of light which fill the cell and even some of its processes; single beads are clearly identifiable by their brilliance and individual diffraction patterns. Areas of unfocused green illumination result from beads located in planes above or below the plane of focus. This ‘glowing’ is noticeable in several photographs of bead-filled cells Že.g., Fig. 2C and E.. Once such a bead-filled cell was located, the filter set was switched to examine whether the cell contained material from both injections. For example, Fig. 2B shows two views of two adjacent PMC cells. Again, distinct bright beads are clearly discernible in both photographs, although the green beads in the cell on the right are slightly out of focus as they were out of the plane of focus of the photograph. During examination of the tissue, however, the microscope lens was raised and lowered to enable clear differentiation of bead from background fluorescence. Other examples of double filled cells are shown in Fig. 2C–E. The cell in Fig. 2C was located near the region containing A5 cells, among the streaming fibers of the rubrospinal tract, and contained both rhodamine- and fluorescein-tagged microspheres Žsee Fig. 2F; rs.. The cell in Fig. 2D was filled with both of the fluorescent emulsions and located in the midline region in the raphe´ magnus ŽRMg.. For the pair of cells illustrated in Fig. 2E, located in the ventral part of the subcoeruleus nucleus ŽsubCV., note that although both cells were filled with fluoresceintagged beads, only the more ventral of these cells is also labeled with rhodamine-tagged microspheres. Upon first examination of brainstem sections under low power Ž=6.3 or =25., the PMC regions light up with explosions of color, revealing label located both within cell bodies and their proximal processes Žfor example, see Fig. 2A.. Under oil at higher power, however, individual cells can be isolated and identified. For example, Fig. 2G consists of two identically positioned photographs of a small part of the PMC region after the injections outlined in Fig. 1D. Although four double-labeled cells in the two photographs are marked by white square symbols, several singly labeled filled cells are also apparent. Note that several beads overly one another within a 30-m m section and therefore many individual beads may not be in focus. Of all brain areas, this PMC region was the most populated by retrogradely labeled cells, both single- and double-labeled. Several other areas in the brainstem contained cells filled with either or both colors, including the subcoeruleus and A5 regions. In addition, small numbers of labeled cells were also located in the portion of the lateral parabrachial nucleus situated lateral to the superior cerebellar peduncle, as well as scattered within the raphe´ and pontine reticular fields. Fig. 3A shows a plot of one brainstem section demonstrating labeling in these areas after injections of rhodamine-tagged microspheres in the right IML and fluorescein-tagged beads in the left IML. In
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this plot, bead filled cells are denoted by open Žrhodamine. or filled Žfluorescein. circles; double-labeled cells are marked by asterisks. Because the PMC regions Žwithin the polygons. were so packed with filled cells, these areas were redrawn under higher magnification; these plots are shown as insets. These insets show red, green and doublelabeled PMC neurons located on both sides of the brain, with double- and single-labeled cells intermingled throughout the rostrocaudal and dorsoventral extent of the PMC. In addition to these cells in the brainstem, some neurons in the red nucleus were also filled, but they were always singly labeled by fluorescence and only from the contralateral injection ŽFig. 3B.. Some retrogradely labeled cells were located at the ventromedial edge of or just ventral to the locus coeruleus ŽLC.. In addition, some labeled cells were found in the ventrolateral pons, either lying among the dorsomedialventrolaterally oriented fibers of the rubrospinal tract or just dorsal to the lateral superior olive Žsee Fig. 2F.. These latter two groups of cells appear to be located within the regions defined by Byrum and Guyenet w4x as the lateral and dorsal subdivisions, respectively, of the A5 cell group. The placement of all these cells within known noradrenergic nuclei or cell regions, however, does not automatically prove that they contain noradrenaline. In order to demonstrate noradrenergic activity, some sections were examined for cells containing dopamine-b-hydroxylase ŽDBH., the final enzyme used in noradrenaline production. Note that by using an antibody to DBH, this procedure would label noradrenaline as well as adrenaline producing cells. As some adrenergic fibers terminate in the rat spinal cord, theoretically these fibers could take up injection material and also be labeled positive from DBH staining. Previous reports of adrenergic cells in the rat medulla, however, have shown them to be mainly limited to the C1 group or the dorsal motor nucleus of the vagus and to send projections primarily to thoracic cord levels, therefore, the possibility of mislabeling adrenergic cells as noradrenergic is limited w2,8,27x. In brainstem sections examined for noradrenaline containing cells, some neurons within the LC ŽFig. 4A. and A5 regions ŽFig. 4D. consistently displayed DBH positive reactions. In addition, some of these DBH positive cells were found to be retrogradely labeled by one or both fluorescent markers. For example, the DBH positive cell marked by an arrow in Fig. 4A is shown under higher magnification in the top frame of Fig. 4B to be also labeled with rhodamine-tagged microbeads. These microspheres are visible as small, bright points of light, but few are discernible in this photograph through the heavy TRITC labeling. This cell was filled with fluorescein-tagged microspheres as well, as shown in the lower frame of Fig. 4B. Some cells ventral to the LC in either the dorsal or ventral subdivisions of the subcoeruleus were also ‘triplelabeled’ in this way ŽFig. 4C., as were a few cells in the A5 lateral subdivision ŽFig. 4D–E.. Other cells in this A5
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region, although DBH positive, were retrogradely labeled by only one fluorescent marker ŽFig. 4F..
4. Discussion As mentioned in Section 1, there have been earlier demonstrations of the bilateral influence of a single pontine micturition center in the lumbosacral spinal cord or to the nerves innervating the urinary bladder. Although Nadelhaft and Vera w20x previously speculated on the existence of bilateral projections to sacral parasympathetic nuclei ŽSPN. from PMC regions, evidence of individual bilaterally projecting PMC cells was lacking. The results of the current experiments demonstrate, for the first time, that this influence resides in single neurons within the PMC, each of which sends projections to both SPNs in the lumbosacral spinal cord. In addition, these experiments have discovered neurons in other areas of the brainstem that also have bilateral projections to those same segments of the spinal cord. The largest number of brainstem neurons that projected to the sacral IML were found in the PMC. The PMC also contained the greatest number of individual neurons that had projections to both sides of the sacral cord. Because the L6-S1 intermediolateral region contains the sacral parasympathetic nucleus, whose neurons provide excitatory impulses to the urinary bladder, it is understandable that the major projection to that area should be from the PMC. Previous studies have shown that unilateral injections into the IML area of rat sacral cord segments retrogradely labeled PMC cells bilaterally we.g., Refs. w5,19xx, and several studies have demonstrated PMC projections to the sacral spinal cord, but bilateral projections have not been assigned to single PMC neurons. For example, after unilateral injections of anterograde tracers into the PMC, labeled fibers were first located ipsilateral to the site of injection, both in the tractus solitarius in the medulla w14x, and in the C1 segment w7x. But further caudally, labeled fibers were found bilaterally both in the lateral funiculi of segment T1 and at the sacral IML level w7,14x. Thus, the transition from ipsilateral to bilateral projection occurs between the medulla and the thoracic cord. Although we have demonstrated that some of the PMC neurons send projections to both sides of the sacral cord, this property may not apply to all PMC neurons. In fact, other investigators have reported that some PMC neurons
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send projections to the ipsilateral sacral cord and to the ipsilateral locus coeruleus w29x. Yet other researchers note that some PMC neurons send projections to the ipsilateral sacral cord and to the paraventricular thalamic nucleus w25x. All of these PMC neurons have at least one projection to the sacral intermediolateral spinal cord, but they may be divided into subsets based on the location of these second projections. It is conceivable that individual PMC neurons may project to even more than two distinct areas. This supports the idea that the PMC is a rather complex nucleus with multifunctional properties. Our results demonstrated that a small number of neurons in the ventrocaudal LC were labeled after sacral IML bead injections, although very few were double-labeled. These cells did not stand out from adjacent DBH positive cells which did not project to IML and resembled them in both morphology and orientation. This paucity of labeled LC neurons is not surprising given the lack of a predominant LC projection to the sacral spinal cord. Previous injections into the sacral IML have labeled few LC neurons, whereas injections into the lumbar IML have labeled many w7,19x, especially those neurons located in the caudal LC w15x. In addition, unilateral HRP injections into the LC demonstrated a majority of labeled fibers in the lumbar segments, with very few reaching the sacral cord w7x. All these results, plus the fact that bilateral lesions of the PMC resulted in difficulties in micturition, whereas bilateral lesions of the LC did not w28x support the contention that, in the rat, the LC is not involved with micturition. Aside from the PMC, the subcoeruleus and A5 adrenergic region had the largest numbers of bead-labeled neurons and some of these cells were also double-labeled. Although there have been previous reports of projections from the subcoeruleus to lumbosacral cord w5,16x, the terminal sites of spinally projecting A5 cells has been mainly restricted to areas above the lumbar enlargement, where fibers have been found in the intermediolateral regions of cervical and thoracic cord w4,13x. In the latter article, the authors point out that no fibers or terminals were observed in the sacral intermediolateral region. But other studies have reported quite different findings. Westlund et al. w31x noted that DBH positive terminals were especially prevalent in the superficial dorsal horn, ventral horn, and around the central canal and IML of thoracic and sacral cord segments. In fact, the noradrenergic endings in the sacral cord were concentrated in the area of the cells of origin of parasympathetic preganglionic
Fig. 4. Brainstem sections examined for noradrenaline-containing cells retrogradely labeled by one or both fluorescent markers. ŽA. DBH-positive reactions of locus coeruleus neurons. Cell marked by arrow is shown under higher magnification in ŽB.. Scale bar is 150 m m. ŽB. Photomicrographs showing magnification of cell in ŽA. retrogradely labeled with rhodamine Žtop. and fluorescein Žbottom.-tagged microspheres. Scale bar is 25 m m. ŽC. DBH-positive cell ventral to the LC in the subcoeruleus was retrogradely labeled with rhodamine Žtop. and fluorescein Žbottom.-tagged microspheres. Scale bar is 25 m m. ŽD. DBH-positive cells in A5 lateral subdivision region. Cell marked by arrow is shown under higher magnification in ŽE.. Scale bar is 100 m m. ŽE. Photomicrographs showing magnification of cell in ŽD. retrogradely labeled with rhodamine Žtop. and fluorescein Žbottom.-tagged microspheres. Scale bar is 25 m m. ŽF. Two DBH-positive cells in A5 lateral subdivision retrogradely labeled by only one fluorescent marker Žrhodamine.. Scale bar is 25 m m.
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fibers. In addition, after injections of DBH antibody into the sacral intermediolateral region, they report that a substantial number of retrogradely labeled cells were located within the A5 cell group. Finally, PRV injections into pelvic viscera such as the urinary bladder w22x, the urethra w30x, and the penis w17x, have all resulted in the labeling of neurons in the A5 area after only short incubation times. All this evidence suggests that it is likely that A5 neurons are retrogradely labeled directly from the sacral spinal cord.
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Acknowledgements We would like to express our appreciation of the excellent assistance provided by Mr. Gary A. Smith in conducting these experiments and in handling and processing the tissue for analysis. This work was supported by the Veterans Administration.
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References w1x D.G. Amaral, J.L. Price, An air pressure system for the injection of tracer substances into the brain, J. Neurosci. Meth. 9 Ž1983. 35–43. w2x D.M. Armstrong, C.A. Ross, V.M. Pickel, T.H. Joh, D.J. Reis, Distribution of dopamine-, noradrenaline-, and adrenaline-containing cell bodies in the rat medulla oblongata: demonstrated by the immunocytochemical localization of catecholamine biosynthetic enzymes, J. Comp. Neurol. 212 Ž1982. 173–187. w3x F.J.F. Barrington, Lesions of the hind- and mid-brain on micturition in the cat, Quant. J. Exp. Physiol. 15 Ž1925. 81–102. w4x C.E. Byrum, P.G. Guyenet, Afferent and efferent connections of the A5 noradrenergic cell group in the rat, J. Comp. Neurol. 261 Ž1987. 529–542. w5x Y.Q. Ding, M. Takada, H. Tokuno, N. Mizuno, Direct projections from the dorsolateral pontine tegmentum to pudendal motoneurons innervating the external urethral sphincter muscle in the rat, J. Comp. Neurol. 357 Ž1995. 318–330. w6x K. Dong, T. Qu, F.A.K.M. Ahmed, L. Zhang, K. Yamada, N.G. Guison, M. Miller, T. Yamadori, Fluoro-Green and Fluoro-Red: two new fluorescent retrograde tracers with a number of unique properties, Brain Res. 736 Ž1996. 61–67. w7x T. Hida, N. Shimizu, The interrelation between the latero–dorsal tegmental area and lumbosacral segments of rats as studied by HRP method, Arch. Histol. Jpn. 45 Ž1982. 495–504. w8x T. Hokfelt, K. Fuxe, M. Goldstein, O. Johansson, Immunohistochemical evidence for the existence of adrenaline neurons in the rat brain, Brain Res. 66 Ž1974. 235–251. w9x L.C. Katz, A. Burkhalter, W.J. Dreyer, Fluorescent latex microspheres as a retrograde neuronal marker for in vivo and in vitro studies of visual cortex, Nature 310 Ž1984. 498–500. w10x L.C. Katz, D.M. Iarovici, Green fluorescent latex microspheres: a new retrograde tracer, J. Neurosci. 34 Ž1990. 511–520. w11x M. Kuru, H. Yamamoto, Fiber connections of the pontine detrusor nucleus ŽBarrington., J. Comp. Neurol. 123 Ž1964. 161–186. w12x A.D. Loewy, Pseudorabies virus: a transneuronal tracer for neuroanatomical studies. In: M.G. Kaplitt, A.D. Loewy ŽEds.., Viral Vectors: Tools for the Analysis and Genetic Manipulation of the Nervous System. Academic Press, Orlando, 1995, pp. 349–366. w13x A.D. Loewy, S. McKellar, C.B. Saper, Direct projections from the
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A5 catecholamine cell group to the intermediolateral cell column, Brain Res. 174 Ž1979. 309–314. A.D. Loewy, C.B. Saper, R.P. Baker, Descending projections from the pontine micturition center, Brain Res. 172 Ž1979. 533–538. W.E. Lyons, J.-M. Fritschy, R. Grzanna, The noradrenergic neurotoxin DSP-4 eliminates the coeruleospinal projection but spares projections of the A5 and A7 groups to the ventral horn of the rat spinal cord, J. Neurosci. 9 Ž1989. 1481–1489. W.E. Lyons, R. Grzanna, Noradrenergic neurons with divergent projections to the motor trigeminal nucleus and the spinal cord: a double retrograde neuronal labeling study, Neuroscience 26 Ž1988. 681–693. L. Marson, K.B. Platt, K.E. McKenna, Central nervous system innervation of the penis as revealed by the transneuronal transport of pseudorabies virus, Neuroscience 55 Ž1993. 263–280. I. Nadelhaft, A.M. Booth, The location and morphology of preganglionic neurons and the distribution of visceral afferents from the rat pelvic nerve: a horseradish peroxidase study, J. Comp. Neurol. 226 Ž1984. 238–245. I. Nadelhaft, C. Devenyi, Pontine micturition center in rat revealed by retrograde transport of rhodamine-labelled beads injected into the sacral intermediolateral column, Soc. Neurosci. Abstr. 13 Ž1987. 734. I. Nadelhaft, P.L. Vera, Central nervous system neurons infected by pseudorabies virus injected into the rat urinary bladder following unilateral transection of the pelvic nerve, J. Comp. Neurol. 359 Ž1995. 443–456. I. Nadelhaft, P.L. Vera, Neurons in the rat brain and spinal cord labeled after pseudorabies virus injected into the external urethral sphincter, J. Comp. Neurol. 375 Ž1996. 502–517. I. Nadelhaft, P.L. Vera, J.P. Card, R.R. Miselis, Central nervous system neurons labelled following the injection of pseudorabies virus into the rat urinary bladder, Neurosci. Lett. 143 Ž1992. 271– 274. H. Noto, J.R. Roppolo, W.D. Steers, W.C. de Groat, Electrophysiological analysis of the ascending and descending components of the micturition reflex pathway in the rat, Brain Res. 549 Ž1991. 95–105. S.C. Nuding, G. Smith, I. Nadelhaft, Projections of the pontine micturition center to the sacral parasympathetic nucleus in the rat, Soc. Neurosci. Abstr. 22 Ž1996. 95. K. Otake, Y. Nakamura, Single neurons in Barrington’s nucleus projecting to both the paraventricular thalamic nucleus and the spinal cord by way of axon collaterals: a double labeling study in the rat, Neurosci. Lett. 209 Ž1996. 97–100. G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates, Academic Press, San Diego, 1997. C.A. Ross, D.M. Armstrong, D.A. Ruggiero, V.M. Pickel, T.H. Joh, D.J. Reis, Adrenaline neurones in the rostral ventrolateral medulla innervate thoracic spinal cord: a combined immunocytochemical and retrograde transport demonstration, Neurosci. Lett. 25 Ž1981. 257– 262. K. Satoh, N. Shimizu, M. Tohyama, T. Maeda, Localization of the micturition reflex center at dorsolateral pontine tegmentum of the rat, Neurosci. Lett. 8 Ž1978. 27–33. R.J. Valentino, S. Chen, Y. Zhu, G. Aston-Jones, Evidence for divergent projections to the brain noradrenergic system and the spinal parasympathetic system from Barrington’s nucleus, Brain Res. 732 Ž1996. 1–15. M.A. Vizzard, V.L. Erickson, J.P. Card, J.R. Roppolo, W.C. de Groat, Transneuronal labeling of neurons in the adult rat brainstem and spinal cord after injection of pseudorabies virus into the urethra, J. Comp. Neurol. 355 Ž1995. 629–640. K.N. Westlund, R.M. Bowker, M.G. Ziegler, J.D. Coulter, Noradrenergic projections to the spinal cord of the rat, Brain Res. 263 Ž1983. 15–31.
Research report
Bilateral projections of the pontine micturition center to the sacral parasympathetic nucleus in the rat Sarah C. Nuding a , Irving Nadelhaft b
a,b,)
a Veterans Administration Medical Center, Bay Pines, FL 33744, USA Department of Pharmacology and Therapeutics, College of Medicine, UniÕersity of South Florida, Tampa, FL 33612, USA
Accepted 30 October 1997
Abstract Previous work has revealed that pontine micturition center ŽPMC. neurons send projections to the sacral parasympathetic nucleus ŽSPN. of the intermediolateral ŽIML. regions of L6-S1 spinal cord segments in rats. Although unilateral SPN injections will retrogradely label PMC neurons bilaterally, it is not known whether single PMC neurons project bilaterally to the SPN. There may be two different populations of PMC neurons on each side of the brainstem, with both groups independently connecting to the SPNs on opposite sides of the spinal cord. To verify one of these alternatives, a small injection of either rhodamine-labeled latex microspheres or a red fluorescent emulsion was made into the SPN on one side of the cord; a similar injection of either fluorescein-tagged microspheres or a green fluorescent emulsion was made into the other. After at least seven days, the rats were perfused. Inspection of 40 m m cord sections confirmed the similar placement of these injections along the rostrocaudal axis of the cord and that no tracer had spread across midline. Thirty-micron brain sections were examined for filled neurons. Red, green and double labeled neurons were found bilaterally in the PMC, subcoeruleus, and A5 regions. Although some red nucleus cells were also filled, they were only singly labeled and always located contralateral to the injection. Finally, immunohistochemical staining of dopamine-b-hydroxylase ŽDBH. containing cells confirmed that some labeled cells were also noradrenergic. We therefore conclude that some PMC, subcoeruleus, and A5 neurons send axons to the SPN on both sides of the lumbosacral cord. q 1998 Elsevier Science B.V. Keywords: Neuroanatomy; Spinal cord; Autonomic; Parasympathetic; Pontine micturition center
1. Introduction The micturition process involves complex pathways of afferent and efferent connections between the urinary bladder, urethral sphincter, lumbosacral cord, brainstem, and more rostral central nervous system ŽCNS. areas. One technique used in our laboratory to study this network of pathways has been to infect selected organs with pseudorabies virus ŽPRV. Žfor review, see Ref. w12x.. After injections of PRV into the bladder, the sequence of areas with infected cells outlines the temporal spread of the virus across functional synapses and delineates the neural pathways involving the bladder w21,22x. The virus is first transported from the site of infection in the bladder to postganglionic neurons in the major pelvic ganglia where it replicates. It is then exported to and transported within
) Corresponding author. Research and Development Service, VA Medical Center, Bay Pines, FL 33744, USA. Fax: q1-813-398-9467; E-mail: [email protected]
0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 1 3 4 7 - 4
the axons of the pelvic nerves to preganglionic neurons in the sacral parasympathetic nucleus ŽSPN. in the intermediolateral ŽIML. regions of L6-S1 spinal cord segments. The replicationrexport process is repeated in each infected neuron and, at longer postinfection times, the virus infects selected groups of neurons at progressively more rostral brain centers w22x. These include the nucleus of the solitary tract, raphe´ pallidus and obscurus, gigantocellular nucleus, locus coeruleus, subcoeruleus, periaquaductal gray, red nucleus, and especially, the pontine micturition center ŽPMC., originally described by Barrington w3x. Higher brain centers including the paraventricular region, the medial preoptic area and the cortex are also infected, but at times later than those of brainstem regions w22x. Following the injection of PRV into the bladder, all the areas exhibiting viral infected neurons are bilaterally symmetric. The bilateral nature of the PMC infection is maintained even if one pelvic nerve and both hypogastric nerves are transected before the initial PRV injection. This transection effectively restricts viral transfer to the CNS via the one remaining intact pelvic nerve w20x, as viral
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transmission to other areas via the blood stream has been ruled out ŽCard, personal unpublished communication.. This suggests that neurons in one SPN are contacted by neurons from both PMCs. Other experiments in our laboratory have established a monosynaptic connection between one SPN of the L6-S1 spinal cord segments and both PMCs by using retrograde transport of fluorescent microspheres w9,10x. After small Ž20–60 nl. unilateral injections into the SPN, equal numbers of neurons were labeled in the PMCs, suggesting that individual PMC neurons might have direct projections to both SPNs w19x. Electrophysiological stimulation of the PMC on one side of the brainstem induced firing of the contralateral, as well as the ipsilateral, bladder postganglionic nerves w23x, demonstrating a connection from the PMC to both sides of the bladder. Kuru and Yamamoto w11x also showed early evidence of a bilateral projection from Barrington’s nucleus when they noted bilateral degeneration of axons descending to the IML of the lumbosacral division after lesions in the dorsolateral pons of cats. More recently, after unilateral injections of biotinylated dextran amine ŽBDA. into the PMC and the adjacent ventral region, anterogradely labeled cells were found bilaterally in the SPN, suggesting that individual PMC neurons might have direct projections to both SPNs w5x. In addition, unilateral injections of the cholera toxin B subunit w5x into the IML and dorsal horn of rat L6-S1 cord segments retrogradely labeled cells bilaterally in the pontine tegmental area, either clustered in the area of the PMC or scattered ventral to it, again suggesting that the SPN receives input from this area on both sides of the pons. The results of all of these experiments can be explained by two possible hypotheses. There may be individual PMC neurons sending processes to both sides of the L6-S1 cord. Alternatively, there may be two different populations of PMC neurons on each side of the brainstem. Both groups might independently connect to the SPNs on opposite sides of the spinal cord, with no single cell projecting to both sides. The goal of the current experiments was to examine the connections between the PMC and the sacral parasympathetic nuclei ŽSPN. to determine if bilaterally projecting PMC neurons exist or if there are two groups of PMC neurons one of which projects to one SPN and the other to the contralateral SPN. To accomplish this aim, two different colors of either dye-labeled fluorescent latex microspheres w9,10x or fluorescent emulsions w6x were injected into the SPNs. A preliminary report of these results has been presented in abstract form w24x. 2. Materials and methods 2.1. Animal preparation The care and use of animals was approved by the Institutional Animal Care and Use Committee of the Bay
Pines Veterans Administration Medical Center. Adult Sprague–Dawley rats ŽHarlan. weighing between 230 and 300 g were anesthetized with pentobarbital Ž50 mgrkg i.p.. and body temperature was maintained at about 378C by an Aquamatic Heat Therapy unit ŽAmerican Pharmaseal.. A midline slit was made through the skin overlying the spinal column and muscle was detached from the vertebral processes until the L6-S1 vertebral interface was established. Using this bony landmark, the approximate location of the L6-S1 spinal cord segments was determined Ž; 2 mm rostral to the L1 and L2 vertebral boundary. and the surface of the cord exposed. After the dura was slit open, a small amount of 2% lidocaine was dripped on the surface to prohibit sudden movement during subsequent manipulations of the cord and spinal roots. Using a fire-polished glass pipette, any roots overlying the injection areas were gently pushed aside and then a 29-gauge needle was used to scratch superficial tears at the intended injection sites, about 0.5 mm lateral to midline. Without this last step, pipettes would not easily penetrate the cord surface. At this point, the animal was placed in a headholder and then suspended from a stereotaxic frame ŽKopf. by means of a clamp gripping the L2 and L3 spinous processes. In addition, the rat’s tail was draped over and then taped directly to a bar which connected both sides of the stereotaxic frame. This method of steadying the animal helped to reduce spinal movement due to breathing during the injection procedure. Lastly, the height and angle of the tail lift was adjusted so that the dorsal surface of the exposed cord was roughly parallel to the horizontal plane. 2.2. Injections A glass micropipette Žbumped to O.D.; 35 m m; I.D.; 14 m m. was coupled to both a micrometer syringe ŽGilmont. and an air pressure injection system Žmodified from Ref. w1x. via a two-way stopcock and polyvinylchloride tubing ŽTygon. and then clamped to the stereotaxic frame. The pipette was lowered into a 2-m l drop of a solution of either latex microspheres Žrhodamine-labeled or fluorescein-tagged, average diameter 0.050 m m and hereinafter referred to as beads, Lumafluor. or Fluoro-dye ŽFluoro-Red: excitation peak: 540 nm, emission peak: 585 nm; Fluoro-Green: excitation peak: 450 nm, emission peak: 505 nm; Tompow Pencil; see Ref. w6x.. Back suction applied via the syringe slowly filled the tip, shank, and some of the barrel of the micropipette. Once the pipette was sufficiently filled, the stopcock was connected to the injection system and the micropipette moved into position over one of the injection sites on the cord surface. The pipette penetrated the dorsal surface and advanced at an angle perpendicular to the plane defined by the stereotaxic frame and was driven into the cord to a depth of about 1.0 mm. After a short delay to settle the pipette in the tissue Ž1 min., the pipette was slowly withdrawn 0.3 mm. After
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another variable delay Ž1 to 5 min., the injection procedure began. The air pressure system, under stimulator control ŽGrass., delivered short duration pulses of nitrogen to the pipette, thereby effectively ejecting measured amounts of tracer w1x. ŽPrevious calibration of pressure, duration, and micropipette size parameters allowed for precise injections: w40 psi, 80 msx for 20 nl; w40 psi, 150 msx for 35 nl.. During each pulse, observation of the meniscus of fluid in
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the pipette barrel confirmed tracer ejection. If the meniscus did not move, even after repeated pulses, pipette blockage was assumed and a new micropipette was then filled and lowered into approximately the same location. After two or three successful pulses, final injection sizes were 40 to 90 nl in size. Once the injection was complete, the pipette was left in place for at least 30 min to lessen the amount of bead solution suctioned back up the electrode track during micropipette withdrawal. After slowly removing and dis-
Fig. 1. Examples of injections into L6-S1 cord segments. For these two experiments, fluorescein-tagged beads were injected on the left side, rhodamine-labeled beads on the right. A and C are fluorescent photomicrographs of two 40 m m sections showing injection locations for two experiments, with the solid white area demarcating the heaviest injection region. The filter set combination used allowed both injections to be visible simultaneously Žexcitation: BP 450–490; FT 510; emission: LP 520.. The plots of B and D show the extent of the injections over several contiguous sections for these experiments, with the center of each injection denoted by a region of solid color and cross-hatched areas outlining the full extent of the injection. Dorsal is to the top; plot scale bars are 500 m m. A and C: R1339. B and D: R1422.
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carding the micropipette following this injection into the SPN on one side of the cord, a similar injection was made into the other side, using the second fluorescent tracer. Upon completion of both injections, the wound was sutured closed and the skin over the tissue was stapled together. No gel foam or other material was left in place as this was observed to cause spinal tissue damage upon later dissection Žnote Fig. 1B.. 2.3. Histology Following at least a seven-day survival period, the deeply anesthetized animal Žpentobarbital w75 mgrkg i.p.x. was first injected through the heart with 100 units of Heparin, then perfused with a Krebs wash solution followed by a 4% paraformaldehyde fixative solution. The spinal cord Žsegments T13-S2. was then dissected and the L6-S1 region isolated and stored in fixative at 48C at least overnight, before being transferred to a solution of 25% sucrose at 48C. The brain was also removed and placed in fixative at 48C for a period of at least four days, before immersion in the 25% sucrose solution at 48C. Once the tissue had sunk, both samples were ready for sectioning. Forty-micron Žcord. or 30-m m Žbrain. transverse cryostat sections were collected in 0.1 M phosphate buffer and stored at 48C. For the cord, all tissue was mounted on gel-coated slides in order to identify the location and the extent of each injection. For the brain tissue, selected sections were mounted, dried, and then coverslipped with Krystalon ŽHarleco.. All slides were also stored at 48C to preserve bead or fluoro-dye longevity. Some sections were also processed by fluorescent immunohistochemistry to locate bead-filled cells which were also adrenergic by using an antiserum to dopamine-b-hydroxylase ŽDBH., the final enzyme used in noradrenergic production. The 30-mm sections were exposed for about 20 h at 48C to rabbit anti-DBH wEugene Tech Internationalx Ž1r1000 dilution in 1% normal donkey serum. followed by either donkey anti-rabbit fluorescein isothiocyanate ŽFITC. or tetramethyl rhodamine isothiocyanate ŽTRITC. at dilutions of 1r50 for 1 h at room temperature ŽJackson ImmunoResearch Laboratories.. Tissues were then mounted and coverslipped with Krystalon ŽHarleco. before storage at 48C. For each animal used in the analysis, inspection of 40
m m cord sections confirmed that the approximate center of each injection was in about the same rostrocaudal location; usually the core of each injection was offset by only a section or two Ž; 50 mm.. In addition, the injections had to include most, if not all, of the area occupied by the cells of the sacral parasympathetic nucleus within the intermediolateral cell column region. Finally, individual injections in each rat could not encroach upon the midline or the central canal. As individual preganglionic neurons within the rat IML do not extend their dendritic processes across the midline w18x, this stipulation prevented double labeling by inadvertent contralateral spread of the injection. Thirty-micron brain sections were examined under oil using a 40 = Planapochromat lens using a Zeiss standard microscope equipped for epifluorescence using Zeiss filter combinations to allow viewing either rhodaminerred emulsion- Žexcitation: BP 546r12; FT 580; emission: LP 590. andror fluoresceinrgreen emulsion- Žexcitation: BP 450–490; FT 510; emission: BP 520–560. filled neurons. Data was plotted using a computer-assisted mapping system ŽMDPlot, Minnesota Datametrics. andror photographed with either Kodak Tmax 400 Žfor black and white. or Ektachrome 400 = Žfor color. film. The general locations of filled cells were determined by comparing the tissue section with the compact rat brain atlas of Paxinos and Watson w26x.
3. Results Photographic examples and plots of sections illustrating successful fluorescent injections into the IMLs of both sides of the L6-S1 cord are shown in Fig. 1. The photograph of Fig. 1A shows an injection of fluorescein-tagged microspheres into the left side of the cord with the solid white area demarcating the heaviest injection region ŽR1339.. Fig. 1B is a depiction of the extent of this and the rhodamine-tagged microsphere injection over several contiguous sections Žleft: fluorescein; right: rhodamine.. For each injection, the center is denoted by a region of solid color; cross-hatched areas outline the full extent of the injection. Although the section shown in Fig. 1A and B is slightly elongated in the mediolateral dimension due to incidental dorsoventral compression of the cord, note that the IML regions were included in the injection areas of
Fig. 2. Photomicrographs of labeled cells located in different brainstem regions and a section redrawn from Paxinos and Watson w26x for reference. All paired photographs were taken from identical locations, only fluorescent filter set was changed. ŽA. Fluorescein-labeled bead-filled cell which was lying in the dorsal most extent of the PMC, medial to the locus coeruleus ŽLC.. Note the bright spheres of light which fill the cell and even some of its processes; single beads are clearly identifiable by their brilliance and individual diffraction patterns. ŽB. Two views of two adjacent PMC cells. ŽC. Cell located near the region containing A5 cells, among the streaming fibers of the rubrospinal tract, contained both rhodamine- and fluorescein-tagged microspheres. ŽD. Cell double-labeled with both of the fluorescent emulsions and located in the midline region in the raphe´ magnus ŽRMg.. ŽE and F. A small part of the PMC region after the injections outlined in Fig. 1D. Three double-labeled cells are marked by white arrows; several singly labeled filled cells are also apparent. ŽG. A pair of cells located in the ventral part of the subcoeruleus nucleus ŽsubCV.; note that although both cells were filled with fluorescein-tagged beads, only the more ventral of these cells is also labeled with rhodamine-tagged microspheres. All scale bars denote 25 m m. The brainstem section is adapted from Paxinos and Watson w26x; Bregma y9.68 mm.
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both sides and that there was no overlap between the injections. A photograph of a section and the corresponding plot showing tagged latex microsphere injections in another animal ŽR1422. are shown in Fig. 1C and D. The photographs of Fig. 1 were taken using a filter set combination which allowed both injections to be visible simultaneously Žexcitation: BP 450–490; FT 510; emission: LP 520.. Individually labeled cells, however, were
located using filter sets designed specifically for their absorption and emission spectra Žsee Section 2. so that rhodamine, fluorescein, and the individual fluorescent dye emissions would not overlap. Fig. 2 shows photographs of several filled cells located in several brainstem regions; a section redrawn from Paxinos and Watson w26x is included for reference ŽFig. 2F; Bregma y9.68 mm.. Fig. 2A is a close-up of a fluorescein-labeled bead-filled cell which
Fig. 3. Plots of a brainstem section and a section including the red nucleus showing labeled cells. Bead filled cells are denoted by open Žrhodamine. or filled Žfluorescein. circles; double-labeled cells are marked by asterisks. Filled squares are neurons from the mesencephalic trigeminal nucleus. ŽA. Solid-filled areas mark PMC regions; these areas were redrawn under higher magnification and are shown as insets. ŽB. Some neurons in the red nucleus were also filled, but they were always singly labeled by fluorescence and only from the contralateral injection. Dorsal is to the top; scale bars are 1 mm.
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was lying in the dorsal most extent of the PMC, medial to the locus coeruleus ŽLC.. Note the bright spheres of light which fill the cell and even some of its processes; single beads are clearly identifiable by their brilliance and individual diffraction patterns. Areas of unfocused green illumination result from beads located in planes above or below the plane of focus. This ‘glowing’ is noticeable in several photographs of bead-filled cells Že.g., Fig. 2C and E.. Once such a bead-filled cell was located, the filter set was switched to examine whether the cell contained material from both injections. For example, Fig. 2B shows two views of two adjacent PMC cells. Again, distinct bright beads are clearly discernible in both photographs, although the green beads in the cell on the right are slightly out of focus as they were out of the plane of focus of the photograph. During examination of the tissue, however, the microscope lens was raised and lowered to enable clear differentiation of bead from background fluorescence. Other examples of double filled cells are shown in Fig. 2C–E. The cell in Fig. 2C was located near the region containing A5 cells, among the streaming fibers of the rubrospinal tract, and contained both rhodamine- and fluorescein-tagged microspheres Žsee Fig. 2F; rs.. The cell in Fig. 2D was filled with both of the fluorescent emulsions and located in the midline region in the raphe´ magnus ŽRMg.. For the pair of cells illustrated in Fig. 2E, located in the ventral part of the subcoeruleus nucleus ŽsubCV., note that although both cells were filled with fluoresceintagged beads, only the more ventral of these cells is also labeled with rhodamine-tagged microspheres. Upon first examination of brainstem sections under low power Ž=6.3 or =25., the PMC regions light up with explosions of color, revealing label located both within cell bodies and their proximal processes Žfor example, see Fig. 2A.. Under oil at higher power, however, individual cells can be isolated and identified. For example, Fig. 2G consists of two identically positioned photographs of a small part of the PMC region after the injections outlined in Fig. 1D. Although four double-labeled cells in the two photographs are marked by white square symbols, several singly labeled filled cells are also apparent. Note that several beads overly one another within a 30-m m section and therefore many individual beads may not be in focus. Of all brain areas, this PMC region was the most populated by retrogradely labeled cells, both single- and double-labeled. Several other areas in the brainstem contained cells filled with either or both colors, including the subcoeruleus and A5 regions. In addition, small numbers of labeled cells were also located in the portion of the lateral parabrachial nucleus situated lateral to the superior cerebellar peduncle, as well as scattered within the raphe´ and pontine reticular fields. Fig. 3A shows a plot of one brainstem section demonstrating labeling in these areas after injections of rhodamine-tagged microspheres in the right IML and fluorescein-tagged beads in the left IML. In
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this plot, bead filled cells are denoted by open Žrhodamine. or filled Žfluorescein. circles; double-labeled cells are marked by asterisks. Because the PMC regions Žwithin the polygons. were so packed with filled cells, these areas were redrawn under higher magnification; these plots are shown as insets. These insets show red, green and doublelabeled PMC neurons located on both sides of the brain, with double- and single-labeled cells intermingled throughout the rostrocaudal and dorsoventral extent of the PMC. In addition to these cells in the brainstem, some neurons in the red nucleus were also filled, but they were always singly labeled by fluorescence and only from the contralateral injection ŽFig. 3B.. Some retrogradely labeled cells were located at the ventromedial edge of or just ventral to the locus coeruleus ŽLC.. In addition, some labeled cells were found in the ventrolateral pons, either lying among the dorsomedialventrolaterally oriented fibers of the rubrospinal tract or just dorsal to the lateral superior olive Žsee Fig. 2F.. These latter two groups of cells appear to be located within the regions defined by Byrum and Guyenet w4x as the lateral and dorsal subdivisions, respectively, of the A5 cell group. The placement of all these cells within known noradrenergic nuclei or cell regions, however, does not automatically prove that they contain noradrenaline. In order to demonstrate noradrenergic activity, some sections were examined for cells containing dopamine-b-hydroxylase ŽDBH., the final enzyme used in noradrenaline production. Note that by using an antibody to DBH, this procedure would label noradrenaline as well as adrenaline producing cells. As some adrenergic fibers terminate in the rat spinal cord, theoretically these fibers could take up injection material and also be labeled positive from DBH staining. Previous reports of adrenergic cells in the rat medulla, however, have shown them to be mainly limited to the C1 group or the dorsal motor nucleus of the vagus and to send projections primarily to thoracic cord levels, therefore, the possibility of mislabeling adrenergic cells as noradrenergic is limited w2,8,27x. In brainstem sections examined for noradrenaline containing cells, some neurons within the LC ŽFig. 4A. and A5 regions ŽFig. 4D. consistently displayed DBH positive reactions. In addition, some of these DBH positive cells were found to be retrogradely labeled by one or both fluorescent markers. For example, the DBH positive cell marked by an arrow in Fig. 4A is shown under higher magnification in the top frame of Fig. 4B to be also labeled with rhodamine-tagged microbeads. These microspheres are visible as small, bright points of light, but few are discernible in this photograph through the heavy TRITC labeling. This cell was filled with fluorescein-tagged microspheres as well, as shown in the lower frame of Fig. 4B. Some cells ventral to the LC in either the dorsal or ventral subdivisions of the subcoeruleus were also ‘triplelabeled’ in this way ŽFig. 4C., as were a few cells in the A5 lateral subdivision ŽFig. 4D–E.. Other cells in this A5
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region, although DBH positive, were retrogradely labeled by only one fluorescent marker ŽFig. 4F..
4. Discussion As mentioned in Section 1, there have been earlier demonstrations of the bilateral influence of a single pontine micturition center in the lumbosacral spinal cord or to the nerves innervating the urinary bladder. Although Nadelhaft and Vera w20x previously speculated on the existence of bilateral projections to sacral parasympathetic nuclei ŽSPN. from PMC regions, evidence of individual bilaterally projecting PMC cells was lacking. The results of the current experiments demonstrate, for the first time, that this influence resides in single neurons within the PMC, each of which sends projections to both SPNs in the lumbosacral spinal cord. In addition, these experiments have discovered neurons in other areas of the brainstem that also have bilateral projections to those same segments of the spinal cord. The largest number of brainstem neurons that projected to the sacral IML were found in the PMC. The PMC also contained the greatest number of individual neurons that had projections to both sides of the sacral cord. Because the L6-S1 intermediolateral region contains the sacral parasympathetic nucleus, whose neurons provide excitatory impulses to the urinary bladder, it is understandable that the major projection to that area should be from the PMC. Previous studies have shown that unilateral injections into the IML area of rat sacral cord segments retrogradely labeled PMC cells bilaterally we.g., Refs. w5,19xx, and several studies have demonstrated PMC projections to the sacral spinal cord, but bilateral projections have not been assigned to single PMC neurons. For example, after unilateral injections of anterograde tracers into the PMC, labeled fibers were first located ipsilateral to the site of injection, both in the tractus solitarius in the medulla w14x, and in the C1 segment w7x. But further caudally, labeled fibers were found bilaterally both in the lateral funiculi of segment T1 and at the sacral IML level w7,14x. Thus, the transition from ipsilateral to bilateral projection occurs between the medulla and the thoracic cord. Although we have demonstrated that some of the PMC neurons send projections to both sides of the sacral cord, this property may not apply to all PMC neurons. In fact, other investigators have reported that some PMC neurons
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send projections to the ipsilateral sacral cord and to the ipsilateral locus coeruleus w29x. Yet other researchers note that some PMC neurons send projections to the ipsilateral sacral cord and to the paraventricular thalamic nucleus w25x. All of these PMC neurons have at least one projection to the sacral intermediolateral spinal cord, but they may be divided into subsets based on the location of these second projections. It is conceivable that individual PMC neurons may project to even more than two distinct areas. This supports the idea that the PMC is a rather complex nucleus with multifunctional properties. Our results demonstrated that a small number of neurons in the ventrocaudal LC were labeled after sacral IML bead injections, although very few were double-labeled. These cells did not stand out from adjacent DBH positive cells which did not project to IML and resembled them in both morphology and orientation. This paucity of labeled LC neurons is not surprising given the lack of a predominant LC projection to the sacral spinal cord. Previous injections into the sacral IML have labeled few LC neurons, whereas injections into the lumbar IML have labeled many w7,19x, especially those neurons located in the caudal LC w15x. In addition, unilateral HRP injections into the LC demonstrated a majority of labeled fibers in the lumbar segments, with very few reaching the sacral cord w7x. All these results, plus the fact that bilateral lesions of the PMC resulted in difficulties in micturition, whereas bilateral lesions of the LC did not w28x support the contention that, in the rat, the LC is not involved with micturition. Aside from the PMC, the subcoeruleus and A5 adrenergic region had the largest numbers of bead-labeled neurons and some of these cells were also double-labeled. Although there have been previous reports of projections from the subcoeruleus to lumbosacral cord w5,16x, the terminal sites of spinally projecting A5 cells has been mainly restricted to areas above the lumbar enlargement, where fibers have been found in the intermediolateral regions of cervical and thoracic cord w4,13x. In the latter article, the authors point out that no fibers or terminals were observed in the sacral intermediolateral region. But other studies have reported quite different findings. Westlund et al. w31x noted that DBH positive terminals were especially prevalent in the superficial dorsal horn, ventral horn, and around the central canal and IML of thoracic and sacral cord segments. In fact, the noradrenergic endings in the sacral cord were concentrated in the area of the cells of origin of parasympathetic preganglionic
Fig. 4. Brainstem sections examined for noradrenaline-containing cells retrogradely labeled by one or both fluorescent markers. ŽA. DBH-positive reactions of locus coeruleus neurons. Cell marked by arrow is shown under higher magnification in ŽB.. Scale bar is 150 m m. ŽB. Photomicrographs showing magnification of cell in ŽA. retrogradely labeled with rhodamine Žtop. and fluorescein Žbottom.-tagged microspheres. Scale bar is 25 m m. ŽC. DBH-positive cell ventral to the LC in the subcoeruleus was retrogradely labeled with rhodamine Žtop. and fluorescein Žbottom.-tagged microspheres. Scale bar is 25 m m. ŽD. DBH-positive cells in A5 lateral subdivision region. Cell marked by arrow is shown under higher magnification in ŽE.. Scale bar is 100 m m. ŽE. Photomicrographs showing magnification of cell in ŽD. retrogradely labeled with rhodamine Žtop. and fluorescein Žbottom.-tagged microspheres. Scale bar is 25 m m. ŽF. Two DBH-positive cells in A5 lateral subdivision retrogradely labeled by only one fluorescent marker Žrhodamine.. Scale bar is 25 m m.
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fibers. In addition, after injections of DBH antibody into the sacral intermediolateral region, they report that a substantial number of retrogradely labeled cells were located within the A5 cell group. Finally, PRV injections into pelvic viscera such as the urinary bladder w22x, the urethra w30x, and the penis w17x, have all resulted in the labeling of neurons in the A5 area after only short incubation times. All this evidence suggests that it is likely that A5 neurons are retrogradely labeled directly from the sacral spinal cord.
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Acknowledgements We would like to express our appreciation of the excellent assistance provided by Mr. Gary A. Smith in conducting these experiments and in handling and processing the tissue for analysis. This work was supported by the Veterans Administration.
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