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Efferent connections of lobule IX of the posterior cerebellar cortex in the rabbit — some functional considerations
Journal of the Autonomic Nervous System, 36 (1991) 209-224 © 1991 Elsevier Science Publishers B.V. All rights reserved 0165-1838/91/$03.50
209
JANS 01221
Efferent connections of lobule IX of the posterior cerebellar cortex in the rabbit some functional considerations J.F.R. Paton
1, A.
La Noce
1, R.M.
Sykes 1, L. Sebastiani 2, p. Bagnoli and D.J. Bradley 1
2,,
B. Ghelarducci 2
t Department of Physiology, Royal Free Hospital School of Medicine, London, U.K., and 2 Dipartimento di Fisiologia e Biochimica, Universitd di Pisa, Pisa, Italy (Received 10 June 1991) (Revision received 13 August 1991) (Accepted 14 August 1991)
Key words: Cerebellar uvula; Purkinje cell axon; Anterograde tracer; Cardiovascular response Abstract The Purkinje cell projection from the cardiovascular region of sublobule b of the uvula (medial area of zone A) has been investigated using anterograde tracing methods in the rabbit. The importance of the integrity of the identified pathways in mediating the cardiovascular responses from the uvula has been studied in subsequent lesioning experiments. Wheat germ agglutinin-conjugated horseradish peroxidase or tritiated amino acids were microinjected into sublobule IXb. This resulted in anterogradely labelled Purkinje cell axons in both the inferior and superior cerebellar peduncle. In agreement with previous studies in rabbit we also found labelled fibres at the level of the fastigial nucleus and vestibular complex. However, the labelled fibres we observed in the parabrachial nucleus have not been reported in previous studies except in the prosimian primate. Projections from IXb showed terminal-like patterns of label in the ventromedial region of the caudal fastigial nucleus, the dorsal areas of the superior and inferior vestibular nuclei and in the medial and lateral divisions of the parabrachial nucleus. Labelled fibres were also seen coursing in the lateral vestibular nucleus. Lesioning experiments have revealed that the integrity of the superior cerebellar peduncle is essential for the expression of the cardiovascular responses (bradycardia and depressor response) elicited from the uvula in the anaesthetized rabbit. In contrast, the pattern of cardiovascular response evoked in a decerebrate rabbit (tachycardia and pressor response) was abolished when the inferior cerebellar peduncle was lesioned.
Introduction
The efferent connections of the posterior cerebellar cortex have been described in a variety of species using a number of neuroanatomical tracing techniques. In general, these studies have
Correspondence: B. Ghelarducci, Dipartimento di Fisiologica e Biochimica, Universitfi di Pisa, Via S. Zeno 31, 56100 Pisa, Italy. * Present address: P. Bagnoli, Facolt~ di Sciense, Universitfi della Tuscia, Viterbo, Italy.
revealed that there are two major projections from the posterior cortex, one to the fastigial nucleus (FN) and the other to the brainstem vestibular nuclei. It is known that the posterior vermis is topographically represented within the caudal region of the ipsilateral fastigial nucleus [27], and the corticovestibular projections, which descend through the ipsilateral inferior cerebellar peduncle (ICP), innervates a number of vestibular nuclei. In a series of cardiovascular studies, a discrete region of the uvula (lobule IX) of the posterior
210
cerebellar vermis has been shown to exert powerful influences on heart rate and arterial blood pressure during either electrical stimulation [8] or following microinjections of excitatory amino acids intra-cortically [9,11,39]. Two patterns of cardiovascular response can be evoked from the uvula which are dependent on the type of preparation used. Electrical or chemical activation of the uvula in the decerebrate rabbit evokes tachycardia and a pressor effect but in the anaesthetized rabbit (intact or decerebrate) a qualitatively reversed response pattern is evoked (falls in heart rate and arterial blood pressure). This prompts the question as to the identity of the pathway(s) and relay(s) responsible for mediating these two different patterns of response. Previous studies have indicated that cells within the caudal FN do not play a role in mediating the cardiovascular effects from the uvula [9] and this was confirmed subsequently using chemical stimulation techniques [39]. Accordingly, it must be assumed that either the vestibular complex or novel brainstem sites, yet to be identified, are responsible for the cardiovascular changes mediated from the uvula. There is, however, only limited and controversial evidence for the vestibular nuclei playing a role in control of the circulation, and even then this evidence has been obtained by electrical stimulation, which leaves open the possibility that the effects resulted from stimulation of fibres of passage [30,35,42,43,45]. In this study we have used tritiated amino acids and wheat germ agglutinin-conjugated horseradish peroxidase to trace anterogradely the efferent projection from the cardiovascular region of the uvula in the rabbit. Subsequently electrolytic lesions have been placed in these pathways to assess their influence on the expression of the cardiovascular responses elicited from the uvula, in both the anaesthetized and decerebrate, unanaesthetized, rabbits.
Materials and Methods
Experiments were performed on 22 New Zealand white rabbits weighing 2.0-2.5 kg. Of these, 11 were used in the anterograde tracing
experiments and ll were used in functional studies.
Anatomical experiments Rabbits were anaesthetized with sodium pentobarbitone (Sagatal, May and Baker Ltd., U.K., 35-40 m g . k g 1, i.v.), the head placed in a stereotaxic apparatus and the posterior cerebellar vermis exposed by retraction of nuchal muscles and removal of the overlying occipital bone. Through a small hole in the dura the tip of a glass micropipette (dia. 20-40 p~m) filled with either wheat germ agglutinin-conjugated horseradish peroxidase ( W G A - H R P , Sigma Ltd.) or tritiated amino acids (New England Nuclear Ltd.) was lowered with the aid of a dissecting microscope I mm from the pia into the medial region of lobule IXb. Five rabbits received a 200 nl microinjection of a 4% solution of W G A - H R P dissolved in saline and three received 40 /xCi of tritiated k-proline (38.5 m C i / m m o l ) dissolved in 200 nl of saline. In an additional three animals a mixture of tritiated L-proline and e-leucine (50 /xCi/ixl) in saline was used and microinjected in 25 nl volumes at depths of 1.0, 0.75, 0.5 and 0.25 mm from the pia (total microinjected was 5 p~Ci). When delivery was complete the micropipette was left in situ for 10 min before withdrawal in order to avoid any leakage of tracer from the pipette track.
WGA-HRP procedures" A survival time of 24-48 h was allowed before rabbits were deeply anaesthetized (Sagatal, 50 mg- kg-~) and perfused transcardially with 1 1 of warm (38 ° C) saline followed by 1 I of fixative, consisting'of 1% paraformaldehyde and 1.25% glutaraldehyde in 0.1 M phosphate buffer at 4 ° C, and finally with 1 1 of a 10% sucrose solution. Immediately after perfusion, the brain was removed from the skull and equilibrated overnight in 30% sucrose solution at 4 °C. The following day, frozen sections were cut at 30 Ixm either in the frontal plane (n = 3) or in the sagittal plane (n = 2). Sections were treated for peroxidase activity with tetramethylbenzidine according to the protocol of Gibson et al. [22] and counterstained
211 with Neutral Red. Sample injection site sections were treated with 3,3' diaminobenzidine-tetrahydrochloride according to the protocol of G r a h a m and Karnovsky [23] before counterstaining with Cresyl Violet.
Autoradiographic procedures This part of the study employed two different protocols but gave qualitatively identical results. Rabbits were allowed a survival time of 4 - 7 days before being deeply anaesthetized (urethane, 1.5 g . k g -1) and perfused transcardially with 10% formalin or 5% glutaraldehyde. I m m e diately after perfusion the brain was removed and post-fixed. Either frozen sections were cut at 30 /zm or blocks of tissue were placed into molten paraffin wax and sections cut at 15 /zm. Frontal sections were cut in both cases and mounted onto glass slides which were dipped in emulsion and exposed for 4 - 8 weeks in the dark at 4 ° C. Autoradiograms were developed, fixed and counterstained before being examined. All material was examined under the light microscope using bright and dark field illumination. Injection sites and labelled structures were recorded with the aid of a projector and photomicrographs taken of representative sections. The currently accepted nomenclature for the rabbit according to Brodal and Jansen [12] and Meessen and Olszewski [35] has been used.
Electrolytic lesioning experiments O f the 11 rabbits used in this study, four were anaesthetized with urethane (Sigma Ltd., 1.4 g. kg -1 i.v.) and seven with alphaxolone alphadolone (Saffan, Glaxovet Ltd., 4 mg" kg-1 i.v. and supplemented with 4 mg bolus injections as required). In all animals the bladder was drained and cannulated and a tracheostomy performed to allow for artificial ventilation. End tidal CO 2 was sampled, monitored and kept at 4.5_+ 0.5% by altering minute ventilation. Arterial blood pressure was monitored via a cannula placed into the femoral artery and connected to a transducer (Gould Statham, U.S.A., P23Db). H e a r t rate was derived from the pulse frequency using a ratemeter (Neurolog, Digitimer Ltd., module NL250). A
femoral vein was also cannulated to permit administration of anaesthetic supplements and drugs. Rectal t e m p e r a t u r e was recorded and maintained at 38 ° C using a heating lamp. Rabbits anaesthetized with Saffan were paralyzed with decamethonium bromide (Sigma Ltd., 0.5 m g . k g - 1 i.v. and supplemented every 15-20 rain), ventilated artificially before being decerebrated at the pre-collicular level during which time both common carotid arteries were occluded. After decerebrating, one common carotid was released. In all animals the posterior vermis was exposed (see above) and protected with frequent topical application of warm paraffin oil; the midline region of lobule IXb was stimulated intracortically using a metal-filled microelectrode (tip diameter 2 0 / z m ) with a 6-s stimulus train (0.5 ms; 100 Hz) and intensities of 200-400 /~A in anesthetized animals and 50-150 /xA in the decerebrate preparations. Constant current was used and monitored on an oscilloscope. The cardiovascular responses evoked from the uvula in anaesthetized and decerebrate rabbits were compared before and after lesioning areas of the brainstem which our anatomical study had shown to contain labelled Purkinje cell axons originating from the uvula. Electrolytic lesions were made by passing 2 m A anodal current for 40 s through a stainless steel electrode insulated to 300 /~m from its tip. Bilateral lesions of the SCP were placed at 4.0 m m anterior to lambda, 3.0-3.3 m m lateral to midline and 13.5 m m ventral to dura in some animals whereas ICP lesions were placed at 1.5 m m anterior to lambda, 3.7 m m to 4.5 m m lateral to midline and 14 m m ventral to dura. In all cases the head was tilted so that lambda was 1.5 m m ventral to bregma. Following the termination of every experiment the brain was removed and placed in 10% formalin and left for 48 h before serial frontal sections were cut (80 tzm), mounted onto slides and stained with Neutral Red. The exact location and size of each lesion was recorded. A two sample t-test was employed to assess the statistical significance of the data. All data are expressed as mean _+ SEM (standard error of the mean).
212
C A
IX
C
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Fig. 1. Representative W G A - H R P (A) and tritiated amino acid (B) injection sites placed in the cerebellar uvula (lobule 1X a,b,c,d) of the rabbit. A: The injection site is centered in the midline of sublobule b (B) and extends laterally up to 800 ~ m of both brain sides (A and C). B: The injection site is centered on the midline of sublobule b and extends to the upper part of sublobule c. Its caudo-rostral extention is shown from A to C; each section is taken at 900-#m interval. These cases are representative of all W G A - H R P and tritiated amino acids injection sites. Light field micrographs (sagittal sections, 30 tzm, scale bar: 2 mm).
213
MEDIAL
1.2
D
2.2
2.6
3.8 LATERAL
Fig. 2. Schematic diagram showing the distribution of anterogradely labelled axons (dashed lines) in sagittal sections of the cerebellum following W G A - H R P microinjection into the midline region of sublobule b of the uvula (A-D). The illustration shows a single case but is representative of the other experiments of this kind. For each section the distance, in mm, from the midline is indicated. See text for abbreviations.
Results
Anatomical experiments In the present study an area was considered to receive efferent projections from the uvula when it exhibited a comparable terminal-like pattern in
both W G A - H R P and tritiated amino acid studies in either frontal or sagittal sections. The location of retrogradely labelled neurons in the brainstem following W G A - H R P microinjections was consistent with data reported previously [5,11] where the retrograde transport of H R P from the cortex of sublobule IXb had been used. In particular, the labelled cells in the inferior olivary complex were located mainly in the 13 nucleus of the medial accessory olive. This area, according to the zonal pattern of olivocerebellar projections proposed by Voogd [46] in the cat, projects exclusively to the medial portion of the vermis which has been termed zone A in the Voogd scheme [24,46].
Injection sites In all except two animals the injection site was localized to the midline portion of sublobule IXb (comparable to the medial region of zone A in the cat [24,46]). In two animals anterograde tracer was seen to have diffused into the adjacent midline regions of the ventral area of IXa and the dorsal half of IXc. The diffusion of the tracer substances was always restricted to the cerebellar cortex and underlying white matter without involving deeper cerebellar structures (Fig. 1A-B). There was no evidence in any experiment of radioactive label or W G A - H R P in deep cerebel-
")
Fig. 3. Dark and bright field micrographs showing the distribution of labelled Purkinje cell axons and terminal-like grains in the caudal fastigial nucleus (FN) following a microinjection of tritiated L-proline into sublobule IXb. Note the presence of labelled fibres projecting through the ventrolateral part of the nucleus and a terminal-like field in its ventromedial part. Comparable results have been obtained in both W G A - H R P and tritiated amino acids experiments (frontal sections, 30/zm, scale bar: 500/zm).
214
lar nuclei. In all animals, label was represented bilaterally in the cerebellum and brainstem.
Labelling within the cerebellum The distribution of anterogradely labelled axons within the cerebellum is represented schematically in the drawings of Fig. 2. In sagittal sections (Fig. 2A) labelled fibres from the uvula enter the ventromedial region of the caudal fastigial nucleus (FN) and then project rostrally through the ventral part of this nucleus. More laterally (Fig. 2B) a large contingent of fibres separates into two components: the first courses via the ventral-most part of the FN while the second passes through the dorsomedial portion of this nucleus. In more lateral sections the majority of labelled fibres bend caudally and descend into the brainstem by passing through the medial part of the inferior cerebellar peduncle (ICP) (juxtarestiform body) and pass through the lateral vestibular nucleus (LVN), while a small number of fibres branch away from the principal bunch and run rostrally before leaving the cerebellum within and around the superior cerebellar peduncle (SCP) (Fig. 2C). Finally, at the level of the interpositus nucleus (IN) (Fig. 2D), labelled Purkinje cell axons were also found to project around the dorso-rostral border of this nucleus before descending into the lateral region of the ICP (restiform body). As shown in the photomicrograph of Fig. 3 and in the drawing of Fig. 4D, a terminal-like labelling was visible in the ventromedial region of the caudal FN in addition to labelled fibres crossing the ventrolateral part of the FN. Labelling within the brainstem The distribution of labelled fibres and putative termination fields in the brainstem is represented schematically in the drawings of Fig. 4, which is a composite of both W G A - H R P and tritiated amino acid experiments. It appears that the heaviest projection from lobule IXb is directed to the vestibular complex. As seen in Fig. 5, labelled fibres leaving the cerebellum via the ICP enter the superior vestibular nucleus (SVN), where a termination field was visible throughout the rostrocaudal ex-
ROSTRAL
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CAUDAL Fig. 4. Schematic diagram showing the distribution of anterogradely labelled fibres (dashed lines) and putative terminal fields (dots) in frontal sections of the cerebellum and brainstem following microinjections of anterograde tracers into sublobule IXb (A-G). The illustration is a composite of data taken from W G A - H R P and tritiated amino acids experiments. For each section the distance, in mm, from obex is indicated. Abbreviations: DN, dentate nucleus; MVNmc, magnocellular medial vestibular nucleus; MVNpc, parvocellular medial vestibular nucleus; PH, praepositus hypoglossal nucleus; x, groups x of Brodal. See text for other abbreviations.
tent of its dorsal half. In the same figure there is also a sparse projection to the dorsal area of the rostral medial vestibular nucleus (MVN), which
215
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Fig. 5. Dark and bright field micrographs of anterogradely labelled Purkinje cell axons and terminal-like fields in the brainstem following tritiated L-proline microinjection into sublobule IXb. Labelling can be seen in the dorsal half of the superior vestibular nucleus (SVN) and in the rostral region of the medial vestibular nucleus (MVN). Comparable results have been obtained in both WGA-HRP and tritiated amino acids experiments. (frontal section, 30 #m, scale bar: 300/zm). Abbreviations: Cb, cerebellum; V, ventricle. See text for other abbreviations.
shows no evidence of termination. A large population of labelled Purkinje cell axons run caudally through the LVN, without exhibiting any appreciable terminal-like pattern, as confirmed in our sagittal sections. Within the inferior vestibular nucleus (IVN), labelled fibres are organized into typical bundles which are intermingled with cell bodies (Fig. 6). In addition, the caudal half of this nucleus, particularly its dorsolateral part, shows a dense terminal-like labelling (Fig. 6). In this region a small number of fibres arising from the ventral part of the labelling within the IVN turn
medially and run close to the dorsolateral edge of the rostral nucleus tractus solitarius (NTS). In addition to the labelling within the vestibular complex there is also a dense projection to the parabrachial nucleus (PBN). Fig. 7 shows heavy terminal-like labelling within both the dorsal halves of the medial and lateral regions of this nucleus. From the evidence presented in the present study it is clear that this projection is a significant Purkinje cell outflow from sub-lobule IXb. To date the PBN has not been identified as a major target of uvula efferent projections even
216
Fig. 6. Dark and bright field micrographs showing labelled Purkinje cell axons and terminal-like fields in the brainstem following tritiated amino acids microinjection into sublobule IXb. Labelling can be seen within the dorsolateral part of the IVN. Comparable results have been obtained in both W G A - H R P and tritiated amino acids experiments (montage, frontal sections, 15/xm, scale bar: 250/xm). See text for other abbreviations.
though numerous studies have been carried out on a range of species.
Electrolytic lesioning experiments Lesions have been placed bilaterally in two different regions of the brainstem, according to the course of anterogradely labelled Purkinje cell
axons descending from the sublobule b of the uvula as indicated by our W G A - H R P and tritiated amino acids experiments. In one group of rabbits the area of the SCP was lesioned electrolytically, while in the second group, lesions were placed at the level of the ICP which encompassed both the lateral (restiform body) and me-
TABLE 1
Comparison of the cardiocascular responses elicited by m'ula stimulation in anaesthetized (N = 4) and decerebrate (N = 3) rabbits before and after lesioning the SCP (mean + SEM) MAP (mmHg)
A. Anaesthetized Baseline Response
B. Decerebrate Baseline Response
N = number of animals. n = number of tests.
HR ( b e a t s / m i n )
Before
After
Before
After
97 _+ 0.86 (n = 43) -14+_0.94 (n = 3 4 )
106 + 0.83 (n = 37) -9+0.55 (n = 2 1 )
326 ± 3.21 (n = 43) 2 2 ± 2.(14 (n = 34)
258 + 2.1)I (n = 37) - 4 + 1.29 (n 21)
130 4-_ 1.111 (n = 16) +35±4.43 (n = 16)
131 ± 4.38 (n = 13) +25±3.86 (n = 13)
245 + 10.63 (n = 16) + 6 0 + 5.53 (n - 16)
267 + 9.42 (n = 13) +53_+9.51 (n = 13)
!
Fig. 7. Series of dark and bright field micrographs showing dense labelling within the dorsal regions of the medial (mPBN) and lateral (IPBN) parts of the parabrachial nucleus in a rabbit which received a microinjection of tritiated L-proline into sublobule IXb. Three levels extending caudo-rostrally from a to c are shown. The interval between the sections is 180 ~m; a is taken at 9.5 mm from the obex. Comparable results have been obtained in both W G A - H R P and tritiated amino acids experiments (frontal sections, 15 kLm, scale bar: 300/~m). See text for abbreviations.
218
B
A ANAESTHETIZED
C DECEREBRATE
BEFORE LESlONING
I
350
fv~
20O 200
~PBN
5O
AFTER LESIONING
350 [
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200
200
L _ _ I
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Fig. 8. An example of the effect of lesioning the SCP on the cardiovascular response ew)ked by uvula stimulation in an anaesthetized (A) and in a decerebrate rabbit (B). HR, heart rate in beats, min ~; BP, arterial blood pressure in mmHg. C: Schematic drawing showing the location and extent of electrolytic lesions of the SCP. Abbreviations: LC, locus coeruleus; VMES, mesencephalic trigeminal nucleus. See text for other abbreviations.
dial regions (juxtarestiform body ). Figs. 8C and 9B illustrate the location and extent of representative lesions in two rabbits.
SCP lesions The destroyed area e n c o m p a s s e d a large region of the SCP and surrounding PBN. In some animals additional regions d a m a g e d included the lateral part of the locus coeruleus and mesencephalic trigeminal nucleus. The effect of such lesions on the cardiovascular responses evoked from sublobule IXb were studied in both anaesthetized and decerebrate animals.
In anaesthetized rabbits (n = 4), following the lesion, there was a significant fall in heart rate ( P < 0.01) from a control level of 326 +_ 3.2 beats • m i n - 1 to 258 _+ 2.0 beats - min-- ~ and a significant rise in arterial blood pressure ( P < 0 . 0 1 ) from a resting level of 97 +_ 0.9 m m H g to 106 _+ 0.8 m m H g . The changes in heart rate and arterial pressure elicited during uvula stimulation are c o m p a r e d before and after the SCP lesion in Table I. It can be seen that the cardiovascular response (bradycardia and a depressor response) was reduced significantly ( P < 0 . 0 1 ) o r abolished (Fig. 8A) when c o m p a r e d to the response evoked
219
A BEFORE LESIONING MVN
300 I 14:{ IO0
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100 AFTER LESIONING
200 L 175
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BP 75 L
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Fig. 9. A: An example of the effect of lesioning the ICP on the cardiovascular responses elicited from the uvula in a decerebrate rabbit. Abbreviations as in fig. 8. B: Schematic drawing to show the location and extent of electrolytic lesions of the ICP. Abbreviations: RB, rest±form body. See text for other abbreviations.
heart rate or arterial blood pressure (Table I) and had no significant effect on the magnitude of the tachycardia and pressor response elicited from the uvula (Fig. 8B and Table I).
ICP lesions Electrolytic lesions of the ICP area were carried out in four decerebrate rabbits. Large regions of the ICP were destroyed in addition to some damage of the dorsal areas of the SVN and LVN. Table II shows that there were no changes in resting levels of heart rate and arterial blood pressure ( P < 0.01) after lesioning. However, the uvula-evoked tachycardia and pressor response was attenuated severely (Table II; P < 0.01). Interestingly, in one animal with an ICP lesion, uvula stimulation elicited a cardiovascular pattern of response qualitatively similar to that observed from an anaesthetized rabbit (bradycardia and depressor response, Fig. 9A). In this case uvula stimulation evoked a reduction in heart rate of 20 _+ 4.1 beats • m i n - ~ and a fall in arterial pressure of 11.6 _+ 1.6 mmHg. Discussion
in the intact anaesthetized rabbit. In order to assess that the reduction in the cardiovascular response was not secondary to brain function deterioration with time, lobule IXb was stimulated in one rabbit at regular intervals for 1 h without performing any lesions. The cardiovascular responses evoked after 1 h showed no difference in their magnitude ( P < 0.01). In decerebrate rabbits (n = 3) lesioning the SCP failed to produce any baseline changes in TABLE II
CardioL, ascular responses elicited by uvula stimulation in decerebrate rabbits (N = 4) before and after lesioning the ICP
Baseline Response
MAP (mmHg)
HR (beats min)
Before
After
Before
After
122±3.41 (n = 18) +52±2.90 (n = 15)
127± 3.41 (n = 18) +9±2.77 (n = 21)
250± 5.83 (n = 18) +35±3.70 (n = 15)
271 ±9.31 (n = 18) +8±2.39 (n = 21)
N = number of animals. n = number of tests.
The present series of experiments in the rabbit have revealed the Purkinje cell projections from the midline region of sublobule IXb, an area analogous to the medial region of zone A according to Voogd's longitudinal classification in the cat [24,45]. Labelled fibres have been found bilaterally in the caudal region of the FN, and also in the SVN, LVN, IVN and PBN. The limitations of light microscopy do not permit an accurate assessment of terminal structures, but the presence of dense grains and evidence of accumulation of transported material is suggestive of possible termination in all these nuclei with the exception of the LVN. The other part of this study has considered the relative importance of the identified pathways in mediating the cardiovascular responses evoked from the uvula. The data from the lesioning experiments suggest that the integrity of the SCP and the ICP are essential for the expression of the distinct cardiovascular patterns of response in the anaesthetized and decerebrate rabbits respectively.
22(I
Uvula Purkinje cell projections There is good anatomical evidence in a variety of species that the uvula projects to the caudal FN [27]. The present data in the rabbit illustrates a densely labelled pathway to the ventromedial portion of the caudal FN which is consistent with earlier reports [2,6,17,29,31,44,46,47] and is in good agreement with the mediolateral organization of the uvula projection to the caudal FN as described for the rat [3]. The SVN, LVN and IVN also received a projection from sublobule b of the uvula in the present study and this supports previous observations made on a variety of other species using different techniques. In earlier reports it was often emphasised that of all the lobules of the posterior vermis it is the uvula that sends the heaviest projection to the vestibular nuclei [1,26]. In our study the majority of fibres projecting to the vestibular nuclei either coursed through the medial or ventral part of the FN or ran around the dorsolateral edge of the IN before entering the brainstem via both regions of the ICP. Labelled fibres were most dense in the medial region of the ICP (juxtarestiform body). The pathways exiting from the cerebellum in the medial ICP have been well described before in the prosimian primate [26], cat [2,46,47] and opossum [31]. Putative termination was restricted to the dorsal SVN and the caudal region of the dorsolateral IVN. Dense label was also seen in the LVN but did not take the form of a terminal region when seen in both frontal and sagittal sections. In early studies there is some inconsistency as to which of the vestibular nuclei receives projections from the uvula. Dow [17] used degeneration techniques and reported termination in all vestibular nuclei following lesions of lobule IX in the rat. Using similar experimental methods in the cat, Walberg and Jansen [47] found degenerating axons in the dorsal halves of the LVN and IVN whereas Voogd [46] described projections to the MVN, descending vestibular nuclei (VN) (analogous to the IVN) and SVN. The difference between these reports and the data from the present study could be attributed to the exact region of lobule IX studied. Indeed, there is anatomical evidence for a topographical distribu-
tion of Purkinje cell axons from the uvula to the vestibular nuclei [6,41]. In the degeneration studies it is clear that different areas of the uvula were removed and the extent of the lesions varied between animals in any one study. In the present study the area of the uvula examined is highly localized and comparable between animals. Other investigators reported similar findings to those described in our study. For example, in the prosimian primate degenerating fibres were identified in the dorsomedial regions of the SVN, LVN and spinal VN (similar to the IVN in the rabbit, [26]). Using similar methods, the uvula was found to innervate these vestibular nuclei in the opossum [31], cat [2] and rat [1,44]. There have been other studies using retrograde transport of HRP to study the cerebellar corticovestibular projections in both cat and rabbit. It was shown that following a microinjection of HRP into physiologically identified vestibular nuclei in the cat, uvula Purkinje cells were retrogradely labelled only when the SVN and IVN were injected [41] which is consistent with the observations made in the present study. In contrast, in the rabbit, Balaban [4] applied H R P iontophoretically to the SVN, MVN and LVN and reported retrogradely labelled Purkinje cells in the uvula. In these latter studies it is possible that both terminal fibres and damaged axons of passage will have transported the HRP. Thus the differences between this study and that of Balaban [4] may be due to the difference in experimental approach. Another major projection from sublobule b of the uvula revealed in the present study included a relatively dense terminal-like label in the dorsal region of the medial and lateral PBN. This pathway appears not to have been reported previously, except in the prosimian primate and has not been recognized as a major projection from the uvula. It is likely that fibres within the SCP project to the PBN, though the possibility that some fibres within the ICP may project to the caudal PBN cannot be ruled out. Such a projection has not been reported by previous authors concerned with the corticofugal projections of the posterior vermis. In the prosimian primate Haines [26] described 'moderate to sparse preterminal
221 debris' in the medial and lateral PBN but was unable to attribute any physiological significance to this projection. From the density of label found in the present study it is surprising that such a pathway should have been unrecognized by other workers, and we see no reasonable explanation for this discrepancy at present. However, it should be emphasized that in this investigation the Purkinje cell projection studied was from a highly localized region of sublobule b of the uvula which was never specifically studied in previous studies. This suggests that, as earlier studies were concerned primarily with the vestibular connections of the posterior cerebellar cortex, other possible brainstem targets were not reviewed fully. Since the SVN and PBN are anatomically close, though separated rostrocaudally, this may have resulted in some confusion. It is interesting that the lateral PBN has been well documented as a region involved in cardiovascular regulation (for references see later) and in this context it is not surprising that the cardiovascular region of the uvula projects to it. Furthermore, its integrity appears essential for one component of the cardiovascular changes elicited from the uvula (see next section). In addition, in the present study there was also a sparse projection to the dorsomedial region of the caudal brainstem, running very close to the extreme dorsal edge of the rostral NTS. This is consistent with other studies which have described a similar projection in the primate [26], cat [2] and rat [1]. Interestingly, De Camilli et al. [18], using a specific immunocytochemical marker for Purkinje cells, demonstrated projections to the FN, VN, PBN, praepositus hypoglossal nucleus and NTS in the rat. Unfortunately using this type of approach yields no information as to the source of these projections.
Role of identified pathways in mediating the cardiovascular changes elicited from the uvula The injection sites were found to be within the midline region of sublobule IXb and therefore to occupy the exact region from which marked changes in cardiovascular variables can be elicited using either electrical [8] or chemical stimulation [9,11,39]. The fact that excitatory amino acids elicited cardiovascular effects from the uvula sug-
gests that these result from Purkinje cell activation rather than antidromic excitation of mossy or climbing fibres which can occur with electrical stimulation and would lead to activation of precerebellar nuclei. The purpose of this study was to identify the connections of the cardiovascular region of lobule IXb so that putative brainstem site(s) involved in mediating the cardiovascular responses could be identified. Our previous studies have indicated that electrical stimulation of the caudal FN elicits a pattern of response qualitatively identical to that elicited from the uvula [10]. Since Purkinje cells are strictly inhibitory it was anticipated that an opposite pattern of cardiovascular response would be observed if the caudal fastigial neurones are involved in mediating the cardiovascular responses from the uvula. Thus it is reasonable to suggest that those caudal fastigial neurones receiving Purkinje cell axons from sublobule IXb are not involved in relaying the cardiovascular responses from the uvula. Further evidence has revealed that chemical excitants microinjected into the caudal FN fail to elicit any change in heart rate or arterial pressure [39]. Thus the cardiovascular responses mediated from the uvula must be relayed via direct Purkinje cell projections to brainstem nuclei. The data obtained in the lesioning study suggest that the integrity of the SCP and surrounding regions of the PBN is necessary for the expression of the cardiovascular response evoked from the uvula in the anaesthetized rabbit (bradycardia and depressor response). However, in the decerebrate animal, where tachycardia and a rise in arterial pressure are evoked, the integrity of fibres in the ICP is essential for this response. From our anatomical evidence it is likely that fibres within the SCP project to the PBN, thus we propose that this nucleus is important in relaying the bradycardia/depressor response evoked from the uvula in the anaesthetized rabbit. The lateral PBN is known to be important in cardiovascular regulation [7,16,19-21,25,28,3234,38,48] and relays visceral afferent information to higher centres [13-15]. In addition, electrical [28] and chemical [40] stimulation of the lateral
222
PBN in the rabbit evokes a marked pressor response. Our hypothesis that the PBN is the relay nucleus for the uvula response observed in the anaesthetized rabbit is further substantiated by preliminary findings that bicuculline microinjected into the lateral PBN abolishes the depressor effect evoked from the uvula [40]. Since destruction of the SCP appeared not to affect the pressor response elicited from the uvula in the decerebrate rabbit it is unlikely that it is involved in mediating this response. Our evidence suggests that fibres contained within the ICP are essential for mediating the tachycardia/pressor response in the decerebrate rabbit. Despite the paucity of studies concerned with the role of vestibular nuclei in cardiovascular regulation the possibility that these nuclei play an important role cannot be ruled out. In man, strong rotatory stimuli were seen to increase forearm muscle blood flow but heart rate and arterial pressure remained unchanged [43]. Electrical stimulation of the vestibular nerve in the anaesthetized cat evoked a fall in blood pressure [42], which was later shown to be associated with an increase in vagal efferent discharge and an inhibition of splanchnic nerve activity [35]. Such effects resulting from vestibular nerve stimulation were abolished by lesions in the medial vestibular nucleus [45] or during stimulation of the posterior lobe in the anaesthetized cat [37]. Since vestibular nerve and uvula stimulation elicit a qualitatively similar pattern of cardiovascular response in an anaesthetized animal it is unlikely that, without an inhibitory interneurone, the medial vestibular nucleus is important in relaying the cardiovascular effects from the uvula since the Purkinje cells are exclusively inhibitory. Alternatively, the caudal and medial regions of the PBN may receive projections from the uvula via the ICP, as shown in the prosimian primate [26]. In this context it is of interest that activation of neurones within the medial PBN evokes increases in arterial blood pressure in the cat [25].
Acknowledgements We wish to thank the Medical Research Council, the British Heart Foundation and the Con-
siglio Nazionale delle Ricerche for their financial assistance. The technical assistance of Mr A. Bertini, Mr M.A. Goring and Mrs M. Vaglini is gratefully appreciated.
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223 15 Cechetto, D.F. and Calaresu, F.R., Central pathways relaying cardiovascular afferent information to amygdala, Am. J. Physiol., 248 (1985) R38-R45. 16 Darlington, D.N. and Ward, D.G., Rostral pontine and caudal mesencephalic control of arterial blood pressure and iliac, celiac and renal vascular resistance. I. Anatomical regions, Brain Res., 361 (1985) 284-300. 17 Dow, R.S., The fibre connections of the posterior parts of the cerebellum in the rat and cat, J. Comp. Neurol., 63 (1935) 527-548. 18 De Camilli, P., Miller, P.E., Levitt, P., Walter, U. and Greengard, P., Anatomy of cerebellar Purkinje cells in the rat determined by a specific immunohistochemical marker, Neuroscience, 11 (1984) 761-817. 19 Felder, R.B. and Mifflin, S.W., Modulation of carotid sinus afferent input to nucleus tractus solitarius by parabrachial nucleus stimulation, Circ. Res., 63 (1988) 35-49. 20 Fleetwood-Walker, S.M. and Coote, J.H., The contribution of brainstem catecholamine cell groups to the innervation of the sympathetic lateral cell column, Brain Res., 205 (1981) 141-155. 21 Fulwiler, C.E. and Saper, C.B., Subnuclear organization of the efferent connexions of the parabrachial nucleus in the rat, Brain Res. Rev., 7 (1984) 229-259. 22 Gibson, A.R., Hansma, D.I., Houk, J.C. and Robinson, F.R., A sensitive low artifact TMB procedure for the demonstration of WGA-HRP in the CNS, Brain Res., 298 (1984) 235-241. 23 Graham, J.C. Jr. and Karnovsky, M.J., The early stages of absorbtion of injected horseradish peroxidase in the proximal tubules of mouse kidney. Ultrastructural eytochemistry by a new technique, J. Histoehem. Cytochem., 14 (1966) 291-302. 24 Groenewegen, H.J. and Voogd, J., The parasagittal zonation within the olivocerebellar projection. I. Climbing fiber distribution in the vermis of cat cerebellum. J. Comp. Neurol., 174 (1977) 417-488. 25 Hade, J.S., Mifflin, S.W., Donta, T.S. and Felder, R.B., Stimulation of parabrachial neurons elicits sympathetically mediated pressor response in cats, Am. J. Physiol., 255 (1988) H1349-H1358. 26 Haines, D.E., Cerebellar corticovestibular fibres of the posterior lobe in a Prosimian Primate, the Lesser Bushbaby (Galago senegalensis), J. Comp. Neurol., 160 (1975) 363-398. 27 Haines, D.E., Patrick, G.W. and Satrulee, P., Organization of cerebellar corticonuclear fibres systems. Expl. Brain Res. (Suppl. 6). In S.L. Palay and V. Chain-Palay (Eds.), The Cerebellum New Vista, Springer Verlag, Berlin, 1982, pp. 320-371. 28 Hamilton, R.B., Ellenberger, H., Liskowsky, D. and Schniederman, N., Parabrachial area as mediator of bradycardia in rabbits, J. Auton. Nerv. Syst., 4 (1981) 262-281. 29 Jansen, J. and Brodal, A., Experimental studies on the intrinsic fibres of the cerebellum. The cortico-nuclear projection in the rabbit and the monkey, Avh. Norske. VidAkad. I. Mar. Nat. KI.3 (1942) 1-50.
30 Johnson, W.H. and Jongkees, L.B.W., Motion Sickness. III. Autonomic Effects. In H.H. Kornhuber (Ed.), Handbook of sensory physiology. Vestibular System. Vol. VI/2, Springer Verlag, Berlin, 1974, pp. 389-404. 31 Klinchachorn, P.S., Haines, D.E. and Culberson, J.L., Cerebellar cortical efferent fibres in the North American opossum, Didelphis virginia. II. The posterior lobe, J. Comp. Neurol., 227 (1984) 439-451. 32 Loewy, A.D., Descending pathways to the sympathetic preganglionic neurons, Prog. Brain Res., 57 (1982) 267277. 33 Loewy, A.D. and McKellar, S., The neuroanatomical basis of central cardiovascular control, Fedn. Proc., 39 (1980) 2495 -2503. 34 Loewy, A.D. and Nell, J.J., The role of descending monoaminergic systems in central control of blood pressure, Fedn. Proc., 40 (1981) 2778-2785. 35 Megirian, D. and Manning, J.W., Input-output relations of the vestibular system, Arch. Ital. Biol., 105 (1967) 15-30. 36 Meessen, H. and Olszewski, J., A cytoarchitectonic atlas of the rhombencephalon of the rabbit, S. Karger, New York, 1949. 37 Miyazawa, T. and Ishikawa, T., Cerebellar inhibitory action on vestibulo-sympathetic responses, J. Auton. Nerv. Syst., 7 (1983) 185-189. 38 Mraovitch, S., Kumada, M. and Reis, D.J., Role of the nucleus parabrachialis in cardiovascular regulation in cat, Brain Res., 232 (1980) 57-75. 39 Paton, J.F.R. PhD Thesis: Cerebellar control of cardiovascular function. (1987). 40 Paton, J.F.R. and Spyer, K.M., Brain stem regions mediating the cardiovascular responses elicited from the posterior cerebellar cortex in the rabbit, J. Physiol., 247 (1990) 533-552. 41 Shojaku, H., Sato, Y., Ikarashi, K. and Kawasaki, T., Topographical distribution of Purkinje cells in the uvula and the nodulus projecting to the vestibular nuclei in the cats, Brain Res., 416 (1987) 100-112. 42 Spiegel, E.A., Effect of labyrinthine reflexes on the vegetative nervous system, Arch. Otolaryng., 44 (1946) 61. 43 Sunahara, F.A., Johnson, W.H. and Taylor, N.B.G., Vestibular stimulation and forearm bloodflow, Can. J. Physiol. Pharmac., 42 (1964) 199-210. 44 Tabuchi, T., Umetani, T. and Yamadori, T., Corticonuclear and corticovestibular projections from the uvula in the albino rat: differential projections from sublobuli of the uvula, Brain Res., 492 (1989) 176-186. 45 Uchino, Y., Kudo, N., Tsuda, K. and lwamura, Y., Vestibular inhibition of sympathetic nerve activities, Brain Res., 22 (1970) 195-206. 46 Voogd, J., The cerebellum of the cat. Droogleever Fortuyn, J. and Verhaart, W.J.C. (Eds.) Royal Van Gorcum Ltd., Assen, 1964. 47 Walberg, F. and Jansen, J., Cerebellar corticovestibular fibres in the cat, Exp. Neurol., 3 (1961) 32-52. 48 Ward, D.G., Stimulation of the parabrachial nuclei with monosodium glutamate increases arterial pressure, Brain Res., 46 (1988) 383-390.
209
JANS 01221
Efferent connections of lobule IX of the posterior cerebellar cortex in the rabbit some functional considerations J.F.R. Paton
1, A.
La Noce
1, R.M.
Sykes 1, L. Sebastiani 2, p. Bagnoli and D.J. Bradley 1
2,,
B. Ghelarducci 2
t Department of Physiology, Royal Free Hospital School of Medicine, London, U.K., and 2 Dipartimento di Fisiologia e Biochimica, Universitd di Pisa, Pisa, Italy (Received 10 June 1991) (Revision received 13 August 1991) (Accepted 14 August 1991)
Key words: Cerebellar uvula; Purkinje cell axon; Anterograde tracer; Cardiovascular response Abstract The Purkinje cell projection from the cardiovascular region of sublobule b of the uvula (medial area of zone A) has been investigated using anterograde tracing methods in the rabbit. The importance of the integrity of the identified pathways in mediating the cardiovascular responses from the uvula has been studied in subsequent lesioning experiments. Wheat germ agglutinin-conjugated horseradish peroxidase or tritiated amino acids were microinjected into sublobule IXb. This resulted in anterogradely labelled Purkinje cell axons in both the inferior and superior cerebellar peduncle. In agreement with previous studies in rabbit we also found labelled fibres at the level of the fastigial nucleus and vestibular complex. However, the labelled fibres we observed in the parabrachial nucleus have not been reported in previous studies except in the prosimian primate. Projections from IXb showed terminal-like patterns of label in the ventromedial region of the caudal fastigial nucleus, the dorsal areas of the superior and inferior vestibular nuclei and in the medial and lateral divisions of the parabrachial nucleus. Labelled fibres were also seen coursing in the lateral vestibular nucleus. Lesioning experiments have revealed that the integrity of the superior cerebellar peduncle is essential for the expression of the cardiovascular responses (bradycardia and depressor response) elicited from the uvula in the anaesthetized rabbit. In contrast, the pattern of cardiovascular response evoked in a decerebrate rabbit (tachycardia and pressor response) was abolished when the inferior cerebellar peduncle was lesioned.
Introduction
The efferent connections of the posterior cerebellar cortex have been described in a variety of species using a number of neuroanatomical tracing techniques. In general, these studies have
Correspondence: B. Ghelarducci, Dipartimento di Fisiologica e Biochimica, Universitfi di Pisa, Via S. Zeno 31, 56100 Pisa, Italy. * Present address: P. Bagnoli, Facolt~ di Sciense, Universitfi della Tuscia, Viterbo, Italy.
revealed that there are two major projections from the posterior cortex, one to the fastigial nucleus (FN) and the other to the brainstem vestibular nuclei. It is known that the posterior vermis is topographically represented within the caudal region of the ipsilateral fastigial nucleus [27], and the corticovestibular projections, which descend through the ipsilateral inferior cerebellar peduncle (ICP), innervates a number of vestibular nuclei. In a series of cardiovascular studies, a discrete region of the uvula (lobule IX) of the posterior
210
cerebellar vermis has been shown to exert powerful influences on heart rate and arterial blood pressure during either electrical stimulation [8] or following microinjections of excitatory amino acids intra-cortically [9,11,39]. Two patterns of cardiovascular response can be evoked from the uvula which are dependent on the type of preparation used. Electrical or chemical activation of the uvula in the decerebrate rabbit evokes tachycardia and a pressor effect but in the anaesthetized rabbit (intact or decerebrate) a qualitatively reversed response pattern is evoked (falls in heart rate and arterial blood pressure). This prompts the question as to the identity of the pathway(s) and relay(s) responsible for mediating these two different patterns of response. Previous studies have indicated that cells within the caudal FN do not play a role in mediating the cardiovascular effects from the uvula [9] and this was confirmed subsequently using chemical stimulation techniques [39]. Accordingly, it must be assumed that either the vestibular complex or novel brainstem sites, yet to be identified, are responsible for the cardiovascular changes mediated from the uvula. There is, however, only limited and controversial evidence for the vestibular nuclei playing a role in control of the circulation, and even then this evidence has been obtained by electrical stimulation, which leaves open the possibility that the effects resulted from stimulation of fibres of passage [30,35,42,43,45]. In this study we have used tritiated amino acids and wheat germ agglutinin-conjugated horseradish peroxidase to trace anterogradely the efferent projection from the cardiovascular region of the uvula in the rabbit. Subsequently electrolytic lesions have been placed in these pathways to assess their influence on the expression of the cardiovascular responses elicited from the uvula, in both the anaesthetized and decerebrate, unanaesthetized, rabbits.
Materials and Methods
Experiments were performed on 22 New Zealand white rabbits weighing 2.0-2.5 kg. Of these, 11 were used in the anterograde tracing
experiments and ll were used in functional studies.
Anatomical experiments Rabbits were anaesthetized with sodium pentobarbitone (Sagatal, May and Baker Ltd., U.K., 35-40 m g . k g 1, i.v.), the head placed in a stereotaxic apparatus and the posterior cerebellar vermis exposed by retraction of nuchal muscles and removal of the overlying occipital bone. Through a small hole in the dura the tip of a glass micropipette (dia. 20-40 p~m) filled with either wheat germ agglutinin-conjugated horseradish peroxidase ( W G A - H R P , Sigma Ltd.) or tritiated amino acids (New England Nuclear Ltd.) was lowered with the aid of a dissecting microscope I mm from the pia into the medial region of lobule IXb. Five rabbits received a 200 nl microinjection of a 4% solution of W G A - H R P dissolved in saline and three received 40 /xCi of tritiated k-proline (38.5 m C i / m m o l ) dissolved in 200 nl of saline. In an additional three animals a mixture of tritiated L-proline and e-leucine (50 /xCi/ixl) in saline was used and microinjected in 25 nl volumes at depths of 1.0, 0.75, 0.5 and 0.25 mm from the pia (total microinjected was 5 p~Ci). When delivery was complete the micropipette was left in situ for 10 min before withdrawal in order to avoid any leakage of tracer from the pipette track.
WGA-HRP procedures" A survival time of 24-48 h was allowed before rabbits were deeply anaesthetized (Sagatal, 50 mg- kg-~) and perfused transcardially with 1 1 of warm (38 ° C) saline followed by 1 I of fixative, consisting'of 1% paraformaldehyde and 1.25% glutaraldehyde in 0.1 M phosphate buffer at 4 ° C, and finally with 1 1 of a 10% sucrose solution. Immediately after perfusion, the brain was removed from the skull and equilibrated overnight in 30% sucrose solution at 4 °C. The following day, frozen sections were cut at 30 Ixm either in the frontal plane (n = 3) or in the sagittal plane (n = 2). Sections were treated for peroxidase activity with tetramethylbenzidine according to the protocol of Gibson et al. [22] and counterstained
211 with Neutral Red. Sample injection site sections were treated with 3,3' diaminobenzidine-tetrahydrochloride according to the protocol of G r a h a m and Karnovsky [23] before counterstaining with Cresyl Violet.
Autoradiographic procedures This part of the study employed two different protocols but gave qualitatively identical results. Rabbits were allowed a survival time of 4 - 7 days before being deeply anaesthetized (urethane, 1.5 g . k g -1) and perfused transcardially with 10% formalin or 5% glutaraldehyde. I m m e diately after perfusion the brain was removed and post-fixed. Either frozen sections were cut at 30 /zm or blocks of tissue were placed into molten paraffin wax and sections cut at 15 /zm. Frontal sections were cut in both cases and mounted onto glass slides which were dipped in emulsion and exposed for 4 - 8 weeks in the dark at 4 ° C. Autoradiograms were developed, fixed and counterstained before being examined. All material was examined under the light microscope using bright and dark field illumination. Injection sites and labelled structures were recorded with the aid of a projector and photomicrographs taken of representative sections. The currently accepted nomenclature for the rabbit according to Brodal and Jansen [12] and Meessen and Olszewski [35] has been used.
Electrolytic lesioning experiments O f the 11 rabbits used in this study, four were anaesthetized with urethane (Sigma Ltd., 1.4 g. kg -1 i.v.) and seven with alphaxolone alphadolone (Saffan, Glaxovet Ltd., 4 mg" kg-1 i.v. and supplemented with 4 mg bolus injections as required). In all animals the bladder was drained and cannulated and a tracheostomy performed to allow for artificial ventilation. End tidal CO 2 was sampled, monitored and kept at 4.5_+ 0.5% by altering minute ventilation. Arterial blood pressure was monitored via a cannula placed into the femoral artery and connected to a transducer (Gould Statham, U.S.A., P23Db). H e a r t rate was derived from the pulse frequency using a ratemeter (Neurolog, Digitimer Ltd., module NL250). A
femoral vein was also cannulated to permit administration of anaesthetic supplements and drugs. Rectal t e m p e r a t u r e was recorded and maintained at 38 ° C using a heating lamp. Rabbits anaesthetized with Saffan were paralyzed with decamethonium bromide (Sigma Ltd., 0.5 m g . k g - 1 i.v. and supplemented every 15-20 rain), ventilated artificially before being decerebrated at the pre-collicular level during which time both common carotid arteries were occluded. After decerebrating, one common carotid was released. In all animals the posterior vermis was exposed (see above) and protected with frequent topical application of warm paraffin oil; the midline region of lobule IXb was stimulated intracortically using a metal-filled microelectrode (tip diameter 2 0 / z m ) with a 6-s stimulus train (0.5 ms; 100 Hz) and intensities of 200-400 /~A in anesthetized animals and 50-150 /xA in the decerebrate preparations. Constant current was used and monitored on an oscilloscope. The cardiovascular responses evoked from the uvula in anaesthetized and decerebrate rabbits were compared before and after lesioning areas of the brainstem which our anatomical study had shown to contain labelled Purkinje cell axons originating from the uvula. Electrolytic lesions were made by passing 2 m A anodal current for 40 s through a stainless steel electrode insulated to 300 /~m from its tip. Bilateral lesions of the SCP were placed at 4.0 m m anterior to lambda, 3.0-3.3 m m lateral to midline and 13.5 m m ventral to dura in some animals whereas ICP lesions were placed at 1.5 m m anterior to lambda, 3.7 m m to 4.5 m m lateral to midline and 14 m m ventral to dura. In all cases the head was tilted so that lambda was 1.5 m m ventral to bregma. Following the termination of every experiment the brain was removed and placed in 10% formalin and left for 48 h before serial frontal sections were cut (80 tzm), mounted onto slides and stained with Neutral Red. The exact location and size of each lesion was recorded. A two sample t-test was employed to assess the statistical significance of the data. All data are expressed as mean _+ SEM (standard error of the mean).
212
C A
IX
C
,
.......
•
A !
!
I
Fig. 1. Representative W G A - H R P (A) and tritiated amino acid (B) injection sites placed in the cerebellar uvula (lobule 1X a,b,c,d) of the rabbit. A: The injection site is centered in the midline of sublobule b (B) and extends laterally up to 800 ~ m of both brain sides (A and C). B: The injection site is centered on the midline of sublobule b and extends to the upper part of sublobule c. Its caudo-rostral extention is shown from A to C; each section is taken at 900-#m interval. These cases are representative of all W G A - H R P and tritiated amino acids injection sites. Light field micrographs (sagittal sections, 30 tzm, scale bar: 2 mm).
213
MEDIAL
1.2
D
2.2
2.6
3.8 LATERAL
Fig. 2. Schematic diagram showing the distribution of anterogradely labelled axons (dashed lines) in sagittal sections of the cerebellum following W G A - H R P microinjection into the midline region of sublobule b of the uvula (A-D). The illustration shows a single case but is representative of the other experiments of this kind. For each section the distance, in mm, from the midline is indicated. See text for abbreviations.
Results
Anatomical experiments In the present study an area was considered to receive efferent projections from the uvula when it exhibited a comparable terminal-like pattern in
both W G A - H R P and tritiated amino acid studies in either frontal or sagittal sections. The location of retrogradely labelled neurons in the brainstem following W G A - H R P microinjections was consistent with data reported previously [5,11] where the retrograde transport of H R P from the cortex of sublobule IXb had been used. In particular, the labelled cells in the inferior olivary complex were located mainly in the 13 nucleus of the medial accessory olive. This area, according to the zonal pattern of olivocerebellar projections proposed by Voogd [46] in the cat, projects exclusively to the medial portion of the vermis which has been termed zone A in the Voogd scheme [24,46].
Injection sites In all except two animals the injection site was localized to the midline portion of sublobule IXb (comparable to the medial region of zone A in the cat [24,46]). In two animals anterograde tracer was seen to have diffused into the adjacent midline regions of the ventral area of IXa and the dorsal half of IXc. The diffusion of the tracer substances was always restricted to the cerebellar cortex and underlying white matter without involving deeper cerebellar structures (Fig. 1A-B). There was no evidence in any experiment of radioactive label or W G A - H R P in deep cerebel-
")
Fig. 3. Dark and bright field micrographs showing the distribution of labelled Purkinje cell axons and terminal-like grains in the caudal fastigial nucleus (FN) following a microinjection of tritiated L-proline into sublobule IXb. Note the presence of labelled fibres projecting through the ventrolateral part of the nucleus and a terminal-like field in its ventromedial part. Comparable results have been obtained in both W G A - H R P and tritiated amino acids experiments (frontal sections, 30/zm, scale bar: 500/zm).
214
lar nuclei. In all animals, label was represented bilaterally in the cerebellum and brainstem.
Labelling within the cerebellum The distribution of anterogradely labelled axons within the cerebellum is represented schematically in the drawings of Fig. 2. In sagittal sections (Fig. 2A) labelled fibres from the uvula enter the ventromedial region of the caudal fastigial nucleus (FN) and then project rostrally through the ventral part of this nucleus. More laterally (Fig. 2B) a large contingent of fibres separates into two components: the first courses via the ventral-most part of the FN while the second passes through the dorsomedial portion of this nucleus. In more lateral sections the majority of labelled fibres bend caudally and descend into the brainstem by passing through the medial part of the inferior cerebellar peduncle (ICP) (juxtarestiform body) and pass through the lateral vestibular nucleus (LVN), while a small number of fibres branch away from the principal bunch and run rostrally before leaving the cerebellum within and around the superior cerebellar peduncle (SCP) (Fig. 2C). Finally, at the level of the interpositus nucleus (IN) (Fig. 2D), labelled Purkinje cell axons were also found to project around the dorso-rostral border of this nucleus before descending into the lateral region of the ICP (restiform body). As shown in the photomicrograph of Fig. 3 and in the drawing of Fig. 4D, a terminal-like labelling was visible in the ventromedial region of the caudal FN in addition to labelled fibres crossing the ventrolateral part of the FN. Labelling within the brainstem The distribution of labelled fibres and putative termination fields in the brainstem is represented schematically in the drawings of Fig. 4, which is a composite of both W G A - H R P and tritiated amino acid experiments. It appears that the heaviest projection from lobule IXb is directed to the vestibular complex. As seen in Fig. 5, labelled fibres leaving the cerebellum via the ICP enter the superior vestibular nucleus (SVN), where a termination field was visible throughout the rostrocaudal ex-
ROSTRAL
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CAUDAL Fig. 4. Schematic diagram showing the distribution of anterogradely labelled fibres (dashed lines) and putative terminal fields (dots) in frontal sections of the cerebellum and brainstem following microinjections of anterograde tracers into sublobule IXb (A-G). The illustration is a composite of data taken from W G A - H R P and tritiated amino acids experiments. For each section the distance, in mm, from obex is indicated. Abbreviations: DN, dentate nucleus; MVNmc, magnocellular medial vestibular nucleus; MVNpc, parvocellular medial vestibular nucleus; PH, praepositus hypoglossal nucleus; x, groups x of Brodal. See text for other abbreviations.
tent of its dorsal half. In the same figure there is also a sparse projection to the dorsal area of the rostral medial vestibular nucleus (MVN), which
215
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Fig. 5. Dark and bright field micrographs of anterogradely labelled Purkinje cell axons and terminal-like fields in the brainstem following tritiated L-proline microinjection into sublobule IXb. Labelling can be seen in the dorsal half of the superior vestibular nucleus (SVN) and in the rostral region of the medial vestibular nucleus (MVN). Comparable results have been obtained in both WGA-HRP and tritiated amino acids experiments. (frontal section, 30 #m, scale bar: 300/zm). Abbreviations: Cb, cerebellum; V, ventricle. See text for other abbreviations.
shows no evidence of termination. A large population of labelled Purkinje cell axons run caudally through the LVN, without exhibiting any appreciable terminal-like pattern, as confirmed in our sagittal sections. Within the inferior vestibular nucleus (IVN), labelled fibres are organized into typical bundles which are intermingled with cell bodies (Fig. 6). In addition, the caudal half of this nucleus, particularly its dorsolateral part, shows a dense terminal-like labelling (Fig. 6). In this region a small number of fibres arising from the ventral part of the labelling within the IVN turn
medially and run close to the dorsolateral edge of the rostral nucleus tractus solitarius (NTS). In addition to the labelling within the vestibular complex there is also a dense projection to the parabrachial nucleus (PBN). Fig. 7 shows heavy terminal-like labelling within both the dorsal halves of the medial and lateral regions of this nucleus. From the evidence presented in the present study it is clear that this projection is a significant Purkinje cell outflow from sub-lobule IXb. To date the PBN has not been identified as a major target of uvula efferent projections even
216
Fig. 6. Dark and bright field micrographs showing labelled Purkinje cell axons and terminal-like fields in the brainstem following tritiated amino acids microinjection into sublobule IXb. Labelling can be seen within the dorsolateral part of the IVN. Comparable results have been obtained in both W G A - H R P and tritiated amino acids experiments (montage, frontal sections, 15/xm, scale bar: 250/xm). See text for other abbreviations.
though numerous studies have been carried out on a range of species.
Electrolytic lesioning experiments Lesions have been placed bilaterally in two different regions of the brainstem, according to the course of anterogradely labelled Purkinje cell
axons descending from the sublobule b of the uvula as indicated by our W G A - H R P and tritiated amino acids experiments. In one group of rabbits the area of the SCP was lesioned electrolytically, while in the second group, lesions were placed at the level of the ICP which encompassed both the lateral (restiform body) and me-
TABLE 1
Comparison of the cardiocascular responses elicited by m'ula stimulation in anaesthetized (N = 4) and decerebrate (N = 3) rabbits before and after lesioning the SCP (mean + SEM) MAP (mmHg)
A. Anaesthetized Baseline Response
B. Decerebrate Baseline Response
N = number of animals. n = number of tests.
HR ( b e a t s / m i n )
Before
After
Before
After
97 _+ 0.86 (n = 43) -14+_0.94 (n = 3 4 )
106 + 0.83 (n = 37) -9+0.55 (n = 2 1 )
326 ± 3.21 (n = 43) 2 2 ± 2.(14 (n = 34)
258 + 2.1)I (n = 37) - 4 + 1.29 (n 21)
130 4-_ 1.111 (n = 16) +35±4.43 (n = 16)
131 ± 4.38 (n = 13) +25±3.86 (n = 13)
245 + 10.63 (n = 16) + 6 0 + 5.53 (n - 16)
267 + 9.42 (n = 13) +53_+9.51 (n = 13)
!
Fig. 7. Series of dark and bright field micrographs showing dense labelling within the dorsal regions of the medial (mPBN) and lateral (IPBN) parts of the parabrachial nucleus in a rabbit which received a microinjection of tritiated L-proline into sublobule IXb. Three levels extending caudo-rostrally from a to c are shown. The interval between the sections is 180 ~m; a is taken at 9.5 mm from the obex. Comparable results have been obtained in both W G A - H R P and tritiated amino acids experiments (frontal sections, 15 kLm, scale bar: 300/~m). See text for abbreviations.
218
B
A ANAESTHETIZED
C DECEREBRATE
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Fig. 8. An example of the effect of lesioning the SCP on the cardiovascular response ew)ked by uvula stimulation in an anaesthetized (A) and in a decerebrate rabbit (B). HR, heart rate in beats, min ~; BP, arterial blood pressure in mmHg. C: Schematic drawing showing the location and extent of electrolytic lesions of the SCP. Abbreviations: LC, locus coeruleus; VMES, mesencephalic trigeminal nucleus. See text for other abbreviations.
dial regions (juxtarestiform body ). Figs. 8C and 9B illustrate the location and extent of representative lesions in two rabbits.
SCP lesions The destroyed area e n c o m p a s s e d a large region of the SCP and surrounding PBN. In some animals additional regions d a m a g e d included the lateral part of the locus coeruleus and mesencephalic trigeminal nucleus. The effect of such lesions on the cardiovascular responses evoked from sublobule IXb were studied in both anaesthetized and decerebrate animals.
In anaesthetized rabbits (n = 4), following the lesion, there was a significant fall in heart rate ( P < 0.01) from a control level of 326 +_ 3.2 beats • m i n - 1 to 258 _+ 2.0 beats - min-- ~ and a significant rise in arterial blood pressure ( P < 0 . 0 1 ) from a resting level of 97 +_ 0.9 m m H g to 106 _+ 0.8 m m H g . The changes in heart rate and arterial pressure elicited during uvula stimulation are c o m p a r e d before and after the SCP lesion in Table I. It can be seen that the cardiovascular response (bradycardia and a depressor response) was reduced significantly ( P < 0 . 0 1 ) o r abolished (Fig. 8A) when c o m p a r e d to the response evoked
219
A BEFORE LESIONING MVN
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Fig. 9. A: An example of the effect of lesioning the ICP on the cardiovascular responses elicited from the uvula in a decerebrate rabbit. Abbreviations as in fig. 8. B: Schematic drawing to show the location and extent of electrolytic lesions of the ICP. Abbreviations: RB, rest±form body. See text for other abbreviations.
heart rate or arterial blood pressure (Table I) and had no significant effect on the magnitude of the tachycardia and pressor response elicited from the uvula (Fig. 8B and Table I).
ICP lesions Electrolytic lesions of the ICP area were carried out in four decerebrate rabbits. Large regions of the ICP were destroyed in addition to some damage of the dorsal areas of the SVN and LVN. Table II shows that there were no changes in resting levels of heart rate and arterial blood pressure ( P < 0.01) after lesioning. However, the uvula-evoked tachycardia and pressor response was attenuated severely (Table II; P < 0.01). Interestingly, in one animal with an ICP lesion, uvula stimulation elicited a cardiovascular pattern of response qualitatively similar to that observed from an anaesthetized rabbit (bradycardia and depressor response, Fig. 9A). In this case uvula stimulation evoked a reduction in heart rate of 20 _+ 4.1 beats • m i n - ~ and a fall in arterial pressure of 11.6 _+ 1.6 mmHg. Discussion
in the intact anaesthetized rabbit. In order to assess that the reduction in the cardiovascular response was not secondary to brain function deterioration with time, lobule IXb was stimulated in one rabbit at regular intervals for 1 h without performing any lesions. The cardiovascular responses evoked after 1 h showed no difference in their magnitude ( P < 0.01). In decerebrate rabbits (n = 3) lesioning the SCP failed to produce any baseline changes in TABLE II
CardioL, ascular responses elicited by uvula stimulation in decerebrate rabbits (N = 4) before and after lesioning the ICP
Baseline Response
MAP (mmHg)
HR (beats min)
Before
After
Before
After
122±3.41 (n = 18) +52±2.90 (n = 15)
127± 3.41 (n = 18) +9±2.77 (n = 21)
250± 5.83 (n = 18) +35±3.70 (n = 15)
271 ±9.31 (n = 18) +8±2.39 (n = 21)
N = number of animals. n = number of tests.
The present series of experiments in the rabbit have revealed the Purkinje cell projections from the midline region of sublobule IXb, an area analogous to the medial region of zone A according to Voogd's longitudinal classification in the cat [24,45]. Labelled fibres have been found bilaterally in the caudal region of the FN, and also in the SVN, LVN, IVN and PBN. The limitations of light microscopy do not permit an accurate assessment of terminal structures, but the presence of dense grains and evidence of accumulation of transported material is suggestive of possible termination in all these nuclei with the exception of the LVN. The other part of this study has considered the relative importance of the identified pathways in mediating the cardiovascular responses evoked from the uvula. The data from the lesioning experiments suggest that the integrity of the SCP and the ICP are essential for the expression of the distinct cardiovascular patterns of response in the anaesthetized and decerebrate rabbits respectively.
22(I
Uvula Purkinje cell projections There is good anatomical evidence in a variety of species that the uvula projects to the caudal FN [27]. The present data in the rabbit illustrates a densely labelled pathway to the ventromedial portion of the caudal FN which is consistent with earlier reports [2,6,17,29,31,44,46,47] and is in good agreement with the mediolateral organization of the uvula projection to the caudal FN as described for the rat [3]. The SVN, LVN and IVN also received a projection from sublobule b of the uvula in the present study and this supports previous observations made on a variety of other species using different techniques. In earlier reports it was often emphasised that of all the lobules of the posterior vermis it is the uvula that sends the heaviest projection to the vestibular nuclei [1,26]. In our study the majority of fibres projecting to the vestibular nuclei either coursed through the medial or ventral part of the FN or ran around the dorsolateral edge of the IN before entering the brainstem via both regions of the ICP. Labelled fibres were most dense in the medial region of the ICP (juxtarestiform body). The pathways exiting from the cerebellum in the medial ICP have been well described before in the prosimian primate [26], cat [2,46,47] and opossum [31]. Putative termination was restricted to the dorsal SVN and the caudal region of the dorsolateral IVN. Dense label was also seen in the LVN but did not take the form of a terminal region when seen in both frontal and sagittal sections. In early studies there is some inconsistency as to which of the vestibular nuclei receives projections from the uvula. Dow [17] used degeneration techniques and reported termination in all vestibular nuclei following lesions of lobule IX in the rat. Using similar experimental methods in the cat, Walberg and Jansen [47] found degenerating axons in the dorsal halves of the LVN and IVN whereas Voogd [46] described projections to the MVN, descending vestibular nuclei (VN) (analogous to the IVN) and SVN. The difference between these reports and the data from the present study could be attributed to the exact region of lobule IX studied. Indeed, there is anatomical evidence for a topographical distribu-
tion of Purkinje cell axons from the uvula to the vestibular nuclei [6,41]. In the degeneration studies it is clear that different areas of the uvula were removed and the extent of the lesions varied between animals in any one study. In the present study the area of the uvula examined is highly localized and comparable between animals. Other investigators reported similar findings to those described in our study. For example, in the prosimian primate degenerating fibres were identified in the dorsomedial regions of the SVN, LVN and spinal VN (similar to the IVN in the rabbit, [26]). Using similar methods, the uvula was found to innervate these vestibular nuclei in the opossum [31], cat [2] and rat [1,44]. There have been other studies using retrograde transport of HRP to study the cerebellar corticovestibular projections in both cat and rabbit. It was shown that following a microinjection of HRP into physiologically identified vestibular nuclei in the cat, uvula Purkinje cells were retrogradely labelled only when the SVN and IVN were injected [41] which is consistent with the observations made in the present study. In contrast, in the rabbit, Balaban [4] applied H R P iontophoretically to the SVN, MVN and LVN and reported retrogradely labelled Purkinje cells in the uvula. In these latter studies it is possible that both terminal fibres and damaged axons of passage will have transported the HRP. Thus the differences between this study and that of Balaban [4] may be due to the difference in experimental approach. Another major projection from sublobule b of the uvula revealed in the present study included a relatively dense terminal-like label in the dorsal region of the medial and lateral PBN. This pathway appears not to have been reported previously, except in the prosimian primate and has not been recognized as a major projection from the uvula. It is likely that fibres within the SCP project to the PBN, though the possibility that some fibres within the ICP may project to the caudal PBN cannot be ruled out. Such a projection has not been reported by previous authors concerned with the corticofugal projections of the posterior vermis. In the prosimian primate Haines [26] described 'moderate to sparse preterminal
221 debris' in the medial and lateral PBN but was unable to attribute any physiological significance to this projection. From the density of label found in the present study it is surprising that such a pathway should have been unrecognized by other workers, and we see no reasonable explanation for this discrepancy at present. However, it should be emphasized that in this investigation the Purkinje cell projection studied was from a highly localized region of sublobule b of the uvula which was never specifically studied in previous studies. This suggests that, as earlier studies were concerned primarily with the vestibular connections of the posterior cerebellar cortex, other possible brainstem targets were not reviewed fully. Since the SVN and PBN are anatomically close, though separated rostrocaudally, this may have resulted in some confusion. It is interesting that the lateral PBN has been well documented as a region involved in cardiovascular regulation (for references see later) and in this context it is not surprising that the cardiovascular region of the uvula projects to it. Furthermore, its integrity appears essential for one component of the cardiovascular changes elicited from the uvula (see next section). In addition, in the present study there was also a sparse projection to the dorsomedial region of the caudal brainstem, running very close to the extreme dorsal edge of the rostral NTS. This is consistent with other studies which have described a similar projection in the primate [26], cat [2] and rat [1]. Interestingly, De Camilli et al. [18], using a specific immunocytochemical marker for Purkinje cells, demonstrated projections to the FN, VN, PBN, praepositus hypoglossal nucleus and NTS in the rat. Unfortunately using this type of approach yields no information as to the source of these projections.
Role of identified pathways in mediating the cardiovascular changes elicited from the uvula The injection sites were found to be within the midline region of sublobule IXb and therefore to occupy the exact region from which marked changes in cardiovascular variables can be elicited using either electrical [8] or chemical stimulation [9,11,39]. The fact that excitatory amino acids elicited cardiovascular effects from the uvula sug-
gests that these result from Purkinje cell activation rather than antidromic excitation of mossy or climbing fibres which can occur with electrical stimulation and would lead to activation of precerebellar nuclei. The purpose of this study was to identify the connections of the cardiovascular region of lobule IXb so that putative brainstem site(s) involved in mediating the cardiovascular responses could be identified. Our previous studies have indicated that electrical stimulation of the caudal FN elicits a pattern of response qualitatively identical to that elicited from the uvula [10]. Since Purkinje cells are strictly inhibitory it was anticipated that an opposite pattern of cardiovascular response would be observed if the caudal fastigial neurones are involved in mediating the cardiovascular responses from the uvula. Thus it is reasonable to suggest that those caudal fastigial neurones receiving Purkinje cell axons from sublobule IXb are not involved in relaying the cardiovascular responses from the uvula. Further evidence has revealed that chemical excitants microinjected into the caudal FN fail to elicit any change in heart rate or arterial pressure [39]. Thus the cardiovascular responses mediated from the uvula must be relayed via direct Purkinje cell projections to brainstem nuclei. The data obtained in the lesioning study suggest that the integrity of the SCP and surrounding regions of the PBN is necessary for the expression of the cardiovascular response evoked from the uvula in the anaesthetized rabbit (bradycardia and depressor response). However, in the decerebrate animal, where tachycardia and a rise in arterial pressure are evoked, the integrity of fibres in the ICP is essential for this response. From our anatomical evidence it is likely that fibres within the SCP project to the PBN, thus we propose that this nucleus is important in relaying the bradycardia/depressor response evoked from the uvula in the anaesthetized rabbit. The lateral PBN is known to be important in cardiovascular regulation [7,16,19-21,25,28,3234,38,48] and relays visceral afferent information to higher centres [13-15]. In addition, electrical [28] and chemical [40] stimulation of the lateral
222
PBN in the rabbit evokes a marked pressor response. Our hypothesis that the PBN is the relay nucleus for the uvula response observed in the anaesthetized rabbit is further substantiated by preliminary findings that bicuculline microinjected into the lateral PBN abolishes the depressor effect evoked from the uvula [40]. Since destruction of the SCP appeared not to affect the pressor response elicited from the uvula in the decerebrate rabbit it is unlikely that it is involved in mediating this response. Our evidence suggests that fibres contained within the ICP are essential for mediating the tachycardia/pressor response in the decerebrate rabbit. Despite the paucity of studies concerned with the role of vestibular nuclei in cardiovascular regulation the possibility that these nuclei play an important role cannot be ruled out. In man, strong rotatory stimuli were seen to increase forearm muscle blood flow but heart rate and arterial pressure remained unchanged [43]. Electrical stimulation of the vestibular nerve in the anaesthetized cat evoked a fall in blood pressure [42], which was later shown to be associated with an increase in vagal efferent discharge and an inhibition of splanchnic nerve activity [35]. Such effects resulting from vestibular nerve stimulation were abolished by lesions in the medial vestibular nucleus [45] or during stimulation of the posterior lobe in the anaesthetized cat [37]. Since vestibular nerve and uvula stimulation elicit a qualitatively similar pattern of cardiovascular response in an anaesthetized animal it is unlikely that, without an inhibitory interneurone, the medial vestibular nucleus is important in relaying the cardiovascular effects from the uvula since the Purkinje cells are exclusively inhibitory. Alternatively, the caudal and medial regions of the PBN may receive projections from the uvula via the ICP, as shown in the prosimian primate [26]. In this context it is of interest that activation of neurones within the medial PBN evokes increases in arterial blood pressure in the cat [25].
Acknowledgements We wish to thank the Medical Research Council, the British Heart Foundation and the Con-
siglio Nazionale delle Ricerche for their financial assistance. The technical assistance of Mr A. Bertini, Mr M.A. Goring and Mrs M. Vaglini is gratefully appreciated.
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