Dynorphin A-containing neural elements in the nucleus of the solitary tract of the rat. Light and electron microscopic immunohistochemistry

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Brain Research, 522 (1990) 251-258 Elsevier BRES 15685

Dynorphin A-containing neural elements in the nucleus of the solitary tract of the rat. Light and electron microscopic immunohistochemistry Mariann Fodor, Attila Csiff~iry, P6ter Kiss and Mikl6s Palkovits 1st Department of Anatomy, Semmelweis University Medical School, Budapest (Hungary) (Accepted 16 January 1990) Key words: Dynorphin A; Immunohistochemistry; Immunoelectron microscopy; Nucleus tractus solitarii; Vagus nerve

Distribution of dynorphin A (DyA) immunoreactivity in the nucleus of the solitary tract (NTS) was examined in rats after various surgical transections by light and electron microscopic immunohistochemistry. In colchicine-treated animals DyA immunostained perikarya were seen in each subdivision of the NTS. In intact rats, dense network of immunopositive nerve fibers was localized light microscopically, and synaptic contacts were found between DyA immunopositive structures (axo-axonic, axo-dendritic synapses), electron microscopically. Surgical transections medial, caudal or rostral to the nucleus did not alter the distribution pattern of DyA in the NTS. Lesion immediately lateral to the nucleus resulted in an ipsilateral appearance of immunostained cell bodies. Vagal and glossopharyngeal afferents (including baroreceptor fibers) terminate in the medial and commissural subnucleus of the NTS. Two days after extracranial vagotomy, synaptic contacts between degenerated presynaptic boutons and DyA immunopositive postsynaptic elements were observed in both medial and commissural part of the NTS. These observations provide morphological evidence suggesting that (1) axons of dynorphin A-containing cell bodies form an intrinsic network inside the nucleus; (2) these DyA cells receive direct peripheral inputs through the vagus nerve, and (3) projecting DyA neurons may exist in the NTS, they may innervate medullary, rather than forebrain, higher brainstem or spinal cord neurons. INTRODUCTION An increasing number of data indicate that dynorphins may influence several autonomic functions including analgesia 9,12,16, feeding 19, thermoregulation 2°, stress 2°, central stimulation of gastric acid secretion 18 and central cardiovascular regulatory mechanisms 8'1°. The exact sites of these effects have not been localized yet, but a number of central autonomic nuclei, including the nucleus of the solitary tract (NTS) are among the possible target regions. The NTS is located in the dorsomedial medulla, receiving peripheral afferents through the Vth, VIIth, IXth and Xth nerves. Baroreceptor fibers arising from peripheral baroreceptors via carotid sinus (glossopharyngeal) and aortic (vagus) nerves terminate mainly in the commissural (NTSc) and medial (NTSm) parts of the NT52,3,6.26,33. A fairly high number among several thousands of neurons in the NTS is peptidergic: more than 30 neuropeptides have been reported in cell bodies or nerve terminals 27. Immunohistochemical analysis revealed a wide distribution of immunoreactive dynorphin A-containing cells in the NTS, with an especially high density in the commissural and medial parts of the nucleus 7' 14,34-37. In good agreement with the immunohistochem-

ical finding dynorphin A ( D y A ) has been detected here in relatively high concentrations by radioimmunoassay TM 13,21,36,39 According to their innervation patterns, neurons in the NTS can be divided into two major groups: local, intrinsic cells and projecting neurons 25'26. NTS efferents project to several brain regions innervating forebrain, hypothalamic, brainstem and spinal cord nuclei 4,17'23' 26,28,30-32. The present study has been performed to find out whether dynorphin neurons in the NTS are intrinsic or extrinsic. The second question we asked ourselves was whether dynorphin A-containing cells might be part of the baroreceptor reflex arc. Therefore, immunohistochemistry in combination with various types of surgical transections in the lower brainstem were performed to interrupt the possible neuronal efferents from the NTS, and vagal inputs to the nucleus. MATERIALS AND METHODS Adult, 200 + 20 g Wistar (CFY strain) rats of either sex (n = 20) were used. The animals were divided into 5 groups: (1) Medullary transections. Under ether anesthesia, the heads of the animals were fixed in a stereotaxic device in a 5° nose-down position. The brainstem was unilaterally transected rostral (a) and caudal (b) to the NTS to interrupt possible ascending and de-

Correspondence: M. Fodor. 1st Department of Anatomy, Semmelweis University Medical School, Budapest, T/izolt6 u. 58, H-1450, Hungary.

252 scending fibers from the NTS to other brain areas. Glass microknives (0.16 mm thick) cut of coverslips were used for the transections. (2) Medullary knife cuts in and lateral to the NTS. The heads of the animals in this group were fixed in a maximal nose-down position. The dorsal surface of the medulla oblongata was exposed by means of transection of the atlanto-occipital membrane. A long, unilateral cut was made by glass microknives along the lateral border of the NTS (a), and through the NTS dividing the medial and lateral subdivisions of the nucleus (b). (3) Extracranial (cervical) vagotomies. A midsagittal hide cut was made at the level of the hyoid bone, and the right vagus nerve was

transected from a parapharyngeal approach. (4) Colchicine injections. Animals received a single injection of colchicine (100/~g/10/d) into the cerebello-medullary cystern, 2 days before perfusion. (5) Controls. This group consisted of (a) normal, intact animals, and (b) sham-operated: penetration through the skull (similar to group 1), or through the atlanto-occipital membrane (similar to group 2) without any damage in the brain. On the 2nd, 5th, 7th and 14th postoperative days (the last 3 periods in groups 1 and 2), the animals were reanesthetized and perfused intracardially with physiological saline followed by a fixative solution (4% paraformaldehyde, 0.2% picric acid, 0.1%

Fig. 1. Coronal sections from the dorsomedial medulla oblongata of the rat. Dynorphin A-immunostained nerve fibers in intact (A,C,E), cell bodies and fibers in colchicine-treated rats (B,D,F). Distances from the bregma are given in mm. Scale bar = 1000/~m. Abbreviations: AP, area postrema; C, cuneate nucleus; dX, dorsal vagal nucleus; NTSc, NTSI and NTSm, commissural, lateral and medial parts of the NTS; TS, solitary tract; IV, fourth ventricle; XII, hypoglossal motor nucleus. The central canal is indicated by arrows.

253 glutaraldehyde in 0.1 M phosphate buffer, pH 7.35). The brains were removed and placed in the same, but glutaraldehyde free, fixative overnight. Serial sections of 50/am thickness were cut in the coronal plane with a Vibratome (Oxford Instruments). To wash out the fixative completely the sections were rinsed 5 times with phosphate buffer for 2 h. Before immunostaining the vials containing sections in 30% sucrose (dissolved in physiological saline) were put in liquid nitrogen until the thin fluid layer just covering the sections was frozen and then thawed to room temperature. The endogenous peroxidase activity of the tissue was suppressed by 3% H20 2 for 10 min and then the sections were incubated in 0.1 M phosphate buffer containing 10% normal goat serum (Miles Lab.; Napperville) for 1 h, at room temprature. The immunohistochemical reactions were performed on free floating sections. First they were incubated in 1:4000 (rabbit) dynorphin A(1-I7) antiserum (generous gift from Dr. N. Zamir, NIMH, Bethesda, MD, U.S.A.). After rinsing with phosphate buffer, sections were incubated in a 1:500 dilution of biotinylated anti-rabbit IgG, followed by biotinylated avidin-peroxidase complex (1:250) (Vector Lab., CA) each for 1 h at room temperature. Sections were developed in 3, 3"-diaminobenzidine (DAB) 50 rag% (in 0.05 M Tris-HC1 buffer, pH 7.6) with 0.01% H202. For light microscopy sections were mounted on gelatin-coated slides, dehydrated and coverslipped. The specificity of the antiserum for dynorphin A(1-17) has been described elsewhere 3s. For electron microscopy tissue sections were postfixed in 1% osmium tetroxide for 1 h, dehydrated in graded ethanols and embedded in Durcupan (Fluka). Ultrathin sections were cut from the medial and commissural regions of the NTS containing dynorphin A immunopositive cell bodies and presumed degenerated vagal terminals two days after extracranial vagotomy. Sections from selected blocks were collected on Formwar-coated single slot grid and stained with lead nitrate and examined with a TESLA BS 413 or JEOL 100 electron microscope. RESULTS

Light microscopy In intact, non-treated animals a moderately dense dynorphin A-immunoreactive network of fibers and varicosities was observed in the nucleus (Fig. 1A,C,E). The highest density of fibers was seen in the medial part of the NTS, just dorsomedial to the dorsal vagal nucleus (Fig. 1C), and in the whole territory of the commissural

NTS (Fig. 1C,E). These immunoreactive fibers were varicose and much thinner than those in the adjacent motor hypoglossal nucleus (Fig. 1C,E). Two days after colchicine injection, DyA-containing cell bodies appeared along the whole rostrocaudal length of the NTS (Fig. 1B,D,F). In rostral sections of the nucleus, a number of DyA-immunoreactive cells were present in the lateral portion of the medial subdivisions of the NTS and cells were also scattered in the lateral subdivisions (Fig. 1B). Immediately caudal to the obex, D y A cells consisted of two small groups, one lying along the area p o s t r e m a - N T S border, the other group of cells located immediately medial to the solitary tract (Fig. 1D). Several DyA-positive cells occurred in the caudal sections of the commissural part of the NTS (Fig. 1F). Neurons here were mainly fusiform in shape; they were clustered mediolaterally over the central canal. No D y A cells were seen in the neighboring (gracile, cuneate, motor hypoglossal, paramedian reticular) nuclei. Short-term lesions. Medullary hemisections or knife cuts rostrai, caudal or lateral to the NTS, as well as extracranial vagotomy did not alter the distribution pattern of the DyA-immunopositive network of fibers inside the NTS. No accumulations or diminutions in the D y A fiber density were observed in the nucleus during any postoperation periods (2, 4, 7 and 14 days). In contrast to this, DyA-immunostained cell bodies became visible in the NTS without colchicine treatment in rats with a knife cut along the lateral border of the NTS (Fig. 2). Neurons (10-15 per section) appeared only ipsilateral to the knife cut immediately medial to the solitary tract, partly in the commissural part of the NTS. This population of cells consists of medium-sized, multipolar neurons (Fig. 2). No immunostained cells were observed in the contralateral NTS or in any other nuclei in the medulla oblongata. Neither rostral or caudal hemisections (group

Fig. 2. A: dynorphin A-containing cell bodies after unilateral transection along the lateral border of the NTS. The surgical transection is indicated by arrowheads. Scale bar = 1000/zm. B: a higher magnification of the circled area in Fig. A. Abbreviations: see Fig. 1.

254 1), nor extracraniai vagotomy (group 3) resulted in perikaryal accumulation of immunostaining in any part of the NTS.

Electron microscopy Dynorphin A-immunostained perikarya and dendrites established synaptic contact with both DyA-immunopositive and immunonegative axons. DyA-DyA synaptic contacts were found in each part of the NTS, more

frequently in the medial subdivision. However, the vast majority of synaptic contacts on postsynaptic DyApositive neuronal elements were established by immunonegative presynaptic boutons. These synaptic contacts seemed to be both symmetrical and asymmetrical types. Labeled spines also formed synaptic contacts with immunonegative nerve terminals (Fig. 4). Two days after unilateral cervical transection of the vagal nerve, cytolysosomal and a dark type of nerve

Fig. 3. A: degenerated vagal afferent (thick black arrow) is in synaptic contact with unlabeled dendrite (asterisk). The curved arrows show a symmetrical synapse. B: cytolysosomaldegeneration (arrow) in the NTS 2 days after unilateral extracranial vagotomy. Scale bars = 1 gm).

255 degenerations appeared in the commissural part of the NTS (Fig. 3). Several degenerated axon terminals occurred still in synaptic contact with DyA labeled dendrites (Fig. 5) indicative of the existence of direct vagal neuronal inputs to DyA-containing neurons in the NTS. DISCUSSION The nucleus of the solitary tract, the well-known primary relay center in the autonomic nervous system, is very rich in neuropeptides including the opioid peptides 15"25. Dynorphin A-containing neuronal perikarya and fibers are unevenly distributed over each subdivision

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of the nucleus 7"14'35-37, the cell bodies being concentrated mainly in the commissural and medial NTS. The density of DyA-containing nerve fibers and varicosities is much higher here than in the neighboring medullary nuclei. The present study was undertaken to localize the possible sources of DyA-containing fibers in the NTS, and the possible targets of DyA-positive projecting neurons of the NTS. Cervical transections of the vagus nerve were performed to verify the existence of direct vagal neuronal inputs to DyA cells in the NTS. None of the surgical interventions applied in this study resulted in a visible alteration in the density of the DyA-containing neuronal network in the NTS. This

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Fig. 4. A: cytoplasmic fragment from dynorphin A-containing neuron. The arrowheads indicate the peroxidase reaction product. Nu, nucleus. B: axo-somatic synaptic contacts (arrows) between an immunolabeled axon terminal (~-) and immunopositive perikaryon (*) in the commissural part of the NTS. C: dynorphin A-immunopositive axon terminal is in synaptic contact (arrow) with a labeled dendrite (asterisk). Scale bars = lpm.

256 observation indicates that the majority of the D y A containing n e t w o r k in the NTS is most p r o b a b l e intrinsic. This finding is s u p p o r t e d by electron microscopic obser-

vations: intact D y A - D y A synaptic contacts r e m a i n e d in the NTS of o p e r a t e d animals. F r o m experience it is known, however, that small changes in immunostaining

Fig. 5. Intact and degenerated nerve terminals in the medial part of the NTS establish synaptic contacts with labeled and unlabeled neurons 2 days after unilateral cervical transection of the vagal nerve. A: intact, unlabeled axon terminal is establishing a symmetric synapse (black arrow) with an unlabeled perikaryon (asterisk). B: axo-somatic connection (black arrow) between a degenerated, autophagous cytolysosomecontaining bouton (black arrowhead) and an unlabeled, immunonegative perikaryon (asterisk). C: degenerated axon terminal forms synaptic contact (black arrow) with an immunopositive cytoplasmatic fragment (asterisk). Note, that in this case the dense clumps of DAB-reaction products are associated with polyribosomes (arrowheads). Scale bars = 1 l~m.

257 (up to 25-40% of the total immunostaining in a certain area) cannot be estimated by microscopic investigation without systematic quantitative analysis. Therefore, the existence of DyA-containing fibers, as a minor portion of the DyA network in the NTS, cannot be excluded. Indeed, reportedly, dynorphin A-immunopositive neurons in the lateral hypothalamus project to the NTS 4°. A number of neurons in the NTS project to the major brain areas including the forebrain, the brainstem and the spinal cord 4'17'23'26'28"30-32. Limited information is available about the chemical character of these fibers. Since no retrograde accumulation of DyA immunoreactivity could be observed in NTS neurons 2-4 days after medullary hemisections rostral or caudal to the nucleus, the existence of ascending or descending projections of NTS DyA-neurons seems to be unlikely. The retrograde accumulation of transmitter substances in the proximal portion of transected axons or in the perikarya following transections is a characteristic sign of the mechanical blockade of the axonal transport and, in combination with immunocytochemistry is an excellent neuromorphological tool to visualize neurotransmitter substances in projecting neurons 5. This is the case in the present study following knife cuts immediately lateral to the NTS. Two to four days after the knife cut, DyAimmunoreactive substance accumulated in several cell bodies of the NTS. This accumulation was ipsilateral to the lesion, no immunopositive cells were visible on the contralateral side. The number of DyA-immunostained neurons that became visible after lateral knife cuts is much lower than that of colchicine treated rats: only a group of cells appeared in the lateral portion of the commissural NTS, in the close vicinity of the solitary tract. The exact projection of these neurons cannot be localized on the basis of surgical cuts, but the target area may be circumscribed. At the level of the NTS, an arc of fibers runs between the dorsomedial and ventrolateral medulla. This arc is constituted by fibers with various origins and destinations. Fibers interconnect the NTS and the ventrolateral medulla vice versa, some of the descending fibers from higher brain areas run through the ventrolateral corner of the medulla and turn dorsomedially to reach the vagal nuclei through this arc, and afferent vagal fibers from the periphery to the NTS, and efferent fibers from the dorsal vagal nucleus to the periphery all use this common arc. Among several others, DyA-containing fibers of NTS origin are present in this arc 17"23"24"28"30. A dynorphin-containing efferent vagal projection may be excluded since (a) no DyAimmunoreactive neurons are present in the dorsal vagal nucleus, and (b) no retrograde accumulation of DyA occurred in any NTS neuron after cervical vagotomy. The possible projection fields of NTS DyA cells are the ventrolateral medulla and the ambiguus nucleus. Neuro-

nal cell groups in both rostral (vasomotor and caudal (vasodepressor) ventrolateral medulla have bidirectional neuronal connections with the NTS 29'3°. Fibers in both directions run through the arc which was transected by the lateral NTS cut. NTS neurons also project to the ambiguus nucleus17'23,28,3°; these fibers also run in the arc. A few additional brain areas cannot be theoretically excluded as possible targets of NTS DyA-cells, but no connections have yet been reported from the NTS to the parvicellular reticular nucleus, the nucleus of the spinal Vth, the cuneate nuclei or inferior olive, which are located ventral or ventrolateral to the knife cuts along the lateral edge of the NTS. Further rostral or caudal regions can be excluded since neither rostral nor caudal hemisections resulted in any retrograde accumulation of DyA in NTS neurons. Practically no data are available about the neuronal inputs to the DyA-containing cells in the NTS. A number of fibers arising in cells of various brain regions terminate on NTS neurons (for ref. see Nakamura22), many of them aminergic and peptidergic. Some of them may innervate DyA neurons. The second group of NTS afferents arise in the periphery and reach the nucleus through the Vth, VIIth, IXth and Xth cranial nerves. In the present study, peripheral afferents through the vagus nerve were investigated. Unilateral cervical vagotomy resulted in several degenerating nerve terminals both in the medial and commissural parts of the NTS. Some of these degenerating axons constitute synaptic connections with DyAimmunopositive dendrites indicating the existence of direct peripheral vagal inputs to DyA cells in the NTS. Cells in the NTS may serve as relay neurons in autonomic reflex mechanisms transferring cardiovascular, respiratory, taste and different visceral signals from the periphery to higher centers in the CNS. The functional role of DyA cells in these mechanisms has not been established yet. Previous experiments demonstrated that intravenously administered DyA caused hypotension and bradycardia which could be arrested by bilateral vagotomy ~°. Dynorphins seem to be endogenous ligand for the ~c-opiate receptor subclass ], and the cardiovascular effects may be carried out via r-receptors 8. Accordingly, dynorphin-induced hypotension and bradycardia could be prevented by administration of ~c-opiate receptor antagonists ~°. The aim of the present study was to clarify whether dynorphin A-containing neurons in the NTS having a primarily peripheral input may participate in a vegetative reflex arc as central interneurons.

Acknowledgements. The authors wish to thank Mrs. R. Ferv~igner for her excellent technical assistance, and Mrs. M. Kiss for her careful secretarial work.

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