THE JOURNAL OF COMPARATIVE NEUROLOGY 324~1-13 (1992)
Quantitative Analysis of Bulbospinal Projections From the Rostra1 Ventrolateral Medulla: Contribution of Cl-Adrenergic and Nonadrenergic Neurons IWONA JESKE AND KEVIN E. McKENNA Department of Physiology, Northwestern University Medical School, Chicago, Illinois 60611
ABSTRACT The contribution of C1-adrenergic and nonadrenergic neurons to the spinal projection from the rostral ventrolateral medulla (RVLM) and their relative innervation density throughout thoracic spinal segments were examined by combining the Fluorogold (FG) retrograde tracing technique with immunofluorescent labeling for the epinephrine-synthesis enzyme phenylethanolamine N-methyltransferase (PNMT). The results indicate that the RVLM-spinal projection is comprised of both PNMT-positive and PNMT-negative neurons located in the subretrofacial area of the RVLM, approximately 1 to 1.7 mm rostral to obex. The bulbospinal projection from the RVLM is predominantly ipsilateral, and bulbospinal neurons do not appear to be organized within the RVLM in a manner indicating their segmental termination site. Eighty-one percent (+-4%, n = 2) of the PNMT-positive cells in the ipsilateral subretrofacial RVLM were retrogradely labeled after unilateral FG injections into multiple thoracic levels of the intermediolateral cell column (IML). Following single level FG injections, the number of retrogradely labeled PNMT-positive neurons in the subretrofacial RVLM decreased with injections in more caudal thoracic segments, indicating a heavier innervation of the upper thoracic IML by C1 neurons. PNMT-negative neurons were the main component of the RVLM-spinal population with 63 8% (n = 7) of the non-PNMT-containing neurons within the ipsilateral subretrofacial RVLM innervating all thoracic levels of the IML. The results indicate that both C1-adrenergic and nonadrenergic neurons in the RVLM make a substantial contribution to the innervation of the IML. o 1992 Wiley-Liss, Inc.
Key words: cardiovascular regulation, phenylethanolamine N-methyltransferase, intermediolateral cell column, retrograde transport
The rostral ventrolateral medulla (RVLM) plays a major role in the regulation of cardiovascular function as it maintains vasomotor tone (Dampney et al., '82; Ross et al., '84b; Barman and Gebber, '85; Morrison et al., '881, forms the efferent limb of cardiorespiratory and somatosensory reflexes (Barman and Gebber, '85; Brown and Guyenet, '85; Granata et al., '85; Sun and Guyenet, '86a; Terui et al., '86; McAllen, '86; Morrison et al., '88; Morrison and Reis, '89a), and processes cardiovascular-related information from other brain regions (Sun and Guyenet, '86b; Terui et al., '86; Barman and Gebber, '87; McCall, '88b). RVLM neurons form excitatory synaptic contacts with sympathetic preganglionic neurons in the intermediolateral cell column (IML) (Morrison et al., '91a), and thereby exert a direct influence on sympathetic nerve discharge (Dembowsky et al., '89; Morrison and Reis, '91b). On the basis of correlative physiological and anatomical observations, it has been proposed that cardiovascular c 1992 WILEY-LISS, INC.
actions of the RVLM are mediated by epinephrine-synthesizing neurons of the C 1 cell group. Specifically, the region from which L-glutamate or electrical stimulation evokes increases in arterial pressure, heart rate, and plasma levels of catecholamines and vasopressin contains neurons immunoreactive for the enzyme converting norepinephrine to epinephrine, phenylethanolamine N-methyltransferase (PNMT) (Dampney et al., '82; Ross et al., '84b). In addition, RVLM neurons identified by the criteria of antidromic activation from the thoracic spinal cord, inhibition to baroreceptor activation, synchronous discharge with the cardiac cycle, and locking to the rhythmic component of
Accepted June 10,1992, Iwona Jeske's current address is Clinical and Scientific Affairs, Pharmaceuticals Group, 235 E. 42nd Street, New York, NY 10017.
1. JESKE AND K.E. McKII:NNA
2 sympathetic nerve discharge are localized in proximity to PNMT-positive cell bodies (Morrison et al., '88). Anatomical studies have demonstrated that many RVLM reticdospinal neurons contain PNMT immunoreactivity (Ross et al., '84a; Tucker et al., '87; Strack et al., '89; Minson et al., '90). Several other neurotransmitter markers are also contained within RVLM-spinal neurons (Mantyh and Hunt, '84; Leibstein et al., '85; Charlton and Helke, '87; Hirsch and Helke, '88; Strack et al., '89), including some which contain more than one marker (Blessing et al., '87; Menetrey and Basbaum, '87; Millhorn et al., '87; Sasek et al., '90). Unresolved, however, are: 1)the number of cells in the C1 group which project to the spinal cord and, more importantly; 2 ) how the C 1 bulbospinal contribution compares to that of nonadrenergic neurons located in the RVLM. Furthermore, although RVLM neurons are essential for cardiovascular control, it is unclear how these sympathoexcitatory reticulospinal projections are organized for the selective innervation of vascular beds. We addressed these issues in the present study by using retrograde tract-tracing and immunohistochemistry to compare the proportion of spinally projecting PNMT-positive to PNMT-negative neurons within the RVLM. Since there is evidence that functional specificity in the sympathetic nervous system may be reflected in its neuroanatomical organization (Appel and Elde, '88; Strack et al., '88; Carrive et al., '891, we also examined the descending organization of the RVLM projection by retrograde tracer injections into different rostrocaudal levels of the IML.
MATERIALS AND METHODS Surgery Male Sprague-Dawley rats (300-350 g, n = 37), anesthetized with ketamine (8.7 mgilOO gm)/xylazine (1.63 mg/ 100 gm i.ni.; additional dosages supplemented when necessary), were mounted in a stereotaxic frame. The vertebral column was stabilized and thoracic spinal segments (T3 through T13) were identified superficially by prominent landmarks such as T2 rostrally, or T9 caudally. Once the vertebra overlaying an intended spinal cord segment was located (Hebel and Stromberg, '76), surrounding muscle was reflected laterally and a laminectomy was performed. A slit was cut in the dura near the dorsolateral sulcus of the spinal cord and a glass micropipette was stereotaxically lowered into the IML, approximately 0.5 mm lateral to the midline and 0.9 mm ventral to the pial surface.
Micropressure injection Single barrel glass micropipettes (10-20 pm, outer tip diameter), connected by polyethylene tubing to a regulated, solenoid valve-controlled pressure source were used to discretely deposit anatomical tracers into the IML. Ejectate volumes (typically 50-80 nl) were measured directly by monitoring the movement of the fluid meniscus in the pipette barrel with a compound microscope fitted with a fine reticule. The pipette tip was left in place 5 minutes after injection to reduce reflux of the tracer into the pipette tract. To examine RVLM-spinal projections, rats were unilaterally injected with Fluorogold (FG, 50-80 nl, 4%, Fluorochrome Inc., Englewood, CO) into one thoracic segment of the upper (T3-T5), middle (T7-T9), or lower (Tll-T13) level of the IML. To representatively label the entire
RVLM-spinal projection, another group of rats was unilaterally injected with FG (50 nl per site) into multiple segments (either two sites at T3 and T13 or five sites at T3, T5, T7, T9, and T11) of the IML. After a 2 week survival period, animals were reanesthetized with ketamineixylazine, and colchicine (150 pg/lO p1) was micropressure injected over a 5 minute period into the lateral ventricle, ipsilateral to the spinal cord injection(s1.The colchicine was applied to enhance immunohistochemical detection, and thus reduce the problem of false negatives. Animals were sacrificed 24-48 hours later.
Tissue preparation Animals were deeply anesthetized with sodium pentabarbitol (50 mgikg) and perfused transcardially with isotonic saline, followed by 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS, pH 7.4). The brainstem and thoracic spinal cord segments were blocked from the neuraxis, postfixed in 4% paraformaldehyde in PBS for 1 hour, and immersed overnight at 4°C in PBS with 25% sucrose. Coronal brainstem sections were cut (30 pm thick) on a cryostat (Hacker Instruments Inc., Fairfield, NJ) and collected in serial order in wells containing cold 0.1 M PBS. A nick on the dorsolateral brainstem surface marked the side ipsilateral to the injection. Alternate tissue sections were processed for anti-PNMT immunofluorescence. The reniaining sections were coverslipped with Fluoromount (Atomergic Chemetals, Farmingdale, NY)and later compared agifinst immunohistochemically processed sections to verify that retrogradely transported FG had not faded or leached out of labeled cells during processing. Spinal cord segment(s) containing the injection site were also cut into coronal sections, and alternate sections were counterstained with thionin to verify the size and location of the FG injection with respect to the IML.
Immunohistochemistry Incubations in primary (anti-PNMT) and secondary 1 fluorescein-conjugated anti-rabbit) antisera were in 0.1 M PBS with 0.5-0.75% Triton x-100 and 3 4 % normal goat serum (NGS) with gentle agitation. Between each step, sections were washed thoroughly in PBS (three times, 5 minutes each of rinse). Tissue sections of the rostra1 medulla from the approximate level of obex to the pons were immersed free floating in PBS with 3% NGS and incubated overnight at room temperature in antiserum raised in rabbits against PNMT (1:1,000, Eugene Tech., Allendale, NJ). Sections were then incubated ( I hour) in fluorescein-conjugated anti-rabbit secondary antibody raised in goats (1:800, Arne1 Products Inc., Cherokee Station, NY)Mounted sections were coverslipped with buffered glycerol containing p-phenylencdiamine (pH 9.0) (Platt and Michael, '83).
Data collection and analysis Tissue sections were examined under an epifluorescent microscope (Leitz Ploempak) with a D filter mirror cube (excitation BP 355-451 nm, suppression LP 460 nm), and a I213 cube (excit. BP 450-490 nm, supp. LP 515 nm) to visualize FG- and Fluorescein (FS)-tagged immunoreactivity, respectively. Double-labeled cells were readily identified by color in each histological section by alternating between the two filter cubes in the microscope without moving the slide. A cell was double-labeled if it was visualized under
QUANTITATION OF RVL BULBOSPINAL PROJECTIONS
Fig. 1. Transverse section of the rat spinal cord at T3 demonstrating that the typical injection site (FG, 50 nl) was centered within the intermediolateral cell column. Scale bar = 500 wm.
each filter system in the same focal plane, with a prominent soma and at least one discernable process. Typically, the boundaries of a double-labeled cell were uniformly filled by each fluorescent label (Figs. 2, 3). Although not as readily apparent in photographs, peak-through of FG labeling under the FS filter conditions was easily distinguished by color under the microscope. The exact location of PNMT-positive, retrogradely labeled PNMT-positive, and retrogradely labeled PNMTnegative neurons was recorded using an x-y plotter driven by microscope stage-position transducers. Once the cell distributions were plotted, tissue sections were counterstained with ethidium bromide. Cytoarchitectural landmarks were examined under epifluorescent illumination in relationship to FG- and FS-labeled cells in the RVLM. Sections and gross landmarks were outlined using a microprojector. Data were analyzed by circling C1 group boundaries (based on the position of PNMT-positive cells in the RVLM) within each plotted section. The number of PNMT-positive, retrogradely labeled PNMT-positive, and retrogradely labeled PNMT-negative neurons was totaled for each section of C1 area, starting at the level of obex to 2 mm rostral to obex. Final cell counts were corrected using the method of Abercrombie ('46). The total number of PNMT-positive neurons in the RVLM was calculated in each animal from 34 tissue sections starting at the level of obex to the facial nucleus (2.0 mm rostral to obex). Cell counts for the
subretrofacial RVLM were calculated in each animal from 12 sections containing the retrofacial division of nucleus ambiguus, starting approximately 1 mm to 1.7 mm rostral to obex. Results are expressed as mean 2 SD. The relationship between cell counts and the segmental IML level injected with tracer was examined with simple linear regression. Significance was determined at a P value less than 0.05.
RESULTS Injection site Fluorogold (FG) injection sites were centered in the IML of the thoracic sympathetic preganglionic cell column (Fig. 1).Dye deposits frequently followed the pipette tract into the dorsal horn, and sometimes spread medially into lamina VII. Necrosis was evident at the immediate perimeter of the injection site, and a diffuse zone of local spinal neuropil retrogradely labeled from the injection site was present in the immediate surrounding area including the ipsilateral ventral horn, lamina X, and the contralateral lamina VII, extending up to 1mm in the rostrocaudal plane.
PNMT-positive neurons in the RVLM In agreement with the reports of others (Ross et al., '84a; Ruggiero et al., '85; Tucker et al., '87; Ellenberger et al., '90; Minson et al., 'go), we found PNMT-positive cells (C1
I. JESKE AND K.E. McKENNA
group) within the paragigantocellular reticular formation starting at levels caudal to the area postrema and extending to the facial nucleus. The rostrocaudal distribution of PNMT-positive cells is illustrated in Figure 4. Intermediate levels of RVLM (1to 1.7 mm rostral to obex) paralleled the compact division of nucleus ambiguus (NA, Bieger and Hopkins, ’87), and the number of PNMT-positive cells averaged 20-25 per section, spread dorsoventrally from the brainstem surface to the NA (Figs. 2, 3, 8).The number of PNMT-containing neurons decreased significantly ( > 1.5 mm rostral to obex) to approximately five per section, and disappeared ( > 1.8mm rostral to obex) with the appearance of the facial nucleus. The rostrocaudal distribution of PNMT-positive cell counts was consistent within animals (ipsilateral and contralateral side, Fig. 4, Tables 1, 21, and between experimental animals (Figs. 4,5,9, Tables 1 , 2 ) . PNMT-positive cells were also observed in ventrolateral medullary levels caudal to obex. In number, they ranged from 15 to 20 cellsisection immediately caudal to obex and decreased to 1-2 cellsisection by 1 mm caudal to obex. These counts have not been included in line graphs of this study. Counts of PNMT-positive cells in the C1 group from the level of obex to the 2.0 mm rostral to obex were averaged from ten animals injected with FG (Tables 1, 2). A total of 540 ~fr40 PNMT-positive cells were identified on the side ipsilateral, and 542 & 58 on the side contralateral to the injection. Given that another 91 t 9 (n = 2) PNMTpositive cells are located per side of the ventrolateral medulla at levels caudal to obex, we conclude that the C1 population in the rat consists of approximately 1,200 PNMT-positive neurons.
Therefore, although the C 1 cell column extends from approximately 1mm caudal to obex to the facial nucleus (2 mm rostral to obex), only the subset of these PNMTcontaining neurons within the subretrofacial RVLM projects predominantly to the IML. In this study we use the term “subretrofacial” RVLM (McAllen, ’86) to distinguish this RVLM area from more caudal or rostral portions of the C1 cell group.
Retrogradely labeled PNMT-positive neurons in the RVLM
Segmental differences in the C1 innervation of the IML
Retrogradely labeled, PNMT-positive neurons were selectively located within intermediate levels of the C1 cell column which paralleled the retrofacial division of nucleus ambiguus. The majority of the total number of RVLMspinal, PNMT-positive cells on both the ipsilateral(80 t 7%, n = 10) and the contralateral sides (77 ? 8%,n = 10, Tables 1, 2) were contained in the subretrofacial RVLM area which was 0.7 mm in length. After a T3 injection, for example, an average of 12 ? 5 of 20 t 7 PNMT-positive neurons per section were retrogradely labeled with FG within the ipsilateral subretrofacial RVLM (data from Fig. 4). Rostra1 medullary levels, ( > 1.7 mm rostral to obex), also contained a large proportion of double-labeled cells, even though the PNMT-containing population was greatly diminished. Only a moderate number of RVLM-spinal, PNMT-positive neurons was found at the caudal pole of the C1 column from obex to 1 mm rostral to obex (Figs. 4, 5). This agrees with a previous study suggesting that PNMTcontaining neurons at these caudal levels primarily innervate hypothalamic sites (Tucker et al., ’87). RVLM-spinal, PNMT-positive neurons were bilaterally distributed in the RVLM. The projection, however, was predominantly ipsilateral, as evidenced by the greater number of double-labeled cells per section in the ipsilateral compared to the contralateral C1 column (Tables 1,2). The number of double-labeled neurons within the contralateral RVLM similarly peaked at intermediate levels of the column (Fig. 41, where, for example, an average of 6 t 4 of 17 i 6 PNMT-positive cells were retrogradely labeled per section of the contralateral subretrofacial RVLM following a FG injection into T3 (data from Fig. 4).
The number of RVLM-spinal, PNMT-positive neurons in the subretrofacial RVLM depended on which thoracic segment had been injected with retrograde tracer. The trend was for double-labeling of progressively fewer cells after injections into more caudal segments of the IML column (Fig. 5). Linear regression analysis revealed a significant negative correlation between the number of retrogradely labeled, PNMT-positive cells and the thoracic segment injected for the ipsilateral ( P < 0.001, r = 0.94, n = 7) and contralateral ( P < 0.009, r = 0.86, n = 7) sides (Fig. 6). The analysis predicts that after each progressively caudal IML injection, the number of RVLM-spinal, PNMT-positive neurons decreases by 9 cellsisegment on the ipsilateral side (y = - 8 . 9 ~+ 167) and by 8 cellsisegment on the contralatera1 side (y = - 8 . 0 ~+ 111). The C1 bulbospinal contribution therefore appears segmentally directed to the upper thoracic IML. Calculations indicate that 56 ? 3% (n = 3) of the total PNMT-containing neurons in the ipsilateral subretrofacial RVLM are retrogradely labeled after T3 or T5 injections, in comparison to 35 t 6% (n = 2) after middle level (T7 or T9), or 25 t 8% (n = 2) after lower level (T11 or T13) injections (Fig. 7). The contribution of C1 neurons to the RVLM-spinal projection from the contralateral subretrofacial RVLM was also related to the IML injection level (Fig. 6), amounting to approximately one-third of the total contralateral PNMTpositive input to the innervation of upper thoracic segments (Fig. 7). The amount of retrograde labeling within RVLM levels outside the subretrofacial RVLM depended on which thoracic segment had been injected. A substantial number of
PNMT-positive cell counts in the subretrofacial RVLM Within the subretrofacial RVLM, a total of 246 t 19 (n = 10) PNMT-positive neurons were found on the side ipsilateral, and 253 t 30 (n = 10) on the side contralateral to FG injections (Tables 1,2). This observation was consistent among animals (Fig. 6) and allowed us to make direct (i.e., nonnormalized) comparisons of the total number of PNMT-positive, retrogradely labeled neurons in the subretrofacial RVLM within our experimental group.
Proportion of the C1 cell group innervating the IML Using injections of FG in several levels of the IML column, the number of PNMT-positive, retrogradely labeled neurons was found to approach the total number of PNMT-positive neurons per section of the subretrofacial RVLM (Figs. 3, 9, Table 2). Specifically, at least 81 t 4% (n = 2) of the PNMT-positive population in the ipsilateral, and 60 ~fr 1%(n = 2) of C1 neurons in the contralateral subretrofacial RVLM contribute to the innervation of the IML in thoracic segments (Fig. 7).
Fig. 2. Phenylethanolamine N-methyltransferase (PNMT)-positive neurons in a subretrofacial rostra1 ventrolateral medulla (RVLM) section (A) retrogradely labeled (B)after a fluorogold (FG)injection into T3. Lateral is to the left. Scale bar = 75 Lm.
I. JESKE AND K.E. McKENNA
QUANTXTATXON OF RVL BULBOSPINAL PROJECT101VS 4o
TABLE 2. Cell Counts After Multiple Thoracic Segment Injection'
T3 + 13 PNMT+ 497 455 254 213 RGPNMT+ 219 168 169 129 RGPNMT392 357 195 179 TH 535? 16 560 2 45 2 3 3 ? 17 2 6 1 ? 13 PNMT+ RGPNMT+ 2 2 4 ? 4 185? 11 1 8 9 ? 4 155 2 4 RGPNMT- 359 i 82 277 2 32 204 ? 52 161 i 23
sRF-C1 (1-1.7 mm)
ROSTROCAUDALEXTENT (MM RELATIVE TO OEEX)
Fig. 4. Distribution of PNMT-positive (filled circles), retrogradely labeled PNMT-positive (thick line, open circles), and retrogradely labeled PNMT-negative (dotted line, open squares) neurons within the RVLM from obex to 2 mm rostral to obex on the side ipsilateral (upper graph) and contralateral (lower graph) to a unilateral injection of FG into the IML at T3. Data from one animal. Histological landmarks on the upper graph: OBEX; RF, retrofacial nucleus ambiguus; FN, facial nucleus.
RG PNMT+ sRF-Cl RG PNMT+ C1 ("I Ipsil
'Cell counts of PNMT-positive, retrogradely labeled PNMT-positive, and retrogradely labeled PNMT-negative neurons within the C1 area and subretrofacial C1 area after FG injections into multlple thoracic segments, and the percent of retrogradely labeled PNMT-positive neurons in the suhretrofacial C1 area out ofthe total retrogradely labeled PNMT-positive neurons in the C1 area. Abbreviations same as Table 1 Results are mean k SD for cell counts from multiple thoracic (TH, T3 + T.5 + T7 + T9 and TI11 injection In = 2).
TABLE 1. Cell Counts After Single Thoracic Segment Injection'
c1 IObex-2mml Injection site
sRF-C1 (1-1.7 mm) Ipsil
T3 PNMT+ 525 ? 29 524 ? 36 236 ? 11 242 ? 57 RG PNMT+ 188 ? 18 111 ? 18 138 2 10 9 1 ? 25 RG PNMT- 365 ? 13 262 ? 12 186 2 13 115 ? 21 T5 255 251 557 517 PNMT+ 135 126 82 196 RG PNMT+ 162 114 380 267 RG PNMTT7 248 286 490 559 PNMT+ 97 30 RG PNMT+ 111 37 141 88 RG PNMT209 146 T9 250 285 PNMT+ 629 569 74 38 89 50 RG PNMT+ 248 111 348 201 RG PNMTT11 287 256 623 620 PNMT+ 88 15 RG PNMT+ 107 22 181 151 RG PNMT306 229 T13 231 223 PNMT+ 549 488 21 43 49 29 RG PNMT+ 53 80 117 76 RG PNMT -
RG PNMT+ sRF-C1 RG PNMT+ C1 ") Ipsil
ROSTROCAUDAL EXTENT fMM RELATIVE TO OEEXI
'Cell counts of PNMT-positive, retrogradely labeled PNMT-positive, and retrogradely labeled PNMT-negative neurons within the C1 area and subretrofacial C1 area after FG injections into a single thoracic segment, and the percent of retrogradely labeled PNMT-positive neurons m the subretrofacial C1 area out of the total retrogradely labeled PNMT-positive neurons in the C1 area. PNMT+, PNMT-positive neurons; RG PNMT+ retrogradely labeled PNMT-positive neurons; RG PNMT-, retrogradely labeled PNMTnegative neurons, Ipsil, ipsilateral; Ctrl, contralateral; sRF, subretrofacial. Results are mean 2 SD for cell counts from T3 injection site (n = 2).
both PNMT-positive and PNMT-negative, retrogradely labeled cells were observed in the ventrolateral medulla at caudal levels (obex to 1 mm rostral to obex) following an upper thoracic injection (Fig. 5). In contrast, labeling in the caudal regions was nearly absent following FG injections in the IML of lower thoracic segments (Fig. 5 ) . It has been suggested that some sympathetic preganglionic neurons are organized into target-specific subnuclei within the rostrocaudal extent of the IML column (Appel and Elde, '88; Strack et al., '88). In this regard, we found no
Fig. 5. Distribution of PNMT-positive (filled circles), retrogradely labeled PNMT-positive (thick line, open circles), and retrogradzly labeled PNMT-negative (dotted line, open squares) neurons within the RVL from obex to 2 mm rostral to obex in three experimental animals injected unilaterally with FG into an upper (T3), middle (T9),or lower (T13)level of the IML. Abbreviations same as Figure 3.
evidence for a segmentally directed organization of reticulospinal neurons in the subretrofacial RVLM. Neurons projecting to sympathetic preganglionic neuronal pools at two rostrocaudal extremes of the IML (T3 and T13) were not segregated in the mediolateral plane of the RVLM, but apparently arose from comparable and/or intermingled sites (Fig. 8). Similarly, RVLM-spinal, PNMT-positive, and PNMT-negative neurons were uniformly distributed within the rostrocaudal extent of the subretrofacial RVLM, regardless of the thoracic segment injected with tracer (Fig. 5 ) . These results imply that the location of a reticulospinal neuron in the RVLM is not anatomically related to the location of the thoracic segment it may innervate.
Retrogradely labeled PNMT-negative neurons in the RVLM PNMT immunoreactivity was not detected in a number of neurons located bilaterally in the RVLM adjacent to, and intermingled with RVLM-spinal, PNMT-positive cells. (Fig. 8). The number of RVLM-spinal, PNMT-negative neurons was consistently greater than the number of retrogradely labeled PNMT-positive neurons (Fig. 4, Tables 1,2),suggest-
I. JESKE AND K.E. McKENNA
- - _ _ ----_
l o T13
Fig. 6. Linear regression analysis of the number of PNMT-positive (solid line), retrogradely labeled PNMT-positive (thick solid line), and retrogradely labeled PNMT-negative (dotted line) within the histologically identified subretrofacial RVL and the thoracic segments (T3, T5, T9, T11, T13) unilaterally injected with FG.
Fig. 8. PNMT-positive (open circles), retrogradely labeled PNMTpositive (filled triangles), and retrogradely labeled PNMT-negative (filled squares) neurons within the ipsilateral and contralateral medulla in corresponding sections at the subretrofaciallevel from two experimental animals, one injected with FG at T3 (upper section),the other at T13 (lower section). 1 0 , inferior olive; PYR, pyramids.
Fig. 7. Percent of retrogradely (RG) labeled PNMT-positive neurons out of the total PNMT-positive population (black bars), and retrogradely labeled PNMT-negative neurons out of the total retrogradelylabeled population (whitebars) from counts within the histologically identified levels of the subretrofacial RVL on the side ipsilateral (left) and contralateral (right) to unilateral FG injections of a single thoracic level (T3, T5, Ti', T9, T11, or T13), and multiple levels of the IML IT3 + 13 and T3 + 5 + 7 + 9 + 11 (TH)]. T3 and TH counts are mean i- SD (n = 2).
ing that RVLM neurons using nonadrenergic neurotransmitters contribute to the innervation of the IML and are likely involved in cardiovascular regulation. As in the case of RVLM-spinal, PNMT-positive neurons, the number of retrogradely labeled PNMT-negative neurons was maximal in sections from 1to 1.7 mm rostral to obex (Figs. 4 , 5 , 9 ) .In
contrast to RVLM-spinal, PNMT-positive neurons, a significant number of retrogradely labeled PNMT-negative neurons were present at caudal medullary levels from obex to 1 mm rostral to obex (Fig. 5). Both PNMT-positive and PNMT-negative RVLM-spinal neurons were intermingled within the subretrofacial C1 area. Both populations were morphologically similar, with predominantly fusiform cell bodies, approximately 30 bm diameter in size. In contrast, at caudal levels ( < 1 mm rostral to obex) retrogradely labeled PNMT-negative neurons were also contained in the immediate area 0.2-0.3 mm dorsal to the C1 boundaries defined in this study (see Materials and Methods), and therefore were not included in the counts. As such, RVLM-spinal, PNMT-negative cell counts at these levels might underestimate the nonadrenergic input to the IML.
Contribution of PNMT-negative neurons to the innervation of the spinal cord Following a single thoracic injection, 63 2 8%(n = 7) of the FG-filled cells in the subretrofacial RVLM on the ipsilateral side and 69 2 14% (n = 7) on the contralateral side consisted of PNMT-negative cells (Fig. 7). This was a consistent finding in each of the experimental animals and did not depend on which thoracic level was injected. Even after multiple retrograde tracer injections, when practically the entire C1 column was double-labeled (Fig. 9), 52 t 67r (n = 2) of the total RVLM-spinal population in the ipsilatera1 C1 area consisted of PNMT-negative neurons (Fig. 7). Nonadrenergic population(s) therefore are clearly a major component of the RVLM innervation of the IML. While the PNMT-positive bulbospinal projection appeared t o be segmentally directed to upper thoracic segments (Fig. 6), cell counts of retrogradely labeled PNMTnegative neurons in the RVLM of the same animals remained relatively constant, or at least did not decrease in number with each progressively caudal injection (Table 1). Statistical analysis revealed a lack of significant correlation between the number of retrogradely labeled PNMTnegative neurons and the thoracic segment injected on the ipsilateral ( P > 0.40, n = 7), and contralateral ( P > 0.51,
QUANTITATION OF RVL BULBOSPINAL PROJECTIONS
might have contributed to the RVLM labeling. FG, like most other retrograde tracers, is transported by fibers of passage damaged from either the path of the pipette or volume distortion of the tissue. However, after control injections into the dorsal horn, an area consistently damaged by pipette tracts into the IML, retrogradely labeled cells were never observed in the RVLM. This procedure, as well as eliminating experimental cases in which the tracer had invaded the dorsolateral funiculus, were steps taken to minimize the incidence of nonspecific uptake. 2. Estimates will be affected by the sensitivity of the methods used to identify and label C l cells in the RVLM. For this reason, colchicine was applied prior to immunohisto00 05 1.o 1.5 20 chemical detection to eliminate false negatives which would underestimate the PNMT-positive population in the RVLM. ROSTROCAUOALEXTENT (MM RELATIVE TO OBEX) Dual-fluorescent labeling was chosen to study the C1 contribution to the RVLM-spinal projection because FG Fig. 9. Distribution of PNMT-positive (thin line), retrogradely labeled PNMT-positive (thick line), and retrogradely labeled PNMT- and FS are histologically compatible, and the method negative (dotted line) neurons within the ipsilateral RVL from obex to 2 provides rapid and reliable identification of double-labeled mm rostral to obex after unilateral injections of FG into five segments cells by color. False positives are reduced because of a of the IML. Data from one animal. significant difference in the bandpass of the two fluorochromes. FS never fluoresced under the filter used for n = 7) sides (Fig. 6). Taken together, the data suggest that visualizing FG, though the converse was true with intense the RVLM-spinal PNMT-negative projection may be widely FG labeling. FG peak-through was obvious by color under the microscope, and rarely interfered in the analysis since distributed along the IML column. colchicine pretreatment and processing with a high titer of primary antibody had resulted in intense immunofluoresDISCUSSION cent labeling of neuronal processes, which were always The following observations were made in the present examined to establish which FG cells also contained PNMT. 3. The mediolateral boundaries of the RVLM were destudy: 1 ) PNMT-positive and PNMT-negative neurons within the subretrofacial RVLM (approximately 1 to 1.7 fined in each section by the location of PNMT-positive cells. mm rostral to obex) form the RVLM-spinal innervation of C 1 cells are a reliable morphological marker for the symthe IML; 2) the RVLM-spinal projection is predominantly pathoexcitatory reticulospinal RVLM area (Morrison et al., ipsilateral, but the population has no obvious segmentally ’881, and most respiratory neurons of the Boztinger group directed organization within the RVLM; 3) approximately are located outside of the immediate C1 area (Ellenberger et 80% of the PNMT-positive neurons in the subretrofacial al., ’90). Therefore, boundaries delineated by the PNMTRVLM are retrogradely labeled after multiple injections positive population helped to limit “noncardiovascular” into the IML levels, suggesting that most of the C1 neurons PNMT-negative bulbospinal cells in the immediate surin the subretrofacial RVLM contribute to the innervation of rounding area from our cell counts. An obvious disadvanthe IML, and may be involved in cardiovascular regulation; tage is that potential “cardiovascular” PNMT-negative 4) the C1 bulbospinal projection appears to be segmentally bulbospinal cells located adjacent to, though outside the directed to upper thoracic spinal cord segments, with 56% defined C1 group boundaries, might also be excluded from of the neurons retrogradely labeled after upper, 35% after counts, thus potentially underestimating the PNMTmiddle, and 25% after lower thoracic segment injections; negative bulbospinal contribution from the area. The reand 5) PNMT-negative neurons are the main component of sults, however, indicate that the number of RVLM-spinal, the RVLM-spinal population in the subretrofacial RVLM, PNMT-negative neurons within the C1 area was consiswith at least 50% of the nonadrenergic neurons innervating tently greater than spinal PNMT-positive neurons of the RVLM, and that the former provide the major component all levels of the IML. of the RVLM bulbospinal innervation of the IML. Methodological concerns 4. The rostrocaudal limits of RVLM were defined based The present anatomical study on RVLM efferents sought on results that RVLM-spinal, PNMT-positive, and PNMTto quantitate the number of PNMT-positive and PNMT- negative neurons are topographically organized within the negative neurons contributing to the RVLM-spinal projec- RVLM, within levels easily distinguished from others based tion. The accuracy of the estimates rests on several method- on histological criteria. Therefore, the PNMT-positive and PNMT-negative bulbospinal contributions were calculated, ological factors: not from cell counts in the entire RVLM, as this would 1. It was assumed that RVLM cells retrogradely labeled greatly underestimate the spinal contribution, but from with FG represent neurons which project to the IML. those at subretrofacial levels from 1 to 1.7 mm rostral to Anterograde transport studies have shown that the RVLM- obex. Such boundaries 1) were easily and reproducibly spinal projections are exclusively directed to the sympa- localized in each animal from counterstained tissue secthetic preganglionic cell column (Ross et al., ’84a). The tions; 2) distinctly corresponded with the peak increase in injection sites of the present experiments were centered in the bulbospinal cell population; and 3) correlated well with the IML, but the presence of necrosis at the typical injection the 0.5-0.7 mm sympathoexcitatory reticulospinal RVLM perimeter, indicates that uptake in adjacent spinal regions area of functional (Benarroch et al., ’86)and electrophysioT3+5+7+9+11
I. JESKE AND ICE. McKENNA
logical studies (Brown and Guyenet, '85; Terui et al., '86). A clear disadvantage is that such anatomical boundaries might not overlap exactly with the functional cardiovascular zone. Another design would be to establish coordinates of the RVLM pressor area by chemical stimulation in the same animal which was injected with retrograde tracer and processed subsequently for PNMT immunoreactivity. However, this approach could increase the probability of falsenegative findings, especially since serial volume injections into a region as discrete as the RVLM (0.5-0.7 mm in length) could easily disrupt FG labeling of neurons. Thus we found it incompatible with the goals of this particular study.
C1 contribution to the RVLM-spinal projection
pathetic rhythms (Sun et al., '88a,b). PNMT-positive neurons, for example, did not exhibit the pacemaker potentials of neighboring PNMT-negative neurons during global glutamate receptor blockade (Sun et al., '88b). Furthermore, local application of epinephrine in the IML depresses rather than excites sympathetic preganglionic activity (Guyenet and Cabot, '81), although the effect may well depend on its site of action (Milner et al., '88).
Contribution of nonadrenergic neurons to the RVLM-spinal projection The results of the present study (that PNMT-negative neurons comprise at least 50% of the descending population) indicate that nonadrenergic neurons have a significant contribution to the RVLM-spinal control of arterial pressure. The neurochemical identity of the PNMTnegative populationh) was beyond the scope of these experiments, although candidates can be found in the literature. For example, it is known that RVLM neurons immunoreactive for cholecystokinin (Mantyh and Hunt, '841, dynorphin (Menetrey and Basbaum, '871, enkephalin (Menetrey and Basbaum, '87; Millhorn et al., '87; Sasek and Helke, '891, glutamate (Nicholas et al., '901, neuropeptide-Y (Blessing et al., '871, substance P (Charlton and Helke, '87; Stracket al., '89), somatostatin (Millhorn et al., '87; Strack et al., '89) thyrotropin-releasing hormone (Hirsch and Helke, '88; Sasek et al., '901, and vasoactive intestinal peptide (Leibstein et al., '851, project to the thoracic spinal cord, and that several of these neurotransmitters are contained within axons making synaptic contact with sympathetic preganglionic neurons (Chiba and Masuko, '87; Bacon and Smith, '88; Llewellyn-Smith et al., '90; Morrison et al., '89b, '91a). A cardiovascular role has been suggested for several of these neurotransmitters since their application in the IML excites the activity of sympathetic preganglionic neurons (for reviews see: Nishi et al., '87; McCall, '88a, Kumada et al., '90). Functionally, it was shown that RVLM stimulation which produces a sustained elevation of arterial pressure is accompanied by a significant release of neuropeptide Y (Morris et al., '87) and substance P (Takano et al., '84) into the spinal subarachnoid space.
Functional studies indicate that the area containing sympathoexcitatory RVLM-spinal neurons has its rostral boundary at the caudal pole of the facial nucleus, and is less than 1 mm in length (Brown and Guyenet, '85; Benarroch et al., '86; Terui et al., '86; Morrison et al., '88). In the present study, we found that the equivalent area, defined histologically, contains PNMT-synthesizing neurons, 80% of which are retrogradely labeled after injections into five levels of the IML. It is thus reasonable to suggest that 100% of the cells would have been labeled if it were technically possible to fill the entire length of the IML column with tracer. Estimates obtained here and in other anatomical studies (Tucker et al., '87; Minson et al., '90) provide quantitative evidence that PNMT-positive neurons are a significant component of the RVLM-spinal projection. Previous observations show that: 1) the C1 area overlaps topographically with the L-glutamate-sensitive pressor area of the RVLM (Dampney et al., '82; Ross et al., '84b; Benarroch et al., '86); 2) lesions of epinephrine neurons or their descending axons abolish responses evoked from the application of drugs to the ventral surface (Benarroch et al., '86); 3) sites of extracellulary identified neurons with cardiovascular-related properties lie in close proximity to PNMT-immunoreactive cell bodies (Morrison et al., '88); 4) the number of PNMT cells (Howe et al., '81) and PNMTenzyme activity (Saavedra et al., '78) are higher in the Segmental organization of the RVLM-spinal ventral medulla of young spontaneously hypertensive compopulation pared to normotensive rats; 5) PNMT activity is elevated in the ventral medulla and spinal cord of normotensive rats In agreement with others (Tucker and Saper, '85; Beluli after baroreceptor denervation (Minson et al., ' 8 5 ) ; and 6) and Weaver, '911, we found an absence of target-specific catecholamine metabolism in the RVLM is increased as a organization among the RVLM reticulospinal neurons proresult of either antidromic stimulation from the IML, jecting to different sympathetic preganglionic levels; rather, hypotension, or hemorrhage (Gillon et al., '90). Thus the they are intermingled in the RVLM. Such an organization cumulative evidence favors a role for C1 epinephrine- resembles that of the superior cervical ganglion, where containing cells in sympathetic vasomotor control. postganglionic neurons with different sympathetic actions In the present study we used an antibody against the (pupil dilatation, piloerection, vasoconstriction) are anato mconverting enzyme PNMT to identify immunohistologically ically commingled, without apparent distinction (Nja and a population of neurons in the rostral medulla that synthe- Purves, '77). It contrasts with the viscerotopic organization size, though they may not actually store, epinephrine (Sved, of the projection from the midbrain periaqueductal gray to '90). This is not to suggest that epinephrine is the neuro- the RVLM (Carrive et al., '89), descending spinal pathways transmitter released from RVLM-spinal terminal endings in the dorsolateral funiculus (Barman and Wurster, '75), under physiological circumstances. Neurotransmitters may and sympathetic preganglionic neurons localized in targetbe coreleased, as indicated by the finding that PNMT is specific groups within segments of the intermediolateral colocalized in some RVLM cells with either glutamate cell column (Appel and Elde, '88; Strack et al., '88). (Nicholas et al., '901, neuropeptide Y (Blessing et al., '87; The lack of a segmentally directed organization within Murukami et al., '891, enkephalin (Ceccatelli et al., '89; the RVLM does not conflict with the physiological findings Murukami et al., '891, or angiotensin I1 (Covenas et al., '90). that the RVLM exerts differential control over sympathetic There has also been some speculation that neurons in the nerves (Barman et al., '84; Hayes and Weaver, '90) and that RVLM other than the C1 group function to generate sym- separate anatomical regions in the RVLM have preferential
QUANTITATION OF RVL BULBOSPINAL PROJECTIONS control over vascular beds (Lovick, ' 8 7 ) ,and postganglionic fibers (Dampney and McAllen, '88). The indications are that while RVLM reticulospinal neurons may not be grouped anatomically depending on which segment they innervate, the organization may relate to the sympathetic function that they mediate.
Segmental organization of the RVLM-spinal projection A greater number of PNMT-positive cells were retrogradely labeled after upper versus lower spinal cord injections, indicating that the C1-spinal projection may be segmentally directed to upper thoracic segments of the spinal cord. In support of this hypothesis is the finding that the density of PNMT-positive terminal-like labeling is greater at upper thoracic levels, and gradually diminishes at lower thoracic segments (Ross et al., '84a). The C1-spinal segmental differences cannot be entirely explained by 1)a limit of the retrograde tracer technique, since only the number but not the intensity of labeling in the RVLM was reduced with lower thoracic injections, nor 2) by uptake by fibers of passage since the dorsolateral funiculus containing descending axons was not damaged or included as part of the injection site and 3) the contribution of nonadrenergic RVLM-spinal neurons in the same area remained relatively constant, and did not depend on the thoracic segment being injected with retrograde tracer. It is possible that factors such as intragriseal projections (Fritschy and Grzanna, '90) or alternatively, inherent cytoarchitectural differences, contributed to the segmental differences. With respect to the latter, the spinal cord lengthens out a t lower thoracic segments, which may be accompanied by a lengthening of the distance between the aggregates of sympathetic preganglionic neurons. Also, three times as many sympathetic preganglionic neurons are contained per millimeter in upper thoracic versus lower thoracic segments of the IML (Henry and Calaresu, '72; Strack et al., '88). Taken together, it could be that while equal volumes of FG were deposited at each IML level, more C1-spinal neurons were labeled from upper thoracic injections because the segments contain densely interspaced and populated cell clumps of sympathetic preganglionic neurons in comparison to lower levels. Nonetheless, the finding that the C1-spinal, but not the nonadrenergic RVL-spinal contribution is segmentally directed to upper thoracic regions, suggests the possibility that functional specificity in the sympathetic nervous system is neurochemically encoded. There is precedence from previous studies that the distribution of terminal-like labeling of serotonin and the neuropeptides is nonuniform, with peaks of terminal density within distinct segments, and even subdivisions of the sympathetic preganglionic cell column (Krukoff, '87; Krukoff et al., '85). Such an organization could form the substrate for neuropeptide-specific modulation of target-specific groups of sympathetic preganglionic neurons. For example, Holets and Elde ('82) found that sympathetic preganglionic neurons projecting to the adrenal medulla are surrounded by somatostatin terminallike labeling, but never by oxytocin and neurophysin terminal-like labeling which abundantly surrounds nonadrenal preganglionics in the IML at other segmental levels.
ACKNOWLEDGMENTS The authors gratefully acknowledge Drs. S.F. Morrison and A.C. Bonham for their critical evaluations of the
manuscript, and Cindy Polchow for excellent photographic assistance.
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