Efferents of the opiocortin-containing region of the commissural nucleus tractus solitarius

Peptides, Vol. 15, No. I, pp. 169-174, 1994 Copyright © 1994 Elsevier Science Ltd Printed in the USA. All rights reserved 0196-9781/94 $6.00 + .00

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Efferents of the Opiocortin-Containing Region of the Commissural Nucleus Tractus Solitarius L A U R A J. SIM l A N D S H I R L E Y A. J O S E P H 2

Neuroendocrine Unit, University o f Rochester School of Medicine and Dentistry, Rochester, N Y 14642 Received 7 J u n e 1993 SIM, L. J. AND S. A. JOSEPH. Efferents of the opiocortin-containing region of the commissural nucleus tractus solitarius. PEPTIDES 15( 1) 169-174, 1994.--Efferentprojectionsof the commissuralnucleustractus solitarius(cNTS) in the regioncontaining opiocortin-immunoreactive(-IR) neurons were identifiedusingPhaseolusvulgarisleucoagglutinin(PHA-L). Efferentswere identified in the bed nucleus of the stria terminalis, preoptic area, amygdala, hypothalamus, periaqueductal gray, parabrachial nucleus, locus coeruleus, medullary catecholaminergicgroups, and NTS. The PHA-L-IR varicositiesin lateral parabrachial nucleus were identified in close association with CRF-IR and enkephalin-iR cells. These data on cNTS projections are consistent with our previous immunocytochemical and lesion studies on opiocortin connectivity and provide anatomical evidence that neurons in the cNTS may influence cardiovascular and sympathetic nervous system function via connectivity with nuclei in the lateral brain stem. Opiocortin

Cardiovascular

Phaseohtsvulgaris leucoagglutinin

Brain stem

the commissural region of the NTS to provide neuroanatomical data regarding its efferent connectivity.

THE nucleus tractus solitarius (NTS) consists of two major subdivisions that extend dorsally throughout the medulla. The rostral NTS is a bilateral structure that is juxtaposed to the solitary tract throughout its extent and receives major gustatory inputs from cranial nerves VII, IX, and X (13,26). At the level of the obex, the NTS becomes a midline structure that is referred to as the commissural NTS (cNTS). McRitchie and Tork (17) have recently reported that the commissural region of the NTS can also be delineated in the human, which underscores the importance of understanding this nucleus as a separate neuroanatomical and functional region. Several distinctly separate neuronal populations can be recognized within the cNTS, suggesting numerous multidirectional efferent pathways. Immunohistochemically, opiocortin peptides (12), angiotensin II (16), cholecystokinin (CCK) (14), somatostatin (9), neurotensin (8), corticotropin-releasing factor (CRF) (24), substance P, enkephalin, and catecholamines (2) have been identified in the cNTS. Although the numerous afferent and efferent connections and neurochemical diversity of the cNTS would indicate a diversity of function, emphasis has been placed on the catecholaminergic autonomic pathway(s). In addition, several reports suggest that this region may contribute to nociceptive responses as well (1,6,19,23), but the connectivity implementing this function has not been elucidated. Our previous studies identifying opiocortin neurons in the cNTS (12) and subsequent immunocytochemical lesion (30) and deafferentation (11) studies are consistent with the concept that this nucleus may contribute to a nociceptive modulatory system. Thus, due to recent information regarding the structure of the cNTS and its possible role in sensory and autonomic homeostasis, we sought to isolate

METHOD

Sixteen male Sprague-Dawley rats weighing 175-225 g were anesthetized (80 mg ketamine + 8 mg acepromazine maleate/ kg body weight) and iontophoretically injected with PHA-L into the cNTS. A 2.5% solution of PHA-L (Vector) was stereotaxically (27) iontophoresed into the cNTS via 10-15-~tm glass micropipettes using 5 ~A alternating positive current over 15 min. The micropipette was left in situ for an additional 5 min to minimize tracking of the tracer. After 15-25 days, the animals were infused with 100 tzg of colchicine in 20/~1 of saline into the lateral ventricle to enhance peptide staining in subsequent immunocytochemical procedures. Twenty-four hours after colchicine administration, animals were deeply anesthetized with pentobarbital and perfused intracardially with 300 ml saline, followed by 4% paraformaldehyde in 0.1 M acetate buffer (pH 6.5) and 4% paraformaldehyde in 0.1 M borate buffer (pH 9.5). Brains were postfixed overnight at 4°C in 4% paraformaldehyde in borate buffer with 10% sucrose (pH 9.5). Frozen sections were cut at 50/~m on a sliding microtome. Sections were collected and rinsed in 0.02 M potassium phosphate-buffered saline (KPBS) (pH 7.4). Tissue was incubated for 48-60 h in rabbitanti-PHA-L (Dakopatts) diluted at 1:2500 in KPBS with 0.4% Triton X- 100 and 1% bovine serum albumin. Sections were processed using the avidin-biotin protocol (Vector), with a 1-h incubation in biotinylated goat-anti-rabbit IgG diluted 1:200 in

J Present address: Department of Physiologyand Pharmacology, Bowman Gray School of Medicine, Winston-Salem, NC 27157. 2 Requests for reprints should be addressed to Dr. S. A. Joseph, Neuroendocrine Unit, Box 609, University of Rochester School of Medicine and Dentistry, 601 Elmwood Ave., Rochester, NY 14642. 169

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m

FIG. 1. (A) Photomicrograph of a representative PHA-L injection site in the cNTS. Arrows indicate PHA-L-IR neurons that have incorporated the lectin (bar = 100 #m). (B) Schematic diagram depicting the location of a representative PHA-L injection site in the cNTS. Stippling indicates the presence of PHA-L-IR neurons. Abbreviations: AP, area postrema; C, central canal; sol, solitary tract; 10, dorsal motor nucleus of the X cranial nerve; 12, nucleus of the XII cranial nerve. KPBS + 0.04% Triton X-100 + 1.5% normal goat serum, followed by a KPBS rinse. Sections were incubated for 1 h in avidin HRP diluted 1:200 in KPBS, then rinsed in 0.1 M T r i s buffer (pH 7.4) and reacted in nickel-enhanced 3',3'-diaminobenzidine tetrahydrochloride (DAB). The reaction solution contained 0.187 g nickel ammonium sulfate, 50 ul of 3% H202, and 7.5 mg DAB in 37.5 ml Tris buffer. Brain stem sections were dual stained to identify the neurochemical content of cells in cNTS-derived putative terminal fields. Sections were incubated for 48-60 h in antiserum generated in rabbits against CRF (1:8000), Met-enkephalin (Met-ENK) (1:10,000), or tyrosine hydroxylase (Eugene Tech; 1:10,000) diluted in 0.05 M sodium phosphate-buffered saline (PBS; pH 7.5) + 0.4% Triton X-100 and 1% bovine serum albumin. The CRF and Met-ENK antisera were generated in our laboratory. Staining of the CRF antiserum is completely eliminated by preabsorption with 50 tzg of rat/human CRF (Bachem) per ml diluted antiserum. The staining of the MetENK antiserum is eliminated by preincubation with 50 #g of Met-ENK (Bachem) per ml diluted antiserum, and unaffected by equimolar amounts of Leu-enkephalin, /3-endorphin, substance P, dynorphin, neurotensin, or vasopressin. Immunocytochemistry was performed using the avidin-biotin procedure as described above, using 0.05 M PBS + 0.04% Triton X-100 as the diluent and rinse. Sections were reacted in 7.5 mg DAB, 100/~1 of H202 in 10 ml of PBS, to produce a brown homogeneous reaction product. RESULTS

Injection Sites The results are reported from 12 brains in which PHA-L was iontophoresed into the cNTS (Fig. 1). The cNTS injections were

confined to the NTS at the commissural level and did not include more rostral subdivisions of the nucleus. Numerous subpopulations of neurons with different neurochemical signatures exist within the cNTS. For example besides the ACTH containing neurons, other subpopulations include neurons containing enkephalon, somatostatin, tyrosine hydroxylase, and CRF. Although we attempted to confine the PHA-L injections to the ACTH-ir perykarya, other neurons, because of their proximity, were also infiltrated with the PHA-L. Also, because of the small area targeted, six injections included some diffusion of the tracer into the adjacent dorsal motor nucleus of the vagus (DMNX). Four animals in which PHA-L was injected into the nucleus of t h e XII cranial nerve were used as control injections. Control injections in this region resulted in extremely limited PHA-L labeling that was confined to the nucleus of the VII cranial nerve, parvocellular reticular nucleus, and rostral continuation of the nucleus of the XII cranial nerve. One injection included only the area postrema and subpostrema region. No PHA-L-IR fibers were identified in the telencephalon or diencephalon after this injection; however, the distribution of terminal fields in the brain stem was similar to that found with cNTS injections.

PHA-L-IR Fibers and Varicosities After cNTS injections, PHA-L-IR fibers with varicosities were identified in the horizontal nucleus of the diagonal band, median and lateral preoptic area, bed nucleus of the stria terminalis (ventral, lateral ventral, lateral dorsal, and medial anterior subdivisions), central and medial amygdala, organum vasculosum lamina

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FIG. 2. Schematic diagrams depicting the distribution of PHA-L-IR fibers and varicositiesin the brain stem after PHA-L injections into the cNTS. Abbreviations:Arab, nucleus ambiguus; AP, area postrema; LC, locus coeruleus; LPB, lateral parabrachial nucleus; LRt, lateral reticular nucleus; mlf, medial longitudinal fasciculus;NTS, nucleus tractus solitarius;sol, solitary tract; 7, nucleus of cranial nerve VII; 7n, VII cranial nerve; 10, dorsal motor nucleus of the X cranial nerve; 12, nucleus of the XII cranial nerve.

terminalis, lateral hypothalamus, paraventricular nucleus of the hypothalamus (PVN), supraoptic nucleus, arcuate nucleus, paraventricular nucleus of the thalamus, and periventricular gray. PHA-L-IR fibers with enlargements were identified in the periaqueductal gray (PAG) at the levels of the superior and inferior colliculi, with the greatest number of fibers in the ventral lateral and lateral PAG. A smaller number of PHA-L-IR fibers with varicosities was identified in the dorsal raphe nucleus (DRN), mainly in the dorsal region of the nucleus, and lateral to the DRN in A8. The distribution of PHA-L-IR fibers with varicosities in the brain stem is depicted schematically in Fig. 2, which is a composite drawing based on the 12 animals with cNTS injections. Numerous fibers with many varicosities were identified in the lateral parabrachial nucleus (LPB). At the level of the inferior colliculus, PHA-L-IR fibers were identified throughout the LPB, with a small cluster of PHA-L-IR elements found in the dorsal portion of the nucleus. Caudal to the inferior colliculus, PHA-L-IR fibers and varicosities were identified in a dense cluster in the dorsal LPB, as well as in the central LPB and external LPB. Fibers with enlargements were found ventral to the LPB in K~Jlliker-Fuse nucleus and A7. Fibers were sometimes seen passing across the superior cerebellar peduncle between the LPB and medial parabrachial nucleus or nucleus locus coeruleus, although the medial parabrachial nucleus contained only sparse fibers. Few PHA-L-IR elements were identified at the caudal extent of the LPB and these were distributed evenly

throughout the nucleus. PHA-L-IR fibers with varicosities were also identified in the nucleus locus coeruleus and medial to the locus coeruleus in Barrington's nucleus. PHA-L-IR fibers with few enlargements were found in the lateral aspect of the brain stem along the V and VII cranial nerves. A cluster of PHA-L-IR fibers with varicosities was identified in the A5 catecholaminergic group, as shown in tyrosine hydroxylasestained sections. Few fibers were identified at the level of the pontine-medullaryjunction, and these were found mainly in the parvocellular reticular nucleus and ventral lateral brain stem. In the ventral lateral medulla, fine PHA-L-IR fibers with enlargements were identified in net-like clusters, particularly in catecholaminecontaining cell groups. Fine PHA-L-IR fibers with varicosities were identified in the rostral medial and lateral NTS and in the NTS at C2. The most dense accumulation of fibers was identified in the medial NTS, adjacent to the fourth ventricle, although PHA-L-IR fibers were also found along the lateral edge of the nucleus. PHA-L-IR elements were rarely identified in the medullary raphe nuclei. In the ventral lateral medulla, a small group of fibers with varicosities was identified in and around the nucleus ambiguus and a larger group of PHA-L-IR fibers with enlargements was located in the Cl region. This ventral lateral medullary group continued caudally and was identified in the region of the lateral reticular nucleus and A 1. PHA-L-IR fibers with enlargements were identified adjacent to the injection site, extending into the medullary reticular nucleus and A 1.

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FIG. 3. Photomicrographs of(A-I) CRF-IR and (J) Met-ENK-IR neurons in the LPB in putative contact with PHA-L-IR varicosities after PHA-L injections into the cNTS. Arrows indicate putative contacts between the peptidergic neurons and PHA-L-IR elements (bar = 12.5 ~m).

Dual lrnmunocytochemistry Dual immunocytochemical procedures were used to identify the neurochemical content of neurons in cNTS terminal fields. This was particularly important in verifying that cNTS-derived projections were located in the A l - A 8 nuclear groups because these nuclei are most easily identified in sections stained for catecholamines. C R F - I R neurons were identified in c N T S terminal fields in the LPB and Barrington's nucleus, and in some cases close associations were identified [Fig. 3(A-I)]. Associations between C R F - I R neurons and PHA-L-IR varicosities were most

often identified in the dorsal LPB, where both a dense cluster of PHA-L-IR fibers with varicosities and a group of C R F - I R neurons were found in the same region. C R F - I R neurons and PHA-L-IR enlargements were also identified in juxtaposition in the superior LPB at the level of the inferior colliculus and in the external LPB caudal to the inferior colliculus. Met-ENK-IR neurons were also identified in association with PHA-L-IR varicosities in the LPB [Fig. 3(J)]; however, no clear regional pattern of distribution was identified. This may be due to the more ubiquitous distribution of M e t - E N K - I R cells compared to C R F - I R neurons.

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DISCUSSION We have identified a specific efferent system from the cNTS to diencephalic and lateral brain stem regions that is crucial to understanding the unique anatomy and function of the commissural region of the NTS. The cNTS is the caudal midline portion of the NTS that can be functionally and anatomically differentiated from rostral portions of the NTS in both animals and humans (17). Its neurochemical diversity and dense innervation from ascending and descending systems provides a unique substrate for the maintenance of autonomic and sensory homeostasis. Numerous studies have demonstrated the importance ofopiocortin and catecholaminergic mechanisms in autonomic functions of the cNTS (5,20,28,38,40) and provide functional support for our hypothesis that cNTS terminal fields in the LPB, ventral lateral medulla, nucleus ambiguus, Krlliker-Fuse nucleus, and hypothalamus comprise important autonomic pathway(s). A particularly important pathway in this regard may be cNTS connectivity with medullary catecholaminergic groups known to regulate autonomic function (32). Further, by identifying cNTS-derived fibers with putative terminals in association with CRF-IR cells in the LPB, we begin to correlate specific neurochemical substances with cNTS anatomy and function. An ascending cNTS pathway that may include opiocortin peptides and catecholamines may be important in modulating CRFmediated autonomic responses to stress (3,4,22). An interesting component of such an autonomic system is the relationship between between ACTH-IR neurons and CRF-IR cells in the LPB (29). The importance ofcNTS to LPB connectivity has functional implications for autonomic regulation [the LPB is a well-described cardiovascular and respiratory center (21)], as well as sensory homeostasis, because LPB has been shown to influence nociception via spinal projections (7). In addition to the role of the cNTS in autonomic function, some data suggest that this region may contribute to nociceptive responses as well (1,6,19,23). The results of the present study demonstrate that the cNTS may be involved in antinociception through connectivity with the PAG, brain stem catecholaminergic groups, or LPB, although it is not clear which cNTS neurotransmitter substances may be involved. The phenomena of stressinduced analgesia (SIA) is well characterized and may be due to both opioid and nonopioid components in the brain, as well as an opioid-mediated hormonal component (15). The NTS is thought to be involved in hormonally mediated SIA via vagal connectivity with brain stem nuclei (31 ). The results of this study provide anatomical evidence that this may occur and, further, indicate that the NTS may also be involved in opioid and/or nonopioid SIA via central connectivity, possibly with PAG, LPB, or catecholaminergic cell groups. Commissural NTS terminal fields were identified in lateral pontine nuclei that have been implicated in nociception (7,18,34,39) and provide an anatomical substrate by which the cNTS may modulate nociception in the brain stem in response to sensory and visceral afferents. We have been particularly interested in the opiocortin system localized to the cNTS in which neurons contain pro-opiome-

lanocortin-derived peptides including ACTH, 16K,/3-endorphin, and/3-1ipotropin (12,33). Surgical or chemical isolation of the medullary opiocortin pool has previously been used to determine the contribution of the cNTS to opiocortin innervation of the brain. Immunocytochemical analyses following deafferentation (11) or monosodium glutamate lesion (30) of the arcuate nucleus have shown that ACTH-IR neurons in the cNTS project to the LPB, locus coeruleus, paragigantocellular reticular nucleus, lateral reticular formation, and NTS. Radioimmunoassay analysis following brain stem hemisection has also demonstrated that the/3-endorphin and ACTH in the NTS, A l, A5, and lateral reticular nucleus originate from opiocortin-IR cells in the cNTS (25). Although PHA-L injections were confined to the cNTS, the size and neurochemical diversity of this region made it difficult to confine the PHA-L injections to a neurochemically homogeneous cell group, such as the opiocortin system. PHA-L was incorporated by cNTS neurons in the opiocortin-containing region of the nucleus and the terminal field distribution, particularly in the LPB, NTS, and ventral lateral medulla, resembles the discrete localization ofopiocortin-IR fibers derived from the cNTS (l 1,30), so we conclude that a subset of the PHA-L-IR fibers emanating from this nucleus contain opiocortin peptides. In light of the results of previous lesion studies (l 1,30) and our previous tract tracing studies (35-37), as well as the present results, it is concluded that the arcuate nucleus innervates telencephalic and diencephalic nuclei, PAG, and midline brain stem nuclei; whereas cNTS opiocortin-IR neurons innervate ventral lateral brain stem nuclei, particularly catecholaminergic cell groups. In addition, the arcuate and cNTS opiocortin systems provide dual innervation of the NTS, LPB, and locus coeruleus. This may reflect different roles of the two opiocortin-containing nuclei in both autonomic function and antinociception. Opiocortin-IR neurons in the arcuate nucleus may modulate the activity of forebrain neuroendocrine and limbic nuclei, mesencephalic antinociceptive systems, and descending serotonergic pathways from the medial brain stem; whereas opiocortin-IR neurons in the cNTS may modulate the activity of descending noradrenergic pathways from lateral brain stem nuclei that mediate autonomic and sensory homeostasis. Immunocytochemical and cytoarchitectural studies, as well as physiological reports, have provided evidence that cNTS neurons modulate autonomic function and nociception. By confining our PHA-L injections to the cNTS, we have specifically identified the efferents from this region that comprise an ascending pathway to forebrain and lateral brain stem regions with well-established autonomic functions and provided anatomical evidence that the cNTS may contribute to other functions as well. These results highlight the importance of the cNTS in visceral and sensory homeostasis and emphasize the need to consider the cNTS as a discrete anatomical and functional unit. ACKNOWLEDGEMENTS This work was supported by USPHS #DA 07232 (L.J.S.) and NIH #NS 21323 (S.A.J.).

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