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Nitric oxide synthase immunoreactive neurons anatomically define a longitudinal dorsolateral column within the midbrain periaqueductal gray of the rat: analysis using laser confocal microscopy
Brain Research, 610 (1993) 317-324 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00
317
BRES 25622
Nitric oxide synthase immunoreactive neurons anatomically define a longitudinal dorsolateral column within the midbrain periaqueductal gray of the rat: analysis using laser confocal microscopy Donna Onstott a Bernd Mayer b and Alvin J. Beitz a a Department of Veterinary PathoBiology, University of Minnesota, St. Paul, MN 55108 (USA) and blnstitute for Pharmacology and Toxicology, University of Graz, Graz (Austria)
(Accepted 19 January 1993)
Key words: Nitric oxide; Central gray; Brainstem; Longitudinal column; Antinociception
Nitric oxide has recently been proposed as a neuronal messenger in both the central and peripheral nervous system. Antibodies against nitric oxide synthase (NOS), the synthesizing enzyme for nitric oxide, were used in combination with immunocytochemistryand confocal laser microscopyto analyze the distribution of this enzyme in the midbrain periaqueductal gray (PAG) of the rat. NOS immunoreactive neurons were localized predominantly in a longitudinally oriented column in the dorsolateral PAG. NOS immunoreactive fibers and processes were scattered throughout the PAG but were most prevalent in the dorsolateral column and in the juxta-aqueductal column. This study provides neurochemical support for the existence of longitudinal columns in the PAG which are postulated to underlie the functional organization of this complex brainstem region.
Nitric oxide (NO) has recently been shown to act as a neuronal messenger t3'46. It appears to mediate transmission from autonomic neurons to smooth muscle in the intestine, to cerebral arteries and to arteries to the penis, as well as between neurons within enteric ganglia 46. Within the central nervous system N O has been implicated in long-term potentiation 9'26'4°, in long-term depression in the cerebellum 43, in spinal nociceptive mechanisms 33'37 and in neurotoxicity 13't9. Several immunocytochemical studies have shown that nitric oxide synthase (NOS) is selectively associated with neuronal elements in many areas of the brain and periphery l°'H't8, although its precise distribution in most areas has not yet been studied in detail. Ultrastructural studies have determined that, within enteric neurons of the guinea-pig small intestine, NOS-immunoreactivity is distributed in a patchy fashion within the cytoplasm and is not found either in synaptic vesicles or in association with the plasma membrane 34. There have been no extensive ultrastructural studies of NOS in the central nervous system to date but light
microscopic studies suggest that NOS is found in neuronal cell bodies, dendrites and axons, where it appears to be colocalized with NADPH-diaphorase 1°. The distribution of NOS within the rodent midbrain in general and in the periaqueductal gray in particular has not been described in any detail and this forms the basis of the present study. The midbrain periaqueductal gray ( P A G ) h a s been shown to play a role in pain modulation, respiratory and cardiovascular control, defensive reactions, vocalization and female reproductive behavior 3'4'7 and has recently been postulated to be involved in integrating these various sensory, motor and autonomic roles in response to changes in the environment or to stressful and potentially threatening stimuli 2'4'7'44. Furthermore, the results of anatomical tract-tracing experiments, as well as studies indicating that many of the functions of the P A G show site specificity, have suggested that the P A G is organized into longitudinal columns of various lengths which extend along the rostrocaudal axis of the P A G 2'7'44. It has been further proposed that this struc-
Correspondence: A.J. Beitz, Department of Veterinary PathoBiology,Universityof Minnesota, Room 295, Animal Sci./Vet. Medicine Bldg., 1988 Fitch Avenue, St. Paul, MN 55108, USA. Fax: (1) (612) 625-0204.
318 tural arrangement may underlie the functional organization of this region and may serve to coordinate and integrate various functional resoonses to stressful stim-
uli or to environmental changes 2'3'7'44. Thus the entire rostrocaudal extent of the PAG must be considered in investigations of its structural or functional characteris-
Fig. 1. Low-magnification fluorescence confocal images of NOS immunoreactivity in the PAG. A: at the level of the posterior commissure NOS-IR cells and processes are visible lateral to the aqueduct (AQ) in a juxta-aqueductal position (the area indicated by the rectangle is shown at higher magnification in Fig. 3A). A few cells (arrows) are also evident at the dorsolateral boundary of the P A G and represent the rostralmost extent of the dorsolateral column. B: at a more caudal level of the rostral PAG, the dorsolateral group of cells has become more numerous and has expanded ventrally and medially. NOS-immunoreactivity is now also visible in the juxta-aqueductal region along the dorsal edge of the aqueduct (the area indicated by the rectangle is shown at higher magnification in Fig. 3B) Aq, mesencephalic aqueduct. C,D: proceeding caudally through the middle one-third of the PAG, the dorsolateral population of cells continues its expansion toward the aqueduct. The four rectangles indicated in D are illustrated at higher magnification in Fig. 2. E: at caudal levels, beginning in the peripheral and ventralmost areas of the dorsolateral PAG, the number of immunostained cells becomes reduced. Many labeled cells can be seen in the dorsal raphe nucleus (DR) at this level. F: as the aqueduct opens into the fourth ventricle (4V), only light immunostaining is evident in the P A G dorsolateral to the ventricle at this low magnification. However, some neurons are still present in this dorsolateral column at this caudal level as illustrated in Fig. 3D which represents a higher-magnification photomicrograph of the area designated by the box. Many intensely stained NOS-IR cells are present in the dorsolateral tegmental nucleus (DLT) at this caudalmost level of the PAG. Bar = 500/xm.
319 tics. As part of our efforts to elucidate the neurochemical organization of this midbrain region, we have examined the distribution of nitric oxide synthase iramunoreactivity ( N O S - I R ) along the longitudinal extent of the P A G in the rat using laser confocal microscopy, Seven adult, male S p r a g u e - D a w l e y rats were deeply anesthetized with chloral hydrate and perfused transcardially with 120 ml ice-cold calcium-free Tyrode's solution followed by 500 ml 4% paraformaldehyde in 0.1 M S0rensen's phosphate buffer. The brains were removed and postfixed for 6 h in 4% paraformaldehyde. Transverse 50 Ixm vibratome sections of the midbrain were cut and stored overnight in PBS at 4°C. After preincubation at room t e m p e r a t u r e for 1 h in PBS containing 1% Triton X-100 (PBS-T) and 2% normal donkey serum, sections were incubated for 24 h at 4°C in rabbit anti-NOS (see K u m m e r et al. 32 for characterization) diluted 1 : 500 to 1 : 6,000 in the same buffer. Sections were then processed for immunofluorescence by first rinsing in PBS for 8 h at room temperature and then incubating overnight in Cy5-1abeled
, ,t ~
donkey anti-rabbit I g G (Jackson ImmunoResearch, West Grove, PA) diluted 1 : 50 in PBS-T. Sections were rinsed as previously described, mounted onto gel-coated slides and coverslipped in fade-retarding medium. Sections were examined with a laser scanning confocal microscope (BioRad MRC-600 equipped with an arg o n / k r y p t o n laser). The overall pattern of NOS immunostaining in the P A G was determined by collecting a single, low magnification image, approximately 10 /zm thick, at the level of the strongest immunoreactivity in each section. Details of immunostaining in areas of interest were then determined by collecting a higher magnification series of either 2 or 0.8/~m thick optical sections, at 2.0 or 1.0 /zm intervals, respectively, through the entire 50 ~ m thickness of the sections. Alternatively, some NOS-treated sections were processed for avidin-biotin-peroxidase labeling by rinsing in PBS, incubating in biotinylated goat anti-rabbit I g G (Vector Laboratories, Burlingame, CA; diluted ! :3 in PBS-T) for 1 h, rinsing in PBS and incubating in avidin-biotin-peroxidase complex. Sections were then
,~
D
Fig. 2. Higher-magnification fluorescence confocal images of areas outlined in Fig. 1D. Projections of 18 (A-C) or 9 (D) consecutive images collected at 1 /zm or 2 Izm intervals, respectively. A,B: dorsolateral columns: many brightly immunostained somas of varying morphologywith their proximal processes (arrows) are seen. At this magnification, a plexus of fine immunoreactive fibers is also visible (arrowheads). C: ventrolateral region: only a few fibers and very lightly stained somas are visible. D: dorsomedial column: except for a moderate number of fibers immediately dorsal to the aqueduct, only a limited number of immunoreactivecells and fibers are present in this area. Bars = 50 tzm.
320 r e a c t e d with d i a m i n o b e n z i d i n e as previously desc r i b e d 36 a n d v i e w e d u n d e r b r i g h t - f i e l d microscopy, T h e most r e m a r k a b l e o b s e r v a t i o n in N O S - i m m u n o t r e a t e d sections was the localized collection of N O S - p o s i t i v e n e u r o n s in t h e d o r s o l a t e r a l P A G along its r o s t r o c a u d a l extent (Fig. 1). A l t h o u g h a few imm u n o r e a c t i v e n e u r o n s w e r e p r e s e n t in o t h e r P A G regions, the majority of N O S i m m u n o s t a i n e d cells were c o n c e n t r a t e d in a l o n g i t u d i n a l c o l u m n localized in the d o r s o l a t e r a l P A G . A t r o s t r a l m o s t m i d b r a i n levels this d o r s o l a t e r a l c o l u m n was r e p r e s e n t e d by a few imm u n o s t a i n e d cells localized at the l a t e r a l e d g e of the dorsal P A G (Fig. 1A, arrows). M o v i n g in a c a u d a l d i r e c t i o n t h r o u g h the P A G , this d o r s o l a t e r a l p o p u l a tion of cells i n c r e a s e d in n u m b e r a n d e x p a n d e d ventrally a n d m e d i a l l y t o w a r d t h e a q u e d u c t , r e a c h i n g its m a x i m u m extent in the m i d d l e t h i r d o f the P A G (Fig. 1 B - D ) . I m m u n o s t a i n e d cells w e r e not m o r p h o l o g i c a l l y
u n i f o r m a n d i n c l u d e d b o t h large a n d small b i p o l a r and m u l t i p o l a r cells (Fig. 2A,B). M o s t N O S - I R cells in this d o r s o l a t e r a l p o p u l a t i o n were highly fluorescent, but s o m e e x h i b i t e d a w e a k immunoreactivity. A large n u m b e r of i m m u n o r e a c t i v e fibers a n d d e n d r i t e s of varying d i a m e t e r s was also p r e s e n t in the d o r s o l a t e r a l P A G (Fig. 3C) at m i d - P A G levels. A t c a u d a l levels (Fig. 1E), the a r e a of t h e d o r s o l a t e r a l c o l u m n o c c u p i e d by N O S I R cells b e c a m e r e d u c e d , b e g i n n i n g at its d o r s o l a t e r a l a n d ventral b o r d e r s . In t h e c a u d a l m o s t P A G (Fig. IF), only a r e m n a n t of this N O S positive n e u r o n a l p o p u l a tion was p r e s e n t b e i n g l o c a t e d in a p o s i t i o n i m m e d i ately d o r s o l a t e r a l to the a q u e d u c t . H o w e v e r , fine imm u n o r e a c t i v e fibers a n d weakly s t a i n e d p e r i k a r y a w e r e o b s e r v e d m o r e p e r i p h e r a l l y in the d o r s o l a t e r a l P A G at this level (Fig. 3D). T h e p a t t e r n of i m m u n o s t a i n i n g o b s e r v e d in t h e d o r s o l a t e r a l c o l u m n was identical in b o t h i m m u n o f l u o r e s c e n t a n d avidin-biotin p e r o x i d a s e
Fig. 3. Higher-magnification fluorescence confocal images of NOS immunoreactivity in selected regions of the PAG. Projections constructed as in Fig. 2. A: the area designated by the rectangle in Fig. 1A: in addition to a few neuronal somas, fine NOS-IR fibers can now also be seen in the area immediately lateral to the aqueduct (arrows) and NOS immunoreactive punctate staining (arrowheads) also appears to be present in ependymal cells. B: the area designated by the rectangle in Fig. 1B: NOS-IR fibers can be seen coursing along the dorsal portion of the aqueduct and a plexus of fine fibers is visible in the adjacent neuropile (arrowheads). C: the area designated by the rectangle in Fig. 1C: many coarse immunoreactive processes (arrowheads) are visible dorsolateral to the aqueduct. D: the area designated by the rectangle in Fig. IF: a few NOS-IR cells (arrows) and fibers are still present in this caudal remnant of the dorsolateral column. Some NOS positive cells are present in a juxta-aqueductal location in the dorsolateral PAG (arrowheads). In addition, several large highly immunoreactive cells are visible just outside the PAG in the central nucleus of the inferior colliculus (IC). Bars = 5(I tzm.
321 preparations. However, the use of the laser confocal microscope in conjunction with Cy5-1abeled secondary antibodies allowed greater resolution of immunolabeled fibers and processes and provided a more threedimensional appearance to the immunostaining, Outside of the dorsolateral column a few NOS-IR neurons were found scattered throughout the PAG, but the majority of NOS-positive structures present in the ventral, lateral, dorsomedial and juxta-aqueductal PAG columns consisted of scattered fibers and long neuronal dendrites. At rostral PAG levels, for example, a few immunostained neurons were observed along the lateral edge of the aqueduct in the juxta-aqueductal region (Fig. 1A). At higher magnification (Fig. 3A), these neurons can be seen to better advantage and several immunoreactive fibers and varicosities can also be observed (arrows). In addition punctate immunofluorescence was present within the ependymal cells that line the aqueduct at this level (Fig. 3A, arrowheads). At a somewhat more caudal level (Fig. 1B), NOS-IR fibers were especially visible in the dorsal juxta-aqueductal area (Fig. 3B). In general throughout most of the rostral-caudal extent of the PAG (Fig. 1B-E), many fine immunostained fibers (Figs. 2D,3B) were found in this area surrounding the aqueduct, Within the rostral one-third of the PAG some weakly immunostained cells and fibers were present in the areas ventral and ventrolateral to the aqueduct. However, in the caudal two-thirds of the PAG little iramunostaining was observed in the ventral half of the PAG, especially at its more peripheral portions (Fig. 2C). Throughout the rostrocaudal extent of the PAG the dorsomedial column displayed very little immunoreactivity. A few weakly stained neurons were present in this column (2D, arrows), especially at midPAG levels, and a small number of immunoreactive dendrites and fibers extended into this region from the dorsolateral columns (2D, arrowheads), but generally immunoreactivity was low in this region. In addition to the immunostaining observed in the PAG proper, a large number of brightly immunostained cells were observed in the dorsal raphe nucleus and the dorsolateral tegmental nuclei, which are found embedded in the ventral PAG (Fig. 1E-F). In the past, several schemes for subdividing the PAG have been proposed based upon cytoarchitectonics 6'7'27 and upon its patterns of connectivity5. Recent studies of afferent ~nd efferent projections 44'45'47 as well as functional studies 15'16'35, have led to the proposal that the PAG is organized into several longitudihal neuronal c o l u m n s T M . Thus dorsomedial, dorsolateral, lateral, ventrolateral and juxta-aqueductal columns have been proposed as the structural frame-
work which underlies PAG function2'7. Examination of a series of transverse sections through the rostro-caudal extent of the PAG (Fig. 1) certainly indicates that the large numbers of NOS-IR cells and fibers present in the dorsolateral PAG form a wedge-shaped longitudihal column. It is noteworthy that nicotinamide dinucleotide phosphate diaphorase (NADPH-d) positive cells have also recently been described in this dorsolateral region of the PAG 24. This NOS immunostained, NADPH-d positive column, appears to correspond to the dorsolateral column proposed by Bandler and colleagues 2. Moreover, the juxta-aqueductal column can be defined in the present study by the presence of a moderate level of NOS-IR fibers and processes throughout its rostrocaudal extent. On the other hand, the dorsomedial column is defined in the present material by the very low level of NOS immunoreactivity in comparison to the adjacent dorsolateral columns which are heavily labeled (Fig. 1). Our finding of a NOS-defined dorsolateral column, which mimics the anatomical location of the functional column defined by Bandler and coworkers 2, is not the first example of chemical specificity within PAG columns. Previous studies have shown that this dorsolateral region can also be defined by the presence of muscarinic cholinergic receptors 48, excitatory amino acid receptors 1, opiate receptors 29 and substance P immunostaining (see Beitz 7 for review). In addition this region contains a concentration of GABA-immunoreactive ceils 41, somatostatin immunoreactive fibers z2, and a higher density of GABAA/benzodiazepine 25, and neurotensin 39'49 binding sites than do other columns of the PAG. Moreover a review of the literature suggests that noradrenergic and adrenergic fibers are predominantly localized to the lateral and ventrolateral columns7'28, while LHRH- and ACTH-containing fibers are concentrated in the juxta-aqueductal column 47. Although these are but a few examples of an increasing number of neurochemicals that appear to be selectively associated with PAG columns, this selective chemical localization within the PAG certainly provides biochemical support for the existence of longitudinal columns in this region In the cerebellum 12 and hippocampus 23 NO has been shown to act as a messenger that mediates the stimulation of cGMP formation by glutamate acting at the NMDA receptor. While glutamate immunoreactive neurons and fibers are present throughout the PAG 8 the dorsolateral PAG has been found to contain the greatest density of binding sites for several excitatory amino acid agonists, including NMDA ~. The dorsolateral PAG also receives selective inputs from several areas including the cingulate cortex 45, the ventrome-
322 dial hypothalamic nucleus 5'47, the posterior hypothalamic nucleus 5, the cuneiform nucleus 5, the anterior pretectal nucleus 5 and the zona incerta 5, all of which provide major sources of excitatory amino acid inputs to the PAG 8. Thus, it is possible that the production of NO in the dorsolateral PAG involves excitatory amino acid stimulation of N M D A receptors, subsequent calcium-dependent activation of NOS, NO production and ultimately activation of guanylate cyclase and cGMP formation as has been described for other neural systems. In the cerebellum, for example, where this physiological cascade was first described, the enhancement of cGMP formation by glutamate is particularly strong ~2. However, it should be noted that NOS immunoreactivity occurs within the brain in many areas that do not demonstrate high levels of guanylate cyclase ~°, suggesting that additional mechanisms of action are likely to be discovered, In order to examine the role of NO as a potential neuronal messenger in the PAG, it will be important to define the potential colocalization of NOS with neuropeptides and neurotransmitters in this region as well as to study the potential relationship of excitatory amino acid-containing axons and terminals to NOS positive neurons. NOS is known to coexist with peptides such as neuropeptide Y and somatostatin in the corpus striatum and cerebral cortex ~s and with VIP and acetylcholine in neurons innervating cerebral arteries 32, raising the question of whether NO may act in conjunction with other substances. The questions of whether these or other neuromodulatory substances coexist with NOS and of the relationships among NOSIR perikarya, fibers and terminals and neuronal structures identified according to their chemical coding a n d / o r their projections to or from the PAG remain to be investigated. Several neuronal roles for NO have been postulated. Immunohistochemical and pharmacological studies have indicated that NO mediates non-adrenergic, non-cholinergic transmission from enteric inhibitory motor neurons and between n e u r o n s in the enteric and m a y participate in the regulation of hormone release ~. Several studies have indicated that
g a n g l i a 14'2°
NO may serve as a mediator of long-term depression in the cerebellum and may function presynaptically to maintain long-term potentiation in the hippocamRecent studies have also indicated that NO plays a role in nociceptive mechanisms at the level of the spinal cord. Thus Meller et al. 37 have shown that NO mediates the thermal hyperalgesia produced under conditions of neuropathic pain, while our laboratory p u s 9'40'42'43.
has shown that NO mediates the spinal cord expression of Fos induced by noxious mechanical stimulation 33.
The specific role of NO in the PAG, however, remains to be defined. The PAG is known to play a role in pain modulation, respiratory and cardiovascular control, defensive reactions, vocalization and female reproductive behavior 7'15-~7'21'35 and has recently been postulated to be involved in integrating these various sensory, motor and autonomic roles in response to stressful and potentially threatening stimuli 2'4'44"45. Although relatively littie is known about the specific functions of the dorsolateral PAG column, we have recently demonstrated that this column is specifically activated by linear acceleration 31. This region is known to receive spatial orientation inputs from the superior colliculus, the substantia nigra pars reticulata and prepositus hypoglossi 3° and thus the dorsolateral column may help control appropriate responses to novel environments or situations, including autonomic and somatomotor responses to changes in gravity or acceleration 7'3°'3~. Whether NO plays a role in the dorsolateral PAG's involvement in adaptation to novel environments requires further investigation. This work is supported by NIH Grants DA06687, DC01086 and DE06682 to AJB and Fonds zur F6rderung der Wissenschaftlichen Forschung in Osterreich to BM. We thank Richard Price for his technical assistance during this study and Todd Brelje for his assistance with the laser confocal microscope.
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30 Holstege, G. and Cowie, R.J., Nucleus prepositus hypoglossi projects to the dorsolateral PAG: a link between visuomotor and limbic systems, Soc. Neurosci. Abstr., 16, (1990) 303.3. 31 Kaufman, G.D., Anderson, J.H. and Beitz, A.J., Fos-defined activity in rat brainstem following centripetal acceleration. J. Neurosci., 12 (1992) 4489-4500. 32 Kummer, W., Fischer, A., Mundel, P., Mayer, B., Hoba, B., Philippin, B. and Preissler, U., Nitric oxide synthase in VIP-containing cholinergic vasodilator nerves, Neuroreport, 3 (1992) 653657. 33 Lee, J.-H., Wilcox, G.L. and Beitz, A.J., Nitric oxide mediates Fos expression in the spinal cord induced by mechanical noxious stimulation, Neuroreport, 3 (1992) 841-844. 34 Llewellyn-Smith, I.J., Song, Z.-M., Costa, M., Bredt, D.S. and Snyder, S.H., Ultrastructural localization of nitric oxide synthase immunoreactivity in guinea-pig enteric neurons, Brain Res., 577 (1992) 337-342. 35 Lovick, T.A., Interactions between descending pathways from the dorsal and ventrolateral periaqueductal gray matter in the rat. In A. Depaulis and R. Bandler (Eds.), The Midbrain Periaqueductal Gray Matter: Functional, Anatomical and Neurochemical Organization, Plenum, New York, 1991, pp. 101-120. 36 Magnusson, K.R., Madl, J.E., Wu, J.Y., Larson, A.A. and Beitz, A.J., Colocalization of taurine- and cysteine sulfinic acid decarboxylase-like immunoreactivity in the cerebellum of the rat with the use of monoclonal antibodies against taurine. J. Neurosci., 8 (1988) 4551-4564. 37 Meller, S.T., Pechman, P.S., Gebhart, G.F. and Maves, T.J., Nitric oxide mediates the thermal hyperalgesia produced in a model of neuropathic pain in the rat, Neuroscience, 50 (1992) 7-10. 38 Morgan, M.M. and Liebeskind, J.C., Site specificity in the development of tolerance to stimulation-produced analgesia from the periaqueductal gray matter of the rat, Brain Res., 425 (1987) 356-359. 39 Moyse, E., Rostene, W., Vial, M., Leonard, K., Mazella, J., Kitabgi, P., Vincent, J.-P. and Beaudet, A. Distribution of neurotensin binding sites in rat brain: a light microscopic radioautographic study using monoiodo [125I]Tyr3-neurotensin, Neuroscience, 22 (1987) 525-536. 40 O'Dell, I.J., Hawkins, R.D., Kandel, E.R. and Arancio, O., Tests on the roles of two diffusable substances in LTP: evidence for nitric oxide as a possible early retrograde messenger, Proc. Natl. Acad. Sci., 88 (1991) 11285-11289. 41 Reichling, D.B., Kwiat, G.C. and Basbaum, A.I., Anatomy, physiology and pharmacology of the periaqueductal gray contribution to antinociceptive controls. In H.L. Fields and J.M. Besson (Eds.), Progress in Brain Research, Vol. 77, Elsevier, Amsterdam, 1988, pp. 31-46. 42 Schuman, E.M. and Madison, D.V., A requirement for the intercellular messenger nitric oxide in long-term potentiation, Science, 254 (1991) 1503-1506. 43 Shibuki, K. and Okada, D., Endogenous nitric oxide release required for long-term synaptic depression in the cerebellum, Nature, 349 (1991) 326-328. 44 Shipley, M.T., Bandler, R., Beitz, A.J., Lovick, T. and Fields. H., Emerging principles of organization within the midbrain periaqueductal gray matter, Soc. Neurosci. Abstr., 18 (1992) 1, No. 2. 45 Shipley, M.T., Ennis, M., Rizvi, T.A. and Behbehani, M.M., Topographical specificity of forebrain inputs to the midbrain periaqueductal gray: evidence for discrete longitudinally organized input columns. In A. Depaulis and R. Bandler (Eds.), The Midbrain Periaqueductal Gray Matter." Functional, Anatomical and Neurochemical Organization Plenum Press, New York, 1991, pp. 417-448. 46 Snyder, S.H,. Nitric Oxide: first in a new class of neurotransmitters, Science, 257 (1992) 494-496. 47 Veening, J., Buma, P., Ter Horst, G.J., Roeling, T.A.P., Luiten, P.G.M. and Nieuwenhuys, R., Hypothalamic projections to the PAG in the rat: topographical, immuno- electronmicroscopical and functional aspects. In A. Depaulis and R. Bandler (Eds.),
324 The Midbrain Periaqueductal Gray Matter: Functional Anatomical and Neurochemical Organization, Plenum Press, New York, 1991, pp. 387-415. 48 Wamsley, J.K., Lewis, M.S., Young lII, W.S. and Kuhar, M.J., Autoradiographic localization of muscarinic cholinergic receptors in rat brainstem, J. Neurosci., 1 (1981) 176-191.
49 Young, W.S, and Kuhar, M.J., Neurotensin receptors: Autoradiographic localization in rat CNS, Eur. J. PharmacoL, 59 (1979) 161-163.
317
BRES 25622
Nitric oxide synthase immunoreactive neurons anatomically define a longitudinal dorsolateral column within the midbrain periaqueductal gray of the rat: analysis using laser confocal microscopy Donna Onstott a Bernd Mayer b and Alvin J. Beitz a a Department of Veterinary PathoBiology, University of Minnesota, St. Paul, MN 55108 (USA) and blnstitute for Pharmacology and Toxicology, University of Graz, Graz (Austria)
(Accepted 19 January 1993)
Key words: Nitric oxide; Central gray; Brainstem; Longitudinal column; Antinociception
Nitric oxide has recently been proposed as a neuronal messenger in both the central and peripheral nervous system. Antibodies against nitric oxide synthase (NOS), the synthesizing enzyme for nitric oxide, were used in combination with immunocytochemistryand confocal laser microscopyto analyze the distribution of this enzyme in the midbrain periaqueductal gray (PAG) of the rat. NOS immunoreactive neurons were localized predominantly in a longitudinally oriented column in the dorsolateral PAG. NOS immunoreactive fibers and processes were scattered throughout the PAG but were most prevalent in the dorsolateral column and in the juxta-aqueductal column. This study provides neurochemical support for the existence of longitudinal columns in the PAG which are postulated to underlie the functional organization of this complex brainstem region.
Nitric oxide (NO) has recently been shown to act as a neuronal messenger t3'46. It appears to mediate transmission from autonomic neurons to smooth muscle in the intestine, to cerebral arteries and to arteries to the penis, as well as between neurons within enteric ganglia 46. Within the central nervous system N O has been implicated in long-term potentiation 9'26'4°, in long-term depression in the cerebellum 43, in spinal nociceptive mechanisms 33'37 and in neurotoxicity 13't9. Several immunocytochemical studies have shown that nitric oxide synthase (NOS) is selectively associated with neuronal elements in many areas of the brain and periphery l°'H't8, although its precise distribution in most areas has not yet been studied in detail. Ultrastructural studies have determined that, within enteric neurons of the guinea-pig small intestine, NOS-immunoreactivity is distributed in a patchy fashion within the cytoplasm and is not found either in synaptic vesicles or in association with the plasma membrane 34. There have been no extensive ultrastructural studies of NOS in the central nervous system to date but light
microscopic studies suggest that NOS is found in neuronal cell bodies, dendrites and axons, where it appears to be colocalized with NADPH-diaphorase 1°. The distribution of NOS within the rodent midbrain in general and in the periaqueductal gray in particular has not been described in any detail and this forms the basis of the present study. The midbrain periaqueductal gray ( P A G ) h a s been shown to play a role in pain modulation, respiratory and cardiovascular control, defensive reactions, vocalization and female reproductive behavior 3'4'7 and has recently been postulated to be involved in integrating these various sensory, motor and autonomic roles in response to changes in the environment or to stressful and potentially threatening stimuli 2'4'7'44. Furthermore, the results of anatomical tract-tracing experiments, as well as studies indicating that many of the functions of the P A G show site specificity, have suggested that the P A G is organized into longitudinal columns of various lengths which extend along the rostrocaudal axis of the P A G 2'7'44. It has been further proposed that this struc-
Correspondence: A.J. Beitz, Department of Veterinary PathoBiology,Universityof Minnesota, Room 295, Animal Sci./Vet. Medicine Bldg., 1988 Fitch Avenue, St. Paul, MN 55108, USA. Fax: (1) (612) 625-0204.
318 tural arrangement may underlie the functional organization of this region and may serve to coordinate and integrate various functional resoonses to stressful stim-
uli or to environmental changes 2'3'7'44. Thus the entire rostrocaudal extent of the PAG must be considered in investigations of its structural or functional characteris-
Fig. 1. Low-magnification fluorescence confocal images of NOS immunoreactivity in the PAG. A: at the level of the posterior commissure NOS-IR cells and processes are visible lateral to the aqueduct (AQ) in a juxta-aqueductal position (the area indicated by the rectangle is shown at higher magnification in Fig. 3A). A few cells (arrows) are also evident at the dorsolateral boundary of the P A G and represent the rostralmost extent of the dorsolateral column. B: at a more caudal level of the rostral PAG, the dorsolateral group of cells has become more numerous and has expanded ventrally and medially. NOS-immunoreactivity is now also visible in the juxta-aqueductal region along the dorsal edge of the aqueduct (the area indicated by the rectangle is shown at higher magnification in Fig. 3B) Aq, mesencephalic aqueduct. C,D: proceeding caudally through the middle one-third of the PAG, the dorsolateral population of cells continues its expansion toward the aqueduct. The four rectangles indicated in D are illustrated at higher magnification in Fig. 2. E: at caudal levels, beginning in the peripheral and ventralmost areas of the dorsolateral PAG, the number of immunostained cells becomes reduced. Many labeled cells can be seen in the dorsal raphe nucleus (DR) at this level. F: as the aqueduct opens into the fourth ventricle (4V), only light immunostaining is evident in the P A G dorsolateral to the ventricle at this low magnification. However, some neurons are still present in this dorsolateral column at this caudal level as illustrated in Fig. 3D which represents a higher-magnification photomicrograph of the area designated by the box. Many intensely stained NOS-IR cells are present in the dorsolateral tegmental nucleus (DLT) at this caudalmost level of the PAG. Bar = 500/xm.
319 tics. As part of our efforts to elucidate the neurochemical organization of this midbrain region, we have examined the distribution of nitric oxide synthase iramunoreactivity ( N O S - I R ) along the longitudinal extent of the P A G in the rat using laser confocal microscopy, Seven adult, male S p r a g u e - D a w l e y rats were deeply anesthetized with chloral hydrate and perfused transcardially with 120 ml ice-cold calcium-free Tyrode's solution followed by 500 ml 4% paraformaldehyde in 0.1 M S0rensen's phosphate buffer. The brains were removed and postfixed for 6 h in 4% paraformaldehyde. Transverse 50 Ixm vibratome sections of the midbrain were cut and stored overnight in PBS at 4°C. After preincubation at room t e m p e r a t u r e for 1 h in PBS containing 1% Triton X-100 (PBS-T) and 2% normal donkey serum, sections were incubated for 24 h at 4°C in rabbit anti-NOS (see K u m m e r et al. 32 for characterization) diluted 1 : 500 to 1 : 6,000 in the same buffer. Sections were then processed for immunofluorescence by first rinsing in PBS for 8 h at room temperature and then incubating overnight in Cy5-1abeled
, ,t ~
donkey anti-rabbit I g G (Jackson ImmunoResearch, West Grove, PA) diluted 1 : 50 in PBS-T. Sections were rinsed as previously described, mounted onto gel-coated slides and coverslipped in fade-retarding medium. Sections were examined with a laser scanning confocal microscope (BioRad MRC-600 equipped with an arg o n / k r y p t o n laser). The overall pattern of NOS immunostaining in the P A G was determined by collecting a single, low magnification image, approximately 10 /zm thick, at the level of the strongest immunoreactivity in each section. Details of immunostaining in areas of interest were then determined by collecting a higher magnification series of either 2 or 0.8/~m thick optical sections, at 2.0 or 1.0 /zm intervals, respectively, through the entire 50 ~ m thickness of the sections. Alternatively, some NOS-treated sections were processed for avidin-biotin-peroxidase labeling by rinsing in PBS, incubating in biotinylated goat anti-rabbit I g G (Vector Laboratories, Burlingame, CA; diluted ! :3 in PBS-T) for 1 h, rinsing in PBS and incubating in avidin-biotin-peroxidase complex. Sections were then
,~
D
Fig. 2. Higher-magnification fluorescence confocal images of areas outlined in Fig. 1D. Projections of 18 (A-C) or 9 (D) consecutive images collected at 1 /zm or 2 Izm intervals, respectively. A,B: dorsolateral columns: many brightly immunostained somas of varying morphologywith their proximal processes (arrows) are seen. At this magnification, a plexus of fine immunoreactive fibers is also visible (arrowheads). C: ventrolateral region: only a few fibers and very lightly stained somas are visible. D: dorsomedial column: except for a moderate number of fibers immediately dorsal to the aqueduct, only a limited number of immunoreactivecells and fibers are present in this area. Bars = 50 tzm.
320 r e a c t e d with d i a m i n o b e n z i d i n e as previously desc r i b e d 36 a n d v i e w e d u n d e r b r i g h t - f i e l d microscopy, T h e most r e m a r k a b l e o b s e r v a t i o n in N O S - i m m u n o t r e a t e d sections was the localized collection of N O S - p o s i t i v e n e u r o n s in t h e d o r s o l a t e r a l P A G along its r o s t r o c a u d a l extent (Fig. 1). A l t h o u g h a few imm u n o r e a c t i v e n e u r o n s w e r e p r e s e n t in o t h e r P A G regions, the majority of N O S i m m u n o s t a i n e d cells were c o n c e n t r a t e d in a l o n g i t u d i n a l c o l u m n localized in the d o r s o l a t e r a l P A G . A t r o s t r a l m o s t m i d b r a i n levels this d o r s o l a t e r a l c o l u m n was r e p r e s e n t e d by a few imm u n o s t a i n e d cells localized at the l a t e r a l e d g e of the dorsal P A G (Fig. 1A, arrows). M o v i n g in a c a u d a l d i r e c t i o n t h r o u g h the P A G , this d o r s o l a t e r a l p o p u l a tion of cells i n c r e a s e d in n u m b e r a n d e x p a n d e d ventrally a n d m e d i a l l y t o w a r d t h e a q u e d u c t , r e a c h i n g its m a x i m u m extent in the m i d d l e t h i r d o f the P A G (Fig. 1 B - D ) . I m m u n o s t a i n e d cells w e r e not m o r p h o l o g i c a l l y
u n i f o r m a n d i n c l u d e d b o t h large a n d small b i p o l a r and m u l t i p o l a r cells (Fig. 2A,B). M o s t N O S - I R cells in this d o r s o l a t e r a l p o p u l a t i o n were highly fluorescent, but s o m e e x h i b i t e d a w e a k immunoreactivity. A large n u m b e r of i m m u n o r e a c t i v e fibers a n d d e n d r i t e s of varying d i a m e t e r s was also p r e s e n t in the d o r s o l a t e r a l P A G (Fig. 3C) at m i d - P A G levels. A t c a u d a l levels (Fig. 1E), the a r e a of t h e d o r s o l a t e r a l c o l u m n o c c u p i e d by N O S I R cells b e c a m e r e d u c e d , b e g i n n i n g at its d o r s o l a t e r a l a n d ventral b o r d e r s . In t h e c a u d a l m o s t P A G (Fig. IF), only a r e m n a n t of this N O S positive n e u r o n a l p o p u l a tion was p r e s e n t b e i n g l o c a t e d in a p o s i t i o n i m m e d i ately d o r s o l a t e r a l to the a q u e d u c t . H o w e v e r , fine imm u n o r e a c t i v e fibers a n d weakly s t a i n e d p e r i k a r y a w e r e o b s e r v e d m o r e p e r i p h e r a l l y in the d o r s o l a t e r a l P A G at this level (Fig. 3D). T h e p a t t e r n of i m m u n o s t a i n i n g o b s e r v e d in t h e d o r s o l a t e r a l c o l u m n was identical in b o t h i m m u n o f l u o r e s c e n t a n d avidin-biotin p e r o x i d a s e
Fig. 3. Higher-magnification fluorescence confocal images of NOS immunoreactivity in selected regions of the PAG. Projections constructed as in Fig. 2. A: the area designated by the rectangle in Fig. 1A: in addition to a few neuronal somas, fine NOS-IR fibers can now also be seen in the area immediately lateral to the aqueduct (arrows) and NOS immunoreactive punctate staining (arrowheads) also appears to be present in ependymal cells. B: the area designated by the rectangle in Fig. 1B: NOS-IR fibers can be seen coursing along the dorsal portion of the aqueduct and a plexus of fine fibers is visible in the adjacent neuropile (arrowheads). C: the area designated by the rectangle in Fig. 1C: many coarse immunoreactive processes (arrowheads) are visible dorsolateral to the aqueduct. D: the area designated by the rectangle in Fig. IF: a few NOS-IR cells (arrows) and fibers are still present in this caudal remnant of the dorsolateral column. Some NOS positive cells are present in a juxta-aqueductal location in the dorsolateral PAG (arrowheads). In addition, several large highly immunoreactive cells are visible just outside the PAG in the central nucleus of the inferior colliculus (IC). Bars = 5(I tzm.
321 preparations. However, the use of the laser confocal microscope in conjunction with Cy5-1abeled secondary antibodies allowed greater resolution of immunolabeled fibers and processes and provided a more threedimensional appearance to the immunostaining, Outside of the dorsolateral column a few NOS-IR neurons were found scattered throughout the PAG, but the majority of NOS-positive structures present in the ventral, lateral, dorsomedial and juxta-aqueductal PAG columns consisted of scattered fibers and long neuronal dendrites. At rostral PAG levels, for example, a few immunostained neurons were observed along the lateral edge of the aqueduct in the juxta-aqueductal region (Fig. 1A). At higher magnification (Fig. 3A), these neurons can be seen to better advantage and several immunoreactive fibers and varicosities can also be observed (arrows). In addition punctate immunofluorescence was present within the ependymal cells that line the aqueduct at this level (Fig. 3A, arrowheads). At a somewhat more caudal level (Fig. 1B), NOS-IR fibers were especially visible in the dorsal juxta-aqueductal area (Fig. 3B). In general throughout most of the rostral-caudal extent of the PAG (Fig. 1B-E), many fine immunostained fibers (Figs. 2D,3B) were found in this area surrounding the aqueduct, Within the rostral one-third of the PAG some weakly immunostained cells and fibers were present in the areas ventral and ventrolateral to the aqueduct. However, in the caudal two-thirds of the PAG little iramunostaining was observed in the ventral half of the PAG, especially at its more peripheral portions (Fig. 2C). Throughout the rostrocaudal extent of the PAG the dorsomedial column displayed very little immunoreactivity. A few weakly stained neurons were present in this column (2D, arrows), especially at midPAG levels, and a small number of immunoreactive dendrites and fibers extended into this region from the dorsolateral columns (2D, arrowheads), but generally immunoreactivity was low in this region. In addition to the immunostaining observed in the PAG proper, a large number of brightly immunostained cells were observed in the dorsal raphe nucleus and the dorsolateral tegmental nuclei, which are found embedded in the ventral PAG (Fig. 1E-F). In the past, several schemes for subdividing the PAG have been proposed based upon cytoarchitectonics 6'7'27 and upon its patterns of connectivity5. Recent studies of afferent ~nd efferent projections 44'45'47 as well as functional studies 15'16'35, have led to the proposal that the PAG is organized into several longitudihal neuronal c o l u m n s T M . Thus dorsomedial, dorsolateral, lateral, ventrolateral and juxta-aqueductal columns have been proposed as the structural frame-
work which underlies PAG function2'7. Examination of a series of transverse sections through the rostro-caudal extent of the PAG (Fig. 1) certainly indicates that the large numbers of NOS-IR cells and fibers present in the dorsolateral PAG form a wedge-shaped longitudihal column. It is noteworthy that nicotinamide dinucleotide phosphate diaphorase (NADPH-d) positive cells have also recently been described in this dorsolateral region of the PAG 24. This NOS immunostained, NADPH-d positive column, appears to correspond to the dorsolateral column proposed by Bandler and colleagues 2. Moreover, the juxta-aqueductal column can be defined in the present study by the presence of a moderate level of NOS-IR fibers and processes throughout its rostrocaudal extent. On the other hand, the dorsomedial column is defined in the present material by the very low level of NOS immunoreactivity in comparison to the adjacent dorsolateral columns which are heavily labeled (Fig. 1). Our finding of a NOS-defined dorsolateral column, which mimics the anatomical location of the functional column defined by Bandler and coworkers 2, is not the first example of chemical specificity within PAG columns. Previous studies have shown that this dorsolateral region can also be defined by the presence of muscarinic cholinergic receptors 48, excitatory amino acid receptors 1, opiate receptors 29 and substance P immunostaining (see Beitz 7 for review). In addition this region contains a concentration of GABA-immunoreactive ceils 41, somatostatin immunoreactive fibers z2, and a higher density of GABAA/benzodiazepine 25, and neurotensin 39'49 binding sites than do other columns of the PAG. Moreover a review of the literature suggests that noradrenergic and adrenergic fibers are predominantly localized to the lateral and ventrolateral columns7'28, while LHRH- and ACTH-containing fibers are concentrated in the juxta-aqueductal column 47. Although these are but a few examples of an increasing number of neurochemicals that appear to be selectively associated with PAG columns, this selective chemical localization within the PAG certainly provides biochemical support for the existence of longitudinal columns in this region In the cerebellum 12 and hippocampus 23 NO has been shown to act as a messenger that mediates the stimulation of cGMP formation by glutamate acting at the NMDA receptor. While glutamate immunoreactive neurons and fibers are present throughout the PAG 8 the dorsolateral PAG has been found to contain the greatest density of binding sites for several excitatory amino acid agonists, including NMDA ~. The dorsolateral PAG also receives selective inputs from several areas including the cingulate cortex 45, the ventrome-
322 dial hypothalamic nucleus 5'47, the posterior hypothalamic nucleus 5, the cuneiform nucleus 5, the anterior pretectal nucleus 5 and the zona incerta 5, all of which provide major sources of excitatory amino acid inputs to the PAG 8. Thus, it is possible that the production of NO in the dorsolateral PAG involves excitatory amino acid stimulation of N M D A receptors, subsequent calcium-dependent activation of NOS, NO production and ultimately activation of guanylate cyclase and cGMP formation as has been described for other neural systems. In the cerebellum, for example, where this physiological cascade was first described, the enhancement of cGMP formation by glutamate is particularly strong ~2. However, it should be noted that NOS immunoreactivity occurs within the brain in many areas that do not demonstrate high levels of guanylate cyclase ~°, suggesting that additional mechanisms of action are likely to be discovered, In order to examine the role of NO as a potential neuronal messenger in the PAG, it will be important to define the potential colocalization of NOS with neuropeptides and neurotransmitters in this region as well as to study the potential relationship of excitatory amino acid-containing axons and terminals to NOS positive neurons. NOS is known to coexist with peptides such as neuropeptide Y and somatostatin in the corpus striatum and cerebral cortex ~s and with VIP and acetylcholine in neurons innervating cerebral arteries 32, raising the question of whether NO may act in conjunction with other substances. The questions of whether these or other neuromodulatory substances coexist with NOS and of the relationships among NOSIR perikarya, fibers and terminals and neuronal structures identified according to their chemical coding a n d / o r their projections to or from the PAG remain to be investigated. Several neuronal roles for NO have been postulated. Immunohistochemical and pharmacological studies have indicated that NO mediates non-adrenergic, non-cholinergic transmission from enteric inhibitory motor neurons and between n e u r o n s in the enteric and m a y participate in the regulation of hormone release ~. Several studies have indicated that
g a n g l i a 14'2°
NO may serve as a mediator of long-term depression in the cerebellum and may function presynaptically to maintain long-term potentiation in the hippocamRecent studies have also indicated that NO plays a role in nociceptive mechanisms at the level of the spinal cord. Thus Meller et al. 37 have shown that NO mediates the thermal hyperalgesia produced under conditions of neuropathic pain, while our laboratory p u s 9'40'42'43.
has shown that NO mediates the spinal cord expression of Fos induced by noxious mechanical stimulation 33.
The specific role of NO in the PAG, however, remains to be defined. The PAG is known to play a role in pain modulation, respiratory and cardiovascular control, defensive reactions, vocalization and female reproductive behavior 7'15-~7'21'35 and has recently been postulated to be involved in integrating these various sensory, motor and autonomic roles in response to stressful and potentially threatening stimuli 2'4'44"45. Although relatively littie is known about the specific functions of the dorsolateral PAG column, we have recently demonstrated that this column is specifically activated by linear acceleration 31. This region is known to receive spatial orientation inputs from the superior colliculus, the substantia nigra pars reticulata and prepositus hypoglossi 3° and thus the dorsolateral column may help control appropriate responses to novel environments or situations, including autonomic and somatomotor responses to changes in gravity or acceleration 7'3°'3~. Whether NO plays a role in the dorsolateral PAG's involvement in adaptation to novel environments requires further investigation. This work is supported by NIH Grants DA06687, DC01086 and DE06682 to AJB and Fonds zur F6rderung der Wissenschaftlichen Forschung in Osterreich to BM. We thank Richard Price for his technical assistance during this study and Todd Brelje for his assistance with the laser confocal microscope.
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