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Direct projection from the cardiovascular control region of the cerebellar cortex, the lateral nodulus-uvula, to the brainstem in rabbits
Neuroscience Research 36 (2000) 15 – 26 www.elsevier.com/locate/neures
Direct projection from the cardiovascular control region of the cerebellar cortex, the lateral nodulus-uvula, to the brainstem in rabbits Kaori Sadakane a,b,*, Makiko Kondo a, Naoko Nisimaru a b
a Department of Physiology, Oita Medical Uni6ersity, Oita 879 -5593, Japan Department of Health Sciences, Oita Uni6ersity of Nursing and Health Sciences, Oita 870 -1201, Japan
Received 27 August 1999; accepted 8 October 1999
Abstract In decerebrate unanesthetized rabbits, electrical stimulation of the lateral nodulus-uvula in the cerebellar vermal cortex evoked an increase in renal sympathetic nerve activity, an increase in blood pressure and a decrease in renal arterial blood flow, which were all in contrast to the effects reported previously in the anesthetized rabbits. In order to identify the pathway mediating these responses, we investigated the Purkinje cell projection from the lateral nodulus-uvula using both anterograde (biotinylated dextran amine, BDA) and retrograde (horseradish peroxidase, HRP) tracing methods in rabbits. When BDA was iontophoretically injected into the lateral nodulus-uvula, labeled Purkinje cell axons were found within and around the superior and inferior cerebellar peduncles (SCP and ICP, respectively). Furthermore, terminal-like fields were found in the dentate and vestibular nuclei as reported in previous studies. However, the terminal-like patterns that we observed in the parabrachial nucleus (PB) in the rabbit have not been reported yet. When HRP was microinjected into the lateral PB, retrogradely labeled Purkinje cells were found in the lateral nodulus-uvula. These results indicate that Purkinje cells in the lateral nodulus-uvula project into the vestibular nuclei via the ICP and to the lateral PB via the SCP. We suggest that these two pathways mediating cardiovascular responses have different sensitivities to anesthetics. © 2000 Published by Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cerebellar cardiovascular control; Nodulus; Decerebrate unanesthetized rabbit; Purkinje cell projection; Biotinylated dextran amine; Horseradish peroxidase; Parabrachial nucleus
1. Introduction The medial part of the cerebellar vermal cortex is known to be involved in cardiovascular control (Moruzzi, 1950; Nisimaru and Yamamoto, 1977; Ito, 1984; Nisimaru et al., 1984; Bradley et al., 1991). We have further shown that electrical stimulation of the lateral nodulus-uvula in the cerebellum resulted in the inhibition of renal sympathetic nerve activity (RSNA) and a decrease in blood pressure (BP) in anesthetized rabbits (Nisimaru and Watanabe, 1985). Recently, we have also found in conscious rabbits that this region is involved in the control of timing and duration of the transient increase in RSNA and in rapid adaptation of * Corresponding author. Tel.: +81-97-586-4432; fax: + 81-97-5864386. E-mail address: [email protected] (K. Sadakane)
BP during changes in head position and body posture (Nisimaru et al., 1998). Under anesthesia this rapid adaptation of BP is abolished. Therefore, it is suggested that the lateral nodulus-uvula has an important role in the control of cardiovascular responses during postural changes, and that this region is greatly affected by anesthesia. Bradley et al. (1987) demonstrated in rabbits the presence of two different pathways mediating cardiovascular responses elicited from the medial uvula, one of which might be blocked by anesthetics. In the present study we confirmed the difference in cardiovascular responses between anesthetized rabbits and decerebrate unanesthetized rabbits resulting from the stimulation of the lateral nodulus-uvula. To understand the mechanism of cardiovascular control by the lateral nodulus-uvula, it is essential to identify the afferent and efferent connections of this cerebellar region. Our preceding electrophysiological
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and anatomical studies on rabbits have showed that the lateral nodulus-uvula receives cardiovascular afferent fibers from the contralateral vagal and aortic nerves via climbing fibers and the ipsilateral vagal nerve via mossy fibers (Nisimaru and Katayama, 1995). There are two major efferent projections from the lateral nodulusuvula, one to the dentate nucleus and the other to the brainstem vestibular nuclei in rabbits (Van Rossum, 1969; Epema et al., 1985; Henry et al., 1989; Wylie et al., 1994). It has been shown previously that electrical stimulation of the vestibular nerve, as well as a head
rotation, elicit changes in sympathetic nerve activity (Uchino et al., 1970; Yates, 1992; Yates and Miller, 1994). It is not clear yet, however, whether the vestibular nucleus plays a role in controlling cardiovascular responses to stimulation of the cerebellar region. In this study we used biotinylated dextran amine (BDA) and horseradish peroxidase (HRP) for anterograde and retrograde tracing, respectively, and found that not only the vestibular nuclei but also the parabrachial nucleus (PB) receives direct projection from the lateral nodulus-uvula in rabbits.
Fig. 1. Effects of stimulation of the lateral nodulus-uvula on BP and RSNA in anesthetized rabbits. (A) Effects of electrical stimulation on BP (upper trace) and RSNA (lower trace). (B) Effects of injection of L-glutamate (0.2 M, 100 nl). The stimulation sites ( ) on the parasagital sections of the cerebellum at 4.2 and 3.2 mm lateral from the midline for (A) and (B), respectively. II-X: lobules of the cerebellar vermis according to Larsell (1970). Horizontal bars show the time when the electrical stimulus in (A) (interval, 5 ms; pulse width, 100 ms) and the chemical stimulus in (B) were applied.
Table 1 Comparison of the cardiovascular changes during lateral nodulus-uvula stimulation in five anesthetized and five decerebrate rabbitsa Anesthetized
Mean arterial pressure (mmHg) Renal blood flow (ml/min) Femoral blood flow (ml/min) a
Decerebrate
Control level
Peak response
Control level
Peak response
102.79 3.1 19.0 90.6 5.79 0.7
−27.89 3.5* (n =13) +2.690.9† (n =8) +2.490.5 (n = 8)
104.09 5.2 18.2 9 1.6 13.19 0.8
+40.292.6* (n= 15) −12.49 1.1* (n= 15) +2.99 2.0 (n =9)
Mean9 S.E. of mean, n= number of tests, (+) indicates an increase and (−) a decrease in the variable. * PB0.01. † PB0.05.
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electrophysiological experiments, seven were used in the anterograde tracing, and the remaining ten in the retrograde tracing experiments. In the electrophysiological experiments, nine rabbits were anesthetized with a-chloralose and urethane (30 and 600–800 mg/kg, respectively, Tokyo Kasei), while 16 were decerebrated at the precollicular level under anesthesia by aspiration with a short-acting anesthetic, thiopental sodium (initial dose 25 mg/kg, i.v., supplemented with 12 mg bolus doses as required). The rabbits were immobilized by intravenous injection of pancuronium bromide (Mioblock, Sankyo) and artificially respired with intermittent positive pressure. In the tracing experiments the animals were anesthetized with a-chloralose and urethane at the same dose as above administered intravenously and supplemented as required. Each rabbit was mounted on a stereotaxic frame in a prone position with the head fixed rigidly with a mouth-piece and two pointed screws piercing the zygomatic arch from both sides. The cerebellar vermis was exposed by partial craniotomy in the occipital region. The animals were warmed with an electric heating pad. The atlas of the rabbit’s cerebellum was according to Larsell (1970).
2.1. Electrophysiological studies
Fig. 2. Effects of electrical stimulation of the lateral nodulus-uvula on BP (upper traces) and RSNA (lower traces) in the decerebrate unanesthetized rabbit. (A) Responses in the same rabbit as in Fig. 1A. (B, C) Stimulation effects before and after injection of sympathetic ganglion blocker, hexamethonium, respectively. Horizontal bars show the time when the electrical stimulus was applied (interval, 5 ms; pulse width, 100 ms).
2. Materials and methods Experiments were performed on 40 albino rabbits weighting 2.5–3.4 kg. Of these, 23 were used in the
The procedure of stimulating the lateral nodulusuvula of the cerebellar vermis has been previously described (Nisimaru and Watanabe, 1985). In order to exclude the possibility that our results obtained by stimulating the lateral nodulus-uvula were due to current spread or activation of passing fibers, we injected L-glutamate (0.2 M, 100 nl) as a chemical stimulant into the lateral nodulus-uvula of five rabbits using a microsyringe. The L-glutamate injection site was determined stereotaxically and it was marked electrolytically at the end of each experiment (current intensity, 400 mA DC; duration, 20 s) and was located later by histological examination. The surgical procedures for isolation of the left renal sympathetic nerve bundle and for recording efferent discharges from these nerves have already been described (Nisimaru et al., 1984). Blood flow to the kidney and to the hindlimb of the same side was monitored using electromagnetic flowmeters (MF-27, Nihon Kohden). Probes were placed distal to the profound branch in the femoral artery and close to the abdominal aorta in the renal artery. Arterial pressure at the abdominal aorta was recorded with a pressure gauge (MP-3, Nihon Kohden) through a heparin-filled polyethylene tube inserted into the left femoral artery. The integrated RSNA, arterial BP and blood flow were recorded simultaneously on a chart recorder (Nihon Kohden) and stored on a data recorder (RD-101T, TEAC) for off-line analysis.
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2.2. Morphological studies 2.2.1. Anterograde axonal transport of BDA A micropipette (tip diameter, 30 mm), containing a 10% solution of BDA (10 kDa mol. wt. lysine fixable, Molecular Probes) dissolved in saline, was stereotaxically inserted through the uvula (sublobule IXb) into
the nodulus. The solution of BDA was then injected iontophoretically into the nodulus with application of a 2–4 mA DC current (tip positive) for 11–20 min. After a survival period of 8–11 days, the rabbits were deeply anesthetized and perfused transcardially with a mixture of 4% paraformaldehyde and 0.05% glutaraldehyde dissolved in 0.1 M phosphate buffer solution at pH 7.4.
Fig. 3. Effects of electrical stimulation of the lateral nodulus-uvula on regional blood flow in rabbits. (A) Stimulation effects on BP (top trace), renal arterial blood flow (middle trace) and femoral arterial blood flow (bottom trace) under the anesthetized condition. (B) Similar parameters shown in (A), but under a decerebrate unanesthetized condition. Horizontal bars show the time when the electrical stimulus was applied.
Fig. 4. Effects of stimulation of the lateral nodulus-uvula on the renal arterial blood flow before and after dissection of the renal nerve fiber in the decerebrate unanesthetized rabbit. (A) Responses to stimulation before the ipsilateral (left) renal nerve dissection as shown in Fig. 3. (B) Parameters similar to those shown in (A) but after dissection of the ipsilateral renal nerve. Horizontal bars show the time when the electrical stimulus was applied.
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Fig. 5. Schematic drawing showing the sites of BDA injection in the nodulus and uvula of the cerebellar cortex in six rabbits (R50, R51, R54, R55, R58 and R60). (A) Surface view of the uvula (sublobule IXd) and the nodulus (lobule X). For this illustration, the cerebellar cortex is unfolded around the borderline between the dorsal and ventral parts of each lobule (dashed line) and compressed along the sagittal line at the ratio of 1/3. The injection area is enclosed. (B) Illustration of the parasagittal cerebellar section 2.94 mm lateral from the midline.
The cerebellum and brainstem were removed and 60mm-thick frozen serial sections were cut parasagittally. These brain slices were treated according to the procedure of Brandt and Apkarian (1992) with modification. BDA in the sections was visualized by incubation with a mixture of 0.02% diaminobenzidine, 0.003% H2O2 and 0.5% nickel ammonium sulfate in Tris buffer solution (pH 7.6). The sections were then counterstained with Neutral Red. To compare the results obtained from the seven rabbits, the mediolateral extent of the nodulus was normalized to the average width (3.3 9 0.3 mm, mean9 S.D., n= 7). 2.2.2. Retrograde axonal transport of HRP A double-barreled glass micropipette (tip diameter of each, 30 mm), with one of the barrels filled with 15% HRP (Grade 1-C, Toyobo) dissolved in 50 mM Tris buffer solution (pH 7.5) and 0.1 M KCl for HRP injection, and the other with 2 M NaCl for electrical stimulation, was inserted stereotaxically into the PB via the anterior cerebellar vermis. Previous reports (Mraovitch et al., 1982; Ward 1988) showed that the electrical stimulation of the PB resulted in an increase in BP (pressor response). Therefore, to determine the localization of the PB for injection, the BP was recorded continuously and the pressor response to the electrical stimulation via one of the barrels of the glass micropipette was examined. After the PB was thus identified, HRP was iontophoretically injected with application of a 4–6 mA DC current (tip positive) for 20 – 40 min. Following a survival period of 24 – 48 h, the rabbits were deeply anesthetized and perfused transcardially with 1% paraformaldehyde and 1.25% glutaraldehyde in 0.1 M phosphate buffer solution at pH 7.4. Histological procedures for examination of HRP transport have been previously described in detail (Katayama and Nisimaru, 1988). To compare the HRP injection site
among the ten rabbits, the distance of the brainstem from the inferior colliculus to the obex was normalized rostrocaudally to the average length (10.79 1.2 mm, mean9S.D., n=10). HRP-labeled cells were drawn on the normalized nodulus and uvula, the same as in the BDA experiments.
3. Results
3.1. The cardio6ascular responses to stimulation of the lateral nodulus-u6ula in anesthetized rabbits and decerebrate unanesthetized rabbits 3.1.1. RSNA and BP In rabbits anesthetized with a-chloralose and urethane, inhibition of RSNA and a decrease in BP (depressor response) were observed following electrical stimulation of the lateral nodulus-uvula with a series of brief current pulses for 10 s (intensity, 50–200 mA; interval, 5 ms; pulse width, 100 ms) as shown in Fig. 1A. Falls in mean blood pressure (MBP) were 27.893.5 mmHg from control values of 102.79 3.1 mmHg in five rabbits (Table 1, PB 0.01). These responses were induced from sites which fell within a small region extending about 1 mm longitudinally through the dorsal nodulus encroaching the border to the ventral uvula,2.9–3.7 mm lateral to the midline as demonstrated in a previous study (Nisimaru and Watanabe, 1985). When 0.2 M L-glutamate (100 nl) was injected into the same region in the lateral nodulus-uvula, inhibition of RSNA and a depressor response were also induced in three anesthetized rabbits (Fig. 1B). This observation confirms that the effects of stimulation of the lateral nodulus-uvula arise from the activation of cerebellar Purkinje cells in this region. We performed similar experiments as described above in decerebrate unanesthetized rabbits. The elec-
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trical stimulation of the lateral nodulus-uvula with repetitive pulses for 10 s (intensity, 50 – 200 mA) evoked a significant increase followed by inhibition of RSNA and a marked pressor response (Fig. 2A and B). Changes in RSNA were transient increases for the first 3 – 6 s of the 10 s stimulus period, but an inhibition at a point where the increase in MBP had reached 10–15 mmHg. Increases in MBP were 40.2 92.6 mmHg from
control values of 104 9 5.2 mmHg in five rabbits (Table 1). These responses were in contrast to those seen in the anesthetized rabbits. Following i.v. injection of hexamethonium, a sympathetic nerve ganglion blocker, RSNA was inhibited and the pressor response disappeared during stimulation of the lateral nodulus-uvula (Fig. 2C). These results showed that the pressor response was induced by the increase in RSNA.
Fig. 6. Terminal-like patterns in the PB anterogradely labeled by BDA into lobule X for R60. (A) Bright-field photomicrograph of the injection site in the dorsolateral region of lobule X (2.88 mm sagittal section right from the midline). (B, C) Bright-field photomicrographs showing BDA-labeled terminal-like patterns (arrowheads) in the lateral (B) and medial (C) PB for R60 (Fig. 7Ae and Ac, respectively). Calibration bars 200 mm in (A), 25 mm in (B) and 20 mm in (C).
3.1.2. Renal and femoral arterial blood flow In five anesthetized rabbits the depressor response was associated with changes in renal and femoral arterial blood flow. The renal blood flow showed an initial increase followed by a decrease during the stimulation as shown in Fig. 3A. In three rabbits the renal blood flow showed increases in mean blood flow (MBF) of 2.69 0.9 ml/min from control values of 199 0.6 ml/ min during nodulus stimulation (Table 1). The initial increase in renal blood flow showed a time course similar to the inhibition of RSNA (Figs. 1A and 3A). This indicates that the kidney vascular resistance transiently decreased due to the inhibition of RSNA. The subsequent decrease in renal blood flow, however, showed a time course similar to the depressor response (Fig. 3A). Therefore, it appears that the subsequent decrease in renal blood flow is a passive effect secondary to the depressor response. The changes in femoral blood flow showed a similar pattern to those in renal blood flow but were less prominent during the stimulation as shown in Fig. 3A and Table 1. In decerebrate rabbits, changes in renal and femoral arterial blood flow were studied during the lateral nodulus-uvula stimulation. The renal blood flow markedly decreased following stimulation with repetitive pulses for 10 s (Fig. 3B). In five rabbits renal blood flow showed significant falls in MBF of 12.491.1 ml/min from control values of 18.29 1.6 ml/min (Table 1). The time course of this decrease in the renal blood flow is similar to that of the increase in RSNA (Figs. 2A and 3B). Following dissection of the left renal nerve fiber, BP showed no significant change but renal blood flow decreased. Under this condition the pressor response to nodulus stimulation was still seen, but the response in terms of blood flow of the ipsilateral renal artery was reversed (an increase instead of a decrease) as shown in Fig. 4. These results confirm that the decrease in the renal blood flow following stimulation of the lateral nodulus-uvula is the direct effect of RSNA. On the other hand, the femoral blood flow initially decreased but immediately increased following a time course similar to the pressor response during stimulation (Fig. 3B and Table 1). This indicates that the changes in hindlimb blood flow are mainly passive effects secondary to the increase in BP.
K. Sadakane et al. / Neuroscience Research 36 (2000) 15–26 Fig. 7. Distribution of labeled axons and terminal-like deposits in the cerebellum and brainstem following injection of BDA into the lateral nodulus-uvula in three out of six rabbits shown in Fig. 5. (A) R60, (B) R51 and (C) R58. Line drawings of the normalized sagittal sections of the ipsilateral cerebellum and brainstem between 1.2 and 4.8 mm from the midline at 0.6 mm intervals (1.5, 2.1, 2.7, 3.3, 3.9 and 4.5 mm for a, b, c, d, e and f, respectively). Labeled axons and terminal-like deposits contained in 10 sections (60 mm thick each) are collectively plotted in each illustration. Dashed lines in each illustration represent the labeled axons. Arrows (*) show the terminal fields. Abbreviations: N.V.mes, mesencephalic nucleus of the trigeminal nerve; CG, central gray matter; PH, prepositus hypoglossal nucleus; F, fastigial nucleus; N.X, dorsal nucleus of the vagus nerve; N.XII, hypoglossal nucleus; MV, medial vestibular nucleus; SCP, superior cerebellar peduncle; ICP, inferior cerebellar peduncle; l.PB, lateral parabrachial nucleus; m.PB, medial parabrachial nucleus; SV, superior vestibular nucleus; LV, lateral vestibular nucleus; IP, interpositus nucleus; IV, inferior vestibular nucleus; D, dentate nucleus; and IC, infracerebellar nucleus.
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Fig. 8. Distribution of labeled cells in the ipsilateral nodulus and uvula following injection of HRP into the entire area of right PB. (A) Line drawing of the brainstem coronal section showing the HRP injection sites for R13 (bold dotted line) and R17 (bold line) on the right side. (B) Illustration of the sagittal section of the cerebellum and brainstem indicated by a dashed line through PB and SCP in (A) also showing the sites of HRP injection. (C) Line drawings of sagittal sections of the nodulus and uvula. Left diagrams: entire view of the sections. Upper and lower illustrations represent the lateral half and the medial half of the nodulus and uvula, respectively. Labeled cells contained in half of the serial sections (each 60 mm thick) are collectively plotted in each illustration (upper panels, at 2.46 mm; lower panels, at 0.84 mm). A large dot represents 10 cells and a small dot represents one cell. Abbreviations: LC, locus ceruleus; MCP, middle cerebellar peduncle. See Fig. 7 for the other abbreviations.
These results suggest that the cardiovascular responses evoked by stimulation of the lateral nodulusuvula are mediated by two different pathways, the dominance of which is changed by anesthesia.
3.2. Morphological studies 3.2.1. Distribution of labeled fibers and terminals following BDA injection In six rabbits the injection sites were localized to the lateral part of the nodulus-uvula as shown in Fig. 5 and Fig. 6A, the stimulation of which resulted in the changes in RSNA, BP and blood flow as described above. All of these injection sites were restricted to the cerebellar cortex and underlying white matter excluding cerebellar nuclei (Fig. 5). The distribution of anterogradely labeled axons is represented schematically in the drawings in Fig. 7 in three typical cases (R60, R51 and R58) out of six. The labeled fibers are identified in the cerebellum and the brainstem between 1.2 and 4.8 mm from the midline. In the medial sagittal sections labeled fibers originate from the lateral nodulus-uvula, and extend to within and
around the fastigial nucleus (Fig. 7Aa, Ab, Ca, and Cb). More laterally, labeled fibers separate into two components as shown in Fig. 7Cc. The first component appears to descend into the brainstem by passing through the medial part of the inferior cerebellar peduncle (ICP) and extends into the superior vestibular nucleus (SV) and medial vestibular nucleus (MV) as shown in Fig. 7Bc and Cc. The second component appears to extend rostrally within and around the superior cerebellar peduncle (SCP) as shown in Fig. 7Ad and Cd. In more lateral sections this second component passes within and around the dorsal SCP and extends into the PB (Fig. 7Ae and Ce). Labeled fibers also pass through the vicinity of the interpositus nucleus as shown in Fig. 7Bc, Bd and Cc. In one rabbit, a few labeled fibers were seen in the dorsal area proximate to the contralateral SCP (data not shown). The distribution of labeled terminal fields is also represented schematically in the drawings in Fig. 7. It appears that the heaviest projection from the lateral nodulus-uvula is seen in the dorsal parts of the SV and the MV (Fig. 7Bb and Bc). On the other hand, no
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labeled terminal-like pattern was observed in the lateral vestibular nucleus and inferior vestibular nucleus. In addition, we found two new terminal regions, the dorsal region of the lateral PB (Figs. 6B and 7Ae), and an area immediately dorsal to the medial PB (Figs. 6C and 7Ac). In the cerebellum a few terminal fields were found in the infracerebellar nucleus as shown in Fig. 7Be. In the more lateral sections, terminal-like deposits were found in the dentate nucleus between 5.7 and 6.1 mm from the midline (data not shown). In this experiment, no labeled terminal fields were found in the fastigial and interpositus nuclei. These results indicate that Purkinje cells in the lateral nodulus-uvula directly project into three regions; the dentate and infracerebellar nuclei via the lateral portion of the cerebellar white matter, the vestibular nuclei complex passing through the ICP, and the PB passing within and around the SCP via the ventral region to the interpositus nucleus.
3.2.2. Distribution of labeled Purkinje cells following HRP injection To study the projection from the lateral nodulusuvula to the PB, we injected a retrograde tracer, HRP, into the PB. The double-barreled micropipette was inserted from the anterior cerebellar vermis to the PB. There was neither histological damage caused by mi-
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cropipette penetrations nor formation of HRP reaction products in the white matter of the posterior cerebellum including cerebellar nuclei and in the SCP. In two experiments HRP was injected into both the lateral and medial PB (Fig. 8A and B). In R13, a large amount of HRP was injected into the PB, and a small amount, therefore, was distributed over the rostral SV. Retrogradely labeled Purkinje cells were found in the entire area of the nodulus and uvula as shown in Fig. 8C. On the other hand, in R17, only a small amount of HRP was injected restrictedly in the PB except for a small region of the adjacent middle cerebellar peduncle and SV as shown in Fig. 8A and B. Labeled Purkinje cells were found only in the nodulus. In five rabbits, the sites for HRP injection were localized in the lateral PB and the tracer was not observed to spread to the SV or the medial PB as shown in Fig. 9A and B. In the cases of R20 and R23, distribution of the tracer was restricted within the lateral PB, and retrogradely labeled Purkinje cells in the medial region of the cerebellar vermis were found extensively in the nodulus and uvula as shown in Fig. 9C. On the other hand, labeled cells in the lateral region of the cerebellar vermis were localized in the dorsal aspect of the nodulus and the ventral aspect of sublobule IXd (Fig. 9C). In three rabbits, HRP was injected into the medial PB and no labeled Purkinje cells were observed
Fig. 9. Distribution of labeled cells in the nodulus and uvula following injection of HRP into the lateral area of right PB. Similarly illustrated to Fig. 8 expect for R14, R20, R23, R33 and R35. Labeled cells in the nodulus and uvula from two typical cases (R20 and R23) out of five rabbits.
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in the nodulus and uvula (data not shown). From these results, it can be deduced that Purkinje cells in the lateral nodulus-uvula, which regulates the cardiovascular system, project directly to the lateral PB.
4. Discussion The present study, using BDA and HRP tracing methods in the rabbit, revealed new direct projections of Purkinje cells from the lateral nodulus-uvula to the PB, especially the lateral PB. On the other hand, Paton et al. (1991) have reported that Purkinje cell axons in the medial uvula (sublobule IXb) project to the PB in the rabbit using anterograde tracing methods with wheat germ agglutin in conjugated HRP. Furthermore, Supple and Kapp (1994) demonstrated that Purkinje cells in the anterior cerebellar vermis (lobules III and IV) terminate to the PB in the rabbit. From these results, it is apparent that Purkinje cells from several regions in the cerebellar vermis project directly into the PB. The PB has been reported to be involved in cardiovascular regulation in various mammalian species as follows. The electrical and chemical stimulations of the PB, particularly the lateral PB, evoke the pressor response, and this nucleus receives afferent inputs from the nucleus tractus solitarius, which is known to receive primary barosensory input (Hamilton et al., 1981; Mraovitch et al., 1982; Ward, 1988; Miura and Takayama, 1991). The PB stimulation also modulates the barorecepter reflex in cats (Felder and Mifflin, 1988). Our previous study showed that the lateral nodulus-uvula in the cerebellum is involved in cardiovascular control (Nisimaru and Watanabe, 1985). Taken together, the present results suggest that the PB is a relay nucleus from Purkinje cells in the lateral nodulus-uvula to the sympathetic nerves which control the cardiovascular functions. On the other hand, in the present study using BDA, the heaviest projection from Purkinje cells in the lateral nodulus-uvula is directed to the SV and the MV. Efferent connections from lobules IX and X to the vestibular nuclei complex were reported in the rabbit (Van Rossum, 1969; Epema et al., 1985; Henry et al., 1989; Wylie et al., 1994), in the cat (Angaut and Brodal, 1967; Voogd, 1967; Dietrichs et al., 1983; Shojaku et al., 1987; Walberg and Dietrichs, 1988), in the rat (Bernard, 1987) and in the prosimian primate (Haines, 1977). Using tracing methods, Henry et al. (1989) and Wylie et al. (1994) demonstrated the termination of the projection from Purkinje cells in the lateral nodulus into the SV, the MV and the group y in the rabbit. In the present study, however, labeled terminals were found not in the group y but in the infracerebellar .
nucleus. The cells of the infracerebellar nucleus are loosely arranged and located between the group y and the dentate nucleus in rabbits as described by Epema et al. (1988). Because of the ambiguous location of this nucleus, other reports might have included the infracerebellar nucleus in the group y and/or the dentate nucleus. Therefore, our results on the projection to the vestibular nuclei complex from the lateral nodulusuvula are nearly in agreement with these previous reports. Uchino et al. (1970) reported that an inhibition of RSNA resulted following electrical stimulation of the vestibular nerve. Yates and Miller (1994) showed that nose-up tilting resulted in an increase in splanchnic nerve activity while nose-down tilting reduced this activity, and these responses were abolished by application of a lesion of the MV and the inferior vestibular nucleus. They suggested that the vestibular system is involved and participated in compensating for posturally related changes in BP. The observed projection to the dentate nucleus from the lateral nodulusuvula in this experiment nearly corresponds with those in previous studies (Van Rossum, 1969; Bernard, 1987). In the present study electrical stimulation of the lateral nodulus-uvula in the decerebrate unanesthetized rabbit evoked a pressor response and a decrease in the renal arterial blood flow associated with a transient increase in RSNA. These responses were in contrast to those in the anesthetized rabbit (Nisimaru and Watanabe, 1985). As the responses observed in the decerebrate unanesthetized rabbit were abolished by anesthesia, as shown in Figs. 1A and 2A, this complete alteration of evoked cardiovascular responses was not caused by decerebration itself but by anesthesia. Bradley et al. (1987) reported similar results in the medial uvula of the cerebellum as follows. In decerebrate unanesthetized rabbits the stimulation of the medial uvula (sublobule IXb) in the posterior cerebellar vermis evoked a marked tachycardia, an increase in BP, a sustained increase in RSNA, and a decrease in both renal and femoral conductances. These cardiovascular responses were abolished by a small dose of anesthetic. Bradley et al. (1987) also suggested the presence of two different pathways mediating cardiovascular function, dominance of which is altered by anesthesia. The present study using the anterograde tracing methods also suggests that labeled fibers of Purkinje cells in the lateral nodulus-uvula are separated into two parts, one of which extends across the cerebellar white matter and enters around and within the SCP, and another which extends into the ICP. It appears that the fibers around and within the SCP project into the PB, and those within the ICP, to the vestibular complex; however, a possibility remains that a part of the former projects
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into the vestibular nuclei via the SCP. Paton et al. (1991) demonstrated two pathways of Purkinje cell axons, that is, via the SCP and the ICP, to the brainstem as described above. They also reported that the presence of a lesion in the SCP abolished the cardiovascular responses to stimulation of the medial uvula in the anesthetized rabbit, but not in the decerebrate unanesthetized one. On the other hand, the presence of a lesion in the ICP abolished these cardiovascular responses only in the decerebrate unanesthetized rabbit. They suggested that these cardiovascular responses in the anesthetized rabbit were mediated by fibers within the SCP and relayed to the PB, while those in the decerebrate unanesthetized rabbit were mediated by ICP and relayed to the vestibular nuclei. These similarities between our results and Paton’s in the Purkinje cell projection and in the effect of anesthesia indicate that both the lateral nodulus-uvula and the medial uvula have two pathways, that is, via the PB and vestibular nuclei, which control the cardiovascular functions. The present study, furthermore, showed that there are dominant terminal fields from the lateral nodulus-uvula in the SV and the MV, as compared with those in the PB. These results indicate that the pathway involved in cardiovascular control via vestibular nuclei is stronger than that via the PB, and that the former pathway might be dominant under the unanesthetized condition and sensitive to anesthetics. Recently Nisimaru et al. (1998) showed that this cerebellar region has an important role in a conscious rabbit during tilting the head up, and is involved in the control of timing and duration of the transient increase in RSNA and in the rapid adaptation of BP during postural changes. Therefore, the present results suggest that the pathway via the ICP and vestibular nuclei may mediate the adaptive cardiovascular control by the lateral nodulus-uvula during postural changes in conscious animals.
Acknowledgements We would like to thank Professor Kazuhiro Yamada (Department of Physiology, Oita Medical University) for his constant encouragement during the course of this study, and Professor Jinzo Yamada (Department of Anatomy, Tokyo Medical University) and Dr Soichi Nagao (Department of Physiology, Jichi Medical School) for their valuable discussions regarding the anatomical aspect of this study. We are grateful to Dr Taiko Kitamura (Department of Anatomy, Tokyo Medical University) for her helpful advice on BDA and HRP tracing techniques. We thank Eriko Hayashi, Hiroaki Kawazato and Aiko Yasuda for their technical assistance.
References Angaut, P., Brodal, A., 1967. The projection of the ‘vestibulocerebellum’ onto the vestibular nuclei in the cat. Arch. Ital. Biol. 105, 441 – 479. Bernard, J.F., 1987. Topographical organization of olivocerebellar and corticonuclear connections in the rat — an WGA-HRP study: I. Lobules IX, X, and the flocculus. J. Comp. Neurol. 263, 241 – 258. Bradley, D.J., Ghelarducci, B., Paton, J.F., Spyer, K.M., 1987. The cardiovascular responses elicited from the posterior cerebellar cortex in the anaesthetized and decerebrate rabbit. J. Physiol. 383, 537 – 550. Bradley, D.J., Ghelarducci, B., Spyer, K.M., 1991. The role of the posterior cerebellar vermis in cardiovascular control. Neurosci. Res. 12, 45 – 56 Review, 38 Refs. Brandt, H.M., Apkarian, A.V., 1992. Biotin – dextran: a sensitive anterograde tracer for neuroanatomic studies in rat and monkey. J. Neurosci. Methods 45, 35 – 40. Dietrichs, E., Zheng, Z.H., Walberg, F., 1983. The cerebellar corticovestibular projection in the cat as studied with retrograde transport of horseradish peroxidase. Anat. Embryol. 166, 369– 383. Epema, A.H., Guldemond, J.M., Voogd, J., 1985. Reciprocal connections between the caudal vermis and the vestibular nuclei in the rabbit. Neurosci. Lett. 57, 273 – 278. Epema, A.H., Gerrits, N.M., Voogd, J., 1988. Commissural and intrinsic connections of the vestibular nuclei in the rabbit: a retrograde labeling study. Exp. Brain Res. 71, 129 – 146. Felder, R.B., Mifflin, S.W., 1988. Modulation of carotid sinus afferent input to nucleus tractus solitarius by parabrachial nucleus stimulation. Circ. Res. 63, 35 – 49. Haines, D.E., 1977. Cerebellar corticonuclear and corticovestibular fibers of the flocculonodular lobe in a prosimian primate (Galago senegalensis). J. Comp. Neurol. 174, 607 – 630. Hamilton, R.B., Ellenberger, H., Liskowsky, D., Schneiderman, N., 1981. Parabrachial area as mediator of bradycardia in rabbits. J. Auton. Nerv. Syst. 4, 261 – 281. Henry, R.T., Connor, J.D., Balaban, C.D., 1989. Nodulus-uvula depressor response: central GABA-mediated inhibition of alphaadrenergic outflow. Am. J. Physiol. 256, H1601 – 1608. Ito, M., 1984. The Cerebellum and Neuronal Control. Raven Press, New York. Katayama, S., Nisimaru, N., 1988. Parasagittal zonal pattern of olivo-nodular projections in rabbit cerebellum. Neurosci. Res. 5, 424 – 438. Larsell, O., 1970. The Comparative Anatomy and Histology of the Cerebellum from Monotremes through Apes. University Minnesota Press, Minneapolis, MN, pp. 116 – 267. Miura, M., Takayama, K., 1991. Circulatory and respiratory responses to glutamate stimulation of the lateral parabrachial nucleus of the cat. J. Auton. Nerv. Syst. 32, 121 – 133. Moruzzi, G., 1950. Problems in Cerebellar Physiology. Thomas, Springfield, IL, pp. 74 – 96. Mraovitch, S., Kumada, M., Reis, D.J., 1982. Role of the nucleus parabrachialis in cardiovascular regulation in cat. Brain Res. 232, 57 – 75. Nisimaru, N., Katayama, S., 1995. Projection of cardiovascular afferents to the lateral nodulus-uvula of the cerebellum in rabbits. Neurosci. Res. 21, 343 – 350. Nisimaru, N., Watanabe, Y., 1985. A depressant area in the lateral nodulus-uvula of the cerebellum for renal sympathetic nerve activity and systemic blood pressure in the rabbit. Neurosci. Res. 3, 177 – 181. Nisimaru, N., Yamamoto, M., 1977. Depressant action of the posterior lobe of the cerebellum upon renal sympathetic nerve activity. Brain Res. 133, 371 – 375.
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Nisimaru, N., Yamamoto, M., Shimoyama, I., 1984. Inhibitory effects of cerebellar cortical stimulation on sympathetic nerve activity in rabbits. Jpn. J. Physiol. 34, 539–551. Nisimaru, N., Okahara, K., Yanai, S., 1998. Cerebellar control of the cardiovascular responses during postural changes in conscious rabbits. Neurosci. Res. 32, 267–271. Paton, J.F., La Noce, A., Sykes, R.M., et al., 1991. Efferent connections of lobule IX of the posterior cerebellar cortex in the rabbit — some functional considerations. J. Auton. Nerv. Syst. 36, 209 – 224. Shojaku, H., Sato, Y., Ikarashi, K., Kawasaki, T., 1987. Topographical distribution of Purkinje cells in the uvula and the nodulus projecting to the vestibular nuclei in cats. Brain Res. 416, 100 – 112. Supple, W. Jr, Kapp, B.S., 1994. Anatomical and physiological relationships between the anterior cerebellar vermis and the pontine parabrachial nucleus in the rabbit. Brain Res. Bull. 33, 561 – 574. Uchino, Y., Kudo, N., Tsuda, K., Iwamura, Y., 1970. Vestibular inhibition of sympathetic nerve activities. Brain Res. 22, 195 – 206. Van Rossum, J., 1969. Corticonuclear and Corticovestibular Projec-
tions of the Cerebellum. Thesis, State University, Leiden, Netherlands. Voogd, J., 1967. Comparative aspects of the structure and fibre connexions of the mammalian cerebellum. Prog. Brain Res. 25, 94 – 134. Walberg, F., Dietrichs, E., 1988. The interconnection between the vestibular nuclei and the nodulus: a study of reciprocity. Brain Res. 449, 47 – 53. Ward, D.G., 1988. Stimulation of the parabrachial nuclei with monosodium glutamate increases arterial pressure. Brain Res. 462, 383 – 390. Wylie, D.R., De Zeeuw, C.I., DiGiorgi, P.L., Simpson, J.I., 1994. Projections of individual Purkinje cells of identified zones in the ventral nodulus to the vestibular and cerebellar nuclei in the rabbit. J. Comp. Neurol. 349, 448 – 463. Yates, B.J., 1992. Vestibular influences on the sympathetic nervous system. Brain Res. Rev. 17, 51 – 59. Yates, B.J., Miller, A.D., 1994. Properties of sympathetic reflexes elicited by natural vestibular stimulation: implications for cardiovascular control. J. Neurophysiol. 71, 2087 – 2092.
Direct projection from the cardiovascular control region of the cerebellar cortex, the lateral nodulus-uvula, to the brainstem in rabbits Kaori Sadakane a,b,*, Makiko Kondo a, Naoko Nisimaru a b
a Department of Physiology, Oita Medical Uni6ersity, Oita 879 -5593, Japan Department of Health Sciences, Oita Uni6ersity of Nursing and Health Sciences, Oita 870 -1201, Japan
Received 27 August 1999; accepted 8 October 1999
Abstract In decerebrate unanesthetized rabbits, electrical stimulation of the lateral nodulus-uvula in the cerebellar vermal cortex evoked an increase in renal sympathetic nerve activity, an increase in blood pressure and a decrease in renal arterial blood flow, which were all in contrast to the effects reported previously in the anesthetized rabbits. In order to identify the pathway mediating these responses, we investigated the Purkinje cell projection from the lateral nodulus-uvula using both anterograde (biotinylated dextran amine, BDA) and retrograde (horseradish peroxidase, HRP) tracing methods in rabbits. When BDA was iontophoretically injected into the lateral nodulus-uvula, labeled Purkinje cell axons were found within and around the superior and inferior cerebellar peduncles (SCP and ICP, respectively). Furthermore, terminal-like fields were found in the dentate and vestibular nuclei as reported in previous studies. However, the terminal-like patterns that we observed in the parabrachial nucleus (PB) in the rabbit have not been reported yet. When HRP was microinjected into the lateral PB, retrogradely labeled Purkinje cells were found in the lateral nodulus-uvula. These results indicate that Purkinje cells in the lateral nodulus-uvula project into the vestibular nuclei via the ICP and to the lateral PB via the SCP. We suggest that these two pathways mediating cardiovascular responses have different sensitivities to anesthetics. © 2000 Published by Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cerebellar cardiovascular control; Nodulus; Decerebrate unanesthetized rabbit; Purkinje cell projection; Biotinylated dextran amine; Horseradish peroxidase; Parabrachial nucleus
1. Introduction The medial part of the cerebellar vermal cortex is known to be involved in cardiovascular control (Moruzzi, 1950; Nisimaru and Yamamoto, 1977; Ito, 1984; Nisimaru et al., 1984; Bradley et al., 1991). We have further shown that electrical stimulation of the lateral nodulus-uvula in the cerebellum resulted in the inhibition of renal sympathetic nerve activity (RSNA) and a decrease in blood pressure (BP) in anesthetized rabbits (Nisimaru and Watanabe, 1985). Recently, we have also found in conscious rabbits that this region is involved in the control of timing and duration of the transient increase in RSNA and in rapid adaptation of * Corresponding author. Tel.: +81-97-586-4432; fax: + 81-97-5864386. E-mail address: [email protected] (K. Sadakane)
BP during changes in head position and body posture (Nisimaru et al., 1998). Under anesthesia this rapid adaptation of BP is abolished. Therefore, it is suggested that the lateral nodulus-uvula has an important role in the control of cardiovascular responses during postural changes, and that this region is greatly affected by anesthesia. Bradley et al. (1987) demonstrated in rabbits the presence of two different pathways mediating cardiovascular responses elicited from the medial uvula, one of which might be blocked by anesthetics. In the present study we confirmed the difference in cardiovascular responses between anesthetized rabbits and decerebrate unanesthetized rabbits resulting from the stimulation of the lateral nodulus-uvula. To understand the mechanism of cardiovascular control by the lateral nodulus-uvula, it is essential to identify the afferent and efferent connections of this cerebellar region. Our preceding electrophysiological
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and anatomical studies on rabbits have showed that the lateral nodulus-uvula receives cardiovascular afferent fibers from the contralateral vagal and aortic nerves via climbing fibers and the ipsilateral vagal nerve via mossy fibers (Nisimaru and Katayama, 1995). There are two major efferent projections from the lateral nodulusuvula, one to the dentate nucleus and the other to the brainstem vestibular nuclei in rabbits (Van Rossum, 1969; Epema et al., 1985; Henry et al., 1989; Wylie et al., 1994). It has been shown previously that electrical stimulation of the vestibular nerve, as well as a head
rotation, elicit changes in sympathetic nerve activity (Uchino et al., 1970; Yates, 1992; Yates and Miller, 1994). It is not clear yet, however, whether the vestibular nucleus plays a role in controlling cardiovascular responses to stimulation of the cerebellar region. In this study we used biotinylated dextran amine (BDA) and horseradish peroxidase (HRP) for anterograde and retrograde tracing, respectively, and found that not only the vestibular nuclei but also the parabrachial nucleus (PB) receives direct projection from the lateral nodulus-uvula in rabbits.
Fig. 1. Effects of stimulation of the lateral nodulus-uvula on BP and RSNA in anesthetized rabbits. (A) Effects of electrical stimulation on BP (upper trace) and RSNA (lower trace). (B) Effects of injection of L-glutamate (0.2 M, 100 nl). The stimulation sites ( ) on the parasagital sections of the cerebellum at 4.2 and 3.2 mm lateral from the midline for (A) and (B), respectively. II-X: lobules of the cerebellar vermis according to Larsell (1970). Horizontal bars show the time when the electrical stimulus in (A) (interval, 5 ms; pulse width, 100 ms) and the chemical stimulus in (B) were applied.
Table 1 Comparison of the cardiovascular changes during lateral nodulus-uvula stimulation in five anesthetized and five decerebrate rabbitsa Anesthetized
Mean arterial pressure (mmHg) Renal blood flow (ml/min) Femoral blood flow (ml/min) a
Decerebrate
Control level
Peak response
Control level
Peak response
102.79 3.1 19.0 90.6 5.79 0.7
−27.89 3.5* (n =13) +2.690.9† (n =8) +2.490.5 (n = 8)
104.09 5.2 18.2 9 1.6 13.19 0.8
+40.292.6* (n= 15) −12.49 1.1* (n= 15) +2.99 2.0 (n =9)
Mean9 S.E. of mean, n= number of tests, (+) indicates an increase and (−) a decrease in the variable. * PB0.01. † PB0.05.
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electrophysiological experiments, seven were used in the anterograde tracing, and the remaining ten in the retrograde tracing experiments. In the electrophysiological experiments, nine rabbits were anesthetized with a-chloralose and urethane (30 and 600–800 mg/kg, respectively, Tokyo Kasei), while 16 were decerebrated at the precollicular level under anesthesia by aspiration with a short-acting anesthetic, thiopental sodium (initial dose 25 mg/kg, i.v., supplemented with 12 mg bolus doses as required). The rabbits were immobilized by intravenous injection of pancuronium bromide (Mioblock, Sankyo) and artificially respired with intermittent positive pressure. In the tracing experiments the animals were anesthetized with a-chloralose and urethane at the same dose as above administered intravenously and supplemented as required. Each rabbit was mounted on a stereotaxic frame in a prone position with the head fixed rigidly with a mouth-piece and two pointed screws piercing the zygomatic arch from both sides. The cerebellar vermis was exposed by partial craniotomy in the occipital region. The animals were warmed with an electric heating pad. The atlas of the rabbit’s cerebellum was according to Larsell (1970).
2.1. Electrophysiological studies
Fig. 2. Effects of electrical stimulation of the lateral nodulus-uvula on BP (upper traces) and RSNA (lower traces) in the decerebrate unanesthetized rabbit. (A) Responses in the same rabbit as in Fig. 1A. (B, C) Stimulation effects before and after injection of sympathetic ganglion blocker, hexamethonium, respectively. Horizontal bars show the time when the electrical stimulus was applied (interval, 5 ms; pulse width, 100 ms).
2. Materials and methods Experiments were performed on 40 albino rabbits weighting 2.5–3.4 kg. Of these, 23 were used in the
The procedure of stimulating the lateral nodulusuvula of the cerebellar vermis has been previously described (Nisimaru and Watanabe, 1985). In order to exclude the possibility that our results obtained by stimulating the lateral nodulus-uvula were due to current spread or activation of passing fibers, we injected L-glutamate (0.2 M, 100 nl) as a chemical stimulant into the lateral nodulus-uvula of five rabbits using a microsyringe. The L-glutamate injection site was determined stereotaxically and it was marked electrolytically at the end of each experiment (current intensity, 400 mA DC; duration, 20 s) and was located later by histological examination. The surgical procedures for isolation of the left renal sympathetic nerve bundle and for recording efferent discharges from these nerves have already been described (Nisimaru et al., 1984). Blood flow to the kidney and to the hindlimb of the same side was monitored using electromagnetic flowmeters (MF-27, Nihon Kohden). Probes were placed distal to the profound branch in the femoral artery and close to the abdominal aorta in the renal artery. Arterial pressure at the abdominal aorta was recorded with a pressure gauge (MP-3, Nihon Kohden) through a heparin-filled polyethylene tube inserted into the left femoral artery. The integrated RSNA, arterial BP and blood flow were recorded simultaneously on a chart recorder (Nihon Kohden) and stored on a data recorder (RD-101T, TEAC) for off-line analysis.
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2.2. Morphological studies 2.2.1. Anterograde axonal transport of BDA A micropipette (tip diameter, 30 mm), containing a 10% solution of BDA (10 kDa mol. wt. lysine fixable, Molecular Probes) dissolved in saline, was stereotaxically inserted through the uvula (sublobule IXb) into
the nodulus. The solution of BDA was then injected iontophoretically into the nodulus with application of a 2–4 mA DC current (tip positive) for 11–20 min. After a survival period of 8–11 days, the rabbits were deeply anesthetized and perfused transcardially with a mixture of 4% paraformaldehyde and 0.05% glutaraldehyde dissolved in 0.1 M phosphate buffer solution at pH 7.4.
Fig. 3. Effects of electrical stimulation of the lateral nodulus-uvula on regional blood flow in rabbits. (A) Stimulation effects on BP (top trace), renal arterial blood flow (middle trace) and femoral arterial blood flow (bottom trace) under the anesthetized condition. (B) Similar parameters shown in (A), but under a decerebrate unanesthetized condition. Horizontal bars show the time when the electrical stimulus was applied.
Fig. 4. Effects of stimulation of the lateral nodulus-uvula on the renal arterial blood flow before and after dissection of the renal nerve fiber in the decerebrate unanesthetized rabbit. (A) Responses to stimulation before the ipsilateral (left) renal nerve dissection as shown in Fig. 3. (B) Parameters similar to those shown in (A) but after dissection of the ipsilateral renal nerve. Horizontal bars show the time when the electrical stimulus was applied.
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Fig. 5. Schematic drawing showing the sites of BDA injection in the nodulus and uvula of the cerebellar cortex in six rabbits (R50, R51, R54, R55, R58 and R60). (A) Surface view of the uvula (sublobule IXd) and the nodulus (lobule X). For this illustration, the cerebellar cortex is unfolded around the borderline between the dorsal and ventral parts of each lobule (dashed line) and compressed along the sagittal line at the ratio of 1/3. The injection area is enclosed. (B) Illustration of the parasagittal cerebellar section 2.94 mm lateral from the midline.
The cerebellum and brainstem were removed and 60mm-thick frozen serial sections were cut parasagittally. These brain slices were treated according to the procedure of Brandt and Apkarian (1992) with modification. BDA in the sections was visualized by incubation with a mixture of 0.02% diaminobenzidine, 0.003% H2O2 and 0.5% nickel ammonium sulfate in Tris buffer solution (pH 7.6). The sections were then counterstained with Neutral Red. To compare the results obtained from the seven rabbits, the mediolateral extent of the nodulus was normalized to the average width (3.3 9 0.3 mm, mean9 S.D., n= 7). 2.2.2. Retrograde axonal transport of HRP A double-barreled glass micropipette (tip diameter of each, 30 mm), with one of the barrels filled with 15% HRP (Grade 1-C, Toyobo) dissolved in 50 mM Tris buffer solution (pH 7.5) and 0.1 M KCl for HRP injection, and the other with 2 M NaCl for electrical stimulation, was inserted stereotaxically into the PB via the anterior cerebellar vermis. Previous reports (Mraovitch et al., 1982; Ward 1988) showed that the electrical stimulation of the PB resulted in an increase in BP (pressor response). Therefore, to determine the localization of the PB for injection, the BP was recorded continuously and the pressor response to the electrical stimulation via one of the barrels of the glass micropipette was examined. After the PB was thus identified, HRP was iontophoretically injected with application of a 4–6 mA DC current (tip positive) for 20 – 40 min. Following a survival period of 24 – 48 h, the rabbits were deeply anesthetized and perfused transcardially with 1% paraformaldehyde and 1.25% glutaraldehyde in 0.1 M phosphate buffer solution at pH 7.4. Histological procedures for examination of HRP transport have been previously described in detail (Katayama and Nisimaru, 1988). To compare the HRP injection site
among the ten rabbits, the distance of the brainstem from the inferior colliculus to the obex was normalized rostrocaudally to the average length (10.79 1.2 mm, mean9S.D., n=10). HRP-labeled cells were drawn on the normalized nodulus and uvula, the same as in the BDA experiments.
3. Results
3.1. The cardio6ascular responses to stimulation of the lateral nodulus-u6ula in anesthetized rabbits and decerebrate unanesthetized rabbits 3.1.1. RSNA and BP In rabbits anesthetized with a-chloralose and urethane, inhibition of RSNA and a decrease in BP (depressor response) were observed following electrical stimulation of the lateral nodulus-uvula with a series of brief current pulses for 10 s (intensity, 50–200 mA; interval, 5 ms; pulse width, 100 ms) as shown in Fig. 1A. Falls in mean blood pressure (MBP) were 27.893.5 mmHg from control values of 102.79 3.1 mmHg in five rabbits (Table 1, PB 0.01). These responses were induced from sites which fell within a small region extending about 1 mm longitudinally through the dorsal nodulus encroaching the border to the ventral uvula,2.9–3.7 mm lateral to the midline as demonstrated in a previous study (Nisimaru and Watanabe, 1985). When 0.2 M L-glutamate (100 nl) was injected into the same region in the lateral nodulus-uvula, inhibition of RSNA and a depressor response were also induced in three anesthetized rabbits (Fig. 1B). This observation confirms that the effects of stimulation of the lateral nodulus-uvula arise from the activation of cerebellar Purkinje cells in this region. We performed similar experiments as described above in decerebrate unanesthetized rabbits. The elec-
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trical stimulation of the lateral nodulus-uvula with repetitive pulses for 10 s (intensity, 50 – 200 mA) evoked a significant increase followed by inhibition of RSNA and a marked pressor response (Fig. 2A and B). Changes in RSNA were transient increases for the first 3 – 6 s of the 10 s stimulus period, but an inhibition at a point where the increase in MBP had reached 10–15 mmHg. Increases in MBP were 40.2 92.6 mmHg from
control values of 104 9 5.2 mmHg in five rabbits (Table 1). These responses were in contrast to those seen in the anesthetized rabbits. Following i.v. injection of hexamethonium, a sympathetic nerve ganglion blocker, RSNA was inhibited and the pressor response disappeared during stimulation of the lateral nodulus-uvula (Fig. 2C). These results showed that the pressor response was induced by the increase in RSNA.
Fig. 6. Terminal-like patterns in the PB anterogradely labeled by BDA into lobule X for R60. (A) Bright-field photomicrograph of the injection site in the dorsolateral region of lobule X (2.88 mm sagittal section right from the midline). (B, C) Bright-field photomicrographs showing BDA-labeled terminal-like patterns (arrowheads) in the lateral (B) and medial (C) PB for R60 (Fig. 7Ae and Ac, respectively). Calibration bars 200 mm in (A), 25 mm in (B) and 20 mm in (C).
3.1.2. Renal and femoral arterial blood flow In five anesthetized rabbits the depressor response was associated with changes in renal and femoral arterial blood flow. The renal blood flow showed an initial increase followed by a decrease during the stimulation as shown in Fig. 3A. In three rabbits the renal blood flow showed increases in mean blood flow (MBF) of 2.69 0.9 ml/min from control values of 199 0.6 ml/ min during nodulus stimulation (Table 1). The initial increase in renal blood flow showed a time course similar to the inhibition of RSNA (Figs. 1A and 3A). This indicates that the kidney vascular resistance transiently decreased due to the inhibition of RSNA. The subsequent decrease in renal blood flow, however, showed a time course similar to the depressor response (Fig. 3A). Therefore, it appears that the subsequent decrease in renal blood flow is a passive effect secondary to the depressor response. The changes in femoral blood flow showed a similar pattern to those in renal blood flow but were less prominent during the stimulation as shown in Fig. 3A and Table 1. In decerebrate rabbits, changes in renal and femoral arterial blood flow were studied during the lateral nodulus-uvula stimulation. The renal blood flow markedly decreased following stimulation with repetitive pulses for 10 s (Fig. 3B). In five rabbits renal blood flow showed significant falls in MBF of 12.491.1 ml/min from control values of 18.29 1.6 ml/min (Table 1). The time course of this decrease in the renal blood flow is similar to that of the increase in RSNA (Figs. 2A and 3B). Following dissection of the left renal nerve fiber, BP showed no significant change but renal blood flow decreased. Under this condition the pressor response to nodulus stimulation was still seen, but the response in terms of blood flow of the ipsilateral renal artery was reversed (an increase instead of a decrease) as shown in Fig. 4. These results confirm that the decrease in the renal blood flow following stimulation of the lateral nodulus-uvula is the direct effect of RSNA. On the other hand, the femoral blood flow initially decreased but immediately increased following a time course similar to the pressor response during stimulation (Fig. 3B and Table 1). This indicates that the changes in hindlimb blood flow are mainly passive effects secondary to the increase in BP.
K. Sadakane et al. / Neuroscience Research 36 (2000) 15–26 Fig. 7. Distribution of labeled axons and terminal-like deposits in the cerebellum and brainstem following injection of BDA into the lateral nodulus-uvula in three out of six rabbits shown in Fig. 5. (A) R60, (B) R51 and (C) R58. Line drawings of the normalized sagittal sections of the ipsilateral cerebellum and brainstem between 1.2 and 4.8 mm from the midline at 0.6 mm intervals (1.5, 2.1, 2.7, 3.3, 3.9 and 4.5 mm for a, b, c, d, e and f, respectively). Labeled axons and terminal-like deposits contained in 10 sections (60 mm thick each) are collectively plotted in each illustration. Dashed lines in each illustration represent the labeled axons. Arrows (*) show the terminal fields. Abbreviations: N.V.mes, mesencephalic nucleus of the trigeminal nerve; CG, central gray matter; PH, prepositus hypoglossal nucleus; F, fastigial nucleus; N.X, dorsal nucleus of the vagus nerve; N.XII, hypoglossal nucleus; MV, medial vestibular nucleus; SCP, superior cerebellar peduncle; ICP, inferior cerebellar peduncle; l.PB, lateral parabrachial nucleus; m.PB, medial parabrachial nucleus; SV, superior vestibular nucleus; LV, lateral vestibular nucleus; IP, interpositus nucleus; IV, inferior vestibular nucleus; D, dentate nucleus; and IC, infracerebellar nucleus.
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Fig. 8. Distribution of labeled cells in the ipsilateral nodulus and uvula following injection of HRP into the entire area of right PB. (A) Line drawing of the brainstem coronal section showing the HRP injection sites for R13 (bold dotted line) and R17 (bold line) on the right side. (B) Illustration of the sagittal section of the cerebellum and brainstem indicated by a dashed line through PB and SCP in (A) also showing the sites of HRP injection. (C) Line drawings of sagittal sections of the nodulus and uvula. Left diagrams: entire view of the sections. Upper and lower illustrations represent the lateral half and the medial half of the nodulus and uvula, respectively. Labeled cells contained in half of the serial sections (each 60 mm thick) are collectively plotted in each illustration (upper panels, at 2.46 mm; lower panels, at 0.84 mm). A large dot represents 10 cells and a small dot represents one cell. Abbreviations: LC, locus ceruleus; MCP, middle cerebellar peduncle. See Fig. 7 for the other abbreviations.
These results suggest that the cardiovascular responses evoked by stimulation of the lateral nodulusuvula are mediated by two different pathways, the dominance of which is changed by anesthesia.
3.2. Morphological studies 3.2.1. Distribution of labeled fibers and terminals following BDA injection In six rabbits the injection sites were localized to the lateral part of the nodulus-uvula as shown in Fig. 5 and Fig. 6A, the stimulation of which resulted in the changes in RSNA, BP and blood flow as described above. All of these injection sites were restricted to the cerebellar cortex and underlying white matter excluding cerebellar nuclei (Fig. 5). The distribution of anterogradely labeled axons is represented schematically in the drawings in Fig. 7 in three typical cases (R60, R51 and R58) out of six. The labeled fibers are identified in the cerebellum and the brainstem between 1.2 and 4.8 mm from the midline. In the medial sagittal sections labeled fibers originate from the lateral nodulus-uvula, and extend to within and
around the fastigial nucleus (Fig. 7Aa, Ab, Ca, and Cb). More laterally, labeled fibers separate into two components as shown in Fig. 7Cc. The first component appears to descend into the brainstem by passing through the medial part of the inferior cerebellar peduncle (ICP) and extends into the superior vestibular nucleus (SV) and medial vestibular nucleus (MV) as shown in Fig. 7Bc and Cc. The second component appears to extend rostrally within and around the superior cerebellar peduncle (SCP) as shown in Fig. 7Ad and Cd. In more lateral sections this second component passes within and around the dorsal SCP and extends into the PB (Fig. 7Ae and Ce). Labeled fibers also pass through the vicinity of the interpositus nucleus as shown in Fig. 7Bc, Bd and Cc. In one rabbit, a few labeled fibers were seen in the dorsal area proximate to the contralateral SCP (data not shown). The distribution of labeled terminal fields is also represented schematically in the drawings in Fig. 7. It appears that the heaviest projection from the lateral nodulus-uvula is seen in the dorsal parts of the SV and the MV (Fig. 7Bb and Bc). On the other hand, no
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labeled terminal-like pattern was observed in the lateral vestibular nucleus and inferior vestibular nucleus. In addition, we found two new terminal regions, the dorsal region of the lateral PB (Figs. 6B and 7Ae), and an area immediately dorsal to the medial PB (Figs. 6C and 7Ac). In the cerebellum a few terminal fields were found in the infracerebellar nucleus as shown in Fig. 7Be. In the more lateral sections, terminal-like deposits were found in the dentate nucleus between 5.7 and 6.1 mm from the midline (data not shown). In this experiment, no labeled terminal fields were found in the fastigial and interpositus nuclei. These results indicate that Purkinje cells in the lateral nodulus-uvula directly project into three regions; the dentate and infracerebellar nuclei via the lateral portion of the cerebellar white matter, the vestibular nuclei complex passing through the ICP, and the PB passing within and around the SCP via the ventral region to the interpositus nucleus.
3.2.2. Distribution of labeled Purkinje cells following HRP injection To study the projection from the lateral nodulusuvula to the PB, we injected a retrograde tracer, HRP, into the PB. The double-barreled micropipette was inserted from the anterior cerebellar vermis to the PB. There was neither histological damage caused by mi-
23
cropipette penetrations nor formation of HRP reaction products in the white matter of the posterior cerebellum including cerebellar nuclei and in the SCP. In two experiments HRP was injected into both the lateral and medial PB (Fig. 8A and B). In R13, a large amount of HRP was injected into the PB, and a small amount, therefore, was distributed over the rostral SV. Retrogradely labeled Purkinje cells were found in the entire area of the nodulus and uvula as shown in Fig. 8C. On the other hand, in R17, only a small amount of HRP was injected restrictedly in the PB except for a small region of the adjacent middle cerebellar peduncle and SV as shown in Fig. 8A and B. Labeled Purkinje cells were found only in the nodulus. In five rabbits, the sites for HRP injection were localized in the lateral PB and the tracer was not observed to spread to the SV or the medial PB as shown in Fig. 9A and B. In the cases of R20 and R23, distribution of the tracer was restricted within the lateral PB, and retrogradely labeled Purkinje cells in the medial region of the cerebellar vermis were found extensively in the nodulus and uvula as shown in Fig. 9C. On the other hand, labeled cells in the lateral region of the cerebellar vermis were localized in the dorsal aspect of the nodulus and the ventral aspect of sublobule IXd (Fig. 9C). In three rabbits, HRP was injected into the medial PB and no labeled Purkinje cells were observed
Fig. 9. Distribution of labeled cells in the nodulus and uvula following injection of HRP into the lateral area of right PB. Similarly illustrated to Fig. 8 expect for R14, R20, R23, R33 and R35. Labeled cells in the nodulus and uvula from two typical cases (R20 and R23) out of five rabbits.
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in the nodulus and uvula (data not shown). From these results, it can be deduced that Purkinje cells in the lateral nodulus-uvula, which regulates the cardiovascular system, project directly to the lateral PB.
4. Discussion The present study, using BDA and HRP tracing methods in the rabbit, revealed new direct projections of Purkinje cells from the lateral nodulus-uvula to the PB, especially the lateral PB. On the other hand, Paton et al. (1991) have reported that Purkinje cell axons in the medial uvula (sublobule IXb) project to the PB in the rabbit using anterograde tracing methods with wheat germ agglutin in conjugated HRP. Furthermore, Supple and Kapp (1994) demonstrated that Purkinje cells in the anterior cerebellar vermis (lobules III and IV) terminate to the PB in the rabbit. From these results, it is apparent that Purkinje cells from several regions in the cerebellar vermis project directly into the PB. The PB has been reported to be involved in cardiovascular regulation in various mammalian species as follows. The electrical and chemical stimulations of the PB, particularly the lateral PB, evoke the pressor response, and this nucleus receives afferent inputs from the nucleus tractus solitarius, which is known to receive primary barosensory input (Hamilton et al., 1981; Mraovitch et al., 1982; Ward, 1988; Miura and Takayama, 1991). The PB stimulation also modulates the barorecepter reflex in cats (Felder and Mifflin, 1988). Our previous study showed that the lateral nodulus-uvula in the cerebellum is involved in cardiovascular control (Nisimaru and Watanabe, 1985). Taken together, the present results suggest that the PB is a relay nucleus from Purkinje cells in the lateral nodulus-uvula to the sympathetic nerves which control the cardiovascular functions. On the other hand, in the present study using BDA, the heaviest projection from Purkinje cells in the lateral nodulus-uvula is directed to the SV and the MV. Efferent connections from lobules IX and X to the vestibular nuclei complex were reported in the rabbit (Van Rossum, 1969; Epema et al., 1985; Henry et al., 1989; Wylie et al., 1994), in the cat (Angaut and Brodal, 1967; Voogd, 1967; Dietrichs et al., 1983; Shojaku et al., 1987; Walberg and Dietrichs, 1988), in the rat (Bernard, 1987) and in the prosimian primate (Haines, 1977). Using tracing methods, Henry et al. (1989) and Wylie et al. (1994) demonstrated the termination of the projection from Purkinje cells in the lateral nodulus into the SV, the MV and the group y in the rabbit. In the present study, however, labeled terminals were found not in the group y but in the infracerebellar .
nucleus. The cells of the infracerebellar nucleus are loosely arranged and located between the group y and the dentate nucleus in rabbits as described by Epema et al. (1988). Because of the ambiguous location of this nucleus, other reports might have included the infracerebellar nucleus in the group y and/or the dentate nucleus. Therefore, our results on the projection to the vestibular nuclei complex from the lateral nodulusuvula are nearly in agreement with these previous reports. Uchino et al. (1970) reported that an inhibition of RSNA resulted following electrical stimulation of the vestibular nerve. Yates and Miller (1994) showed that nose-up tilting resulted in an increase in splanchnic nerve activity while nose-down tilting reduced this activity, and these responses were abolished by application of a lesion of the MV and the inferior vestibular nucleus. They suggested that the vestibular system is involved and participated in compensating for posturally related changes in BP. The observed projection to the dentate nucleus from the lateral nodulusuvula in this experiment nearly corresponds with those in previous studies (Van Rossum, 1969; Bernard, 1987). In the present study electrical stimulation of the lateral nodulus-uvula in the decerebrate unanesthetized rabbit evoked a pressor response and a decrease in the renal arterial blood flow associated with a transient increase in RSNA. These responses were in contrast to those in the anesthetized rabbit (Nisimaru and Watanabe, 1985). As the responses observed in the decerebrate unanesthetized rabbit were abolished by anesthesia, as shown in Figs. 1A and 2A, this complete alteration of evoked cardiovascular responses was not caused by decerebration itself but by anesthesia. Bradley et al. (1987) reported similar results in the medial uvula of the cerebellum as follows. In decerebrate unanesthetized rabbits the stimulation of the medial uvula (sublobule IXb) in the posterior cerebellar vermis evoked a marked tachycardia, an increase in BP, a sustained increase in RSNA, and a decrease in both renal and femoral conductances. These cardiovascular responses were abolished by a small dose of anesthetic. Bradley et al. (1987) also suggested the presence of two different pathways mediating cardiovascular function, dominance of which is altered by anesthesia. The present study using the anterograde tracing methods also suggests that labeled fibers of Purkinje cells in the lateral nodulus-uvula are separated into two parts, one of which extends across the cerebellar white matter and enters around and within the SCP, and another which extends into the ICP. It appears that the fibers around and within the SCP project into the PB, and those within the ICP, to the vestibular complex; however, a possibility remains that a part of the former projects
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into the vestibular nuclei via the SCP. Paton et al. (1991) demonstrated two pathways of Purkinje cell axons, that is, via the SCP and the ICP, to the brainstem as described above. They also reported that the presence of a lesion in the SCP abolished the cardiovascular responses to stimulation of the medial uvula in the anesthetized rabbit, but not in the decerebrate unanesthetized one. On the other hand, the presence of a lesion in the ICP abolished these cardiovascular responses only in the decerebrate unanesthetized rabbit. They suggested that these cardiovascular responses in the anesthetized rabbit were mediated by fibers within the SCP and relayed to the PB, while those in the decerebrate unanesthetized rabbit were mediated by ICP and relayed to the vestibular nuclei. These similarities between our results and Paton’s in the Purkinje cell projection and in the effect of anesthesia indicate that both the lateral nodulus-uvula and the medial uvula have two pathways, that is, via the PB and vestibular nuclei, which control the cardiovascular functions. The present study, furthermore, showed that there are dominant terminal fields from the lateral nodulus-uvula in the SV and the MV, as compared with those in the PB. These results indicate that the pathway involved in cardiovascular control via vestibular nuclei is stronger than that via the PB, and that the former pathway might be dominant under the unanesthetized condition and sensitive to anesthetics. Recently Nisimaru et al. (1998) showed that this cerebellar region has an important role in a conscious rabbit during tilting the head up, and is involved in the control of timing and duration of the transient increase in RSNA and in the rapid adaptation of BP during postural changes. Therefore, the present results suggest that the pathway via the ICP and vestibular nuclei may mediate the adaptive cardiovascular control by the lateral nodulus-uvula during postural changes in conscious animals.
Acknowledgements We would like to thank Professor Kazuhiro Yamada (Department of Physiology, Oita Medical University) for his constant encouragement during the course of this study, and Professor Jinzo Yamada (Department of Anatomy, Tokyo Medical University) and Dr Soichi Nagao (Department of Physiology, Jichi Medical School) for their valuable discussions regarding the anatomical aspect of this study. We are grateful to Dr Taiko Kitamura (Department of Anatomy, Tokyo Medical University) for her helpful advice on BDA and HRP tracing techniques. We thank Eriko Hayashi, Hiroaki Kawazato and Aiko Yasuda for their technical assistance.
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