0361-9230/92$5.00 + .OO Copyright0 1992Pergamon Press Ltd.

Brain Research Bulletin, Vol. 29, pp. 821-829, 1992

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TRH Regulation of Tracheal Tension Through Vagal Preganglionic Motoneurons MICHIKO IWASE,* SEIJI SHIODA,? YASUMITSU NAKAI,? KENJI IWATSUKI# AND IKUO HOMMA*’ *Department of Physiology and fDepartment of Anatomy, School of Medicine, Showa University, l-S-8 Hatanodai, Shinagawa-ku, Tokyo 142, Japan $Tsukuba Life Science Laboratory, Nippon Petrochemicals Co., Ltd., 5-9-9 Tokodai, Tsukuba-shi, Ibaraki 300-26, Japan

Received 5 March 1992; Accepted 19 March 1992 IWASE, M., S. SHIODA, Y. NAKAI, K. IWATSUKI AND I. HOMMA. TRH regulafion of tracheal tension through vagal nreaannlionic motoneurons. BRAIN RES BULL 29t61 82 l-829. 1992.-TRH-immunoreactive nerve terminals innervate the ambiGous nucleus in the rabbit. Vagal preganglionic motoneurons’that innervate the trachea, were revealed by HRP h&chemistry in the rostra1 part of the ambiguous nucleus and the dorsal motor nucleus of the vagus. HRP histochemistry plus TRH immunocytochemistry revealed that TRH-immunoreactive axon terminals synapsed on ambiguous nucleus neurons retrogradelylabeled

by HRP injection into tracheal smooth muscle and the superior laryngeal nerve. Microinjection of 50 ng TRH into the rostral ambiguous nucleus caused slight dilation followed by constriction, which was inhibited by atropine and vagotomy. Results show that central TRHcontaining neurons regulatetracheal tension through synapseson vagal preganglionicmotoneurons that innervate tracheal smooth muscle. Trachea Thyrotropin-releasing hormone (TRH) HRP Immunocytochemistry Ultrastracture

Vagal preganglionic motoneuron

TRH in the central nervous system affects autonomic functions; it facilitates respiration (6,8,20), increases blood pressure (33), and stimulates gastrointestinal motility (4,9) and gastric secretion (7,32). Microinjection of TRH into the dorsal motor nucleus of the vagus (dmnX) or the nucleus of the solitary tracts (NTS) directly excites or inhibits neuronal activity, which shows the powerful effects of TRH on vagally mediated functions (19,25,28). Immunohistochemical and autoradiographic studies have revealed TRH-containing neurons and TRH binding sites distributed in the dorsal vagal nuclei, raphe nuclei, and other regions of the reticular formation ( 11,12,13,18). Recently, Rinaman and Miselis (26) reported that TRH-immunoreactive (TRH-I) neurons make synapses on dendrites of gastric vagal motoneurons in the dmnX and the NTS. This provides morphological evidence for the profound stimulatory effect of central TRH on gastric vagal motor activity. Administration of TRH into the fourth ventricle or NTS increased respiratory frequency (6,8,20), but no central effects on tracheal smooth muscle tension have been reported. The trachea is innervated by the recurrent and superior laryngeal nerves, and reflexly causes constriction via vagal C fibers or rapidly adapting receptors (2,27). Brain stem projection of sensory and motor neurons from the trachea was investigated by horse-

Rabbit

radish peroxidase (HRP), and it was shown that the rostral part of the ambiguous nucleus (nA) contributed to motor innervation of the trachea in the cat (14). The rostra1 nA of the rabbit was described by Lawn ( 16) as a compact formation consisting of a principal column, a dorsomedial division, and a medial column, a region now frequently called the retrofacial nucleus (nRF). However, there is no information about the localization of vagal preganglionic motoneurons or central regulation by substances at this level in the rabbit. We focused our attention on the possible role of TRH-containing neurons that terminate on vagal preganglionic motoneurons, using retrograde HRP transport, TRH immunocytochemistry, and double labeling. Central regulation of tracheal tension by TRH was examined physiologically. METHOD Histological Study HRP-retrograde tracing. Ten white rabbits weighing 2.4-3.6 kg were anesthetized with 25 mg/kg pentobarbital sodium IV. The trachea was exposed and incised 2-3 mm parallel to a cartilage ring, 1 cm caudal from the cricoid cartilage. Wheat germ agglutinin-coupled horseradish peroxidase (WGA-HRP) dissolved to 2% in 0.1 M Tris-buffer (pH 7.6) were placed in glass

’ Requests for reprints should be addressed to Ikuo Homma, Department of Physiology, School of Medicine, Showa University, l-5-8 Hatanodai, Shinagawa-ku, Tokyo 142, Japan.

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micropipettes (tip diameter. 20-30 Km) connected to a Hamilton IO ~1 syringe through a polyethylene tube. WGA-HRP was injected through the glass micropipettes into the tracheal smooth muscle layer at three or five points, and the surface was wiped to prevent nonspecific uptake. WGA-HRP was also injected into the superior laryngeal nerve (SLN) and the recurrent laryngeal nerve (RLN) (Fig. 1). After survival for 72-93 h, the rabbits were deeply anesthetized with pentobarbital sodium and perfused transcardially with 0.9% NaCl followed by 4% paraformaldehyde and 0.4% glutaraldehyde in 0.05 M phosphate buffer (pH 7.2). The brains were removed. immersed in the fixative overnight, and serial 50 pm thick sections were then cut on a Vibratome (Oxford Instrument). Retrogradely labeled neurons were visualized using 3,3’.5.5’ tetramethylbenzidine (TMB) in acetate buffer according to the method of Mesulam (22). After TMB staining, some sections were placed on gelatin-coated slide glasses, lightly counterstained with neutral red, dehydrated in ethanol. and mounted. Other sections were stabilized with 3,3’ diaminobenzidine tetrahydrochloride (DAB) (29) after TMB reaction. and then processed for electron microscopy. These sections were postfixed in I%>osmium tetroxide in 0.1 M phosphate buffer, pH 7.2 (PB) for I h at 4”C, dehydrated in ethanol and embedded in an Epon-Araldite mixture. Ultrathin sections were mounted on grids, stained with uranyl acetate and lead citrate, and then examined with Hitachi HS-9, and JEOL JEM-1200EXII electron microscopes. TRIJ-irnrnunocqfochemistr~. Three rabbits were examined by TRH-immunocytochemistry. Animals were anesthetized with 25 mg/kg pentobarbital sodium IV. perfused transcardially with the same fixative and in the same way as that used for the HRP histochemistry. The Vibratome sections (40-50 pm, thick) were washed in 0.1 M PB with 7.5% sucrose, pH 7.2 (sucrose-PB). After incubation in normal goat serum (diluted I :40 in sucrosePB) for 30 min, the sections were incubated in TRH-antiserum (Bioscience Co., diluted I :2000 in sucrose-PB containing 0. I % dimethyl sulfoxide) at 4°C for 48 h. The sections were incubated sequentially in goat antirabbit IgG (Toyo Serum, Japan, diluted I : 100 in sucrose-PB) and in peroxidase antiperoxidase complex (PAP. Toyo Serum, Japan, diluted I :200 in sucrose-PB) for I h in each at room temperature. This incubation series was then repeated to enhance the immunoreaction. Sections were then processed by the DAB reaction procedure previously described (11,12). Sections were osmicated for I h. dehydrated, and embedded in an Epon-Araldite mixture. as described above. The ultrathin sections were doubly stained and examined with electron microscopes as described above. Dot& labeling technique yfHRP histochemistry with TRH immuno~~tochemistr?‘. Some sections that were processed by HRP histochemistry were stabilized with DAB-cobalt and then processed by TRH immunocytochemistry as described elsewhere (30). These sections were immunostained in the following solutions:

I. TRH antiserum (Bioscience Co. l/2000) for 48 h at 4°C; 2. goat antirabbit IgG (Toyo Serum, l/100) for 2 h at room temperature: 3. peroxidase antiperoxidase (PAP) complex (Toyo Serum, I / 100) for 2 h at room temperature. After the DAB reaction, the sections were postfixed in 1% osmium tetroxide solution and then embedded in an EponAraldite mixture. Ultrathin sections were double stained and examined with electron microscopes as described above. Con t rol.5 TRH antiserum was preabsorbed with 100 Kg TRH (Protein Res. Inc., Japan). No TRH-like immunoreactivity (TRH-LI)

IM/‘SE

El‘ AL.

FIG. I. Schema of experimental procedure. Curved arrows: WGA-HRP injection sites into trachea, SLN. and RLN. Details in text.

was detected in sections treated with preabsorbed with sucrose-PB instead of TRH antiserum.

antiserum,

or

Eight rabbits (body weight 2.8-3.5 kg) were anesthetized with urethane-chloralose (450 mg/ml urethane + 45 mg/ml chloralose solution, I ml/kg IV). This anesthetic was additionally applied in doses of 0.1 ml/kg, at 60- 120 min intervals. The trachea was cannulated 3 cm caudal from the cricoid cartilage and connected to an artificial ventilator (Harvard Instrument, USA) (Fig. I). Animals were ventilated artificially and paralyzed with 4 mg/kg gallamine triethiodide IV, initially and at 30 min intervals. The femoral artery was cannulated to measure the blood pressure, and the femoral vein was cannulated for systemic administration of saline containing 5% glucose and test drugs. Trachea tension was estimated from pressure of a 2 cm long balloon inserted into the rostra1 trachea. The balloon was inserted in the rostra1 direction through the incision made for the respirator cannula (Fig. I), filled with distilled water, connected to a pressure transducer (Statham Co., USA), and initially inflated to 10 cmHzO. The trachea was distended passively to decrease the balloon pressure, and was then stabilized at a resting tension of about 3-5 cmHzO. The medulla oblongatas of the rabbits were dorsally exposed and microinjected with TRH (Protein Res., Japan). The TRH was dissolved in artificial cerebrospinal fluid (CSF, pH 7.4) at 100 &ml, and 500 nl was applied from a 1 11 Hamilton syringe over 45-60 s to avoid pressure artifact. The coordinates of the microinjections into the rostra1 nA were 2.3-2.5 mm rostra1 from the area postrema (AP), 2.8-3.0 mm lateral from the midline, and 3.5-3.7 mm ventral from the dorsal surface (2 I). After the physiological investigation, the rabbit medulla oblongata was

TRH REGULATION

OF TRACHEAL

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removed and fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (PB) for at least 48 h. The tissue blocks were transferred to 15% sucrose in 0.1 M PB, and sliced transversely into 40-50 pm thick sections in a cryostat (Sakura Seiki, Japan). They were counterstained with neutral red and the injection sites were examined. RESULTS

trachea

20. Q 0

.

Lo.

HRP Histochemistry Cell bodies retrogradely labeled by WGA-HRP were found in the dmnX and the nA of the medulla oblongata after injection into the trachea, the SLN, and the RLN. Figure 2 shows the distribution of neurons retrogradely labeled by injection into each of the three sites. After injection of WGA-HRP into the trachea, labeled neurons were found bilaterally in the nA from 0.5 mm rostra1 from the AP to the caudal pole of the facial nucleus, and in the dmnX throughout the medulla oblongata. The cell bodies labeled by tracheal injection were scattered in the principal column of the rostra1 nA, with a peak at the most rostra1 part (Fig. 2). These cell bodies, observed by a light microscope, were fusiform or circular, and were 15-30 pm in diameter (Fig. 3a,b). After injection into the SLN, labeled neurons were found ipsilaterally in the rostra1 nA at the same levels as those observed after tracheal injection. The labeled cells were distributed in the principal column as after the tracheal injection, and in the medial column. The labeled cells in the medial column were fusiform with dimensions of 20-35 pm. After injection of WGA-HRP into the RLN, labeled neurons were found ipsilaterally in the nA, throughout the medulla oblongata with the peak at the obex level, and scattered in the dmnX (Fig. 2). The nA neurons that were labeled at the obex level by injection into the RLN were large and polygonal in shape, with maximum dimensions of 35-50 Km. Neurons in the principal and medial columns of the rostra1 nA were also labeled by RLN injection. The ultrastructure of WGA-HRP labeled neurons in the principal column of the rostra1 nA were observed after injection of WGA-HRP into the trachea and SLN. HRP-labeling was identified by the electron-dense, crystalline structures produced by the TMB reaction. Many crystalline structures were seen in the cell somas and dendrites (Fig. 4a, b, c). The HRP-labeled neuronal dendrites (Fig. 4b) and somas (Fig. 4c) received many synaptic inputs from unidentified axon terminals. Of 252 WGAHRP labeled neurons, 184 (73.0%) had synaptic contacts on their dendrites, and the others (27.0%, N = 68) had axo-somatic synapses. Of the axo-dendritic synapses, 38.6% (N = 71) could be identified as symmetrical, 49.5% (N = 91) as asymmetrical, and 12.0% (N = 22) could not be characterized, according to the definitions by Peters et al. (24). Of the 68 axo-somatic synapses, 45.6% (N = 3 1) were symmetrical, 44.1% (N = 30) were asymmetrical, and 7 could not be characterized.

O*

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SLN

0 3 P

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30.

3 20. 0 B

.

P 10.

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-4

-3

-2

-1

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2

dlstancefrotnAPmstralend FIG. 2. Histograms of distribution of retrogradely labeled cell bodies in the medulla oblongata. Labeled neurons were found bilaterally after injection of HRP into trachea smooth muscle (trachea), and ipsilaterahy after injection into the SLN or RLN. Each bar shows counts in fifteen 50 pm thick sections spaced at about 500 pm intervals. White columns show numbers of neurons in nA; shaded columns show numbers in dmnX. Each histogram is based on the total of three rabbits. Abscissas: minus, caudal; plus, rostral; 0, rostra1 end of AP.

TRH Immunocytochemistry TRH-LI was observed in fibers and terminals located in the nA, dmnX, NTS, and other nuclei by light microscope. TRHI nerve fibers formed varicosities, distributed in all levels of the rostra1 nA and surrounding regions extending to the ventral surface (Fig. 5a, b). The density of TRH-I neurons in the nA was less than that in the NTS or dmnX. The ultrastructure of the TRH-I neurons was studied in the principal column of the rostra1 nA. TRH-I axon terminals were 1.O-2.5 pm in diameter, and had large and small granular vesicles. TRH-LI was found mainly in large dense granular vesicles,

about 90-140 nm in diameter. These TRH-I axon terminals made contact with neurons in the rostra1 nA through axo-dendritic (Fig. 6) and axo-somatic synapses. TRH-I axons without synaptic contacts, which seemed to be cross sections of varicosities, were frequently observed in the principal column. Combination of HRP Histochemistry with TRHImmunocytochemistry Combined HRP histochemistry and TRH-immunocytochemistry revealed TRH-I axons and their terminals, and more

IWASt-. 1: I

FIG. 3. (a,b) Light photomicrographs injection into the trachea. Each bar:

of retrogradelv labeled cell bodies (arrows) in the principal 50 grn

frequent HRP-retrogradely labeled neuronal somas and dendrites observed in the same tissue after injection of WGA-HRP into the trachea and SLN. TRH-I axons were distributed among the HRP-labeled neurons and occasionally made synapses on HRPlabeled dendrites (Fig. 7a, b). There were 43 TRH-I axons that did not synapse on nA neurons, and 59 TRH-I axon terminals that did. Of the 59 TRH-I axon terminals, 57 synapsed on dendrites (96.6%) and two on somas (3.4%). Of the axo-dendritic synapses, 47.4% (IL’ = 27) were symmetrical, 17.5% (N = 10) were asymmetrical, and 35.1% (N = 20) could not characterized. The two axo-somatic synapses were symmetrical. Of 59 TRHI axon terminals, 10 ( 16.9%) synapsed on HRP-labeled neurons (Fig. 7a, b). All except one of these synapses were axo-dendritic. Five synapses between TRH-I axon terminals and HRP-labeled neurons were symmetrical, and the remaining five could not be characterized. Physiological

Studio

TRH (50 ng/500 nl) was microinjected into the rostra1 part of the nA. Figure 8A shows that microinjection of 50 ng of TRH into the rostra1 nA produced a tracheal pressure change. The tracheal pressure transiently decreased just after the injection, and then increased about 60 s later. The increase persisted for about 6 min. Intravenous application of atropine (2 mg/kg) remarkably reduced the tracheal pressure increase by TRH (Fig. 8B). The peak pressure increase in the trachea due to TRH was 1.18 f 0.65 cmHzO (N = 8, mean f SD), and that was reduced by intravenous atropine to 0.07 f 0.09 cmHzO (N = 3). Bilateral transection of the vagus nerve and the SLN prevented the tra-

column of the rostra1 nA after

Al

IHRP

cheal pressure increase induced by TRH, but the initial decrease was not blocked. Control injection of 500 nl CSF did not induce pressure change (not shown). The sites at which microinjection affected tracheal pressure were in and around the principal column of the rostra1 nA. Although blood pressure was transiently increased by TRH. the change varied, and was not significant. DISCUSSION

The present study, using the the electron-microscope to examine double labeling, verified that TRH-I neurons synapse on vagal preganglionic motoneurons that innervate the trachea. The morphological evidence was supported by physiological evidence that application of TRH into the nA affects tracheal pressure. The distribution of TRH-I fibers has been previously demonstrated in the rat ( 13,17,26), cat (9) and rabbit (1 1,12). Although they were observed to be dense in the dmnX and NTS, some were also seen in the nA of the rabbit. At present, using a new antiserum to enhance the sensitivity of the immunohistochemical staining, we can show moderate density of TRH-I neurons throughout the nA. We could also find more dense distribution in the dmnX and NTS than previously reported (I I, 12). The electron microscopic study showed that TRH-I axon terminals synapse on the dendrites of neurons in the rostra1 nA. The distribution of vagal preganglionic neurons has been investigated in the cat medulla oblongata (15). The extrathoracic trachea of cat is innervated from the rostral nA, but is not innervated by the dmnX (14). Kalia and Mesulam (14) suggested that the rostra1 region of the nA (2-5 mm rostra1 of the obex) contributes heavily to the motor innervation of the extrathoracic

TRH REGULATION

OF TRACHEAL

TENSION

FIG. 4. (a) Electron photomicrograph of HRP-labeled cell body in the principal column of the rostra1 nA after HRP injection into the SLN. Arrows: TMB reaction products. Bar: 5 pm. N: nucleus. (b) Electron photomicro~ph of axo-dendritic synapses (arrow heads) on an HRP-labeled neuron. Arrow: TMB reaction product. ‘: Unidentifi~ axon terminals. Bar: 0.5 pm. (c) Electron photomicrograph of axo-somatic synapses (arrow heads) to an HRP-labeled ceil soma. Arrows: TMB reaction products. *: Unidentified axon terminals. Bar: I Mm.

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FIG. 5. Light photomicrographs of varicosities with TRH-LI (arrows) in (a) and around (b) the principal column of the rostra1 nA. Bars: 50 Wm.

trachea. Results of this present study of rabbits agree with the report concerning localization of retrogradely labeled cell bodies in the nA in cats, but we also found HRP-labeled cells in the dmnX throughout the medulla oblongata. In rabbits, Getz and

FIG. 6. Electron micrograph on an unidentified neuronal

Sirnes concluded from the degeneration of perikarya after tioning a peripheral nerve, that the dmnX supplies thoracil gans like the trachea, the bronchi, and the oesophagus (5). discrepancy between cats and rabbits seems to be a species

of axon terminal with TRH-LI. TRH-I (TRH) and unknown (*) axon terminals process. Arrows: TRH-I granules. Arrow heads: synapses. Bar: 500 nm.

have synaptic

contacts

secc orThe spe-

TRH REGULATION

OF TRACHEAL

TENSION

FIG. 7. (a,b) Examples of synapses (arrow heads) of TRH-I axon terminals (TRH) and retrograde HRP-labeled neuronal process. Small arrows: TRH-I granules. Large arrows: TMB reaction products Bars: 1 gm. *: unidentified axon terminals.

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(A) PT

-r”----c--[“, -

(B) 5

13 Cldi20

PT -,

m-2

--

-

--------a

i-i” FIG. 8. Effects of 50 ng TRH infused into rostra1 nA. Before (A) and after(B) 2 mg/kg atropine IV. Upper traces (PT), tracheal pressure measured by balloon; lower traces (BP), blood pressure in femoral artery. Underlines. infusion of TRH.

cific difference. Our results indicate that vagal preganglionic motoneurons that innervate the trachea are located in the principal column of the rostra1 nA and scattered throughout the dmnX. The medial column of the rostra1 nA, which is known to innervate the larynx (3) was labeled by SLN and RLN injections, but not by tracheal injection. Another region that was labeled by both SLN and RLN injections, was the principal column of the rostra1 nA. The upper trachea is known to be innervated by the SLN and RLN (1,2). which suggests that some

neurons in the principal column innervate the trachea via the SLN and/or RLN. The HRP-labeled neurons in the principal column of the rostra1 nA received many synaptic inputs on their somas and dendrites, and the combination of HRP histochemistry with TRH immunocytochemistry showed that TRH-I axon terminals synapse on the dendrites and somas of HRP labeled and unlabeled neurons. Of the 59 TRH-I axon terminals examined, 16.9% were found to synapse on HRP-labeled neurons. The crystalline structure that characterizes HRP-labeled neurons may not be evident in all of the HRP-labeled neurons examined. so 16.9’% is the smallest proportion of HRP labeled dendrites that could be identified by the crystalline structure. The observed TRH-1 synapses on HRP-labeled neurons were nearly all on dendrites. The synapses between the TRH-I neurons and HRP-labeled neurons provide evidence that TRH-containing neurons affect vagal preganglionic neurons that innervate the trachea. It has been reported that TRH has both excitatory and inhibitory effects on neuronal activity in the dmnX (9,28). Microinjection shows that TRH affects vagal motoneurons and causes tracheal pressure to first decrease and then increase (Fig. 8). The tracheal pressure increase, but not the decrease. induced by TRH was blocked by bilateral transection of the vagus nerve and SLN. so the increase was mediated by vagal preganglionic neurons. Furthermore, since this stimulatory effect was blocked by atropine administered IV. it was mediated by muscarinic receptors in the tracheal smooth muscle. The dilation just after injection was an equivocal response since it remained after the denervation. TRH-I fibers in the NTS and dmnX are known to project from the raphe nuclei. and the paraolivary and parapyramidal regions in the medulla oblongata. but not from the hypothalamus (10,17,23.3 I ). The neural origin of the TRH-I axon terminals in the rostra1 nA thus seems to be a region in the medulla oblongata in which TRH-I cell bodies were previously found (I 1.12). Further investigation is needed to verify this.

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