THE JOURNAL OF COMPARATIVE NEUROLOGY 311211-288 (1991)

Thyrotropin-Releasing Hormone-ImmunoreactiveProjections to the Dorsal Motor Nucleus and the Nucleus of the Solitary Tract of the Rat RICHARD B. LYNN, MARGARET S. KREIDER, AND RICHARD R. MISELIS Department of Medicine, Jefferson Medical College, Philadelphia 19107 (R.B.L.), and Department of Psychiatry (M.S.K.)and Department of Animal Biology and Institute of Neurological Sciences (R.R.M.),University of Pennsylvania, Philadelphia 19104, Pennsylvania

ABSTRACT Thyrotropin-releasing hormone-immunoreactive nerve terminals heavily innervate the dorsal motor nucleus and nucleus of the solitary tract, whereas cell bodies containing thyrotropin-releasing hormone reside most densely in the hypothalamus and raphe nuclei. By using double-labelingtechniques accomplished by retrograde transport of Fluoro-Gold following microinjection into the dorsal motor nucleus/nucleus of the solitary tract combined with immunohistochemistry for thyrotropin-releasing hormone, it was demonstrated that thyrotropin-releasing hormone-immunoreactive neurons projecting to the dorsal motor nucleus/ nucleus of the solitary tract reside in the nucleus raphe pallidus, nucleus raphe obscurus, and the parapyramidal region of the ventral medulla, but not in the paraventricular nucleus of the hypothalamus. The parapyramidal region includes an area along the ventral surface of the caudal medulla, lateral to the pyramidal tract and inferior olivary nucleus and ventromedial to the lateral reticular nucleus. Varying the position of the Fluoro-Gold injection site revealed a rostra1 to caudal topographic organization of these raphe and parapyramidal projections. Key words: vagus, raphe nuclei, parapyramidal, gastrointestinal,neuropeptide

The cell body source of the extensive thyrotropinreleasing hormone (TRH) containing axonal terminal field in the dorsal motor nucleus of the vagus (DMN) and nucleus of the solitary tract (NTS) is presumably in the raphe system but has not been directly demonstrated in a double-labeling study. In addition, the topographic organization of TRH positive neurons within the raphe system in relation to their projections to the DMN/NTS has not been studied. Palkovits et al. ('86) reported that knife cuts made ventral to the DMNiNTS markedly reduced TRH in the DMN/NTS as assessed by immunohistochemistry. Experiments employing knife cuts through the midbrain, which presumably interrupted pathways from the hypothalamus to the medulla, did not decrease the quantity of TRH in the DMN/NTS as assessed by radioimmunoassay. Similarly, bilateral electrolytic lesions of the paraventricular nucleus of the hypothalamus (PVN) did not decrease TRH in the NTS by immunohistochemistry (Siaud et al., '87). These findings suggest that the TRH innervation of the DMN/ NTS originates not in the hypothalamus? but rather in the ventral medulla. However, this is a limited approach that does not define the specific location of these neurons or o 1991 WILEY-LISS, INC.

their topographicorganization. Double-labelingstudies have demonstrated substance P and serotonin (Thor and Helke, '87), as well as enkephalin and somatostatin (Millhorn et al., '87) in neurons projecting to the DMN/NTS. However, the location of the TRH neurons projecting to the DMN/ NTS has not been demonstrated by these techniques. Thyrotropin-releasing hormone is a neuropeptide in neuronal cell bodies in well-defined locations in the brain, most abundantly in hypothalamic and raphe nuclei (Johansson and Hokfelt, '80; Lechan and Jackson, '82; Kreider et al., '85; Liposits et al., '87; Tsuruo et al., '87; Iwase et al., '88; Merchenthaler et al., '88; Hokfelt et al., '89). TRHimmunoreactive (IR) nerve fibers are widely distributed in the CNS with a high density in the median eminence and DMN/NTS (Hokfelt et al., '75; Kreider et al., '85; Iwase et al., '88; Merchenthaler et al., '88; Rinaman et al., '89a). TRH-IR nerve terminals make asymmetric type synapses Accepted May 15,1991. Address reprint requests to Dr. Richard R. M i d i s , School of Veterinary Medicine, University of Pennsylvania, 3800 Spruce St., Philadelphia, PA 19104-6045.

R.B. LYNN ET AL.

272 (presumably excitatory) onto dendrites of gastric motoneurons (Rinaman and Miselis, '90). Dendrites of DMN motor neurons are known to exist in the NTS and within the DMN (Shapiro and Miselis, '85a; Altschuler et al., '90) and the TRH asymmetric synapses occur in both places as well as on some gastric motoneuron cell bodies in the DMN (Rinaman and Miselis, '90). Gastric sensory terminals, which are predominantly in the gelatinosus subnucleus of the NTS (NTSgel), make monosynaptic contacts with dendrites of gastric motor neurons extending up into the NTSgel (Rinaman et al., '89b). However, we do not know the function of these contacts or whether TRH terminals in the NTSgel modulate them. Receptors for TRH have been demonstrated in the DMN/NTS by autoradiography (Manaker et al., '85). Within the DMN/NTS, the highest TRH receptor concentration was in the medial DMN (Manaker and Rizio, '89), an area that primarily innervates the stomach and particularly the antral-pyloric region (Shapiro and Miselis, '85a; McConnie et al., '88; Okumura and Namiki, '90). There were also high concentrations of TRH receptors in the lateral two-thirds of the DMN, the hypoglossal nucleus and the gelatinosus, centralis, and medial subnuclei of the NTS rostral to the area postrema (AP)(Manaker and Rizio, '89). Thyrotropin-releasing hormone stimulates a multitude of autonomic responses when injected intracerebroventricularly in rats (Koivusalo et al., '79; Tache et al., '80, '89; Hedner et al., '81; Tsay and Lin, '82; Holtman et al., '86a; Paakkari et al., '86; Okuda et al., '871, including stimulation of gastric acid output in excess of the response to pentagastrin intravenous infusion (Tache et al., '83). When TRH is microinjected into the DMN/NTS, there are marked physiologic responses including stimulation of gastric acid secretion (Rogers and Hermann, '85; Okuma et al., '87; Ishikawa et al., '88; Stephens et al., '88; Feng et al., '90; Lynn et al., '91), gastric contractions (Rogers and Hermann, '87; Garrick et al., '89; Hornby et al., '89; Feng et al., 'go), formation of gastric mucosal erosions (Hernandez and Emerick, '881, shortening of respiratory inspiratory time (McCown et al., '861, and an early depressor effect on blood pressure (Feuerstein et al., '83). Thus there is strong evidence supporting the hypothesis that TRH is a neurotransmitter in the DMN/NTS with important influence on gastric and other autonomic functions. To determine the specific location of TRH-IR neurons that project to the DMN/NTS, we combined retrograde neuronal tracing with immunohistochemistry for TRH. The fluorescent neuroanatomical tracer Fluoro-Gold, which

retrogradely labels neuronal cell bodies and remains within the labeled cell, was used in combination with a Texas Red tagged secondary antibody to the TRH primary antibody. We have chosen a double fluorescence technique as it enables confident identification of double-labeled cells.

METHODS Sprague-Dawley rats (250-375 g) were used in this study. The animals were anesthetized with Ketamine (85 mg/kg; ip) and Xylazine (12 mgkg; ip) and were placed in a stereotaxic instrument (David Kopf, Tujunga, CAI. The head was flexed ventrally, the skin and muscle of the dorsal neck were incised in the midline from the nuchal crest to the second cervical vertebra, and the nuchal musculature bluntly dissected laterally to expose the atlanto-occipital membrane. The latter was excised to reveal the dorsal surface of the caudal medulla. The tracer-laden glass micropipette was then lowered into the appropriate brain structures by a micromanipulator under direct visual guidance. The depth of placement below the surface of the brain was controlled with the assistance of the stereotaxic instrument. Tracer was pressure injected via a glass micropipette (tip O.D. 100 pm, beveled) affixed with sealing wax to the tip of a 1.0-pl syringe (Hamilton, Reno, NV). Pressure injections were applied by depressing the plunger approximately 10 nl increments with 30-second pauses between increments. All injections reported in this study were 40 nl, which was found in preliminary trials to be the smallest injection that adequately labeled neurons retrogradely. The tracer used was Fluoro-Gold (Fluorochome, Englewood, CO) 2% solution in saline stored in frozen aliquotes. After a 5-minute wait, the micropipette was withdrawn at a rate of 0.1 mm/minute (to permit the tissue to seal along the pipette tract). The skin was closed with surgical clips. After 5 days, the animal was reanesthetized with Ketamine (85 mg/kg; ip) and Xylazine (12 mg/kg; ip) and placed in the stereotaxic apparatus. Colchicine (Sigma, St. Louis) 50 pg/lOO g body weight (dissolved in 5-10 p1 of saline) was injected unilaterally into the lateral ventricle. The scalp incision was closed with surgical clips. After 48 hours the animal was heparinized, deeply anesthetized with pentobarbitol (30 mg in 0.5 ml; ip), and perfused through the ascending aorta with 300 ml of heparinized phosphatebuffered saline solution (PBS), (pH 7.4) at room temperature. This was followed by 300 ml of ice-cold 8.3% paraformaldehyde, 0.2% picric acid, and 0.05% glutaraldehyde in 0.1

Abbreuiations

3v 4v V VII XI1

AP cc

cu DMN DVC ECu Gr

I0 LPG LRt mNTS

third ventricle fourth ventricle spinal trigeminal nucleus facial nucleus hypoglossal nucleus area postrema central canal cuneate nucleus dorsal motor nucleus of the vagus dorsal vagal complex external cuneate nucleus gracile nucleus inferior olive lateral paragigantocellular nucleus lateral reticular nucleus medial division of the solitary tract

NA-C NAsc NTS NTScen NTScom NTSgel NTSmed PVN PrH

PY RO RP RMg TRH tS Ve

nucleus ambiguus-compact formation nucleus ambiguus-semi-compactformation nucleus of the solitary tract subnucleus centralis of NTS commissural subnucleus of NTS subnucleus gelatinosus of NTS medial subnucleus of NTS paraventricular nucleus of the hypothalamus prepositus hypoglossal nucleus pyramidal tract nucleus raphe obscurus nucleus raphe pallidus nucleus raphe magnus thyrotropin-releasing hormone solitary tract vestibular nucleus

Fig. 1. A. Fluorescence photomicrograph montage of a coronal section of the dorsomedial medulla of case LF 51 showing the microinjection site of Fluoro-Gold in the DMNNTS. The zone of tracer uptake is the dense core of brilliant fluorescence. and is confined to the NTS

and lateral tip of the DMN. B. Brightfield photomicrograph of the same section as in A after thionine staining to allow definition of cell groups. Bar in A = 118 Frn in both A and B.

R.B. LYNN ET AL.

274 TABLE 1. Description of Injection Sites’

~

~

~

~

Zone 3

Zones 1 & 2 (areaof tracer uptake) Center of the injection

Case

Maximal diamete? med-lat/mst-caud

~

LF55

Rostral DMN (850 pm rostral to AP)

Avg distance bevond zone 2

Structures included ~

~~~~

-rostral DMN

0.35 md0.6 mm

0.36 mm

-rostral parts of NTSgel & NTScen

LF 47

NTSgel(100pmrostraltoAP)

0.40 m d 0 . 6 mm

LF 51

Lateral third of DMN (at level of the rostral edgeofAP)

0.30 md0.44 mm

LF 56

Medial third of DMN (at level of the rostral edge of AP)

0.25 mmi0.3 mm

LF 58

Medial third of DMN (200 pm caudal to the rostral edge of AP)

0.26 m d 0 . 4 mm

LF 57

Caudal DMN (850 pm caudal to the rostral

0.25 mmi0.4 mm

medial divieion of the NTS rostral to the NTSgel & NTScen -lateralthird of DMN most of the NTSgel & NT&en -parts of the medial, commissural & interstitial subnuclei of the NTS -partof the solitary tract -nu gracile -lateral third of DMN dormlateral corner of XU -NTScom & NTSmed medial third of DMN -dorsal edge of XI1 -NTseOm & NTSmed -medial edge of contralateral NTSwm -rostrolateral corner of AF’ medial third of DMN -dorsal edge of XI1 -NTScom & NTSmed -medial edge of contralateral NTseom -rostrolateral comer of AF’ medial half of caudal DMN -NTScom bilaterally caudal aspeet of NTSmed -Caudaltip of AP -dorsomedial mrner of XI1

edgeofAP)

0.35 mm

0.35 mm 0.3 mm

0.37 mm

0.42 mm

‘All injections were 40 nl volumes. ‘See Figure 2 for illustrations of extent of maximal diameters

LF55

4v

M PBS. The brain was removed, placed in the same ice-cold fixative for 90 minutes, followed by 30% sucrose in PBS (pH 7.4) for 48 hours. Three animals were serially sectioned through the entire brain. The remaining brains (including those reported in Table 2) were serially sectioned through the medulla and the hypothalamus. Brains were frozen-sectioned on a rotary microtome at 30-pm thickness, and four 1-in-4 sets of serial sections were collected in ice-cold PBS. Sections were incubated (free-floating) in rabbit anti-TRH antiserum (Kreider et al., ’85)diluted 1:800 in a solution of 0.5%BSA, 0.2% Triton-PBS for 48 hours at 4°C. The sections were then rinsed in PBS (3 x 5 min) and incubated in Texas Red (TXR)conjugated to goat anti-rabbit IgG antiserum (Jackson, West Grove, PA) 1:200 in a solution of 1.0% goat serum, 0.3% Triton-PBS at room temperature. Sections were then rinsed in PBS and placed on glass slides, air dried, and coverslippedwith glycerine 19:l in PBS. Sections were examined and photographed with an Olympus BH-2 microscope with fluorescent attachment. FluoroGold was visualized with the “UG1” filter cube (excitation light BP 375; dichroic mirror DM 400; barrier filter LP

TABLE 2. Total Number of Neurons Containing Thymtropin-Releasing Hormone (TRH),Fluoro-Gold (FG),or Double-Labeled with Both TRH and Fluoro-Gold (DL)’ nu. raphe pallidus Case

TRH

FG

DL

nu. raphe obscurus TRH

FG

DL ~

Fig. 2. Schematic illustration depicting the location and size of Fluoro-Gold microinjection sites. The shaded areas represent the maximal size of the core of brilliant fluorescence which represents the zone of uptake of the tracer. The injection sites which are at differing depths are projected onto the drawing of a horizontal section of the medulla at the level of the AP.

LF55 LF47 LF51 LF56 LF58 LF57 LF54

144

20 29

158

40

185

17 19 43 92

199

200 140 175

7 14 12 8

9 19 37

152 126 131 180 134 134 163

‘Absolute #‘s from only every fourth section.

45 52 49 50 21 41 121

14 4

Parapyramidal TRH

16

123 89 180 122 159 130

31

140

10 21 15

FG

_________

27 52 96 25 52 52 119

DL ~

3 7 20 9 23 16 38

TRH PROJECTIONS’TO THE DMN/NTS

275

TABLE 3. Topographic Distribution of the Total # of Neurons Containing Thyrotropin-ReleasingHormone (TRH), Fluoro-Gold (FG), and Double-Labeled with Both TRH and Fluoro-Gold (DL)’ Caudal to the rostral edge of the area postrema nu. raphe pallidus

nu. raphe obscurus

Parapyramidal

Case

TRH

FG

DL

TRH

FG

DL

TRH

FG

LF 55

27

6

2

39

17

4

22

5

1

36 39

10 25

4 15

44 48

10 13

6 11

30 27

30 15

11 11

DL

(I’OStFd)

LF 51,56,58’ LF 57 (caudal)

Rostral to the area postrema nu. raphe pallidus Case LF 55 (FOStrd) LF51,56, 5Sa LF 57 (caudal)

nu. raphe obscurus

Parapyramidal

TRH

FG

DL

TRH

FG

DL

TRH

FG

DL

172

14

5

113

28

10

101

22

2

145 101

16 18

6 4

104 86

30 28

9 5

123 103

27 37

6 5

’Absolute #’s from only every fourth section. ‘Means of counts from three animals with microinjection sites in the dorsal vagal complex at the level of the area postrema

420). Texas Red was visualized with the “G” filter cube (exciting filter BP 545; dichroic mirror DM 580; barrier filter 590). Kodak Tri-X or T-max film (ASA 400) was used for photography. Sections in the glycerine coverslipped group were traced by using an overhead projector. The Fluoro-Gold and Texas Red labeled cells were hand drawn onto the traced outlines. After completion of this stage of study, the slides were uncoverslipped, thionine counterstained, dehydrated, xylene cleared, coverslipped with DPX, and reexamined to determine the location of labeled cells, and to prepare schematic drawings. Data were quantified for (1)the number of TRH-IR neurons, (2) the number of Fluoro-Gold labeled neurons in specific nuclei, and (3) the number of neurons double labeled with both Fluoro-Gold and TRHimmunoreactivity. We were very conservative in calling a cell labeled. The Fluoro-Gold had to be bright enough to be easily visible; thus cells that seemed lightly labeled were not counted. Similarly, the Texas Red labeled TRH-IR cells had to be distinct from the background and distinguishable as an individual cell; thus lightly labeled and overlapping cells were not counted. In some cells a nuclear pallor was visible, but in most fluorescence labeled cells this is obscured. To avoid counting cuts through edges of cells, we restricted our counts to cells that showed nuclear pallor, dendritic or axonal extensions, or seemed to be full-size profiles of cell bodies. These standards improve the confidence of our cell counts but increase the likelihood that we are underestimating the true number of cells in each category. For the specimens undergoing serial sectioning, one set of sections was used for control procedures. The primary anti-TRH antiserum was preabsorbed with TRH peptide (Peninsula, Belmont, CA) 100 p,M for 2 hours before use. These sections were otherwise treated exactly the same as above. The non-preabsorbed anti-TRH antiserum solution was also prepared 2 hours before use. A third set of sections was air dried, dehydrated, cleared with xylenes, and coverslipped with DPX mounting medium. A fourth set of sections was air dried after sectioning, counterstained with thionine, dehydrated, cleared with xylenes, and coverslipped with DPX. The nodose ganglia were removed from 4 rats, sectioned, and stained as above. In 2 additional rats, colchicine-soaked

gauze was placed around one nodose ganglion. After 48 hours, the animals were perfused as above and the nodose ganglia were dissected out. This tissue was frozen sectioned and stained as above.

RESULTS Anatomic definitions and nomenclature Since the hindbrain is the likely source of TRH-IR projections to the DMN/NTS and most hindbrain TRH-IR neurons are located in the midline and ventral surface of the medulla, we review the anatomy of these areas. The medullary raphe nuclei are midline structures whose boundaries have been well described in the rat (Steinbusch and Nieuwenhuys, ’83; Tork, ’85). One fine point is that the rostral edge of the nucleus raphe obscurus ends abruptly at the level of the caudal pole of the facial nucleus and the nucleus raphe magnus extends rostrally from this level. However, the nucleus raphe pallidus overlaps rostrally with the caudal pole of the facial nucleus. In the current study, cells along the most medial aspect of the dorsal surface of the pyramids are included in the nucleus raphe pallidus as these neurons seem to be contiguous lateral extensions of this nucleus. We use the term “parapyramidal region” to describe neurons located close to the pyramidal tract, along its dorsal surface and lateral edge, and extending farther laterally along the ventral surface of the medulla (Helke et al., ’89). We use the rostral edge of the area postrema (AP)as the rostrocaudal zero point as it is easier to locate than the anatomic obex, which is about 80 p,m farther caudal. Distances in our rats may be slightly less than those in published atlases because of tissue shrinkage caused by perfusion with hypertonic fixative.

TRH-immunoreactive (IR) neurons In the brainstem, TRH-IR neurons were found almost exclusively in the nucleus raphe pallidus, nucleus raphe obscurus, nucleus raphe magnus, and in the parapyramidal region (see Table 2). TRH-IR neurons were numerous throughout the nucleus raphe pallidus and the nucleus raphe obscurus. TRH-IR neurons were found only in the

ECu

+1.4mm

ECu

Cu

f0.58mm

-0.14mm

1.1mm 0

0

TRH only Fluoro-Gold only Double labeled for both TRH and Fluoro-Gold

R P' Fig. 3. Drawings of four coronal sections through the caudal medulla of case LF 51 showing the distribution of neurons labeled with Fluoro-Gold only, TRH-IR only and neurons double labeled for both Fluoro-Gold and TRH-immunoreactivity.Each symbol represents one neuron. Levels of the medulla are relative to the rostra1 edge of the AP.

TRH PROJECTIONS TO THE DMN/NTS

Fig. 4. Fluorescence photomicrographs of TRH-IR (A and C)and Fluoro-Gold labeled (B and D) neurons in the nucleus raphe pallidus in coronal sections of the caudal medulla. Paired photomicrographs (A and B, C and D) are of the same brain sections viewed with different fluorescence filters. Arrows point to neurons double labeled for both TRH-immunoreactivity and Fluoro-Gold. Solid arrowheads point to

277

examples of neurons that contain TRH but not Fluoro-Gold, and outlined arrowheads point to neurons that contain Fluoro-Gold but are not TRH-IR. A and B are from a section from case LF 61; C and D are from case LF 54. Both sections are approximately at the same level as the section in Fig. 5B. Bar in D = 20 pm in A and B, and 25 pm in C and D.

Fig. 5. Low-magnification brightfield photomicrographs of two brain sections in the caudal medulla after thionine staining. A is at the level of the rostral edge of the Ap. The rectangle in the nucleus raphe obscurus outlines the area shown in Figure 6A, B. B is at alevel1.9 mm rostral to A at the level of the caudal pole of the facial nucleus.

TRH PROJECTIONS TO THE DMN/NTS caudal aspects of the nucleus raphe magnus. In the parapyramidal region, TRH-IR cells were most numerous along the ventrolateral edge of the pyramidal tract at levels rostral to the AP. There were occasional isolated TRH-IR neurons in the reticular formation. However, we found no TRH-IR neurons in the dorsomedial medulla or in other raphe nuclei. In the forebrain, TRH-IR neurons were densely packed in the parvicellular parts of the paraventricular nucleus, especially the periventricular, anterior, and medial subdivisions. TRH-IR cells encircled the fornix anteriorly in the bed nucleus of the stria terminalis, as well as caudally at the level of the PVN with a cluster medial to the fornix and below the lateral parvicellular subdivision of the PVN. TRH-IR neurons were in the rostral supraoptic nuclei and clustered at the dorsal medial edge of the optic tract dorsal to the supraoptic nuclei, with labeled cells extending dorsally into the lateral hypothalamic area to join the group of cells around the caudal fornix. Preabsorption with TRH peptide in control sections abolished Texas Red labeling of cell bodies in all the above areas in the brainstem and forebrain.

279

DMN at this level of the medulla. Case LF 55 was a more rostral injection at a level about 850 km rostral to the rostral edge of the AP and included the DMN and the surrounding NTS at this level. Case LF 57 was a caudal injection with the dense core mostly caudal to the caudal tip of the AP and centered at about 850 krn caudal to the rostral edge of the AP. This injection site was centered on the DMN, which is rather medial at this level. Cases LF 54, LF 52, and LF 61 received four injections into the DMN, two on each side at both the rostral edge and caudal tip of the AP. Fluoro-Gold labeled cell bodies were in forebrain structures including dense clusters in the PVN especially the anterior, medial, and lateral subdivisions, and the central amygdala nucleus. Scattered cells were in the lateral hypothalamus, the bed nucleus of the stria terminalis, and the insular cortex. In the hindbrain, cells were in the nucleus raphe pallidus, nucleus raphe obscurus, nucleus raphe magnus, dorsal raphe nucleus, parapyramidal region, paratrigeminal nucleus, and scattered throughout the reticular formation with a rare cell in the median raphe nucleus. The parapyramidal labeling was bilateral. The cell counts of Fluoro-Gold cells in Tables 2 and 3 are limited to the nuclei Fluoro-Gold injection sites and labeled or regions where the TRH-IR neurons were located, which are the nucleus raphe pallidus, nucleus raphe obscurus, and neurons parapyramidal region and do not include any Fluoro-Gold The Fluoro-Gold injection site has three distinct zones: cells elsewhere in the medulla. In Table 3 we average the (1) the needle tract and a small but variable region of results of three rats whose injection sites were at the level of necrosis at the center of the injection site, (2) the dense-core the AP (LF 51, 56, and 58) and compare these averages to region of brilliant fluorescence (Fig. l),which is presum- results after the most rostral (LF 55) and caudal (LF 57) ably the zone of active terminal uptake (Schmued and injections. Cells in the caudal aspects of the nucleus raphe Fallon, '86), and (3) peripheral to this is a larger sphere of pallidus were preferentially labeled by the most caudal less intense staining due to diffusion of the dye away from injection site, whereas the rostral aspects of this nucleus the site of injection. It has been reported that the third zone were preferentially labeled by the most rostral injection of diffusion contributes little to the transport of the dye site. The most rostral injection site labeled very few cells in (Schmued and Fallon, '86). the parapyramidal region. In our experiments, all injections were 40 nl and the spherical dense core ranged 400-600 +m in maximal diameDouble-labeledneurons ter. With all injections reported in this study, there was a Neurons double labeled with both Fluoro-Gold and TRHsmall amount of leakage of Fluoro-Gold out the pipette tract spilling onto the dorsal surface of the brain in an area immunoreactivity were located exclusively in the nucleus limited to the immediate surroundings of the injection site raphe pallidus, nucleus raphe obscurus, and the parapyrawith no spread of tracer to the rest of the surface of the midal region (Table 2; Figs. 3-8). In both the nucleus raphe medulla. Control injections onto the surface of the medulla pallidus and nucleus raphe obscurus, the double-labeled mimicking the amount of leakage from the experiment neurons were located throughout the entire extent of the injections did not label neurons in the raphe nuclei or nuclei. In the nucleus raphe pallidus, some double-labeled ventral medulla. Control injections of 40 nl into the fourth cells were very large round neurons about 30 km in ventricle labeled the ependyma of the caudal fourth ventri- diameter, and others were smaller oval cells approx. 18 krn cle and some neurons just below, but did not label any mostly along the ventral surface. In general, no particular neurons in the raphe nuclei or ventral medulla. These size or shape of the TRH-IR neurons appeared to be controls confirm that the small degree of leakage of the preferentially double labeled in the nucleus raphe pallidus. tracer out the injection tract in our study did not contribute In the nucleus raphe obscurus, most double-labeled neuto the retrograde labeling of neurons. rons were bipolar or tripolar, about 22 km long and 8 pm Seven rats were studied quantitatively with serial sec- wide oriented vertically. Some were spherical shape cells tions. Six rats received single microinjections with varied approx. 15 pm in diameter. Double-labeled TRH-IR neurons in the parapyramidal injection sites as illustrated in Figure 2 and described in Table 1. In case LF 51 (Figs. 1, 21, the dense core of the region were most commonly found in the caudal medulla microinjection site was centered on the lateral DMN at the along the ventral surface of the brain (Figs. 7, 8). These level of the AP. In cases LF 56 and LF 58, the micropipette neurons were arranged in a discrete longitudinal column passed through the lateral AP, and the dense core of the extending from the level of the decussation of the pyramids injection site included the medial aspect of the DMN as well to the rostral edge of the AP. This column was located as the surrounding areas such as the commissural and ventromedially to the lateral reticular nucleus and was medial subnuclei of the NTS. In case LF 47, the injection closer to this nucleus than to the pyramids. These neurons site was more rostral and superficial centering on the were typically spindle shaped, 20 pm long and 10 pm wide, subnucleus gelatinosus of the NTS and including the parallel with the direction of the ventral surface of the brain subnucleus centralis of the NTS and the lateral tip of the in the cross-sectionalplane. Additionally, occasional double-

280

R.B. LYNN ET AL.

Figure 6

281

TRH PROJECTIONS TO THE DMN/NTS labeled cells in the parapyramidal region were found rostral to the AP at levels where TRH-IR cells were more numerous. These double-labeled cells were either thin, spindleshape neurons along the dorsal surface of the pyramid, or round, multipolar cells immediately lateral to the pyramid along the ventral surface. Double-labeledneurons in all three areas were dispersed longitudinally such that they were found in many sections rather than clustered at any particular level. Whereas the locations of these double-labeled neurons were the same in all rats studied, the number and distribution varied with the location of the injection site (Table 3). Case LF 57 received the most caudal injection and clearly had a caudal shift in the distribution of double-labeled neurons. For example, in sections caudal to the rostral edge of the AP, there were three times as many double-labeled neurons in the nucleus raphe pallidus and twice as many in the nucleus raphe obscurus when compared to the average counts of the three animals with injections at the level of the AP.Rostra1 to the AP,case LF 57 had fewer double-labeledcells in both the nucleus raphe pallidus and nucleus raphe obscurus than the three averaged cases. Along the caudal aspects of the ventral surface of the medulla of case LF 57, a very high percentage of the Fluoro-Gold labeled cells were double labeled with TRH-IR and these were clustered in the most caudal few sections of the medulla. Case LF 55 received the most rostral injection site and there was a clear rostral shift of the double-labeled neurons in the raphe nuclei. Interestingly, in case LF 55 there was a decrease in the number of double-labeled cells throughout the parapyramidal region with a scarcity of either Fluoro-Gold or double-labeled neurons in this region below the rostral edge of the AP. In all 7 rats in which serial sections were studied and quantified, the number of TRH-IR neurons did not vary significantly. However, the number of the Fluoro-Gold retrogradely labeled cells did vary from animal to animal. In general, the number of double-labeled neurons tended to vary proportionally to the number of Fluoro-Gold cells. In case LF 54, we made multiple injections of tracer and clearly increased the number of Fluoro-Gold cells and thereby increased the yield of double-labeled cells (Table 2). There were no double-labeledneurons in the paraventricular nucleus of the hypothalamus (Fig. 9) or in any forebrain location. Nodose ganglia were removed bilaterally from 4 rats that received colchicine icv, and unilaterally from 2 rats that had colchicine applied directly onto the ganglion. In all cases the entire ganglia were studied. In each case there were numerous cells retrogradely filled with Fluoro-Gold. However, there was no evidence of TRHimmunoreactivity in any nodose ganglia.

DISCUSSION

The present study shows that TRH-IR neurons projecting to the DMN/NTS are located in the nucleus raphe pallidus, nucleus raphe obscurus, and the parapyramidal region of the ventral medulla and have a topographical organization. The nucleus raphe pallidus and nucleus raphe obscurus are the most caudal of the raphe nuclei, have well-defined boundaries, contain most of the TRH-IR neurons in the medulla, and were the expected origin of the TRH-IR neurons projecting to the DMN/NTS (Palkovits et al., '86; Siaud et al., '87). The parapyramidal region has not been previously discussed in the literature as a likely source of the TRH-IR pathways. We have found that a significant proportion of the TRH-IR projections originate in this region (Table 2). Whereas double-labeled cells were scattered throughout this region, most were located caudal to the AP along the ventral surface, lateral to the inferior olivary nucleus, and immediately ventromedial to the lateral reticular nucleus. We found no double-labeledneurons clearly within the nucleus raphe magnus. However, the transition from nucleus raphe pallidus to nucleus raphe magnus can be difficult to define; thus it is possible that some double-labeledneurons were in the later nucleus. The term "parapyramidal region" refers to an area along the ventral surface of the medulla including neurons dorsal and lateral to the pyramidal tracts and neurons near the ventral surface of the medulla lateral to the pyramids. An early study limited the parapyramidal region to the rostral parts of the medulla, at levels of the nucleus raphe magnus (Johansson et al., '80). Helke et al. ('89) have redefined the parapyramidal region to extend caudally to the level of the pyramidal decussation. The parapyramidal region includes or overlaps with various previously identified cell groups or areas of physiologic responsiveness. Since many of these neurons contain serotonin, they have been considered lateral extensions of both B1 in the caudal medulla and B3 more rostrally at levels of the facial nucleus (Dahlstrom and Fuxe, '64; Loewy and McKellar, '81; Steinbusch, '81; Skagerberg and Bjorklund, '85). The parapyramidal region overlaps with the pars alpha of the gigantocellular reticular nucleus as well as the medial and ventral aspects of the lateral paragigantocellular nucleus (Andrezik et al., '81). Other terminology applied to the ventral surface of the medulla includes the nucleus interfascicularis hypoglossi (NIH) (Chan-Palay et al., '78; Loewy et al., '81; Skagerberg and Bjorklund, '85; Ciriello et al., '881, the "paraolivary" region (Johansson et al., 'Sl), and the nucleus ventralis subolivaris (Gorcs et al., '86). We found a cluster of double-labeled neurons along the ventral surface of the most caudal levels of the medulla, lateral to the tip of the inferior olivary nucleus (101, and medial to the magnocellular part of the lateral reticular Fig. 6 . Fluorescence photomicrographs of TRH-IR (A and C) and Fluoro-Gold labeled (B and D) neurons in the nucleus raphe obscurus nucleus. We include this area in the parapyramidal region, in coronal sections of the caudal medulla. Paired photomicrographs (A thereby extending this region more laterally along the and B, C and D) are of the same brain sections viewed with different ventral surface than previously defined. fluorescence filters. Arrows point to neurons double labeled for both Central afferents to the DMN/NTS have been extensively TRH-immunoreactivity and Fluoro-Gold. Solid arrowheads point to examples of neurons that contain TRH but not Fluoro-Gold, and mapped, but most of these reports have not examined the outlined arrowheads point to neurons that contain Fluoro-Gold but are medulla in detail (Sawchenko, '83; Thor and Helke, '87 for not TRH-immunoreactive.A and B are from a section from case LF 54. reviews). Menetrey and Basbaum ('87) reported only on The same brain section after thionine staining is shown in Figure 5A. trigeminal projections to the NTS. AutoradiographicThe solid rectangle in the nucleus raphe obscurus in Figure 5A outlines the area shown in A and B. C and D are from case LF 61 at anterograde tracing studies have reported d e r e n t s to the approximately the same level as the brain section in Figure 5A. Bar = NTS from the rostral raphe nuclei, including the dorsal raphe (Taber-Pierce et al., '76j, the raphe centralis superior 25 pm.

Figure 7

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TRH PROJECTIONS TO THE DMN/NTS (Bobillier et al., '761, and the raphe magnus (Basbaum et al., '78; Ross et al., '81). A retrograde autoradiographic study reported that the magnus, pontis, median, and dorsal raphe also project to the NTS, but this study did not examine the nucleus raphe pallidus or nucleus raphe obscurus @chaffer et al., '88). Anatomic studies have reported projections to the DMN/NTS originating in the nucleus raphe obscurus (Rogers et al., '80; ter Horst et al., '84; Thor and Helke, '87) and nucleus raphe pallidus (Ross et al., '81; Thor and Helke, '87). Projections from the ventral surface of the medulla to the DMN/NTS have been reported for more rostral areas includmg the nucleus paragigantocellularis lateralis (Loewy et al., '81; Guyenet and Young, '873, the nucleus reticularis gigantocellularis (Zemian et al., '84), and the Al/C1 regions (Blessing et al., '81; Thor and Helke, '881, but projections from the caudal parapyramidal region have been reported in only a single study (Thor and Helke, '87). The nodose ganglion is a well-known source of afferents to the DMNiNTS but has never been shown to contain TRH. In this study, we have found evidence that the FluoroGold labeled projections and, consequently, the TRH-IR double-labeled pathways from the nucleus raphe pallidus, nucleus raphe obscurus, and parapyramidal region to the DMN/NTS are topographically organized such that more rostral neurons project to more rostral parts of the DMN/ NTS, and more caudal neurons project to more caudal parts of the DMN/NTS. Additionally, Fluoro-Gold and especially TRH double-labeled neurons in the parapyramidal region project preferentially to the DMN/NTS at levels of the AP and more caudally with few projections to the rostral injection site. The viscerotopic mapping of the NTS has been more clearly and sharply defined (Altschuleret al., '89, '91) providing a means to relate TRH terminals to sensory input from specific viscera. Fluoro-Gold injection sites probably involved the adjacent borders of the hypoglossal nucleus and area postrema (AF').This is unavoidable because the DMN is a thin nucleus adjacent to the hypoglossal nucleus throughout its rostrocaudal extent, and medially it is ventral to the AP. However, neither the hypoglossal nucleus (Loewy et d., '81; Borke et al., '83; Travers and Norgren, '83) nor the AP (Shapiro and Miselis, '85b) receive innervation from the raphe nuclei or the parapyramidal region. Thus tracer uptake from the hypoglossal nucleus or the AP would not retrogradely fill the double-labeled TRH-IR neurons described in this study. The function of the specific TRH-IR pathways projecting to the DMN/NTS is unknown. However, we can attempt to draw inferences by correlating physiologic responses to exogenous TRH with responses to stimulation of specific loci. Microinjection of L-glutamate into the nucleus raphe

Fig. 7. Fluorescence photomicrographs of TRH-IR (A) and FluoroGold labeled (B)neurons in the parapyramidal region along the ventral surface in a coronal section of the caudal medulla of case LF 51. A and B are of the same brain section viewed with different fluorescence filters. Arrows point to neurons double labeled for both TRH-immunoreactivity and Fluoro-Gold. Solid arrowheads point to examples of neurons that contain TRH but not Fluoro-Gold,and outlined arrowheads point to neurons that contain Fluoro-Gold but are not TRH-immunoreactive. C is a low-magnificationbrightfield photomicrograph of the same brain section as A and B, after thionine staining. The rectangle in C outlines the area shown in A and B. Bar in C = 25 +m in A and B and 277 pm in C.

pallidus and nucleus raphe obscurus increased pyloric motility by vagal dependent pathways (McCann et al., '89; Hornby et al., '90). Microinjection of kainic acid into the nucleus raphe obscurus increased gastric secretion of acid and pepsin (White, '91). The evidence is favorable that the gastrointestinal stimulatory effects of TRH in the DMN/ NTS originate in neurons in the nucleus raphe pallidus and nucleus raphe obscurus, if not the parapyramidal region as well. Central TRH also stimulates the respiratory and cardiovascular systems and increases arousal under a variety of circumstances. TRH injected icv increased minute ventilation by increasing respiratory rate with no increase in tidal volume (Hedner et al., '81; Holtman et al., '86a), and increased mean arterial pressure and heart rate (Koivusalo et al., '79; Tsay and Lin, '82; Paakkari et al., '86; Okuda et al., '87). The ventral surface of the medulla is an area that regulates respiratory function and is important in maintaining arterial blood pressure (Millhorn and Eldridge, '86; for review). Whereas most studies place these functions in the rostral medulla, the ventral surface of the caudal medulla also has chemosensitive areas that influence respiration (Loeschcke et al., '70; Schlaefke et al., '70) and areas that regulate arterial pressure (Feldberg and Guertzenstein, '76; Keeler et al., '84). There is substantial evidence that the caudal medullary raphe nuclei also modulate the respiratory and cardiovascular systems (Adair et al., '77; Yen et al., '83; Holtman et al., '86b; Millhorn, '86; Haselton et al., '88).

The caudal raphe and parapyramidal regions influence the respiratory and cardiovascular systems, in part, by projections to the sympathetic nervous system in the spinal cord (Loewy, '81; Helke et al., '82; Lorenz et al., '85; Skagerberg and Bjorklund, '85). TRH has been demonstrated in bulbospinal projections to the intermediolateral cell column in the spinal cord (Hirsch and Helke, '88; Sasek et al., '90). Thus TRH in the caudal medulla may affect autonomic function by projections to the parasympathetic or sympathetic preganglionic neurons. TRH coexists with serotonin, or substance P, or both serotonin and substance P (Johansson et al., '81; Staines et al., '88; Sasek et al., '90) in neurons in the caudal raphe and parapyramidal region. Similarly, serotonin and substance P neurons in the nucleus raphe pallidus, nucleus raphe obscurus, and parapyramidal regions project to the DMN/ NTS (Thor and Helke, 'a?), and it is likely, but not yet demonstrated, that some of these neurons also contain TRH. The double-labeled TRH-IR neurons we have described in the most caudal medulla are very close to the ventral surface of the brain (Figs. 7,8) and may coexist with serotonin-IR neurons on the surface of the brain as described by Gorcs et al. ('85),or serotonin- or substance P-IR neurons in the brain parenchyma close to the surface (Ciriello et al., '88).TRH often coexists with human growth hormone in perikarya in the nucleus raphe pallidus, nucleus raphe obscurus, and parapyramidal region (Lechan et al., '83). Met-enkephalin- (Finley et al., '81; Hunt and Lovick, '82), somatostatin- (Johansson et al., '84; Vincent et al., '85), GABA-IR (Bowker et al., '881, and a few VIPand CCK-IR (Bowker et al., '88) neurons are found in the caudal raphe and parapyramidal regions, but coexistance with TRH-IR has not been reported. Neurons containing both enkephalin and somatostatin immunoreactivityproject to the DMN/NTS (Millhorn et al., '87), but these were located in the paragigantocellular nucleus and in the RMg;

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Fig. 8. Fluorescence photomicrographs of TRH-IR (A) and Fluoro-Gold labeled (B) neurons in the parapyramidal region along the ventral surface in a coronal section of the caudal medulla of case LF 52. A and B are of the same brain section viewed with different fluorescence filters. Arrows point to neurons double labeled for both TRH-immunoreactivity and Fluoro-Gold. Bar in B = 25 pm.

TRH PROJECTIONS TO THE DMN/NTS

Fig. 9. Fluorescence photomicrographs of Fluoro-Gold (A and B) and TRH-IR (C) neurons in the paraventricular nucleus of the hypothalamus of case LF 51, demonstrating a lack of double labeled neurons in this nucleus. All three panels are of the same brain section. Solid arrowheads point to examples of neurons that contain TRH but not Fluoro-Gold, and outlined arrowheads point to neurons that contain Fluoro-Goldbut are not TRH-immunoreactive. Bar in C = 50 km in A and 25 Fm in B and C.

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thus they are less likely to coexist with TRH-IR neurons projecting to the DMN/NTS. Overall, the caudal raphe and parapyramidal regions are rich in neuropeptide immunoreactivity, making it likely that TRH coexists with other neuropeptides in neurons projecting to the DMN/NTS. The significance of this coexistance remains to be determined. In summary, TRH innervation of the DMN/NTS originates in the nucleus raphe pallidus, nucleus raphe obscurus and parapyramidal region. We have identified a topographic organization to these pathways which are the putative neuroanatomic substrate for the important physiologic observations pertaining to TRH. Identification of these pathways is a critical step in understanding the role and significance of TRH in medullary modulation of autonomic activity.

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