INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

THE ROSTRAL VENTROLATERAL medulla (RVLM) plays an important role in the regulation of cardiovascular function (4, 8). Electrical or chemical stimulation of the RVLM produces large increases in arterial pressure and heart rate (HR) (22, 34). Conversely, bilateral electrolytic destruction or chemical inactivation of this area produces a decrease in blood pressure comparable to that seen after cervical spinal transection or total sympathetic blockade (7, 22, 34). In addition, inhibition of the RVLM blocks baroreflexes (7) and other cardiovascular reflexes (25, 32). Thus RVLM neurons appear to play a prominent role in the tonic and reflexive control of sympathetic vasomotor outflow.

Many RVLM neurons directly innervate preganglionic sympathetic neurons in the spinal cord (18, 21). RVLM bulbospinal neurons can be divided into two populations based on neurochemical phenotype: those containing the enzyme phenylethanolamine-N-methyltransferase (PNMT) and other enzymes involved in catecholamine biosynthesis, known as the C1 cell population, and those that are not catecholaminergic. Difficulty has arisen in determining the relative contributions of these two cell populations to cardiovascular regulation, although evidence is available to support a role of each of these cell groups. The most active pressor region of the RVLM is found in an area overlapping the distribution of the C1 cell population (22). Approximately half of the spinally projecting neurons of the RVLM are C1 cells (28, 30). Both C1 and non-C1 spinally projecting RVLM neurons respond to changes in baroreceptor input, as demonstrated by direct electrophysiological recordings (9, 23), as well as by Fos expression in response to hydralazine-induced hypotension (28). Thus it appears likely that both populations contribute in some manner to cardiovascular control.

The lack of a technique for creating selective lesions of either of these populations of RVLM neurons has hindered progress toward a more complete understanding of their particular roles in cardiovascular function. The nonspecificity of electrolytic or excitotoxic (e.g., kainate) lesions makes these techniques unsuitable for destroying specific cell types in an area containing interspersed cell populations, such as the RVLM. The C1 cell population is unaffected by the classical catecholaminergic toxin, 6-hydroxydopamine (6-OHDA) (12); transport of 6-OHDA into catecholaminergic cells is necessary for its toxicity, and C1 cells do not express messenger RNA for known catecholamine transporters (13).

Recently, a novel immunotoxin consisting of an antibody to dopamine -hydroxylase (DH), conjugated to a toxin, saporin, has been shown to selectively destroy DH-containing neurons when injected into the cerebral ventricles (20, 35) or directly into brain tissue (3). DH is associated with the internal membrane of vesicles, and is therefore exposed extracellularly during exocytotic neurotransmitter release. This exposure allows the antibody portion of the exogenously administered immunotoxin to bind to DH. Upon endocytotic recycling of the vesicular membrane, the anti-DH-saporin toxin is taken into the neuron. Once inside the neuron, saporin inactivates the 60s subunit of ribosomes, thus prohibiting protein synthesis and ultimately resulting in cell death (24). Thus saporin toxin, conjugated to an antibody that binds to DH, should effectively and selectively destroy DH-containing neurons. Because DH is present in PNMT-containing neurons as well as noradrenergic neurons, this toxin should destroy PNMT-containing neurons. Indeed, Wrenn et al. (35) have shown this to be the case after injection of the toxin into the cerebral ventricles. The aim of the present study was to determine whether injections of anti-DH-saporin directly into the RVLM can selectively destroy neurons of the C1 cell population and thereby provide an approach to examine the role of C1 neurons in cardiovascular regulation.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Animals. All procedures conformed to National Institutes of Health guidelines and were approved by the University of Pittsburgh Animal Care and Use Committee. Male Sprague-Dawley rats (Zivic Laboratories, Zelienople, PA) weighing 250-470 g at the time of injections into the RVLM were used in these studies. Rats were singly housed and given ad libitum access to pelleted rat chow (Purina, St. Louis, MO) and water. The colony room was maintained at a temperature of 22-23°C and kept on a 12:12-h light-dark cycle.

Anatomical studies. One group of rats (n = 8) received a unilateral injection of anti-DH-saporin [21 ng in 200 nl artificial cerebrospinal fluid (aCSF); 1:20 dilution of concentrated toxin solution provided by D. A. Lappi of Advanced Targeting Systems, San Diego, CA] into the RVLM. This dose was based on results from pilot studies that indicated that a 21-ng dose of anti-DH-saporin in 200 nl aCSF produced a large reduction in the number of PNMT-immunoreactive neurons in the RVLM, whereas smaller effects were seen at lower doses, and no cell loss was observed at doses of 1 ng or less. Doses 50 ng produced obvious necrosis at the injection site (see Fig. 3D). Control rats received a unilateral injection of saporin conjugated to an antibody raised against mouse IgG (21 ng in 200 nl aCSF; Mab-Zap, Advanced Targeting Systems) (n = 6) or a mouse antibody against DH (same antibody used in toxin conjugate) with no saporin conjugated to it (20-50 ng in 200 nl aCSF; Chemicon, Temecula, CA) (n = 2). An additional control group (n = 5) received bilateral injections of aCSF (200 nl) into the RVLM.

To determine the specificity of the immunotoxin for the C1 cell population within the RVLM, one group of rats (n = 4) received a unilateral injection of anti-DH-saporin (21 ng in 200 nl aCSF) into the RVLM followed 2 wk later by bilateral injections of Fluorogold (FG; Fluorochrome, Englewood, CO) into the rostral thoracic spinal cord. Then, 2-3 wk later, to elicit the expression of Fos protein in barosensitive neurons, these rats were injected with a hypotensive dose of hydralazine (10 mg/kg ip) 90-120 min before perfusion (5, 28).

A subset of rats (n = 4) that received only a unilateral injection of 21 ng anti-DH-saporin into the RVLM were used to assess the destruction of other DH-containing cell populations. Data from these rats were compared with data from other rats (n = 3) that received a unilateral injection of FG (4%, 60 nl) into the RVLM to determine which DH-containing neurons project to the portion of the RVLM receiving the toxin injection.

To assess the destruction of DH-containing neurons of the area postrema, one group of rats (n = 5) received bilateral injections of anti-DH-saporin (21 ng in 200 nl aCSF) into the RVLM, and qualitative comparisons of the number of DH-positive area postrema neurons were made between these rats and bilaterally aCSF-injected rats (n = 5). To verify the efficacy of the bilateral toxin injections in this group, the extent of C1 cell depletion also was quantified.

RVLM microinjections (dorsal approach). Animals were anesthetized with halothane (2-5% in 100% oxygen) and placed in a stereotaxic instrument with the incisor bar positioned 11 mm below the interaural line. After partial removal of the occipital bone, the meninges covering the dorsal surface of the brain stem were cut and retracted, and calamus scriptorius was visualized. For all dorsal approach microinjections into the RVLM, a glass micropipette (outer tip diameter, 40-75 µm) was positioned as follows: with the pipette angled 20° rostrally, the pipette tip was placed on the dorsal surface of the brain stem at the caudal tip of the area postrema, then moved 1.8 mm lateral and 1.8 mm rostral to this landmark. The tip was then advanced 2.8 mm through the medulla. These coordinates are based on the region of the RVLM in which the greatest pressor response to L-glutamate is elicited (10, 11). Microinjections were given in a volume of 200 (saporin conjugates) or 60 nl (FG) over a 30- to 120-s period with the use of a PicoPump (WPI, New Haven, CT). After microinjection the pipette was left in place for 1-2 min. Then the pipette was withdrawn, the skin wound was closed, and rats were given an injection of Bicillin (30,000 U im) and returned to their home cage. A minimum of 10 days recovery time was permitted before further procedures were performed. Pilot studies indicated that before 3 days after anti-DH-saporin injection, there was no detectable loss of C1 cells, whereas significant C1 cell loss was seen by 10 days after the toxin injection. Wrenn et al. (35) also have reported that anti-DH-saporin produces maximal cell loss within 2 wk of intraventricular administration.

FG injections into the spinal cord. Two weeks after toxin injection, rats that were to receive FG injections into the spinal cord were anesthetized with halothane (2-5% in 100% oxygen) and placed in a stereotaxic instrument. An incision was made between the shoulder blades, and the musculature was removed from around the vertebral column at the level of the first and second thoracic vertebrae. The spinal cord was stabilized by an attachment of the second thoracic vertebra to the stereotaxic frame, and a laminectomy was performed at the level of the first thoracic vertebra. Four 100-nl microinjections of 4% FG, spaced 1.0 mm apart in the rostrocaudal plane, were made bilaterally (for a total of 8 injections) through a glass micropipette (40-75 µm OD). The coordinates for these microinjections were 0.8 mm lateral to the midline and 1.0 mm ventral to the dorsal surface of the spinal cord. After FG injections, the skin wound was closed and animals were given an injection of Bicillin (30,000 U im) and returned to their home cages. Animals were allowed to recover for 2-3 wk and then were injected with a hypotensive dose of hydralazine (10 mg/kg ip) ~90-120 min before perfusion (5, 28).

Perfusion fixation and tissue collection. At the conclusion of each experiment, rats were deeply anesthetized with urethan (2 g/kg ip) and perfused through the heart with 0.9% NaCl followed by a solution (PLP) of 4% paraformaldehyde, 1.4% lysine, and 0.2% sodium metaperiodate in 0.1 M sodium phosphate buffer (SPB). Brains were removed and postfixed in PLP solution for 2-4 h, then cryoprotected in 20% sucrose solution in 0.1 M SPB. A freezing stage microtome was used to cut coronal brain sections with a thickness of 30 µm. Sections were collected (in 6 serially adjacent sets) and stored in cryopreservant solution (33) at 20°C until processed for immunocytochemical staining.

Immunocytochemical processing. A one-in-six series of brainstem sections from each rat was incubated at 4°C for 48 h in rabbit anti-PNMT (1:500-1:2,000, depending on lot number; Protos Biotech, New York, NY). Primary and secondary antisera were diluted in 0.1 M SPB containing 0.3% Triton-X and 1% donkey serum. Sections were rinsed in SPB and then incubated in biotinylated donkey anti-rabbit IgG (1:500; Jackson ImmunoResearch Laboratories, West Grove, PA). Sections were rinsed in SPB and then processed in accordance with the avidin-biotin immunoperoxidase method as previously described (19) with Elite Vectastain reagents (Vector Laboratories, Burlingame, CA). Tissue from a subset of rats was processed for immunocytochemical localization of DH or tyrosine hydroxylase (TH). The procedures for the detection of DH and TH were the same as those for immunocytochemical detection of PNMT, except that the primary antibody used was a polyclonal rabbit anti-TH (1:500; Protos Biotech), a mouse monoclonal anti-DH (1:30,000; Chemicon) or a polyclonal rabbit anti-DH (1:1,000; Protos Biotech). Tissue sections from a subset of rats were stained with cresyl violet. Sections were dehydrated in a graded ethanol series, defatted in xylene, and coverslipped with Histomount (VWR, Pittsburgh, PA).

A one-in-six series of tissue sections from hydralazine-treated animals was processed for triple localization of stimulus-induced neuronal Fos expression, PNMT immunolabeling, and retrogradely transported FG. For this purpose, sections were first incubated for 48 h at 4°C in rabbit anti-Fos antiserum (provided by P. J. Larsen and J. D. Mikkelsen, Denmark; 1:50,000). Sections were rinsed and processed for immunoperoxidase localization of nuclear Fos labeling with the use of biotinylated donkey anti-rabbit IgG and Elite Vectastain reagents as described above. Tissue sections were then incubated for 48 h at 4°C in rabbit anti-PNMT (1:200), rinsed, and incubated for 2 h at room temperature in Cy3-conjugated donkey anti-rabbit IgG (1:200; Jackson ImmunoResearch Laboratories). Sections were rinsed, mounted onto glass slides, and coverslipped as described above. Slides were examined with bright-field illumination to detect Fos labeling, ultraviolet fluorescent filters to detect retrograde FG labeling, and rhodamine filter combinations to detect Cy3 (red) PNMT immunolabeling.

Counts of labeled neurons were performed in every sixth brain stem section (30 of each 180 µm). All immunolabeled and FG-positive perikaryal profiles were counted, regardless of whether or not the nucleus was visible, with the exception of the counts of triple (Fos, FG, and PNMT)- and double (Fos and FG)-labeled neurons. Cells were counted at ×400 magnification with a Zeiss Axioplan 2 microscope and bright-field or fluorescent illumination. Photomicrographs of representative sections were digitally processed with Adobe Photoshop to optimize brightness and contrast.

Physiological studies. A separate group of rats (n = 6) was used to assess the consequences of unilateral destruction of C1 neurons on changes in mean arterial pressure (MAP) and HR elicited by microinjections of glutamate and tyramine into both sides of the RVLM. Rats received a unilateral injection of anti-DH-saporin (21 ng in 200 nl aCSF) into the RVLM. Two to four weeks later a subset of this group (n = 4) was anesthetized with halothane (2-5% in oxygen) and implanted with arterial and venous catheters as previously described above (10). Immediately after the termination of halothane anesthesia, urethan anesthesia (1.5 g/kg over 30 min iv) was started. The arterial catheter was connected to a pressure transducer (Statham p23xl), and arterial pressure and HR were measured and recorded on a polygraph (Model 7DAG, Grass Instruments, Quincy, MA). Rats were placed in a stereotaxic frame in a supine position with the incisor bar positioned at the level of the interaural line. The trachea was cannulated and rats were paralyzed (0.5 mg/kg D-tubocurare, supplemented every hour with 0.17 mg/kg) and ventilated with 100% oxygen (small animal respirator; Harvard Apparatus, South Natick, MA). The upper trachea, larynx, esophagus, and surrounding musculature were cut and retracted, and the occipital foramen and occipital bone were exposed. The basal aspect of the occipital bone was removed. Coordinates for drug microinjections were 2.5-4.0 mm rostral to the caudal tip of the occipital foramen, 1.3-2.3 mm lateral to the basilar artery, and 0.7 mm below the ventral surface of the medulla [adapted from Willette et al. (34)]. After an initial injection of glutamate (1 nmol in 100 nl aCSF) into the RVLM, the rostrocaudal and mediolateral coordinates of the injection site were adjusted systematically by 0.3 mm in each direction, and additional injections of glutamate (1 nmol in 100 nl) were performed. In most cases, if a larger pressor response was found in any direction, then the coordinates were again adjusted systematically from that site by 0.3 mm in each direction (such that sites from which smaller pressor responses were elicited encircled the final injection site). The tyramine injection (10 nmol in 100 nl aCSF) was performed at the injection site from which glutamate elicited the largest pressor response. Because these microinjections into the RVLM hindered our ability to immunocytochemically verify the lesions produced by the toxin, the remaining two animals from this group were used to verify the efficacy of the toxin. A control group of intact rats (n = 4) received injections of glutamate and tyramine into the RVLM in accordance with the same procedures as described above. At the end of the experiment, rats were given an additional dose of urethan (0.3 g/kg) and then were perfused with fixative as described above.

To control for the possible confounds of the destruction of the A5 cell group in addition to the C1 cell group after injection of anti-DH-saporin into the RVLM, the A5 cell group was bilaterally destroyed in a separate group of rats (n = 4), and the physiological assessments described above were performed. Briefly, rats were anesthetized with halothane (2-5% in oxygen) and placed in a stereotaxic apparatus in the prone position with the incisor bar set 2.5 mm below the level of the interaural line. An incision was made and the skull was exposed. Two small holes were drilled in the skull at points 1.0 mm caudal and ± 2.6 mm lateral to interaural zero. Pargyline (75 mg/kg) was then administered intraperitoneally, a micropipette was lowered into the A5 area (0.2 mm below interaural zero), and 5 µg 6-OHDA in 2 µl vehicle (0.1% ascorbic acid) was infused over 20 min in each side. After a 2-wk recovery period rats were anesthetized and prepared for measuring MAP and HR as described above. Rats received injections of glutamate (1 nmol in 100 nl) and tyramine (10 nmol in 100 nl) into the RVLM as described above. To verify the destruction of the A5 cell group after 6-OHDA infusions into the A5 area, tissue from these animals was processed for the immunohistochemical detection of DH, and the A5 cell population was counted.

Statistical analysis. A one-way ANOVA test with Newman- Keuls post hoc tests was used to compare counts of PNMT-positive neurons of the C1 cell group located within 720 µm caudal to the caudal pole of the facial nucleus in three groups: the right side of bilaterally aCSF-injected rats, the injected side of unilaterally toxin-injected animals, and the noninjected side of unilaterally toxin-injected animals. In addition, a comparison between mean PNMT-positive cell counts in these three groups was performed over the rostrocaudal extent of the C1 cell population by means of a two-way ANOVA. For other cell populations, cell counts from the noninjected and toxin-injected sides of unilaterally toxin-injected animals were compared with each other and with cell counts from aCSF-injected animals by means of one-way ANOVA tests with Newman-Keuls post hoc tests. In animals receiving a unilateral injection of anti-DH-saporin into the RVLM, injections of FG into the spinal cord, and an injection of hydralazine before perfusion, counts of double (Fos positive and FG positive but PNMT negative)- and triple (Fospositive, FG positive, and PNMT positive)-labeled neurons of the C1 area were compared between the toxin-injected and noninjected sides with one-tailed unpaired t-tests. Comparisons of baseline values and peak changes in MAP and HR elicited by injections of glutamate and tyramine into the RVLM between the injected and noninjected sides of unilaterally toxin-injected animals, both sides of intact control rats, and bilaterally A5-lesioned rats were performed with one-way ANOVA tests with planned comparisons between pairs. All statistical tests were done with Prism statistical software (GraphPad Software, San Diego, CA) or SYSTAT statistical software (SYSTAT, Evanston, IL).

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Destruction of the C1 cell population with injection of anti-DH-saporin in the RVLM. PNMT-positive cells were found from ~1 mm caudal to the obex to the caudal pole of the facial nucleus, with the majority present 1-2 mm rostral to the obex (Fig. 1). Two weeks after unilateral injection of 21 ng of anti-DH-saporin into the RVLM (n = 8), the number of PNMT-positive neurons in the area of the RVLM was markedly reduced. In the region extending caudally for 720 µm from the caudal pole of the facial nucleus, which completely encompasses the area from which pressor responses of at least 20 mmHg can be elicited by injection of 1 nmol glutamate in 100 nl aCSF (Ito and Sved, unpublished observations), the number of PNMT-positive neurons was reduced by 88% ipsilateral to the toxin injection and by 24% contralateral to the toxin injection compared with similar counts from aCSF-injected animals (each, P < 0.001; Figs. 1 and 2). Remaining C1 neurons ipsilateral to the toxin injection were never found near the center of the affected region, with the rare exception of one or two morphologically abnormal neurons (Fig. 2C). In most cases, the area of C1 cell depletion appeared relatively normal in Nissl-stained sections, except for the presence of small glia-like cells (Fig. 3C). However, in 6 of the 17 rats that were included in the anatomical studies and received an injection of 21 ng anti-DH-saporin into the RVLM, a small area of necrosis, never exceeding 600 µm in diameter, was present at the site of injection. The C1 area is shown in representative sections from an aCSF-injected and a unilaterally toxin-injected animal in Fig. 2. The reduction in the number of PNMT-positive (C1) neurons appeared to extend rostrocaudally from the site of injection in a graded fashion. The most complete depletion was seen ~900-1,980 µm rostral to obex, whereas a smaller reduction was observed from 720 µm rostral to obex to 1,080 µm caudal to obex (Fig. 1). Two-way ANOVA indicated that both toxin injection and distance from obex had a significant effect on cell count (each, P < 0.0001) and that there was a significant interaction between the effects of toxin injection and distance from obex on cell count (P < 0.0001). A similar profile of C1 cell loss was seen when brain stem tissue sections were immunocytochemically stained for DH or TH instead of PNMT, except that more labeled neurons were observed caudally due to the additional immunostaining of the A1 cell group.

View larger version (24K): [in this window] [in a new window]   Fig. 1.   Effect of anti-dopamine -hydroxylase (DH)-saporin injected into rostral ventrolateral medulla (RVLM) on number of phenylethanolamine-N-methyltransferase (PNMT)-positive cells in RVLM. Number of PNMT-positive cells (unilateral) in ventrolateral quadrant are shown in bilaterally artificial cerebrospinal fluid (aCSF)-injected rats (; n = 5) and in unilaterally toxin-injected rats (n = 8) on toxin-injected () and noninjected () sides. Note graded depletion of C1 cell population after ipsilateral anti-DH-saporin injection. Also note area sustaining largest depletion corresponds to level at which glutamate elicits large pressor responses (unpublished observation). Cell counts were made in every sixth 30-µm section over rostrocaudal extent of C1 area. Values are means ± SE.

View larger version (86K): [in this window] [in a new window]   Fig. 2.   Representative photomicrographs of RVLM from anti-DH-saporin-injected rat and control rat. A: PNMT-positive neurons in C1 area of rat that received bilateral injections of aCSF into RVLM. B and C: photomicrographs of one tissue section from unilaterally toxin-injected animal; RVLM contralateral to toxin injection is shown in B, whereas RVLM ipsilateral to toxin injection is shown in C. All photomicrographs depict similar rostrocaudal level of RVLM (within 720 µm caudal to caudal pole of facial nucleus). Note paucity of PNMT-positive neurons ipsilateral to injection of anti-DH-saporin (C). Scale bar = 200 µm.

View larger version (58K): [in this window] [in a new window]   Fig. 3.   Representative photomicrographs of Nissl-stained sections through RVLM in rats receiving injections of aCSF (A) or increasing doses of anti-DH-saporin: 10.5 (B), 21 (C), or 53 ng (D). Note presence of neuronal profiles and relative absence of stained nucleoli in A and B. In C note presence of neuronal profiles and proliferation of stained nucleoli indicative of glial cells. In contrast note lack of neuronal profiles and intense proliferation of stained nucleoli indicative of glial cells in D. Scale bar = 200 µm.

Control injections were performed to verify that the lesions produced by anti-DH-saporin injection were due specifically to the immunotoxin. The individual components of the immunotoxin did not produce a loss of C1 cells: no reduction in the number of C1 neurons was observed after injection of saporin conjugated to mouse IgG, or after injection of an unconjugated mouse antibody to DH (data not shown). In rats receiving these control injections, there was minimal gliosis in the RVLM; this gliosis was similar to that observed after aCSF injection into the RVLM (Fig. 3A).

Specificity for the C1 cell population: other barosensitive bulbospinal neurons of the RVLM. To determine whether noncatecholaminergic spinally projecting barosensitive neurons of the RVLM were destroyed by the anti-DH-saporin injection, one group of rats that received unilateral injections of toxin also received injections of FG into the spinal cord and were treated with hydralazine to elicit neuronal Fos expression (n = 4). In tissue stained for Fos and PNMT, the neurons containing both FG and Fos but not PNMT were counted in every sixth section in an area defined rostrally by the caudal pole of the facial nucleus and extending caudally for 720 µm. This area was also defined medially by the inferior olive, laterally by the spinal trigeminal tract, dorsally by the nucleus ambiguus, and ventrally by the ventral surface of the brain stem. Cell counts of the toxin-injected and noninjected sides were compared. These counts were not significantly different (P > 0.2, Table 1). Cell counts of neurons labeled for FG, Fos, and PNMT from the same sections were performed, and the toxin-injected and intact sides were compared; a nearly complete loss of spinally projecting, PNMT-positive, Fos-positive neurons was noted on the toxin-injected side (P < 0.003, Table 1).

Specificity for the C1 cell population: other DH-containing cell populations. To identify DH-containing cell populations that project to the portion of the RVLM receiving the toxin injection and are therefore potentially subject to destruction by the toxin, unilateral injections of FG were made into the RVLM, and tissue sections were processed for the detection of DH, TH, or PNMT. These FG injections encompassed a roughly spherical area of the ventrolateral medulla extending caudally for ~2,000 µm from the caudal pole of the facial nucleus. The injection sites were mainly contained within an area defined medially by the inferior olive, laterally by the spinal trigeminal tract, dorsally by the nucleus ambiguus, and ventrally by the ventral surface of the brain stem (Fig. 4). On the side contralateral to the FG injection, 14 ± 3% of the PNMT-positive C1 neurons contained FG labeling. In addition, a small proportion of noradrenergic neurons of the area postrema contained FG. Some neurons of the ipsilateral and contralateral caudal ventrolateral medulla (CVLM) also contained FG. Although most of the labeled cells in the CVLM were located dorsal to the A1 noradrenergic cell population, ipsilateral to the FG injection 20 ± 5% of the A1 cell population contained FG, whereas contralateral to the FG injection 5 ± 2% of the A1 neurons were retrogradely labeled with FG. The medial and commissural regions of the nucleus of the solitary tract also were found to contain FG-labeled neurons, although few of these stained for DH or TH; <4% of A2 neurons contained FG. Neurons of the A5 cell population, as well as neurons slightly dorsal to the A5 area, also contained FG. Ipsilateral to the FG injection 30 ± 6% of the A5 cell population contained FG, whereas contralateral to the FG injection 22 ± 7% of this population contained FG. In addition, sparse FG labeling was observed in the A6 cell population. In tissue stained for PNMT, a number of neurons from the ipsilateral (17 ± 7) as well as the contralateral (8 ± 4) C2 and C3 cell population were found to contain FG; this represents ~14% of the total number of cells within this population.

View larger version (80K): [in this window] [in a new window]   Fig. 4.   Location of Fluorogold (FG) tracer deposit in RVLM. A: schematic of coronal section through C1 area of RVLM adapted from Swanson (29). Box indicates area represented in photomicrograph (B), which illustrates typical FG injection site in RVLM. This injection site corresponds to level of RVLM (~1,200 µm rostral to obex) at which a large number of C1 neurons are located and at which microinjections of glutamate elicit large pressor responses (11, 22). IO, Inferior olive; NA, nucleus ambiguous; NTSm, medial nucleus of the solitary tract; PGRNl, lateral paragigantocellular reticular nucleus; py, pyramidal tract; SPVI, spinal trigeminal nucleus.

In light of these results, we sought to determine whether injection of anti-DH-saporin into the RVLM destroyed other DH-containing cell populations in addition to the C1 cell group. For this purpose, catecholaminergic neurons of the A1, A2, A5, and A7 cell populations were counted in every sixth section through the brain stem of four rats with extensive unilateral C1 lesions and in four rats that received aCSF injections. The cell counts from the injected and contralateral sides of toxin-injected rats were compared with each other and with the counts from aCSF-injected rats (Table 2). For the A1, A2, and A7 cell populations no differences in cell counts were noted between toxin-treated and control rats. In addition, DH-containing neurons remaining in these regions after the toxin injection appeared morphologically normal. However, cell counts of the A5 cell population differed between the injected side of unilaterally toxin-injected rats and aCSF-injected rats, as well as the noninjected side of unilaterally toxin-injected rats and aCSF-injected rats (P < 0.001 and 0.01, respectively). Also, cell counts for the A5 cell population differed between the injected and contralateral sides of unilaterally anti-DH-saporin- injected animals (P < 0.05). Neurons of the A5 cell population remaining after the toxin injection appeared morphologically normal. Representative sections through these areas can be seen in Fig. 5. Due to the density of DH-containing neurons in the A6 cell population, cell counts of these neurons were not performed; however, this area appeared histologically normal in unilaterally toxin-injected rats. Because the area postrema lies along the midline, qualitative assessments of DH-labeled neurons of the area postrema were performed in bilaterally toxin-injected animals and compared with those of bilaterally aCSF-injected rats. Compared with vehicle-injected control rats (n = 4), bilaterally toxin-injected rats (n = 5) were found to have a 94% reduction in the number of PNMT-labeled neurons in an area of the RVLM defined rostrally by the caudal pole of the facial nucleus and extending caudally for 720 µm. In addition, these rats continued to gain weight normally during the 3-4 wk postinjection and exhibited normal health and behavior. In these rats there was a tendency toward larger depletions of the A5 cell group observed in bilaterally anti-DH-saporin-injected animals compared with the injected side of unilaterally anti-DH-saporin-injected animals (55 ± 10 vs. 69 ± 7 cells remaining, respectively). However, no apparent difference was noted in the qualitative assessments of DH-labeled neurons of the area postrema between these two groups. The C2 and C3 cell populations also appeared normal. Due to the relatively small number of C2 and C3 neurons labeled by FG injection into the RVLM, we would expect toxin-induced depletions to be so small as to easily go undetected; therefore, quantitative counts of these populations were not performed.

View larger version (123K): [in this window] [in a new window]   Fig. 5.   Representative photomicrographs of DH-positive cell groups from control rat (left) and rat in which anti-DH-saporin was injected unilaterally into RVLM (right). Photomicrographs depict DH-positive neurons (A-F) or tyrosine hydroxylase (TH)-positive neurons (G, H) in medulla and pons of rats receiving bilateral injections of aCSF (A, C, E, G), or ipsilateral injection of anti-DH-saporin (B, D, F, H) into RVLM. A and B correspond to area containing area postrema and A2 cell population. C and D illustrate area containing A1 neurons. Area containing A5 neurons is depicted in E and F. G and H depict area containing A7 neurons. There are no obvious treatment-related differences in number of DH-containing neurons except in A5 cell group (E and F). Scale bar = 200 µm.

Consequences of the destruction of C1 neurons on MAP and HR responses elicited by administration of pharmacological agents into the RVLM. Baseline values for MAP and HR were similar in urethan-anesthetized, unilaterally toxin-injected rats and control rats prepared for microinjections into the RVLM via a ventral approach (control rats: n = 4, 109 ± 3 mmHg, 418 ± 12 beats/min; unilaterally toxin-injected rats: n = 4, 112 ± 3 mmHg, 410 ± 10 beats/min). Injection of glutamate (1 nmol in 100 nl) unilaterally into the RVLM increased blood pressure in all rats. The peak increase in MAP occurred within 30 s of injection, and blood pressure returned to baseline in ~2.5 min; neither the time of peak response nor the duration of the response differed between control and toxin-injected rats (Fig. 6). However, compared with control rats, the pressor response evoked by glutamate injected into the toxin-treated RVLM was reduced by ~37%, whereas the response elicited from the contralateral RVLM was enhanced by ~69% (Figs. 6 and 7). HR changes evoked by injections of glutamate into RVLM were variable and did not differ among groups. In three of the four control rats, the injection sites from which the maximal pressor responses were elicited were located at the same rostrocaudal and corresponding mediolateral coordinates on both sides of the midline. In the remaining control animal the rostrocaudal coordinates for the injections were the same on both sides, but the mediolateral coordinates differed by 0.3 mm (+2.0 and 2.3 mm from basilar artery). In contrast, the injection sites from which the maximal pressor responses were elicited were located at the same rostrocaudal and corresponding mediolateral coordinates on both sides in only one unilaterally toxin-injected animal. In the other three unilaterally toxin-injected animals the maximal pressor site on the toxin-injected side was located 0.3 mm lateral to the maximal pressor site on the noninjected side in one case, 0.3 mm medial to the maximal pressor site on the noninjected side in a second animal, and 0.3 mm caudal to the maximal pressor site on the noninjected side in the third rat.

View larger version (14K): [in this window] [in a new window]   Fig. 6.   Representative polygraph records illustrating effects of glutamate injected into RVLM in intact control rat (A), into injected side of unilaterally toxin-injected rat (B), and into noninjected side of unilaterally toxin-injected rat (C). Arrows indicate time of injection. Scale bars = 1 min. Note differences in magnitude of evoked pressor responses between 3 panels. Group data are shown in Fig. 7. Top traces, arterial pressure (mmHg); middle traces, MAP (mmHg); bottom traces, heart rate (beats/min).

View larger version (24K): [in this window] [in a new window]   Fig. 7.   Bar graphs depicting effects on mean arterial pressure (MAP) of glutamate or tyramine injected into RVLM on left (open bars) or right (shaded bars) side of intact control rats (n = 4) or on noninjected (hatched bars) or injected (filled bars) side of unilaterally toxin-injected rats (n = 4). Values are means ± SE. Baseline values were not statistically different between control rats (109 ± 3 mmHg) and unilaterally toxin-injected rats (112 ± 3 mmHg). * Statistically different from all other groups, P < 0.05. There were no statistically significant differences in change in heart rate between any groups (P > 0.1), and baseline values were not statistically different between control rats (418 ± 12 beats/min) and unilaterally toxin-injected rats (410 ± 10 beats/min). Representative responses are presented in Figs. 6 and 8.

In intact control animals injection of tyramine into the same sites at which glutamate elicited a maximal pressor response led to a change in MAP of 26 ± 3 and 22 ± 9 mmHg and a change in HR of 15 ± 6 and 21 ± 7 beats/min on the left and right sides, respectively (Figs. 7 and 8). In contrast, the changes in MAP and HR elicited by injection of tyramine into the maximal pressor area were 0.8 ± 0.3 mmHg and 10 ± 7 beats/min on the injected side of unilaterally toxin-injected rats and 34 ± 5 mmHg and 14 ± 7 beats/min on the noninjected side (Fig. 7). In control rats injection of tyramine into the site at which glutamate elicited the maximal pressor response resulted in a decrease in MAP and HR that peaked within 2.5 min and returned to baseline in 3.1 ± 1.1 min (Fig. 8A). The time course of the tyramine response in the noninjected side of unilaterally toxin-injected animals was not significantly different from this response in control rats (Fig. 8, B and C).

View larger version (16K): [in this window] [in a new window]   Fig. 8.   Representative polygraph records illustrating effects of tyramine injected into RVLM in intact control rat (A), into injected side of unilaterally toxin-injected rat (B), and into noninjected side of unilaterally toxin-injected rat (C). Arrows indicate time of injection. Scale bars = 1 min. Note lack of depressor response in B. Group data are shown in Fig. 7. Top traces, arterial pressure (mmHg); middle traces, MAP (mmHg); bottom traces, heart rate (beats/min).

To control for the destruction of neurons of the A5 cell group in addition to the destruction of C1 neurons after injection of immunotoxin into the RVLM, the responses evoked by injection of glutamate or tyramine into the RVLM were assessed in rats in which the A5 cell group alone was bilaterally destroyed (n = 4). Bilateral infusion of 6-OHDA into the A5 area resulted in an ~75% reduction in the number of A5 neurons compared with control animals. Compared with control rats, in A5-lesioned rats there were no differences in baseline values (MAP, 109 ± 3 mmHg; HR, 418 ± 7 beats/min) or changes in MAP or HR evoked by injection of tyramine (MAP, 22 ± 5 mmHg; HR, 3 ± 2 beats/min) or glutamate (MAP, 35 ± 7 mmHg; HR, 23 ± 11 beats/min) (P > 0.6 in all cases).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Validation of the technique as a tool for destroying C1 cells. The primary goal of the present study was to evaluate the utility of intraparenchymal injections of anti-DH-saporin for creating selective lesions of the C1 cell population, specifically the C1 cells in the region of the RVLM involved in cardiovascular regulation. To validate this technique it was necessary to first demonstrate that the C1 cell population was destroyed by the toxin injection. Indeed, injections of anti-DH-saporin into the RVLM produced a marked loss of PNMT-containing neurons in this area. In the region of the RVLM that is most involved in cardiovascular regulation, ~85-90% of the C1 cells were destroyed by unilateral toxin injections. It is also worth noting that bilateral injections of anti-DH-saporin into the RVLM resulted in an apparently more extensive depletion of C1 neurons (~95% depletion). This finding is consistent with our observation of a contralateral projection of C1 neurons; more complete lesions would be expected after bilateral toxin injections due to the additional destruction of C1 neurons with projections to the contralateral injection site. More importantly, this finding demonstrates our ability to bilaterally destroy the C1 cell group, a technique that should be useful in future studies examining the role of these neurons in cardiovascular regulation.

Several lines of evidence support the assertion that the loss of PNMT immunoreactivity is indicative of C1 neuronal death. First, the mechanism of toxicity of the saporin molecule is not consistent with the possibility that these cells are still functional but not able to produce a specific enzyme. Saporin, free within a cell, is known to irreversibly inactivate the 60s subunit of ribosomes, thus prohibiting protein synthesis and ultimately resulting in cell death. In addition, anti-DH-saporin injections eliminated the immunocytochemical detection of multiple enzymes (TH, DH, and PNMT) in C1 cells. Toxin injection into the RVLM also reduced the number of RVLM neurons retrogradely labeled from the spinal cord. Furthermore, the inability to stain C1 neurons for TH, DH, or PNMT immunoreactivity in animals allowed to survive more than 5 wk after the toxin injection indicates that this is a long-lasting change, consistent with cell death. Other studies also have indicated an inability to immunocytochemically detect these enzymes for up to 9 mo after rats have received intracerebral ventricular injections of anti-DH-saporin (35). Prominent gliosis within the area of the toxin injection 2 wk after the injection is also indicative of neuronal death. Considered together, these results support the conclusion that the toxin killed the C1 cells.

A second critical issue that had to be addressed in evaluating this lesion was whether the injection of anti-DH-saporin into the RVLM destroyed non-C1 neurons in the RVLM. The present study provides solid evidence that the non-C1 neurons in the RVLM are unaffected by the toxin injection. Most notably, the number of non-C1 bulbospinal, barosensitive neurons of the RVLM is not significantly reduced by injection of anti-DH-saporin into the RVLM. In addition, several observations support the proposed mechanism of specific uptake of anti-DH-saporin by DH-containing neurons and thereby provide support for the assertion that the toxin did not enter and kill neurons that do not contain DH. These observations include the fact that injection of the saporin toxin molecule conjugated to an antibody against mouse IgG led to no reduction in the C1 cell population. Furthermore, injection of saporin conjugated to mouse IgG into the RVLM, which did not result in the destruction of C1 neurons, also did not result in prominent gliosis, suggesting an absence of nonspecific uptake of the toxin and cell death.

Another key aspect in evaluating this technique was to determine whether the anti-DH-saporin injection into the RVLM was creating lesions of other DH-containing cell populations in addition to the C1 cell population. Given the proposed mechanism of specific uptake of anti-DH-saporin by DH-containing neurons, one would expect that other DH-containing neurons with projections to the RVLM would be destroyed. There is a great deal of inconsistency among previous studies that have investigated the noradrenergic input to the RVLM. One study reported that the only significant noradrenergic input to the RVLM is from the A5 cell population (26). In contrast, another study has reported that in rabbits the only noradrenergic input to the RVLM originates from neurons of the area postrema (2). Yet another study demonstrated that a small number of locus coeruleus neurons were retrogradely labeled by tracer injections into the RVLM (31). In the present study neurons in each of these noradrenergic cell populations, as well as the A1 cell population, were labeled after injections of FG into the RVLM. It should be noted however, that FG can be taken up by damaged fibers of passage and transported retrogradely to the soma; therefore a major limitation of these studies is that neurons labeled by FG may not send projections to the RVLM but may simply have projections through this area. However, due to the presumed lack of exocytotic transmitter release along fiber tracts, anti-DH-saporin injected into the RVLM would not be expected to be taken up by fibers of passage and should therefore destroy only those neurons with projections to the RVLM and not those neurons with projections through this area. The only noradrenergic cell population significantly depleted by toxin injections into the RVLM was the A5 cell population. The 49% ipsilateral loss of A5 cells and the 25% contralateral loss of A5 cells after a unilateral injection of anti-DH-saporin into the RVLM are consistent with the number of A5 neurons found to putatively project to the RVLM. Because cell counts of the A5 cell population in unilaterally toxin-injected rats revealed a bilateral projection from the A5 cell group to the RVLM, counts of A5 cells were performed in bilaterally toxin-injected rats to determine whether different sets of neurons project to the ipsilateral vs. the contralateral RVLM. The 59% depletion of the A5 cell group in bilaterally toxin-injected rats compared with the 49% depletion ipsilateral to the injection in unilaterally toxin-injected rats suggests that a portion of the A5 cells that project to the contralateral RVLM are distinct from those A5 cells that project to the ipsilateral RVLM. The lack of cell loss in the area postrema and locus coeruleus is not surprising; taking into account the large number and high density of DH-containing neurons of the area postrema and the locus coeruleus, as well as the small proportion of neurons in these populations found to project to or through the RVLM, small toxin-induced depletions might easily go undetected. In contrast, a more sizable number of A1 cells were retrogradely labeled from the RVLM, and yet no loss of A1 cells was apparent after toxin injection into the RVLM. As mentioned previously, one explanation for this finding is that neurons of the A1 cell population do not actually terminate within the RVLM (14); their axons coursing through the RVLM might pick up FG injected there, whereas they would not endocytose saporin toxin due to the presumed lack of exocytotic transmitter release along fiber tracts.

Mechanism of uptake of the toxin by neurons: evidence for somatodendritic exocytotic release by catecholaminergic vesicles in C1 cells. The proposed mechanism of saporin toxin uptake in the present experimental paradigm is dependent on exocytosis of DH-containing vesicles and the resulting exposure of DH to the extracellular space. Our results provide empirical evidence that saporin had access to C1 cells via their soma and/or dendrites, although exocytotic release from DH-containing vesicles is classically thought to occur only at axon terminals. Although there are some PNMT-containing axon terminals within the RVLM (16), the origin of these terminals has not yet been determined, and the present retrograde labeling data suggest that at least some of these terminals arise from a small number of C2 and C3 cells and from contralateral C1 cells. Furthermore, the paucity of PNMT-containing terminals within the RVLM (16) does not seem consistent with the extensive lesions produced by injections of toxin into this area. Additionally, when C1 neurons were filled with biotinamide, no recurrent collaterals were seen even though cellular processes were visible several millimeters distal from the soma (23). Hence our results support the possibility that in C1 neurons there is some amount of somal and/or dendritic exocytosis of DH-containing vesicles, similar perhaps to exocytotic release from sympathetic catecholaminergic cell bodies and dendrites (36).

Consequences of the destruction of C1 neurons on MAP and HR responses elicited by administration of pharmacological agents into the RVLM. Having established the utility of anti-DH-saporin for creating selective lesions of the C1 cell population, it is now possible to begin using this technique to address the issue of the functional importance of the C1 cell population in cardiovascular regulation. The depressor effect of tyramine administered into the RVLM is believed to be mediated by the evoked release of endogenous catechols in this region (6), and therefore injection of tyramine should provide a good pharmacological test of the anti-DH-saporin-induced lesion. That is, we would expect the depressor effect of tyramine injected into the RVLM to be abolished by prior treatment with anti-DH-saporin. Indeed, this is what was observed. Sources of the endogenous catechols in this region that are likely responsible for the tyramine-evoked depressor response are terminals arising from neurons of the A5 cell population and C1 cells themselves. Results from the present studies suggest that the terminals arising from neurons of the A5 cell group and synapsing in the RVLM are not critical for the tyramine-evoked depressor response. Furthermore, preliminary data indicate that the depressor effect induced by RVLM microinjection of clonidine, a direct norepinephrine receptor agonist presumably working via the same receptors responsive to the tyramine-evoked norepinephrine release, is attenuated by administration of anti-DH-saporin (27). These data suggest that administration of anti-DH-saporin destroys not only the source of the endogenous norepinephrine, but also many of the neurons containing the receptors on which this norepinephrine acts.

Results from the present study also suggest that the C1 cell population plays a role in the arterial pressure changes elicited by microinjections of glutamate into the RVLM. Because the most active pressor region of the RVLM overlaps with the distribution of the C1 cell group, this cell group has been speculated to play a role in the RVLM pressor response (22). Data from the present study provide the first direct evidence in support of this hypothesis. The destruction of the C1 cell population attenuates the glutamate-evoked pressor response of the RVLM. However, the pressor response elicited by stimulation of the RVLM is blocked by intrathecal injections of glutamate receptor antagonists but not by catecholamine receptor antagonists (1, 15). Thus stimulation of C1 cells may result in a pressor response that is mediated by glutamate release from these cells, a suggestion made previously on the basis of the presence of phosphate-activated glutaminase in C1 neurons (17).

Surprisingly, the pressor response elicited by microinjection of glutamate into the RVLM contralateral to the toxin injection was potentiated. One possible explanation for this finding is that destruction of the C1 cell population attenuates the baroreflex buffering of this response. Consistent with this explanation, the pressor effects of glutamate injected into the RVLM are potentiated by acute inhibition of the contralateral NTS (Ito and Sved, unpublished observations). Such a mechanism would also lead to a potentiated decrease in MAP evoked by tyramine injection into the RVLM contralateral to the toxin injection. In fact, the tyramine-induced decrease in MAP elicited from the RVLM contralateral to the toxin injection was 42% larger than this response in control rats, although this difference did not reach statistical significance. An alternative mechanism to account for the enhanced response to glutamate is that C1 neurons within the RVLM may inhibit the contralateral RVLM either directly, consistent with evidence for a contralateral projection of C1 neurons in the present study, or indirectly. Current information does not allow for a distinction between these possibilities, and it is possible that several mechanisms participate in the potentiation of the glutamate-evoked pressor response.

In conclusion, the present study demonstrates that anti-DH-saporin can be used to selectively lesion the C1 cell population of the RVLM. Injections of anti-DH-saporin into the RVLM produced an extensive reduction in the number of PNMT-containing neurons of the RVLM without decreasing the number of non-C1 RVLM barosensitive spinally projecting neurons. In addition, the present study suggests that the C1 cell population plays a role in the glutamate-evoked pressor response elicited from the RVLM, supporting a role of C1 cells in cardiovascular regulation. Future studies will allow for a more complete characterization of the role of PNMT-containing neurons in the control of blood pressure as well as other physiological and behavioral processes.