Brain Research, 576 (1992) 59-67 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00
59
BRES 17558
Distinct populations of neurons in the ventromedial periaqueductal gray project to the rostral ventral medulla and abducens nucleus Elisabeth J. Van Bockstaele and Gary Aston-Jones Hahnemann University, Department of Mental Health Sciences, Division of Behavioral Neurobiology, Philadelphia PA 19102 (U.S.A.) (Accepted 12 November 1991)
Key words: Paragigantocellularis; Retrograde transport; Supraoculomotor nucleus of the central gray; Oculomotor nucleus; Medulla oblongata; Brainstem; Anterograde transport; Cardiovascular regulation
Following microinjections of a colloidal gold complex into the nucleus paragigantocellularis (PGi) of the ventral medulla, and of latex microspheres into the nucleus abducens (Abd) of the same animal, retrogradely labeled neurons were identified in the region of the contralateral supraoculomotor nucleus (SOM) in the ventromedial periaqueductal gray (PAG). Neurons labeled from the Abd were found in the SOM in the ventromedial PAG throughout the midbrain, as well as scattered ventrally in the oculomotor nucleus. Neurons in the SOM area retrogradely labeled from the PGi were most numerous rostral to the dorsal raphe nucleus and extended throughout the level of the oculomotor nucleus. Direct comparison of the two labels revealed that the neurons that project to the Abd were located slightly more ventrally than PGi-projecting neurons. Almost no doubly labeled neurons were identified, although singly labeled neurons formed adjacent but separate populations. These results indicate that neurons in the SOM area projecting to the PGi are distinct from those projecting to the Abd, and that the PGi-projecting neurons are probably not pre-oculomotor neurons. Given their more dorsal location and the nature of their target neurons in the rostral ventrolateral medulla, PGi-projecting neurons may be related to visceromotor, as opposed to oculomotor functions. INTRODUCTION
pre-oculomotor in function 1°-12.
Retrograde transport studies using a variety of tracers have found that n e u r o n s in the area of the contralateral supraoculomotor nucleus (SOM) project to the nucleus paragigantocellularis (PGi) in the rostral ventral medulla (RV1V[) 19'20. These labeled n e u r o n s lie immediately dor-
In the present study, to determine whether n e u r o n s in the SOM area of the rostral ventromedial P A G that project to the PGi may be part of this pre-oculomotor group, we used double retrograde transport to discern whether these cells also innervate the A b d nucleus. O u r results indicate that PGi- projecting neurons are a slightly
sal to the oculomotor nucleus within the rostral ventromedial aspect of the periaqueductal gray ( P A G ) . A n t e r ograde tracing studies have confirmed and further detailed this projection, indicating a strong and specific projection to the sympathoexcitatory region of the PGi, corresponding to the rostroventrolateral reticular nucleus (RVL) 19. The R V L is located (i) caudal to the facial nucleus, (ii) in the lateral aspect of the PGi ventral to the nucleus ambiguus and will be referred to as the lateral retrofacial PGi. Anatomical and physiological studies have implicated n e u r o n s in the SOM in oculomotor-related activities due to (i) projections to the abducens nucleus in the pons s' 10,11 and (ii) afferents from nuclei with visual-related p•operties such as the frontal eye field s, the superior colliculus7, and the medial fastigial nucleus2'5'6'16. These results have led to the classification of SOM n e u r o n s as
more dorsal, separate population from those innervating the A b d nucleus. MATERIALS AND METHODS Sprague-Dawley rats (n = 12; Taconic Farms) weighing 275-350 g were anesthetized using sodium pentobarbital (50 mg/kg, i.p.) and placed in a stereotaxic apparatus for surgery. Rectal temperature was monitored continuously and maintained at 37°C by a heating pad. Supplementary doses of sodium pentobarbital were administered as needed.
Retrograde transport experiments Microinjections (250-300 nl) of wheat germ agglutinin-conjugated to inactive horseradish peroxidase coupled to 15-nm gold particles (WGA-apoHRP-Au), synthesized according to the protocol of Basbaum and Menetrey (1987)1, were stereotaxically deposited into the PGi15area using precleaned glass micropipettes (15-20 /~m tips; n = 5). Injections were made using a solenoid controlled pneumatic pressure device (Picospritzer; General Valve, Inc.), and the desired volume was obtained by directly measuring
Correspondence: G. Aston-Jones, Hahnemann University, Department of Mental Health Sciences, Division of Behavioral Neurobiology, Broad & Vine Streets, Mail Stop 403, Philadelphia PA 19102, U.S.A.
60 the movement of the fluid column within the pipette. Prior to depositing the tracer, physiologic recordings were made through the injection pipettes and the PGi was tentatively identified as previously described z°. In the same animals, a microinjection (25-50 nl) of fluorescein-coated latex microspheres (n = 4; Tracer Technology), or of rhodamine-coated latex microspheres (n = 1) was similarly deposited into the Abd. The pipette was left in place for five minutes prior to and following injections. In two animals, injections were placed in a region rostral to the Abd nucleus in the rostral dorsal pontine reticular formation corresponding to the level of Plate 56 of the rat brain atlas of Paxinos and Watson (1986) 15. In one of these animals, rhodamine latex microspheres were deposited in the PGi and WGA-apoHRP-Au was deposited in the dorsal pontine reticular formation. In the other animal, WGA-apoHRP-Au was deposited in the PGi and rhodamine-coated latex microspheres were deposited into the dorsal pontine reticular formation. To confirm that the retrograde tracers yielded a similar pattern of retrograde transport regardless of the site of injection, rhodamine-coated latex microspheres were injected into the PGi and WGA-apoHRP-Au was injected into the Abd in one animal. Animals survived for 72 h and were deeply anesthetized with sodium pentobarbital (60 mg/kg, i.p.) and transcardially perfused with 80 ml of saline followed by 1 1 of 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4). All solutions were at room temperature and were prepared immediately prior to perfusion. Brains were removed and immersed in the same fixative for an additional 90 rain at 4°C. Brains were cut at 40/tin thickness on a cryostat (Microm, Zeiss Inc.) and collected into 0.1 M PB. Sections were processed for silver intensification of the colloidal gold complex using a commercially available kit (Amersham Corp.). Briefly, sections were rinsed twice in 0.1 M sodium acetate buffer (pH 5.5) and incubated in the silver intensification kit for 30 rain in the dark. Sections were then rinsed in NaAc twice and the silver was fixed with a solution containing 2.5% sodium thiosulphate in 0.1 M PB. Retrogradely labeled neurons were observed with epi-fluorescence microscopy using either a filter with an emission peak at 380 nm or 490 nm to detect the fluorescein or rhodamine-coated latex microspheres, respectively, or with darkfield microscopy for the WGAapoHRP-Au particles. The silver intensification procedure did not appear to compromise the sensitivity or intensity of the latex microsphere retrograde labeling.
Anterograde transport experiments To further detail the innervation patterns of SOM/ventromedial PAG neurons in the PGi, Abd and dorsal pontine reticular formation, Phaseolus vulgaris-leucoagglutinin (PHA-L; Vector Laboratories; 2.5% in 0.01 M PBS) was iontophoretically deposited into the rostral ventromedial PAG (n = 4). Injections were made with +5 /~A of pulsed current (4 s on, 4 s off) for 30 min 19. Animals survived for 7-14 days and were then deeply anesthetized with sodium pentobarbital (60 mg/kg, i.p.) and perfused transcardially with 200 ml of 0.9% saline solution followed by 1500 ml of 4% paraformaldehyde, 0.1% glutaraldehyde and 0.2% picric acid in 0.1 M PB 21. Brains were postfixed for 2 h and cryoprotected in 20% sucrose overnight. Brains were cut at 30/~m thickness in
Fig. 1. Brightfield-illuminated photomicrograph of a coronal section taken through the midbrain depicting an iontophoretic deposit of Phaseolus vulgaris leucoagglutinin in the rostral ventromedial PAG and SOM. See Fig. 2 for labeling from this injection. Aq, cerebral aqueduct; PAG, periaqueductal gray; III, oculomotor nucleus. Bar = 200/~m.
the coronal plane on a cryostat (Microm, Zeiss, Corp.). Briefly, sections were collected into 0.1 M PBS and incubated with primary antibody for 48 h (Goat anti-PHA-L; 1:4,000; Vector Labs, Burlingame, CA). The avidin-biotin method (Vector Labs, Burlingame, CA) was used to detect immunoreactivity, as previously described 2°.
RESULTS L a r g e P H A - L injections into the S O M / v e n t r o m e d i a l P A G (Fig. 1) which e x t e n d e d v e n t r a l l y b e y o n d the P A G b o u n d a r y into the o c u l o m o t o r nucleus y i e l d e d a n t e r o g r a d e l y l a b e l e d fibers in the dorsal p o n t i n e r e t i c u l a r form a t i o n rostral to the A b d (Fig. 2 A ) , t h e P G i (Fig. 2 B ) , and the A b d a m o n g o t h e r targets. P H A - L
injections
which w e r e r e s t r i c t e d to t h e v e n t r o m e d i a l P A G y i e l d e d f e w e r a n t e r o g r a d e l y l a b e l e d fibers in the A b d nucleus
Fig. 2. A: Brightfield-illuminated photomicrograph of a coronal section taken through the rostral pons depicting PHA-L anterogradely labeled fibers in the dorsal pontine reticular formation following an injection into the rostral ventromedial PAG and SOM. Inset: low-power camera lucida drawing illustrating the level from which the photomicrograph is taken. The box indicates the region in which the fibers are found. B: brightfield-illuminated photomicrograph of a coronal section through the rostral medulla depicting PHA-L anterogradely labeled fibers in the nucleus paragigantocellularis following an injection into the contralateral ventromedial PAG. Inset: low-power camera lucida drawing illustrating the level from which the photomicrograph is taken. The box indicates the region in which the fibers are found, mlf, medial longitudinal fasciculus; PrH, nucleus prepositus hypoglossi; MVe, medial vestibular nucleus; Cu, nucleus cuneatus; ECu, external nucleus cuneatus; SP51, spinal trigeminal nucleus pars interpolaris; icp, inferior cerebellar peduncle; tz, trapezoid body; py, pyramidal tract; LSO, lateral superior olive; Mo5, motor nucleus of the trigeminal nerve; LC, locus coeruleus; scp, superior cerebellar peduncle. Bars = 150 ~um.
61
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Fig. 3. A: epifluorescent-illuminated photomicrograph depicting an injection of fluorescein-coated latex microspheres into the nucleus abducens. B: brightfield-illuminated photomicrograph of a coronal section taken through the same level as shown in A depicting choline acetyltransferase immunoreactive neurons in the abducens nucleus. Inset: low-power camera lucida drawing illustrating the level at which the injection was made. py, pyramidal tract; Sp50, spinal trigeminal nucleus pars oralis; icp, inferior cerebellar peduncle; VCP, ventral cochlear nucleus posterior; VII, facial nucleus; IV, fourth ventricle; mlf, medial longitudinal fasciculus; gVII, genu of the seventh nerve. Bars = 200 pm. Lower panels: brightfield-illuminated coronal section taken from the rostral ventral medulla stained for Nissl substance depicting a microinjection of W G A - a p o H R P - A u (black deposit) into the nucleus paragigantocellularis. Inset: low-power camera lucida drawing illustrating the level of the injection site. mlf: medial longitudinal fasciculus; PrH, nucleus prepositus hypoglossi; MVe, medial vestibular nucleus; SpVe, superior vestibular nucleus; sp5, spinal trigeminal tract; icp, inferior cerebellar peduncle; Amb, nucleus ambiguus; Gi, nucleus gigantocellularis; IO, inferior olive; py, pyramidal tract; Sp51, spinal trigeminal nucleus pars interpolaris. Bar = 250 ~m.
63
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Fig. 4. Camera lucida drawings illustrating the distribution of retrogradely labeled neurons throughout the rostral ventromedial periaqueductal gray/SOM area following an injection of fluoresceincoated latex microspheres into the nucleus abducens and of WGAapoHRP-Au into the nucleus paragigantocellularis of the same animal. The injection sites of this case are shown in Fig. 3. Plot A represents the rostralmost level and plot D the caudalmost. Retrogradely labeled neurons from the nucleus paragigantoceUularis are illustrated as filled squares, and labeled neurons from the abducens nucleus are illustrated as half-stippled circles. Note that PGi-projecting ceils are located just dorsal to neurons labeled from Abd injections. Note also the lack of doubly labeled neurons. PAG, periaqueductal gray; Aq, cerebral aqueduct; III, oculomotor nucleus; IV, trochlear nucleus; mlf, medial longitudinal fasciculus; xscp, crossing of the superior cerebellar peduncle.
although dense anterograde labeling was still observed in the dorsal pontine reticular formation rostral to the Abd and the PGi. Retrograde tracing experiments from the dorsal pontine reticular formation rostral, the Abd and the PGi confirmed the anterograde transport results. Microinjections of fluorescein- (Fig. 3A) or rhodamine-coated latex microspheres, or of W G A - a p o H R P - A u into the region of choline acetyltransferase immunoreactive neurons of the nucleus Abd (Fig. 3B) yielded retrogradely labeled neurons in the contralateral SOM area. These neurons were located immediately dorsal to the oculomotor nucleus in the ventromedial PAG, as described previously by others 9'1° and shown in Fig. 4. The number of labeled neurons from the Abd varied with their rostrocaudal location in the SOM area. Caudally,
at the level of the trochlear nucleus, numerous labeled neurons were present (15-20 neurons per section; Fig. 4C). More rostrally, at the level of the oculomotor nucleus, fewer neurons were retrogradely labeled from the Abd (5-10 neurons per section; Fig. 4A). In addition, neurons labeled from the Abd were not restricted to the P A G boundary but extended ventrally along the lateral borders of the oculomotor nucleus (Fig. 4A,B) and mlf (Fig. 4C,D). Microinjections of WGA-apoHRP-Au, or of latex microspheres into the lateral retrofacial PGi also retrogradely labeled neurons in the area of the contralateral SOM and elsewhere in the PAG. However, injections restricted to the medial aspect of the PGi did not yield retrogradely labeled neurons in the SOM/ventromedial PAG. With injections that encroached on both the medial and lateral aspects of the PGi (Fig. 3C), retrogradely labeled neurons (illustrated as squares in Fig. 4) in the periaqueductal gray were identified bilaterally, with a contralateral predominance in the SOM area, as previously reported with other tracers 19'2°. PGi-projecting cells in the SOM area were most prominent at rostral P A G levels. These neurons were greatest in number (20-25 neurons per section) at the level of the oculomotor nucleus (Fig. 4A,B) and extended to the level of the anterior pole of the trochlear nucleus (Fig. 4C). At caudal P A G levels, few retrogradely labeled neurons (1-5 neurons per section) from the PGi were found in the ventromedial P A G (Fig. 4D) although numerous retrogradely labeled neurons were present in the lateral and ventrolateral aspects of the PAG. The retrograde and anterograde tracing results suggested that the same population of neurons in the SOM/ ventromedial P A G area may innervate the Abd and the PGi. Therefore, the same sections were examined for retrograde tracers from both the Abd and the PGi to determine whether the same neurons projected to both targets. These double labeling experiments revealed that, at rostral P A G levels, neurons labeled from either the Abd or from the PGi formed adjacent but separate populations (Fig. 5). A total of only two doubly labeled neurons were identified in the SOM/ventromedial PAG in all cases examined (n = 5). In other animals containing retrograde tracer injections in the PGi, WGA-apoHRP-Au or rhodaminecoated microspheres were placed into the dorsal pontine reticular formation immediately rostral to the nucleus abducens (Fig. 6). Such injections also yielded retrogradely labeled neurons in the contralateral SOM area. However, following these double injections, 7% of neurons projecting to the PGi also projected to the rostral dorsal pontine reticular formation, as shown in Fig. 7.
64
Fig. 5. A: darkfield-iUuminated photomicrograph depicting WGA-apoHRP-Au retrogradely labeled neurons in the rostral ventromedial PAG/SOM area following an injection into the contralateral nucleus paragigantocellularis. Arrows indicate examples of retrogradely labeled neurons. Filled star indicates a blood vessel which can also be seen in Panel B. Inset: low-power camera lucida drawing illustrating the level where these labeled neurons are found. B: epifluorescent illuminated photomicrograph of the same field shown in panel A depicting neurons retrogradely labeled with fluoresccin-coated microspheres following an injection into the nucleus abducens. Open arrows indicate examples of retrogradely labeled neurons. Note more ventral location of abducens projecting cells, and lack of double labeling. PAG, periaqueductal gray; SuG, superior colliculus; MG, medial geniculate; SN, substantia nigra; IPN, interpeduncular nucleus; III, oculomotor nucleus. Bar = 100/2m.
65
Fig. 6. Brightfield-illuminated photomicrograph of a coronal section stained for Nissl substance through the pons depicting a WGAapoHRP-Au injection into the dorsal pontine reticular formation rostral to the nucleus abducens. Arrow indicates the WGA-apoHRP-Au injection site. LC, locus coeruleus; MoV, motor nucleus of the trigeminal nerve; IV, fourth ventricle. Bar = 250/~m.
DISCUSSION The results of the present study indicate that separate populations of neurons in the SOM region of the rostral ventromedial P A G project to the PGi and the Abd nuclei. The present study confirms the results of previous studies that used other retrograde tracers to show projections from neurons in the SOM/ventromedial P A G area to the PGi 19'2° and the Abd 1~'~2. In addition, the results presented here extend those previous studies by showing that neurons projecting to the Abd and PGi are located in adjacent, but deafly separate regions in the ventromedial PAG. Specifically, PGi-projecting neurons are located dorsal to Abd-projecting neurons. In addition to these new findings, neurons in the SOM area were found to also project to the dorsal pontine reticular formation rostral to the Abd, a heretofore uncharacterized projection. The retrograde tracing results are further supported by anterograde tracing with PHA-L. Large P H A - L in-
jections into the rostral ventromedial P A G and SOM, which extended ventral to the gray matter into the oculomotor nucleus, yielded anterogradely labeled fibers in several regions including the Abd, dorsal pontine reticular formation rostral to the Abd and the rostral ventral medulla. As such injections covered a large region of the ventromedial P A G , they were not useful in determining specificity of innervation from different neuronal populations. However, P H A - L injections that were restricted to the rostral ventromedial P A G and did not extend into the oculomotor nucleus yielded only few anterogradely labeled fibers in the Abd nucleus but produced numerous fibers in both the dorsal pontine reticular formation rostral to the Abd and the lateral aspect of the PGi. The latter results, coupled with the finding that neurons in the ventromedial P A G are doubly labeled from injections into the PGi and dorsal ports, but not into the PGi and Abd, suggest that PGi-projecting neurons are probably not part of the SOM/oculomotor complex. In light of the functions attributed to the subregion of the PGi targeted by neurons in the rostral ventromedial P A G , it is possible that these P A G neurons function in autonomic processes. In particular, projections from the rostral ventromedial P A G innervate a specific region of the PGi that has been strongly implicated in cardiovascular control 17'1s, respiratory-related activities 3'4, and cardioinhibition 14. As this area of the rodent ventromedial P A G has not been thoroughly examined using physiological techniques, it is unknown whether these neurons participate in such activities. It has been well documented that P A G neurons in other subregions of the central gray, such as the dorsal and lateral aspects, participate in such functions. Our preliminary studies using electrical stimulation of the rostral ventromedial P A G have revealed that these neurons may alter respiratory activity (Van Bockstaele, Akaoka and Aston-Jones, unpublished observations), but additional experiments are required to determine the specificity of these effects. Interestingly, Lovick has reported similar effects in the cat 9. Although rostral ventromedial P A G neurons do not project in common to the PGi and Abd, the proximity of the two populations of neurons suggests that they may share common afferents and that this region may be important in the coordination of visceromotor events. Interestingly, pupillary changes have been correlated with changes in blood pressure and respiratory rate 13. The overlap in these two systems may be responsible,for their adjacent positions. Additional studies are needed to elucidate and detail the functional significance of rostral ventromedial P A G projections to the rostral ventral medulla as well as its significance in visceromotor control.
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Fig. 7. A: darkfield-illuminated photomicrograph depicting WGA-apoHRP-Au retrogradely labeled neurons in the rostral ventromedial PAG following an injection of rhodamine-coated latex microspheres into the contralateral nucleus paragigantocellularis. Note retrogradely labeled neuron at arrow which is doubly labeled from the rostral dorsal pontine reticular formation (panel B). Inset: low-power camera lucida drawing illustrating the level from which the photomicrograph was taken. PAG, periaqueductal gray; SuG, superior colliculus; MG, medial geniculate; SN, substantia nigra; IPN, interpeduncular nucleus. B: epifluorescent illuminated photomicrograph of the same field shown in panel A depicting retrogradely labeled neurons following an injection of WGA-apoHRP-Au into the rostral dorsal pontine reticular formation. Arrow indicates same cell labeled in panel A. Bar = 75 ~m.
Acknowledgements. The authors wish to thank Dr. Pierre Luppi for providing tissue sections revealing choline acetyltransferase im-
REFERENCES 1 Basbaum, A. and Menetrey, D., Wheat germ agglutinin-apoHRP gold: a new retrograde tracer for light and electron mi-
munoreactive neurons in the nucleus abducens. This work was supported by PHS Grants NS24698 and DA06214.
croscopic single and double label studies, J. Comp. Neurol., 261 (1987) 306-318. 2 Beitz, A.J., The organization of afferent projections to the midbrain periaqueductal gray of the rat, Neuroseience, 7 (1982)
67 35-159. 3 Ellenberger, H.H., Feldman, J.L. and Goshgarian, H.G., Ventral respiratory group projections to phrenic motoneurons: electron microscopic evidence for monosynaptic connections, J. Comp. Neurol., 302 (1990) 707-714. 4 Feldman, J.L., Neurophysiology of breathing in mammals. In EE. Bloom (Ed.), Handbook of Physiology; Section 1: The
13 14
Nervous System, Volume IV; Intrinsic Regulatory Systems of the Brain, American Physiological Society, 1986, pp. 463-524.
15
5 Gonzalo-Ruiz, A., Leichnetz, G.R. and Smith, D.J., Origin of cerebellar projections to the region of the oculomotor complex, medial pontine reticular formation, and superior colliculus in new world monkeys: a retrograde horseradish peroxidase study, J Comp. Neurol., 268 (1988) 508-526. 6 Gonzalo-Ruiz, A. and Leichnetz G.R., Collateralization of cerebellar efferent projections to the paraoculomotor region, superior colliculus, and medial pontine reticular formation in the rat: a fluorescent doublelabeling study, Exp. Brain Res., 68 (1987) 365-378. 7 Henkel, C.K. and Edwards, S.B., The superior colliculus control of pinna movements in the cat: possible anatomical connections, J. Comp. Neurol., 182 (1978) 763-776. 8 Leichnetz, G.R., Hardy, S.G.P. and Carruth, M.K., Frontal projections to the region of the oculomotor complex in the rat: a retrograde and anterograde HRP study, J. Comp. Neurol., 263 (1987) 387-399. 9 Lovick, T.A., Convergent afferent inputs to nucleus paragigantocellularis lateralis in the cat, Brain Res., 456 (1988) 483-487. 10 Maciewicz, R. and Phipps, B.S., The oculomotor internuclear pathway: a double retrograde labeling study, Brain Res., 262 (1983) 1-8. 11 Maciewicz, R.J., Kaneko, C.R.S., Highstein, S.M. and Baker, R., Morphophysiological identification of interneurons in the oculomotor nucleus that project to the abducens nucleus in the cat, Brain Res., 1975 (1975) 60-65. 12 May, P.J., Baker, H., Vidal, P.-P., Spencer, R.F. and Baker, R., Morphology and distribution of serotoninergic and oculo-
16
17
18
19
20
21
motor internuclear neurons in the cat midbrain, J. Comp. Neurol., 266 (1987) 150-170. McAllen, R.M., Action and specificity of ventral medullary vasopressor neurones in the cat, Neuroscience, 18 (1986) 51-59. Nosaka, S., Yamamoto, T. and Yasunuga, K., Localization of vagal cardioinhibitory preganglionic neurons within rat brain stem, J. Comp. Neurol., 186 (1979) 79-92. Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic, New York, 1986. Person, R.J., Andrezik, J.A., Dormer, K.J. and Foreman, R.D., Fastigial nucleus projections in the midbrain and thalamus in dogs, Neuroscience, 18 (1985) 105-120. Ross, C.A. Ruggiero, D.A., Park, D.H., Joh, T.H., Sun, A.F., Fernandez-Pardal, J., Saavedra, J.M. and Reis, D.J., Tonic vasomotor control by the rostral ventrolateral medulla: effect of electrical stimulation or chemical stimulation of an area containing C1 adrenaline neurons on arterial pressure, heart rate and plasma catecholamines and vasopressin, J. Neurosci., 4 (1984) 474-494. Ruggiero, D.A., Cravo, S.L., Arango, V. and Reis, D.J., Central control of the circulation by the rostral ventrolateral reticular nucleus: anatomical substrates. J. Ciriello, M.M. Caverson and C. Polosa (Eds.), Progress in Brain Research, Vol. 81, Elsevier, New York, 1989, pp. 49-79. Van Bockstaele, E.J., Aston- Jones, G., Pieribone, V.A., Ennis, M. and Shipley, M.T., Subregions of the periaqueductal gray topographically innervate the rostral ventral medulla in the rat, J. Comp. Neurol., 309 (1991) 305-327. Van Bockstaele, E.J., Pieribone, V.A. and Aston-Jones, G., Diverse afferents converge on the nucleus paragigantocellularis in the rat ventrolateral medulla: retrograde and anterograde studies, J. Comp. Neurol., 290 (1989) 561-584. Wouterlood, F.G., Bol, J.G.J.M. and Steinbusch, H.W.M, Double-label immunocytochemistry: combination of anterograde neuroanatomical tracing with Phaseolus vulgaris-leucoagglutinin and enzyme immunocytochemistry of target neurons, J. Histochem. Cytochem., 35 (1987) 817-823.
59
BRES 17558
Distinct populations of neurons in the ventromedial periaqueductal gray project to the rostral ventral medulla and abducens nucleus Elisabeth J. Van Bockstaele and Gary Aston-Jones Hahnemann University, Department of Mental Health Sciences, Division of Behavioral Neurobiology, Philadelphia PA 19102 (U.S.A.) (Accepted 12 November 1991)
Key words: Paragigantocellularis; Retrograde transport; Supraoculomotor nucleus of the central gray; Oculomotor nucleus; Medulla oblongata; Brainstem; Anterograde transport; Cardiovascular regulation
Following microinjections of a colloidal gold complex into the nucleus paragigantocellularis (PGi) of the ventral medulla, and of latex microspheres into the nucleus abducens (Abd) of the same animal, retrogradely labeled neurons were identified in the region of the contralateral supraoculomotor nucleus (SOM) in the ventromedial periaqueductal gray (PAG). Neurons labeled from the Abd were found in the SOM in the ventromedial PAG throughout the midbrain, as well as scattered ventrally in the oculomotor nucleus. Neurons in the SOM area retrogradely labeled from the PGi were most numerous rostral to the dorsal raphe nucleus and extended throughout the level of the oculomotor nucleus. Direct comparison of the two labels revealed that the neurons that project to the Abd were located slightly more ventrally than PGi-projecting neurons. Almost no doubly labeled neurons were identified, although singly labeled neurons formed adjacent but separate populations. These results indicate that neurons in the SOM area projecting to the PGi are distinct from those projecting to the Abd, and that the PGi-projecting neurons are probably not pre-oculomotor neurons. Given their more dorsal location and the nature of their target neurons in the rostral ventrolateral medulla, PGi-projecting neurons may be related to visceromotor, as opposed to oculomotor functions. INTRODUCTION
pre-oculomotor in function 1°-12.
Retrograde transport studies using a variety of tracers have found that n e u r o n s in the area of the contralateral supraoculomotor nucleus (SOM) project to the nucleus paragigantocellularis (PGi) in the rostral ventral medulla (RV1V[) 19'20. These labeled n e u r o n s lie immediately dor-
In the present study, to determine whether n e u r o n s in the SOM area of the rostral ventromedial P A G that project to the PGi may be part of this pre-oculomotor group, we used double retrograde transport to discern whether these cells also innervate the A b d nucleus. O u r results indicate that PGi- projecting neurons are a slightly
sal to the oculomotor nucleus within the rostral ventromedial aspect of the periaqueductal gray ( P A G ) . A n t e r ograde tracing studies have confirmed and further detailed this projection, indicating a strong and specific projection to the sympathoexcitatory region of the PGi, corresponding to the rostroventrolateral reticular nucleus (RVL) 19. The R V L is located (i) caudal to the facial nucleus, (ii) in the lateral aspect of the PGi ventral to the nucleus ambiguus and will be referred to as the lateral retrofacial PGi. Anatomical and physiological studies have implicated n e u r o n s in the SOM in oculomotor-related activities due to (i) projections to the abducens nucleus in the pons s' 10,11 and (ii) afferents from nuclei with visual-related p•operties such as the frontal eye field s, the superior colliculus7, and the medial fastigial nucleus2'5'6'16. These results have led to the classification of SOM n e u r o n s as
more dorsal, separate population from those innervating the A b d nucleus. MATERIALS AND METHODS Sprague-Dawley rats (n = 12; Taconic Farms) weighing 275-350 g were anesthetized using sodium pentobarbital (50 mg/kg, i.p.) and placed in a stereotaxic apparatus for surgery. Rectal temperature was monitored continuously and maintained at 37°C by a heating pad. Supplementary doses of sodium pentobarbital were administered as needed.
Retrograde transport experiments Microinjections (250-300 nl) of wheat germ agglutinin-conjugated to inactive horseradish peroxidase coupled to 15-nm gold particles (WGA-apoHRP-Au), synthesized according to the protocol of Basbaum and Menetrey (1987)1, were stereotaxically deposited into the PGi15area using precleaned glass micropipettes (15-20 /~m tips; n = 5). Injections were made using a solenoid controlled pneumatic pressure device (Picospritzer; General Valve, Inc.), and the desired volume was obtained by directly measuring
Correspondence: G. Aston-Jones, Hahnemann University, Department of Mental Health Sciences, Division of Behavioral Neurobiology, Broad & Vine Streets, Mail Stop 403, Philadelphia PA 19102, U.S.A.
60 the movement of the fluid column within the pipette. Prior to depositing the tracer, physiologic recordings were made through the injection pipettes and the PGi was tentatively identified as previously described z°. In the same animals, a microinjection (25-50 nl) of fluorescein-coated latex microspheres (n = 4; Tracer Technology), or of rhodamine-coated latex microspheres (n = 1) was similarly deposited into the Abd. The pipette was left in place for five minutes prior to and following injections. In two animals, injections were placed in a region rostral to the Abd nucleus in the rostral dorsal pontine reticular formation corresponding to the level of Plate 56 of the rat brain atlas of Paxinos and Watson (1986) 15. In one of these animals, rhodamine latex microspheres were deposited in the PGi and WGA-apoHRP-Au was deposited in the dorsal pontine reticular formation. In the other animal, WGA-apoHRP-Au was deposited in the PGi and rhodamine-coated latex microspheres were deposited into the dorsal pontine reticular formation. To confirm that the retrograde tracers yielded a similar pattern of retrograde transport regardless of the site of injection, rhodamine-coated latex microspheres were injected into the PGi and WGA-apoHRP-Au was injected into the Abd in one animal. Animals survived for 72 h and were deeply anesthetized with sodium pentobarbital (60 mg/kg, i.p.) and transcardially perfused with 80 ml of saline followed by 1 1 of 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4). All solutions were at room temperature and were prepared immediately prior to perfusion. Brains were removed and immersed in the same fixative for an additional 90 rain at 4°C. Brains were cut at 40/tin thickness on a cryostat (Microm, Zeiss Inc.) and collected into 0.1 M PB. Sections were processed for silver intensification of the colloidal gold complex using a commercially available kit (Amersham Corp.). Briefly, sections were rinsed twice in 0.1 M sodium acetate buffer (pH 5.5) and incubated in the silver intensification kit for 30 rain in the dark. Sections were then rinsed in NaAc twice and the silver was fixed with a solution containing 2.5% sodium thiosulphate in 0.1 M PB. Retrogradely labeled neurons were observed with epi-fluorescence microscopy using either a filter with an emission peak at 380 nm or 490 nm to detect the fluorescein or rhodamine-coated latex microspheres, respectively, or with darkfield microscopy for the WGAapoHRP-Au particles. The silver intensification procedure did not appear to compromise the sensitivity or intensity of the latex microsphere retrograde labeling.
Anterograde transport experiments To further detail the innervation patterns of SOM/ventromedial PAG neurons in the PGi, Abd and dorsal pontine reticular formation, Phaseolus vulgaris-leucoagglutinin (PHA-L; Vector Laboratories; 2.5% in 0.01 M PBS) was iontophoretically deposited into the rostral ventromedial PAG (n = 4). Injections were made with +5 /~A of pulsed current (4 s on, 4 s off) for 30 min 19. Animals survived for 7-14 days and were then deeply anesthetized with sodium pentobarbital (60 mg/kg, i.p.) and perfused transcardially with 200 ml of 0.9% saline solution followed by 1500 ml of 4% paraformaldehyde, 0.1% glutaraldehyde and 0.2% picric acid in 0.1 M PB 21. Brains were postfixed for 2 h and cryoprotected in 20% sucrose overnight. Brains were cut at 30/~m thickness in
Fig. 1. Brightfield-illuminated photomicrograph of a coronal section taken through the midbrain depicting an iontophoretic deposit of Phaseolus vulgaris leucoagglutinin in the rostral ventromedial PAG and SOM. See Fig. 2 for labeling from this injection. Aq, cerebral aqueduct; PAG, periaqueductal gray; III, oculomotor nucleus. Bar = 200/~m.
the coronal plane on a cryostat (Microm, Zeiss, Corp.). Briefly, sections were collected into 0.1 M PBS and incubated with primary antibody for 48 h (Goat anti-PHA-L; 1:4,000; Vector Labs, Burlingame, CA). The avidin-biotin method (Vector Labs, Burlingame, CA) was used to detect immunoreactivity, as previously described 2°.
RESULTS L a r g e P H A - L injections into the S O M / v e n t r o m e d i a l P A G (Fig. 1) which e x t e n d e d v e n t r a l l y b e y o n d the P A G b o u n d a r y into the o c u l o m o t o r nucleus y i e l d e d a n t e r o g r a d e l y l a b e l e d fibers in the dorsal p o n t i n e r e t i c u l a r form a t i o n rostral to the A b d (Fig. 2 A ) , t h e P G i (Fig. 2 B ) , and the A b d a m o n g o t h e r targets. P H A - L
injections
which w e r e r e s t r i c t e d to t h e v e n t r o m e d i a l P A G y i e l d e d f e w e r a n t e r o g r a d e l y l a b e l e d fibers in the A b d nucleus
Fig. 2. A: Brightfield-illuminated photomicrograph of a coronal section taken through the rostral pons depicting PHA-L anterogradely labeled fibers in the dorsal pontine reticular formation following an injection into the rostral ventromedial PAG and SOM. Inset: low-power camera lucida drawing illustrating the level from which the photomicrograph is taken. The box indicates the region in which the fibers are found. B: brightfield-illuminated photomicrograph of a coronal section through the rostral medulla depicting PHA-L anterogradely labeled fibers in the nucleus paragigantocellularis following an injection into the contralateral ventromedial PAG. Inset: low-power camera lucida drawing illustrating the level from which the photomicrograph is taken. The box indicates the region in which the fibers are found, mlf, medial longitudinal fasciculus; PrH, nucleus prepositus hypoglossi; MVe, medial vestibular nucleus; Cu, nucleus cuneatus; ECu, external nucleus cuneatus; SP51, spinal trigeminal nucleus pars interpolaris; icp, inferior cerebellar peduncle; tz, trapezoid body; py, pyramidal tract; LSO, lateral superior olive; Mo5, motor nucleus of the trigeminal nerve; LC, locus coeruleus; scp, superior cerebellar peduncle. Bars = 150 ~um.
61
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Fig. 3. A: epifluorescent-illuminated photomicrograph depicting an injection of fluorescein-coated latex microspheres into the nucleus abducens. B: brightfield-illuminated photomicrograph of a coronal section taken through the same level as shown in A depicting choline acetyltransferase immunoreactive neurons in the abducens nucleus. Inset: low-power camera lucida drawing illustrating the level at which the injection was made. py, pyramidal tract; Sp50, spinal trigeminal nucleus pars oralis; icp, inferior cerebellar peduncle; VCP, ventral cochlear nucleus posterior; VII, facial nucleus; IV, fourth ventricle; mlf, medial longitudinal fasciculus; gVII, genu of the seventh nerve. Bars = 200 pm. Lower panels: brightfield-illuminated coronal section taken from the rostral ventral medulla stained for Nissl substance depicting a microinjection of W G A - a p o H R P - A u (black deposit) into the nucleus paragigantocellularis. Inset: low-power camera lucida drawing illustrating the level of the injection site. mlf: medial longitudinal fasciculus; PrH, nucleus prepositus hypoglossi; MVe, medial vestibular nucleus; SpVe, superior vestibular nucleus; sp5, spinal trigeminal tract; icp, inferior cerebellar peduncle; Amb, nucleus ambiguus; Gi, nucleus gigantocellularis; IO, inferior olive; py, pyramidal tract; Sp51, spinal trigeminal nucleus pars interpolaris. Bar = 250 ~m.
63
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Fig. 4. Camera lucida drawings illustrating the distribution of retrogradely labeled neurons throughout the rostral ventromedial periaqueductal gray/SOM area following an injection of fluoresceincoated latex microspheres into the nucleus abducens and of WGAapoHRP-Au into the nucleus paragigantocellularis of the same animal. The injection sites of this case are shown in Fig. 3. Plot A represents the rostralmost level and plot D the caudalmost. Retrogradely labeled neurons from the nucleus paragigantoceUularis are illustrated as filled squares, and labeled neurons from the abducens nucleus are illustrated as half-stippled circles. Note that PGi-projecting ceils are located just dorsal to neurons labeled from Abd injections. Note also the lack of doubly labeled neurons. PAG, periaqueductal gray; Aq, cerebral aqueduct; III, oculomotor nucleus; IV, trochlear nucleus; mlf, medial longitudinal fasciculus; xscp, crossing of the superior cerebellar peduncle.
although dense anterograde labeling was still observed in the dorsal pontine reticular formation rostral to the Abd and the PGi. Retrograde tracing experiments from the dorsal pontine reticular formation rostral, the Abd and the PGi confirmed the anterograde transport results. Microinjections of fluorescein- (Fig. 3A) or rhodamine-coated latex microspheres, or of W G A - a p o H R P - A u into the region of choline acetyltransferase immunoreactive neurons of the nucleus Abd (Fig. 3B) yielded retrogradely labeled neurons in the contralateral SOM area. These neurons were located immediately dorsal to the oculomotor nucleus in the ventromedial PAG, as described previously by others 9'1° and shown in Fig. 4. The number of labeled neurons from the Abd varied with their rostrocaudal location in the SOM area. Caudally,
at the level of the trochlear nucleus, numerous labeled neurons were present (15-20 neurons per section; Fig. 4C). More rostrally, at the level of the oculomotor nucleus, fewer neurons were retrogradely labeled from the Abd (5-10 neurons per section; Fig. 4A). In addition, neurons labeled from the Abd were not restricted to the P A G boundary but extended ventrally along the lateral borders of the oculomotor nucleus (Fig. 4A,B) and mlf (Fig. 4C,D). Microinjections of WGA-apoHRP-Au, or of latex microspheres into the lateral retrofacial PGi also retrogradely labeled neurons in the area of the contralateral SOM and elsewhere in the PAG. However, injections restricted to the medial aspect of the PGi did not yield retrogradely labeled neurons in the SOM/ventromedial PAG. With injections that encroached on both the medial and lateral aspects of the PGi (Fig. 3C), retrogradely labeled neurons (illustrated as squares in Fig. 4) in the periaqueductal gray were identified bilaterally, with a contralateral predominance in the SOM area, as previously reported with other tracers 19'2°. PGi-projecting cells in the SOM area were most prominent at rostral P A G levels. These neurons were greatest in number (20-25 neurons per section) at the level of the oculomotor nucleus (Fig. 4A,B) and extended to the level of the anterior pole of the trochlear nucleus (Fig. 4C). At caudal P A G levels, few retrogradely labeled neurons (1-5 neurons per section) from the PGi were found in the ventromedial P A G (Fig. 4D) although numerous retrogradely labeled neurons were present in the lateral and ventrolateral aspects of the PAG. The retrograde and anterograde tracing results suggested that the same population of neurons in the SOM/ ventromedial P A G area may innervate the Abd and the PGi. Therefore, the same sections were examined for retrograde tracers from both the Abd and the PGi to determine whether the same neurons projected to both targets. These double labeling experiments revealed that, at rostral P A G levels, neurons labeled from either the Abd or from the PGi formed adjacent but separate populations (Fig. 5). A total of only two doubly labeled neurons were identified in the SOM/ventromedial PAG in all cases examined (n = 5). In other animals containing retrograde tracer injections in the PGi, WGA-apoHRP-Au or rhodaminecoated microspheres were placed into the dorsal pontine reticular formation immediately rostral to the nucleus abducens (Fig. 6). Such injections also yielded retrogradely labeled neurons in the contralateral SOM area. However, following these double injections, 7% of neurons projecting to the PGi also projected to the rostral dorsal pontine reticular formation, as shown in Fig. 7.
64
Fig. 5. A: darkfield-iUuminated photomicrograph depicting WGA-apoHRP-Au retrogradely labeled neurons in the rostral ventromedial PAG/SOM area following an injection into the contralateral nucleus paragigantocellularis. Arrows indicate examples of retrogradely labeled neurons. Filled star indicates a blood vessel which can also be seen in Panel B. Inset: low-power camera lucida drawing illustrating the level where these labeled neurons are found. B: epifluorescent illuminated photomicrograph of the same field shown in panel A depicting neurons retrogradely labeled with fluoresccin-coated microspheres following an injection into the nucleus abducens. Open arrows indicate examples of retrogradely labeled neurons. Note more ventral location of abducens projecting cells, and lack of double labeling. PAG, periaqueductal gray; SuG, superior colliculus; MG, medial geniculate; SN, substantia nigra; IPN, interpeduncular nucleus; III, oculomotor nucleus. Bar = 100/2m.
65
Fig. 6. Brightfield-illuminated photomicrograph of a coronal section stained for Nissl substance through the pons depicting a WGAapoHRP-Au injection into the dorsal pontine reticular formation rostral to the nucleus abducens. Arrow indicates the WGA-apoHRP-Au injection site. LC, locus coeruleus; MoV, motor nucleus of the trigeminal nerve; IV, fourth ventricle. Bar = 250/~m.
DISCUSSION The results of the present study indicate that separate populations of neurons in the SOM region of the rostral ventromedial P A G project to the PGi and the Abd nuclei. The present study confirms the results of previous studies that used other retrograde tracers to show projections from neurons in the SOM/ventromedial P A G area to the PGi 19'2° and the Abd 1~'~2. In addition, the results presented here extend those previous studies by showing that neurons projecting to the Abd and PGi are located in adjacent, but deafly separate regions in the ventromedial PAG. Specifically, PGi-projecting neurons are located dorsal to Abd-projecting neurons. In addition to these new findings, neurons in the SOM area were found to also project to the dorsal pontine reticular formation rostral to the Abd, a heretofore uncharacterized projection. The retrograde tracing results are further supported by anterograde tracing with PHA-L. Large P H A - L in-
jections into the rostral ventromedial P A G and SOM, which extended ventral to the gray matter into the oculomotor nucleus, yielded anterogradely labeled fibers in several regions including the Abd, dorsal pontine reticular formation rostral to the Abd and the rostral ventral medulla. As such injections covered a large region of the ventromedial P A G , they were not useful in determining specificity of innervation from different neuronal populations. However, P H A - L injections that were restricted to the rostral ventromedial P A G and did not extend into the oculomotor nucleus yielded only few anterogradely labeled fibers in the Abd nucleus but produced numerous fibers in both the dorsal pontine reticular formation rostral to the Abd and the lateral aspect of the PGi. The latter results, coupled with the finding that neurons in the ventromedial P A G are doubly labeled from injections into the PGi and dorsal ports, but not into the PGi and Abd, suggest that PGi-projecting neurons are probably not part of the SOM/oculomotor complex. In light of the functions attributed to the subregion of the PGi targeted by neurons in the rostral ventromedial P A G , it is possible that these P A G neurons function in autonomic processes. In particular, projections from the rostral ventromedial P A G innervate a specific region of the PGi that has been strongly implicated in cardiovascular control 17'1s, respiratory-related activities 3'4, and cardioinhibition 14. As this area of the rodent ventromedial P A G has not been thoroughly examined using physiological techniques, it is unknown whether these neurons participate in such activities. It has been well documented that P A G neurons in other subregions of the central gray, such as the dorsal and lateral aspects, participate in such functions. Our preliminary studies using electrical stimulation of the rostral ventromedial P A G have revealed that these neurons may alter respiratory activity (Van Bockstaele, Akaoka and Aston-Jones, unpublished observations), but additional experiments are required to determine the specificity of these effects. Interestingly, Lovick has reported similar effects in the cat 9. Although rostral ventromedial P A G neurons do not project in common to the PGi and Abd, the proximity of the two populations of neurons suggests that they may share common afferents and that this region may be important in the coordination of visceromotor events. Interestingly, pupillary changes have been correlated with changes in blood pressure and respiratory rate 13. The overlap in these two systems may be responsible,for their adjacent positions. Additional studies are needed to elucidate and detail the functional significance of rostral ventromedial P A G projections to the rostral ventral medulla as well as its significance in visceromotor control.
66
Fig. 7. A: darkfield-illuminated photomicrograph depicting WGA-apoHRP-Au retrogradely labeled neurons in the rostral ventromedial PAG following an injection of rhodamine-coated latex microspheres into the contralateral nucleus paragigantocellularis. Note retrogradely labeled neuron at arrow which is doubly labeled from the rostral dorsal pontine reticular formation (panel B). Inset: low-power camera lucida drawing illustrating the level from which the photomicrograph was taken. PAG, periaqueductal gray; SuG, superior colliculus; MG, medial geniculate; SN, substantia nigra; IPN, interpeduncular nucleus. B: epifluorescent illuminated photomicrograph of the same field shown in panel A depicting retrogradely labeled neurons following an injection of WGA-apoHRP-Au into the rostral dorsal pontine reticular formation. Arrow indicates same cell labeled in panel A. Bar = 75 ~m.
Acknowledgements. The authors wish to thank Dr. Pierre Luppi for providing tissue sections revealing choline acetyltransferase im-
REFERENCES 1 Basbaum, A. and Menetrey, D., Wheat germ agglutinin-apoHRP gold: a new retrograde tracer for light and electron mi-
munoreactive neurons in the nucleus abducens. This work was supported by PHS Grants NS24698 and DA06214.
croscopic single and double label studies, J. Comp. Neurol., 261 (1987) 306-318. 2 Beitz, A.J., The organization of afferent projections to the midbrain periaqueductal gray of the rat, Neuroseience, 7 (1982)
67 35-159. 3 Ellenberger, H.H., Feldman, J.L. and Goshgarian, H.G., Ventral respiratory group projections to phrenic motoneurons: electron microscopic evidence for monosynaptic connections, J. Comp. Neurol., 302 (1990) 707-714. 4 Feldman, J.L., Neurophysiology of breathing in mammals. In EE. Bloom (Ed.), Handbook of Physiology; Section 1: The
13 14
Nervous System, Volume IV; Intrinsic Regulatory Systems of the Brain, American Physiological Society, 1986, pp. 463-524.
15
5 Gonzalo-Ruiz, A., Leichnetz, G.R. and Smith, D.J., Origin of cerebellar projections to the region of the oculomotor complex, medial pontine reticular formation, and superior colliculus in new world monkeys: a retrograde horseradish peroxidase study, J Comp. Neurol., 268 (1988) 508-526. 6 Gonzalo-Ruiz, A. and Leichnetz G.R., Collateralization of cerebellar efferent projections to the paraoculomotor region, superior colliculus, and medial pontine reticular formation in the rat: a fluorescent doublelabeling study, Exp. Brain Res., 68 (1987) 365-378. 7 Henkel, C.K. and Edwards, S.B., The superior colliculus control of pinna movements in the cat: possible anatomical connections, J. Comp. Neurol., 182 (1978) 763-776. 8 Leichnetz, G.R., Hardy, S.G.P. and Carruth, M.K., Frontal projections to the region of the oculomotor complex in the rat: a retrograde and anterograde HRP study, J. Comp. Neurol., 263 (1987) 387-399. 9 Lovick, T.A., Convergent afferent inputs to nucleus paragigantocellularis lateralis in the cat, Brain Res., 456 (1988) 483-487. 10 Maciewicz, R. and Phipps, B.S., The oculomotor internuclear pathway: a double retrograde labeling study, Brain Res., 262 (1983) 1-8. 11 Maciewicz, R.J., Kaneko, C.R.S., Highstein, S.M. and Baker, R., Morphophysiological identification of interneurons in the oculomotor nucleus that project to the abducens nucleus in the cat, Brain Res., 1975 (1975) 60-65. 12 May, P.J., Baker, H., Vidal, P.-P., Spencer, R.F. and Baker, R., Morphology and distribution of serotoninergic and oculo-
16
17
18
19
20
21
motor internuclear neurons in the cat midbrain, J. Comp. Neurol., 266 (1987) 150-170. McAllen, R.M., Action and specificity of ventral medullary vasopressor neurones in the cat, Neuroscience, 18 (1986) 51-59. Nosaka, S., Yamamoto, T. and Yasunuga, K., Localization of vagal cardioinhibitory preganglionic neurons within rat brain stem, J. Comp. Neurol., 186 (1979) 79-92. Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic, New York, 1986. Person, R.J., Andrezik, J.A., Dormer, K.J. and Foreman, R.D., Fastigial nucleus projections in the midbrain and thalamus in dogs, Neuroscience, 18 (1985) 105-120. Ross, C.A. Ruggiero, D.A., Park, D.H., Joh, T.H., Sun, A.F., Fernandez-Pardal, J., Saavedra, J.M. and Reis, D.J., Tonic vasomotor control by the rostral ventrolateral medulla: effect of electrical stimulation or chemical stimulation of an area containing C1 adrenaline neurons on arterial pressure, heart rate and plasma catecholamines and vasopressin, J. Neurosci., 4 (1984) 474-494. Ruggiero, D.A., Cravo, S.L., Arango, V. and Reis, D.J., Central control of the circulation by the rostral ventrolateral reticular nucleus: anatomical substrates. J. Ciriello, M.M. Caverson and C. Polosa (Eds.), Progress in Brain Research, Vol. 81, Elsevier, New York, 1989, pp. 49-79. Van Bockstaele, E.J., Aston- Jones, G., Pieribone, V.A., Ennis, M. and Shipley, M.T., Subregions of the periaqueductal gray topographically innervate the rostral ventral medulla in the rat, J. Comp. Neurol., 309 (1991) 305-327. Van Bockstaele, E.J., Pieribone, V.A. and Aston-Jones, G., Diverse afferents converge on the nucleus paragigantocellularis in the rat ventrolateral medulla: retrograde and anterograde studies, J. Comp. Neurol., 290 (1989) 561-584. Wouterlood, F.G., Bol, J.G.J.M. and Steinbusch, H.W.M, Double-label immunocytochemistry: combination of anterograde neuroanatomical tracing with Phaseolus vulgaris-leucoagglutinin and enzyme immunocytochemistry of target neurons, J. Histochem. Cytochem., 35 (1987) 817-823.
