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Department of Neuroscience, University of Pittsburgh, Pittsburgh, Pennsylvania 15260
Submitted 18 February 2003 ; accepted in final form 28 July 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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-hydroxylase-saporin into the RVLM. These immunotoxin injections resulted in 32–99% depletion of the RVLM-C1 neurons and 
50% depletion of the A5 cell population. In conscious rats with large (>80%) depletion of the RVLM-C1 cell population, resting arterial pressure was 10 mmHg lower than in control injected rats, although heart rate was not significantly different. Similar results were observed when arterial pressure was recorded in urethan-anesthetized rats, although under anesthesia, heart rate was also reduced in rats with large (>80%) depletion of the RVLM-C1 neuronal population. Sympathoexcitatory responses to baroreceptor unloading, chemoreceptor activation, and electrical stimulation of sciatic nerve afferent fibers were attenuated in rats with >80% depletion of the RVLM-C1 cell population. These effects of RVLM-C1 plus A5 cell populations were not mimicked by either smaller lesions of the RVLM-C1 population or by selective destruction of the A5 cell population with 6-hydroxydopamine. Sympathoinhibitory responses such as decreases in arterial pressure and heart rate evoked by injection of GABA into the RVLM or by intravenous phenylbiguanide administration were not altered by RVLM-C1 plus A5 cell depletion. These data suggest that RVLM-C1 cells contribute to the maintenance of baseline arterial pressure and play an integral role in sympathoexcitatory responses.
immunotoxin; phenylethanolamine-N-methyltransferase; baroreceptor reflex; chemoreceptor reflex; somatic pressor reflex
The RVLM is composed of at least two neurochemically distinct populations of neurons that project to the spinal cord. Approximately 50–70% of spinally projecting RVLM neurons contain the enzymes phenylethanolamine-N-methyltransferase (PNMT) and dopamine--hydroxylase (DH) as well as other enzymes involved in catecholamine biosynthesis (38, 39) and are part of the C1 cell population. The remainder of RVLM spinal neurons lack the enzymes involved in catecholamine biosynthesis. Although the role of the RVLM in cardiovascular regulation is well established, the specific contributions of these two neurochemically defined cell populations within this area are unclear. There is indirect evidence to support a role of the C1 cell population in cardiovascular regulation. The most active pressor region of the RVLM is found in an area overlapping the distribution of the C1 cell population (30). In addition, both direct electrophysiological recordings (8, 31) and c-Fos expression in response to hypotension (2, 38) have demonstrated that spinally projecting C1 neurons are affected by baroreceptor input. However, many spinally projecting non-C1 cells within the most active pressor region of the RVLM are also responsive to baroreceptor input (2, 8, 31, 38).
With the recent development of an immunotoxin consisting of the ribosome-inactivating toxin saporin bound to an antibody to DH (anti-DH-saporin), which can selectively destroy central neurons containing DH (including C1 neurons) (45), several studies (22, 23, 32–34) have begun to directly assess the role of the C1 cell population in sympathetic outflow and cardiovascular function. In these studies, two approaches have been taken to target the C1 cell population; Schreihofer et al. (32–34) injected anti-DH-saporin into the spinal cord (in the vicinity of the terminal fields of the C1 neurons), whereas we (21–23) chose to inject the toxin directly into the RVLM. Both approaches effectively destroy spinally projecting neurons of the C1 cell population (22, 33). However, just as critical to the validation of these lesioning techniques is the selectivity of the lesion for the C1 cell population. Unfortunately, Schreihofer and Guyenet (33) were unable to evaluate the degree of nonspecific damage caused to the spinal cord at the site of the toxin injections, making interpretation of some of their data problematic. On the other hand, our previous experiments have demonstrated that a 21-ng dose of anti-DH-saporin injected directly into the RVLM can create a near-complete reduction in the number of C1 cells within the RVLM while sparing bulbospinal barosensitive noncatecholaminergic neurons of this area (22). In rats in which the C1 cells have been destroyed in this manner, we (22) found that C1 neurons are necessary for the complete expression of increased AP, and presumably sympathetic vasomotor outflow, after stimulation of the RVLM, which has been confirmed by Schreihofer et al. (34). The present study uses this approach to lesion the C1 neuronal population to more fully evaluate the role of this group of neurons in the tonic and reflexive control of cardiovascular function.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Male Sprague-Dawley rats (Zivic Laboratories) weighing 250–350 g at the time of brain stem toxin injections were used in these studies. All rats were singly housed and given ad libitum access to standard rat chow (Purina 5001) and water. The colony room was maintained at a temperature of 22–23°C and kept on a 12:12-h light-dark cycle. All protocols were approved by the Animal Care and Use Committee at the University of Pittsburgh and were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Brain Stem Toxin Injections
RVLM microinjections. Animals were anesthetized with halothane (2–5% in oxygen) and placed in a stereotaxic instrument with the incisor bar positioned 11 mm below the interaural line. A small portion of the occipital bone was removed, the meninges covering the dorsal surface of the brain stem were cut and retracted, and the area postrema was visualized. For microinjections of toxin 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 apex of the area postrema, and the pipette was then positioned 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 coordinates from which the largest pressor response can be elicited by injections of glutamate (11). In addition, we (22) have previously shown that injection of toxin using these coordinates results in large reductions in the number of spinally projecting C1 neurons. All injections were given over a 1- to 2-min period using a PicoPump (WPI; New Haven, CT). To bilaterally destroy the C1 cell population, animals (n = 57) received bilateral injections into the RVLM of anti-DH-saporin [21 ng in 200 nl artificial cerebrospinal fluid (aCSF) or sterile saline per side]; this dose produces the maximal depletion of C1 cells while still maintaining selectivity for the C1 cell population (22). To control for any nonspecific effects of a saporin-conjugated toxin, other rats (n = 21) received bilateral injections into the RVLM of saporin conjugated to an antibody raised against mouse IgG (MabZap; ATS, 21 ng in 200 nl aCSF or sterile saline). To control for nonspecific damage created by microinjections into the RVLM, another group of rats (n = 37) received either no injection or bilateral injections of aCSF or sterile saline into the RVLM. After intraparenchymal injections, the incision was closed, and the animal received an injection of Bicillin (30,000 units im) and was returned to its home cage. A minimum recovery time of 10 days was permitted before further procedures were performed; minimal cell death was noted 3 days after toxin injection, whereas within 10 days the maximal cell loss was observed (22).
A5 area infusions. To control for the depletion of the A5 cell population in anti-DH-saporin-treated rats, other rats (n = 18) received infusions of 6-hydroxydopamine (6-OHDA) into the A5 area. Animals were anesthetized with halothane (2–5% in oxygen) and placed in a stereotaxic instrument with the incisor bar positioned 2.5 mm below 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 administered intraperitoneally, a micropipette was lowered into the A5 area (0.2 mm below interaural zero), and 5 µg of 6-OHDA in 2 µl of vehicle (0.1% ascorbic acid) were infused over 20 min in each side. Finally, the incision was closed, and the animal received an injection of Bicillin (30,000 units im) and was returned to its home cage. A 2-wk recovery period was allowed before any further experimentation was performed on these rats.
Most experiments were performed using conscious unrestrained rats. Before experimentation, rats were anesthetized with halothane (2–3% in oxygen with the exception of a subset of the RVLM-C1-lesioned rats in which a higher concentration of halothane, up to 5%, was required to achieve an adequate level of surgical anesthesia) and implanted with femoral arterial (polyethylene-50 tubing filled with heparinized saline) and venous (polyvinyl-3 tubing filled with heparinized saline) catheters that were tunneled subcutaneously, exteriorized between the scapulae of the rat, and threaded through a tethering harness. Halothane anesthesia was terminated, and rats were allowed at least 1 day to recover before experimentation. To record AP and HR, the arterial catheter was attached to a pressure transducer (Statham P23 ID) and a polygraph recording system (Grass model 7 Physiograph). Experiments involving microinjections into the RVLM or stimulation of the sciatic nerve were conducted in rats that were subsequently anesthetized with urethan (1.5 g/kg infused intravenously over 30 min).
Experimental Protocols
After recovery from the toxin or control injections and implantations of arterial and venous catheters, rats were subjected to the following procedures. All rats were subjected to at least two of the following tests with the exception that a unique group of rats was used for testing the somatic pressor response. The testing procedures were always performed in the order in which they are described below.
Basal cardiovascular variables. Basal cardiovascular variables were assessed in conscious unrestrained rats. After being connected to the polygraph recording system, rats were allowed at least 30 min to acclimate before data collection was initiated. Basal mean AP (MAP) and HR values were calculated by averaging the values taken at 30-s intervals for 5 min.
Baroreceptor reflex, Bezold-Jarisch reflex, and chemoreceptor reflex testing. The baroreceptor reflex, the Bezold-Jarisch reflex, and the chemoreceptor reflex were tested in conscious unrestrained rats. The arterial baroreflex was assessed by increasing AP with intravenous injections of phenylephrine (Phe; 1, 2, 4, 8, and 16 µg/kg of a 10 µg/ml solution in random order; Sigma) and decreasing AP with intravenous injections of sodium nitroprusside (SNP; 2, 4, 8, 20, and 40 µg/kg of a 10 or 100 µg/ml solution in random order; Sigma) and observing the resulting change in HR. Phenylbiguanide (PBG; 16 µg/kg iv of a 10 µg/ml solution; Aldrich Chemicals) was administered to elicit the Bezold-Jarisch reflex. The chemoreflex was stimulated by administration of potassium cyanide (KCN; 80 µg/kg iv of a 100 µg/ml solution; Fischer Scientific) (5). In some rats, the baroreceptor reflex-evoked sympathoexcitation was also assessed by measuring plasma levels of catecholamines in response to hypotension. In these rats, food and water were removed from the cage just before the the test was begun. A baseline blood sample (1.8 ml) was collected into a heparinized tube (2.4 µl of 10,000 U/ml). Blood was then centrifuged at 8,000 revolutions/min for 1 min, and 300-µl aliquots of plasma were mixed with 6 µl of 5 N perchloric acid and stored at –80°C until assayed for catecholamines. Hydralazine (HDZ; 10 mg/kg; Sigma) was then injected intravenously to decrease AP, and 30 min later a second blood sample was taken following the same procedure as described for the baseline sample. At the time of analysis, 3,4-dihydroxybenzylamine was added as an internal standard. Plasma catecholamines were extracted with alumina and measured using HPLC with electrochemical detection (Waters; Marlborough, MA) as previously described (42).
Microinjections into the RVLM. After recovery from intraparenchymal microinjections into the brain stem, exposing the dorsal surface of the brain stem a second time is problematic. Therefore, to perform intraparenchymal microinjections to test responses to locally administered drugs, the RVLM was approached ventrally. The rats were anesthetized with urethan (1.5 g/kg iv) and placed in a stereotaxic frame in the supine position with the incisor bar positioned at the level of the interaural line. Core body temperature was monitored and maintained at 37°C with a heating pad. The trachea was cut and intubated, and rats were paralyzed (0.5 mg/kg D-tubocurarine supplemented every hour with 0.2 mg/kg) and artificially ventilated with 100% oxygen (2.5 ml tidal volume, 70 breaths/min; small animal respirator, Harvard Apparatus; South Natick, MA). The larynx, esophagus, and surrounding musculature were cut and retracted, and the occipital foramen and occipital bone were exposed. Part of the occipital bone was removed, and the coordinates for microinjections were 2.5–4 mm rostral to the caudal tip of the occipital foramen, 1.5–2.1 mm lateral to the basilar artery, and 0.7 mm below the ventral surface of the medulla (44). Microinjections of glutamate (1 nmol in 100 nl aCSF) and GABA (0.1, 1, and 10 nmol in 100 nl aCSF in random order) were performed using a single-barrel glass micropipette (outer tip diameter, 40–75 µm). All microinjections were given over a 5-to 10-s period using a PicoPump (WPI). At least five injections of glutamate were performed to determine the precise area at which glutamate elicited the largest pressor response, as previously reported (22). After this maximal pressor area was localized, the micropipette was withdrawn, rinsed with distilled water, and filled with one of the GABA solutions. The pipette was then positioned at the injection site from which glutamate elicited the largest pressor response, and GABA was injected. MAP was allowed to return to basal levels for at least 5 min between all microinjections.
Sciatic nerve stimulation. Rats were anesthetized with urethan (1.5 g/kg iv infused over 30 min), paralyzed, and artificially ventilated as described above. The left sciatic nerve was isolated and placed on the exposed ends of two Teflon-coated triple-stranded wires separated by 1 mm. The nerve and wires were covered with warm (37°C) mineral oil, and the distal end of the nerve was cut. A constant voltage stimulator (Grass 88) equipped with a photoelectric stimulus isolation unit (Grass PSIU 6B) was used to stimulate the nerve. Stimuli were delivered by passing 1-ms cathodal square wave pulses given at a frequency of 20 Hz for 5 s. Stimulation currents ranging from 100 to 1,000 µA were used to generate stimulus-response curves in each rat.
Histological assessment of lesions. At the conclusion of all experiments, rats were deeply anesthetized (2 g/kg ip urethan) and perfused through the heart with a 0.9% saline solution, followed by a solution of 4% paraformaldehyde, 1.4% lysine, and 0.2% sodium metaperiodate in 0.1 M sodium phosphate buffer (SPB). The brains of all animals were removed and postfixed in this fixative solution for 3–24 h. This tissue was then transferred to a 20% sucrose solution in SPB and stored at 4°C for 12–24 h. A freezing stage microtome was used to cut the brain tissue into coronal sections with a thickness of 30 µm. These tissue sections were stored in cryoprotectant solution (43) at –20°C until immunohistochemical staining was performed. After immunohistochemical staining, all tissue was mounted on slides, dehydrated in a graded ethanol series, defatted in xylene, and coverslipped with Histomount (VWR).
A one-in-six series of brain stem sections from each rat was incubated at 4°C for 48 h in rabbit anti-PNMT or rabbit anti-tyrosine hydroxylase (TH; Protos; dilutions determined by a titration performed on each lot of antibody received). Primary antisera were diluted in SPB containing 0.3% Triton-X and 1% donkey serum. Sections were rinsed in SPB and then incubated in biotinylated donkey anti-rabbit IgG diluted in SPB containing 0.3% Triton-X (Jackson ImmunoResearch Laboratories; 1:500). Sections were rinsed in SPB and then processed according to the avidin-biotin immunoperoxidase method as previously described (28) using Elite Vectastain reagent (Vector Laboratories).
To determine the extent to which the C1, A5, and A1 cell populations were destroyed in each rat, cell counts of PNMT-positive and TH-positive neurons were conducted. Cell counts were performed in every sixth brain stem section (30 µm of each 180 µm) through the entire rostral-caudal extent of the RVLM, the A5 area, and the A1 area. The RVLM was anatomically defined as the area extending 720 µm caudally from the caudal pole of the facial nucleus and bordered 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. TH-positive neurons in the ventrolateral pons extending rostrally from the level of the facial nucleus were considered to belong to the A5 cell population. TH-positive neurons in the ventrolateral medulla found caudal to the obex were considered to belong to the A1 cell population. Cells were counted at x100–200 magnification using bright-field illumination. Cell counts of bilaterally toxin-injected rats, MabZap-injected rats, and 6-OHDA-infused rats were compared with those of intact control rats. All counts presented refer to the numbers of cells counted bilaterally. In all rats, the lesion site was also assessed for signs of local tissue damage as indicated by necrotic holes within the injection site.
Statistical Analyses
All statistics were performed using Systat (version 10, SPSS). Experiments examining one variable across several groups were analyzed using one-way ANOVA. If a significant overall effect was found, then post hoc testing was performed using the Fisher's least-significant difference (LSD) test. For experiments involving several doses of a drug or several stimulation intensities in the same rat, two-way repeated-measures ANOVA was used. If a significant overall effect of the between-subjects variable was found, post hoc testing was performed for this significant main effect using the Fisher's LSD test. If a significant interaction effect was found, then one-way ANOVA was performed at each level of the repeated variable, with significant effects, followed by a Fisher's LSD test. A 
2-analysis was used to compare the proportions of animals with necrosis at the injection site among treatment groups. Values are expressed as means ± SE. Differences were considered significant when P < 0.05. Control, 6-OHDA-infused, and MabZap-injected rats were assigned to groups according to the treatment received; the anti-DH-saporin group, however, was subdivided into rats with small (<80%) depletion of the C1 cell population and rats with large (>80%) depletion of this population. A depletion of 80% was chosen a priori as a division point of the anti-DH-saporin group based on a previous study (46) on the functional effects of lesions of distinct populations of catecholaminergic neurons. However, this cutoff was supported by subsequent regression analysis of the baroreceptor reflex data, which indicated that the inflection point was at 80%.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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The total number of PNMT-containing neurons found in the ventrolateral medulla was 1,000 per side, in agreement with previous reports (14, 22, 38, 39). The majority of PNMT-positive cells were located 1–1.5 mm rostral to the obex, which corresponds to the area just caudal to the caudal pole of the facial nucleus [or 11.6–12.3 mm posterior to the bregma based on the atlas of Paxinos and Watson (27)]. All histological assessments in the present study were found to be similar to our previously reported data (22). Two weeks after the injection of 21 ng anti-DH-saporin into the RVLM, the number of PNMT-positive neurons in the area of the RVLM was reduced by 80 ± 2% (range: 32–99%, n = 57) in the toxin-injected rats compared with the average number of neurons counted in control noninjected animals (Table 1). All lesions were bilaterally symmetrical with the reduction in the number of C1 neurons extending rostrocaudally from the site of injection in a graded fashion. The most complete depletion was seen 900–1,800 µm rostral to the obex, whereas a smaller reduction was observed in the area extending from the obex to 720 µm rostral to the obex, as previously documented (22). A variety of control injections were performed to evaluate the nonselective effects of the immunotoxin injection. After the injection of MabZap into the RVLM, a small (10%) depletion of the C1 cell population was observed (Table 1). Compared with control counts, no reduction in the number of C1 neurons was observed after the infusion of 6-OHDA into the A5 area (Table 1).
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In 13 of 57 rats injected with anti-DH-saporin, a small area of necrosis was noted at the injection site; as we have previously reported (22), this nonspecific damage was typically limited to 360 µm in diameter and never exceeded 600 µm in diameter. A similar area of necrosis at the site of injection was found in a subset of MabZap-injected rats (n = 5 of 21). The percentage of rats with necrosis at the injection site in the anti-DH-saporin-treated group did not differ from that in the MabZap-treated group (P > 0.05).
The numbers of catecholaminergic neurons of the A1 and A5 cell populations were counted in every sixth section through the brain stem of all rats (see Table 1). Compared with all other groups, cell counts of the A1 cell population were significantly reduced in the anti-DH-saporin-injected rats with >80% depletion of the C1 cells within the RVLM (18% reduction compared with counts from control animals, P < 0.05). There were no reductions in the number of A1 neurons counted in any of the other groups compared with control rats. Cell counts for the A5 cell population were significantly reduced (62 ± 3%, range: 36–94%) in the anti-DH-saporin-injected animals with >80% depletion of RVLM-C1 cells compared with control rats (P < 0.001) and in anti-DH-saporin-injected animals with <80% depletion of RVLM-C1 cells compared with control rats (18 ± 7%, range 0–69%, P < 0.001). Compared with control rats, cell counts of the A5 cell population were significantly smaller in rats receiving an infusion of 6-OHDA into the A5 area (75 ± 5% reduction, range: 30–93%, P < 0.001). There was no reduction in the number of A5 neurons counted in rats with MabZap injected into RVLM compared with control rats.
Basal Resting MAP and HR
MAP was 10 mmHg lower in rats with >80% depletion of the RVLM-C1 cell population compared with all other control injection groups (Table 2), although MAP and HR did not differ from noninjected control rats. When recorded while conscious, rats with >80% depletion of the RVLM-C1 cell population had a baseline MAP of 108 ± 2 mmHg (n = 13) compared with 116 ± 1 mmHg (n = 55) for all of the control rats (P < 0.005). A subset of these rats plus additional rats in each group were studied while anesthetized with urethan, and MAP was also significantly lower in rats with large RVLM-C1 lesions compared with control rats when measured under this condition (Table 2). Also, urethan-anesthetized rats with large RVLM-C1 lesions had lower HR than control rats (Table 2).
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Baroreceptor Reflex
Large depletion (>80%) of the C1 cell population attenuated the gain of the baroreceptor reflex determined by the increase in HR in response to SNP-evoked decreases in MAP (Figs. 1, A and B, and 2A). In contrast, the reflex was not altered by smaller depletion (<80%) of the C1 cell population, partial depletion of the A5 cell population, or injection of the immunotoxin Mab-Zap into the RVLM (Figs. 1, A and B, and 2, B and C). In addition, the maximum HR evoked by SNP was significantly smaller in rats with large (>80%) depletion of the C1 cell population (438 ± 7 beats/min) compared with control rats (474 ± 8 beats/min), A5-lesioned rats (488 ± 10 beats/min), Mab-Zap-injected rats (462 ± 7 beats/min), and rats with small (<80%) depletion of the C1 cell group (460 ± 6 beats/min, all P < 0.05; Fig. 2, A–C). The SNP-evoked depressor response was significantly larger in rats with >80% depletion of the C1 cell population compared with control and MabZap-injected rats (Table 3). With the exception of a precipitous fall in HR for the two largest doses of Phe in all treatment groups except the control group, the gain of the baroreceptor reflex evoked by the smaller doses of Phe did not differ between groups (Fig. 2, A–C). In addition, the Pheevoked pressor response did not differ among the groups (Table 4).
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In rats receiving injections of HDZ, resting MAP and HR did not differ significantly among the groups (Table 5), although, as with the larger group, MAP tended to be lower in rats with >80% lesions compared with the rats in the groups receiving control injections. In all rats, injection of HDZ resulted in a long-lasting (>30 min) hypotension that was accompanied by tachycardia. The peak decrease in MAP evoked by HDZ administration did not differ among the groups (Table 5). The peak increase in HR was significantly attenuated in rats with large depletion of the C1 cell population compared with control rats (P < 0.05; Table 5). Baseline plasma levels of norepinephrine and epinephrine were typically below the level of detection of our assay (300 pg/ml). Plasma levels of norepinephrine evoked by HDZ were significantly attenuated in the rats with large depletion of the C1 cell population compared with control rats (Fig. 3). However, HDZ-evoked plasma epinephrine values were highly variable and did not differ among groups (Fig. 3).
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Bezold-Jarisch Reflex
PBG produced a large decrease in MAP and HR in all rats. No significant correlation was found between the extent of C1 cell depletion and the decrease in MAP evoked by administration of PBG. The change in MAP and HR in response to PBG did not differ significantly between any of the groups (Fig. 4).
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Chemoreflex
Administration of KCN resulted in a large increase in MAP in all control and MabZap-injected rats and in most anti-DH-saporin-injected and 6-OHDA-infused rats; however, two anti-DH-saporin-injected rats and one 6-OHDA-infused rat responded to KCN administration with a decrease in MAP. The change in MAP evoked by KCN was significantly blunted in animals with large depletion of C1 cells within the RVLM (Fig. 5). No correlation was found between the extent of A5 cell depletion in 6-OHDA-infused rats and the KCN-evoked change in MAP, and thus all 6-OHDA-infused rats were considered as one group; in these rats, the KCN-evoked change in MAP was not altered compared with control. In most rats, KCN administration resulted in a very rapid and transient increase in HR associated with locomotor activity, followed by a large bradycardia; in rats not showing this response, only a large bradycardia was observed. Rats lacking a tachycardia and locomotor response were observed in all groups and were not preferentially associated with any particular treatment group. The magnitude of the bradycardia observed after KCN administration also did not differ between groups (Fig. 5).
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Somatic Pressor Reflex
Stimulation of the sciatic nerve resulted in an intensity-dependent increase in MAP in all groups (P < 0.001). At the largest stimulation intensities (800 and 1,000 µA), the pressor responses were significantly attenuated in the anti-DH-saporin-injected rats compared with all other groups (Fig. 6). Whereas the increases in MAP resulting from smaller stimulation intensities (up to 600 µA) were not statistically different between groups, these responses tended to be smaller in the rats with depletion of the C1 cell population (Fig. 6A). Sciatic nerve stimulation also resulted in an intensity-dependent increase in HR in all groups (P < 0.001), but no significant effect of group or group by intensity interactions were found (Fig. 6B). In the group of anti-DH-saporin-injected rats used in this experiment, all had >80% depletion of the C1 cell population with the exception of one rat (70% depletion). Because this was the only rat with a small C1 cell depletion (<80%), it was excluded from analysis; however, the responses of this rat closely resembled those seen in all of the anti-DH-saporin-injected rats with >80% depletion of the C1 cell population.
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RVLM-Evoked Responses
Unilateral microinjection of glutamate (1 nmol) into the RVLM produced a marked increase in MAP (Fig. 7A) accompanied by a slight tachycardia. The glutamate-evoked pressor response in all treatment groups was rapid in onset and of short duration; the peak responses occurred within 30 s, and the levels returned to baseline within 1–2 min. Depletion of C1 neurons within the RVLM did not affect the glutamate-evoked pressor response (even rats with >80% depletion of these neurons had responses of normal magnitude; Fig. 7, A and B). The pressor response in rats that received infusions of 6-OHDA into the A5 area was significantly larger than the pressor responses in all of the other groups (P < 0.05; Fig. 7A). No significant differences in the glutamate-evoked pressor response were present among any of the other groups (Fig. 7A). In addition, the various treatments had no apparent effect on the distribution of sites in the RVLM from which pressor responses were elicited (data not shown). The change in HR evoked by glutamate did not differ between any of the groups (Fig. 7).
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Injection of GABA into the RVLM produced a dose-dependent decrease in MAP and HR in all groups (Fig. 8). These decreases in MAP and HR did not differ among the groups (Fig. 8).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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To evaluate the role of C1 neurons of the RVLM in cardiovascular regulation, we have taken advantage of an immunotoxin that selectively targets DH-containing neurons. By infusing this toxin locally into the RVLM, we were able to produce selective loss of RVLM C1 neurons exceeding 80%. This allowed us to study cardiovascular regulation in rats with large depletion of the C1 cell population. In a recent series of experiments, Schreihofer et al. (32–34) also investigated the role of the C1 cell population in sympathetic reflexes and cardiovascular regulation using intraspinal injections of this immunotoxin to destroy C1 neurons. Across the two sets of studies, many aspects of the results are quite similar. However, the lesions are somewhat different, and this difference impacts the conclusions that can be drawn from these data sets.
In the present study, we evaluated the physiological alterations resulting from depletion of the C1 cell population. Previous work (4, 46) has demonstrated that small depletion of other catecholaminergic cell populations have little functional impact, due in a large part to the remarkable compensatory capacity of the remaining neurons of these populations. However, when depletion reach 70–90%, functional impairments are observed (4, 46). Not surprisingly, the magnitude of the C1 cell depletion influenced the alterations seen in several of the physiological variables examined in the present study. For these reasons, we chose to subdivide the anti-DH-saporin-injected group into rats with large (>80%) depletion of RVLM-C1 neurons and rats with smaller (<80%) depletion of this population. In fact, some of the significant effects of C1 cell depletion on physiological function would have been masked had we considered the anti-DH-saporin-injected rats as a single group regardless of the magnitude of C1 cell depletion. For example, the basal MAP was significantly lower in rats with >80% depletion of the C1 cell population compared with the rats with smaller (<80%) depletion of the C1 cell population or control groups receiving injection of MabZap into the RVLM or infusion of 6-OHDA into the A5 area. In addition, there is no effect on the SNP-evoked index of baroreceptor function of the depletion of up to 80% of the RVLM-C1 neurons (Fig. 1A; linear regression r = 0.24), whereas depletion of >80% result in a seemingly linear decline in the SNP-evoked index of baroreceptor function (Fig. 1A; linear regression r = 0.64). Although baroreceptor reflexes were tested in only a few rats with lesions exceeding 95% of the RVLM-C1 neurons, the results from these animals suggest that C1 cells make a sizable contribution to the baroreceptor reflex.
The conclusions of the present study depend on the assertion that, within the RVLM, anti-DH-saporin effectively and selectively destroys the C1 cell population. We (22) and others (33) have previously argued that the inability to immunohistochemically detect C1 neurons after anti-DH-saporin treatment is indicative of a loss of these cells. In particular, after the administration of anti-DH-saporin, there is a marked decrease in the number of cells within the RVLM that are retrogradely labeled from the thoracic spinal cord (22, 33). In addition, our lesions are also quite selective for C1 neurons. We (22) have previously demonstrated that the lesioning methodology used in the present study causes minimal damage to the spinally projecting non-C1 cell population within the RVLM. Because all aspects of the histological assessment in the present study are consistent with those we have reported previously, we assume that the degree of damage inflicted upon the spinally projecting non-C1 cell population of the RVLM is also similar to that found in our previous report. Nonetheless, in 20% of rats injected with the toxin, we saw a small area of necrosis at the injection site. In the present study, we saw no difference in the functional effects of the lesions whether or not they included this small area of necrosis (e.g., see Fig. 5A). In addition, to control for any uptake of the toxin unrelated to the specific DH-mediated uptake, a control group of rats received an injection of MabZap into the RVLM. Approximately 20% of the MabZap-injected rats also had a small area of necrosis at the injection site. As in the anti-DH-saporin-injected group, the necrotic damage in the MabZap-injected rats had no detectable functional effect (see Fig. 5A). Injections of anti-DH-saporin into the RVLM also resulted in a loss of A5 neurons. To control for the destruction of A5 neurons in the anti-DH-saporin-injected rats, we included a group of rats with selective depletion of the A5 cell population.
In conscious and urethan-anesthetized rats with large RVLM-C1 lesions, baseline MAP was modestly decreased, as was HR in urethan-anesthetized rats. [In the experiment examining the effects of HDZ this difference was not statistically significant (Table 5), but this likely results from the small sample size used in that experiment.] The finding of decreased MAP in rats with >80% depletion of the RVLM-C1 cell population is surprising given that these cardiovascular variables were measured at least 10 days after the injections of toxin were performed, allowing plenty of time for compensation to occur. In fact, a previous study (26) has shown that removal of all supraspinal drive to sympathetic preganglionic neurons by cervical spinal transection, although it elicits an initial depressor response, has no long-term effect on resting MAP, attesting to the functional redundancy in this system. Schreihofer and Guyenet (33) reported that rats with depletion of spinally projecting C1 and A5 neurons produced by injection of anti-DBH-saporin into the spinal cord had normal MAP and HR as measured while rats were anesthetized with chloralose. Although the difference with the present study may reflect differences in anesthetic or differences in the extent of the destruction of A5 cells, the most likely explanation is that the C1 cell lesion made by Schreihofer and Guyenet was not of sufficient size. They reported a depletion of the spinally projecting C1 cells of only 73 ± 7% (33); had we treated all rats injected with anti-DBH-saporin as a single group, we would have reported no effect of the toxin on baseline MAP. Thus it appears that RVLM-C1 neurons may be important for the maintenance of resting AP and that extensive destruction of the RVLM-C1 population may be necessary to reveal the impact of their destruction. Linear regression analysis of MAP as a function of the percent depletion of the RVLM-C1 population based on all injected rats reveals a significant correlation (r = 0.30, P < 0.02), although such an analysis likely underestimates the magnitude of the effect because these parameters do not seem to be related in a linear manner, but rather MAP may decrease after some threshold level of depletion of the RVLM-C1 population. Although a decrease in sympathetic vasomotor tone is most likely responsible for the decreased MAP observed in rats with extensive depletion of the RVLM-C1 neurons, this hypothesis remains to be tested. Rats with extensive RVLM-C1 lesions do not have reduced plasma levels of norepinephrine (21), although it is not clear whether plasma norepinephrine levels are a sensitive enough index of sympathetic nerve activity to detect the decrease in sympathetic nerve activity responsible for a decrease in MAP of 10 mmHg.
Depletion of the C1 cell population within the RVLM altered the reflex relationship between MAP and HR. The tachycardia evoked by a decrease in pressure is mediated by increased sympathetic outflow to the heart and by a withdrawal of basal cardiac vagal tone (9, 35). The relationship between an evoked hypotensive response and the resulting tachycardia was altered by the depletion of the C1 plus A5 cell populations with anti-DH-saporin but not by depletion of the A5 cell population alone. Thus, in rats with large lesions of the C1 cell group, there was a decrease in the gain of the baroreceptor reflex and a lowering of the maximum SNP-evoked or HDZ-evoked HR compared with control rats. Similarly, Schreihofer and Guyenet (33) found that the increase in splanchnic sympathetic nerve activity in response to hypotension was blunted in anesthetized rats that had lesions of DH-positive neurons (including C1 neurons) projecting to the thoracic spinal cord. However, because of the nature of expressing sympathetic nerve activity as a percentage of baseline activity and the difficulty of assessing absolute levels of baseline nerve activity, this previous study could not determine whether the attenuation of this response is representative of an absolute difference or is instead a reflection of a change in the baseline activity. With respect to this issue, the present study demonstrated that plasma concentrations of norepinephrine in response to HDZ-evoked hypotension are attenuated in rats with large depletion of RVLM-C1 neurons compared with control rats. Together, these results suggest that the C1 cell population plays a role in the baroreflex-evoked activation of sympathetic nerves. In addition, because hypotension-evoked tachycardia is partially mediated by a withdrawal of cardiac vagal tone, we cannot exclude the possibility that the C1 cell population also plays a role in the withdrawal of basal cardiac vagal tone.
Because the baroreceptor reflex acts to acutely buffer changes in MAP, an attenuation of this reflex should result in larger changes in MAP to given stimuli. Indeed, for a given dose of SNP, larger decreases in MAP were observed in rats with large depletion of the C1 cell population compared with control rats. This observation lends further support to the assertion that neurons of the C1 cell population play a role in the baroreflex-mediated sympathoexcitation. However, a similar phenomenon was not observed when hypotension was evoked by HDZ administration. A possible explanation for this observation is that any compensatory responses induced by the HDZ-evoked hypotension may not be capable of overcoming the longer-duration hypotensive actions of HDZ, whereas compensatory responses may be able to counteract the very acute hypotensive effects of SNP. Because HDZ acts directly on vascular smooth muscle to induce hypotension, one might expect the antihypertensive effects of compensatory responses, such as sympathetic vasomotor activation and vasopressin secretion, to be ineffective, and therefore an attenuation of these mechanisms in C1-lesioned rats would be inconsequential.
In contrast to the apparent role of C1 neurons in the baroreceptor-mediated activation of sympathetic function, C1 lesions seem to have no effect on baroreceptor-mediated parasympathetic activation. The parasympathetic branch of the autonomic nervous system mediates the reflexive bradycardia evoked by an increase in AP (9, 35); this relationship was not altered by depletion of the C1 and/or A5 cell population. It is likely that distinct pathways that do not involve the RVLM mediate these responses (25).
Several previous studies have demonstrated roles of both the RVLM and A5 area in the chemoreceptor reflex. In particular, this reflex is attenuated in anesthetized rats by acute blockade of excitatory amino acid receptors in the RVLM (18), by acute inhibition of the A5 area (17), or by acute inactivation of noradrenergic input to the spinal cord (16). In the present study, activation of the chemoreceptor reflex in conscious rats led to a marked increase in MAP that was attenuated by large depletion of the C1 plus A5 cell populations with anti-DH-saporin but not by destruction of the A5 cell population alone. Similarly, chronic removal of the A5 and C1 cell populations has been demonstrated to attenuate this reflex in 
-chloralose-anesthetized rats (33). In another study (16) involving chronic removal of spinally projecting noradrenergic neurons, the magnitude of the carotid chemoreceptor reflex was not attenuated in urethan-anesthetized rats. The most likely explanation for these data is that the A5 and C1 cell populations play important roles in the carotid chemoreceptor reflex, and thus acute removal of one of these populations (the A5 cell population) attenuates this reflex. However, with chronic removal of the A5 cell population, the C1 cell population is capable of compensating for this lesion, whereas chronic removal of both cell populations leads to an attenuation of this reflex. Because no currently available techniques are capable of removing the C1 cell population without also removing neurons of the A5 cell population, it remains to be determined whether removal of the C1 cell population alone would affect the magnitude of the chemoreflex.
Activation of the somatic pressor reflex by electrical stimulation of the sciatic nerve in anesthetized rats results in intensity-dependent increases in MAP and HR that are dependent upon excitatory amino acid-mediated transmission within the RVLM (15). At the highest stimulation intensities, this somatic pressor response was significantly attenuated by the depletion of C1 plus A5 cell populations. In contrast, depletion of the A5 cell population alone did not alter the sciatic nerve-evoked responses, in agreement with previous data (36). The most straightforward interpretation of these data is that the C1 cell population contributes to the increase in sympathetic outflow evoked by stimulation of somatic afferents.
Thus large depletion of the C1 plus A5 cell populations with anti-DH-saporin, but not depletion of the A5 cell population alone, attenuates sympathoexcitatory responses evoked by baroreceptors, chemoreceptors, and somatic afferents. These observations suggest a general principle that reflex sympathoexcitatory responses are attenuated in rats with large depletion of the C1 cell population. This is consistent with previous reports showing that the pressor response elicited by chemical stimulation of the RVLM is attenuated in rats with unilateral depletion of RVLM-C1 cells (22) and that the pressor response elicited by electrical stimulation of the RVLM is attenuated in rats with bilateral depletion of RVLM-C1 cells (34). However, in the present study, stimulation of cell bodies within the RVLM by local injection of glutamate produced an increase in MAP and HR that was not attenuated in rats with large bilateral depletion of the C1 cell population. There are several potential explanations for this apparently conflicting observation. One possibility is that in the present study only a single, submaximal dose of glutamate was used to evoke an increase in pressure, and this dose could be analogous to the smaller stimulation intensities at which no attenuation was seen in anti-DH-saporin-treated rats in the study of Schreihofer et al. (34) or in the present experiments investigating the SPR (see Fig. 6A). Another possible explanation for the differences could be the use of electrical excitation, as in the study of Schreihofer et al. (34), compared with glutamate-evoked excitation of the RVLM used in the present study. Slight differences in the populations of cells affected by these two methodologies could explain the apparent discrepancy in the results. A final difference that could account for the differing results is the use of unilaterally toxin-injected rats (22) compared with bilaterally toxin-injected rats (present study). In the case of unilateral depletion of the C1 cell population, one might expect a primary effect of attenuating the glutamate-evoked sympathetic output coupled to normal baroreflex buffering of this response; this could result in an attenuated pressor response compared with a control animal. In contrast, one might expect in the case of bilateral depletion of the C1 cell population a primary effect of attenuating the glutamate-evoked sympathetic output but also an attenuated baroreflex buffering of this response, resulting in a response that is larger than observed in rats with unilateral lesions and maybe not significantly different from the control response. Indeed, blunting the baroreceptor reflex would be expected to potentiate the pressor response elicited by injection of glutamate into RVLM (12).
Rats with selective destruction of the A5 cell population had significantly larger pressor responses to RVLM injections of glutamate compared with all other groups. One possible explanation of this unexpected response is that the A5 cell population normally blunts the RVLM-evoked pressor response. It has been reported that stimulation of the A5 area results in a depressor response (10), and this depressor response does appear to be partially dependent on the A5 cell group (19). In addition, A5 cells are known to project to the intermediolateral cell column (20). Thus it is conceivable that this projection provides some inhibitory influence that attenuates the responsiveness of preganglionic neurons to input from the RVLM. This may help to explain the lack of an attenuation of the pressor response to injection of glutamate into RVLM in the C1/A5-lesioned rats. If depletion of the C1 cell group was to attenuate the pressor response to stimulation of the RVLM and the effect of depletion of the A5 cell group was to augment this response, then rats with partial depletion of both of these cell groups may exhibit a response of normal magnitude.
In contrast to the sympathoexcitatory responses that appear to be attenuated in rats with large depletion of RVLM-C1 neurons, sympathoinhibitory responses are apparently unaffected by these lesions. The decrease in MAP and HR evoked by administration of PBG is mediated in part by inhibition of RVLM sympathetic premotor neurons (40). In the present study, the PBG-evoked depressor response and bradycardia were not attenuated by depletion of the C1 cell population. These data demonstrate that basal sympathetic vasomotor outflow to the heart and vasculature is maintained in rats with near-complete depletion of the C1 cell population of the RVLM. Our interpretation of these data is that non-C1 neurons of the RVLM alone are capable of supporting basal levels of sympathetic outflow. However, an alternative interpretation of these data is that, in rats treated with anti-DH-saporin, inhibition of sympathetic outflow in response to PBG occurs through a site other than the RVLM. This interpretation seems unlikely given that the RVLM is still the major site responsible for the generation of sympathetic vasomotor tone in rats treated with anti-DH-saporin (present study and Ref. 34). Thus, while there is some evidence to suggest that C1 cells may contribute to the PBG-sensitive component of basal sympathetic outflow under normal conditions (41), these cells do not appear to be necessary for the expression of this sympathoinhibitory reflex.
In summary, it appears that rats with extensive lesions of the RVLM C1 cell population have a slightly reduced baseline AP and impaired sympathoexcitatory cardiovascular reflexes. Thus RVLM C1 neurons play a substantial role in cardiovascular regulation by contributing to several sympathoexcitatory reflexes such as the baroreceptor reflex, the carotid chemoreceptor reflex, and the somatic pressor reflex.
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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ACKNOWLEDGMENTS |
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Present address of C. J. Madden: Oregon Health and Science Univ., Neurological Sciences Institute, 505 NW 185th Ave., Beaverton, OR 97006.
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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H-saporin. Am J Physiol Regul Integr Comp Physiol 277: R1063–R1075, 1999.H-saporin. Am J Physiol Regul Integr Comp Physiol 279: R729–R742, 2000.This article has been cited by other articles:
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  A.-J. Li, Q. Wang, T. T. Dinh, and S. Ritter Simultaneous Silencing of Npy and Dbh Expression in Hindbrain A1/C1 Catecholamine Cells Suppresses Glucoprivic Feeding J. Neurosci., January 7, 2009; 29(1): 280 - 287. [Abstract] [Full Text] [PDF]
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 G. Wang, T. A. Milner, R. C. Speth, A. C. Gore, D. Wu, C. Iadecola, and J. P. Pierce Sex differences in angiotensin signaling in bulbospinal neurons in the rat rostral ventrolateral medulla Am J Physiol Regulatory Integrative Comp Physiol, October 1, 2008; 295(4): R1149 - R1157. [Abstract] [Full Text] [PDF]
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T.-K. Lee, J. H. Lois, J. H. Troupe, T. D. Wilson, and B. J. Yates Transneuronal tracing of neural pathways that regulate hindlimb muscle blood flow Am J Physiol Regulatory Integrative Comp Physiol, April 1, 2007; 292(4): R1532 - R1541. [Abstract] [Full Text] [PDF]
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 G. R. Pedrino, I. Maurino, D. S. de Almeida Colombari, and S. L. Cravo Role of catecholaminergic neurones of the caudal ventrolateral medulla in cardiovascular responses induced by acute changes in circulating volume in rats Exp Physiol, November 1, 2006; 91(6): 995 - 1005. [Abstract] [Full Text] [PDF]
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C. J. Madden, S. D. Stocker, and A. F. Sved Attenuation of homeostatic responses to hypotension and glucoprivation after destruction of catecholaminergic rostral ventrolateral medulla neurons Am J Physiol Regulatory Integrative Comp Physiol, September 1, 2006; 291(3): R751 - R759. [Abstract] [Full Text] [PDF]
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; n = 17), anti-dopamine-
; n = 27), anti-D
; n = 1), 6-hydroxydopamine (6-OHDA)-infused rats with depletion of the A5 cell population (A5X; 
; n = 11), saporin conjugated to an antibody raised against mouse IgG (MabZap)-injected rats (
; n = 10), and MabZap-injected rats with localized necrosis at the injection site (
; n = 2). Solid lines represent regression analyses of the rats injected with anti-D
MAP; B: 
