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Segmental distribution of thyrotropin releasing hormone in rat spinal cord
&win Rr,w~rck
Bullrtirr.
Vol. 17. pp. I l-19,
1986. ’ Ankho
International
Inc.
Printed
0361.9?30/86
in the U.8.A
$3.00 + .OO
Segmental Distribution of Thyrotropin Releasing Hormone in Rat Spinal Cord DONALD
H. HARKNESS’.’
Received
AND MARK S. BROWNFIELD:’
7 February 1986
HARKNESS. D. H. AND M. S. BROWNFIELD. S~,,~I?IOII~(//cli.strihri,m ~~f’th~rotn~pi~r w/crr.ti~~g /zotwrr~~rc~in ror .\piutr/ cord. BRAIN RES BULL 17(I) I l-19. 1986.-The segmental and laminar distribution of thyrotropin releasing hormone (TRH) was determined in the rat spinal cord using radioimmunoassay (RIA) and immunocytochemistry (ICC). Immunoreactive TRH was found in sensory, autonomic. and motor spinal columns. Dorsal horn TRH-containing fibers and cell bodies in lamina II and along the lamina II/III border were seen by ICC in all spinal cord segments. ICC showed dense TRH immunoreactivity in the sympathetic areas of the thoracic cord. Densely staining TRH-containing fibers were seen in the ventral horn of all spinal segments. RIA of whole segment extracts showed high concentrations in CGTI and Tl2-L6. Low levels were seen in C2-C4 and TS-T6. Other segments were intermediate in concentration. RIA and ICC results were comparatively evaluated. Thyrotropin
releasing
hormone
(TRH)
Spinal cord
THYROTROPIN releasing hormone (TRH) has been localized in several areas of the central nervous system (CNS), including the spinal cord, by both immunocytochemistry [ 15. 18, 19, 24, 27, 281 and radioimmunoassay [7, 23, 26, 36, 471. Immunocytochemical studies of rat spinal cord generally show immunoreactivity in the intermediolateral (IML) cell column and in the ventral horn, but not in the dorsal horn [l5, 16, 18, 191. However, radioimmunoassay studies [26,29] and recent immunocytochemical reports [8.17] do show the presence of TRH in the dorsal horn. Most approaches to neuropeptide spinal cord localization have involved an analysis of laminar distribution at selected segments of the spinal cord rather than a longitudinal segmental distribution. While the laminar approach is useful in predicting the general function a peptide may have, a spinal cord segmental approach to neuropeptide localization provides an anatomical understanding of the importance of a peptide to the innervation of certain peripheral structures. lmmunocytochemistry provides a good qualitative evaluation, while a quantitative method such as radioimmunoassay is useful in determining where a substance is most concentrated in various regions of the spinal cord, and consequently, in what physiological processes it may have a role. Spinal cord segmental localization of TRH by both ICC and RIA is of interest in that TRH appears to elicit effects on the autonomic nervous system [5, 6, 431 and to have an excitatory action on ventral horn motoneurons [33,46]. TRH localization is also of interest in providing a basis for the site
lmmunocytochemistry
Radioimmunoassay
Rat
of action for potential therapeutic uses in spinal cord trauma [ 12-141 and degenerative spinal cord diseases [I I]. To determine if these putative physiological actions have an anatomical correlation, a segmental and laminar study of the distribution of TRH in the rat spinal cord using both immunocytochemistry and radioimmunoassay was undertaken. METHOD
Ten male Sprague-Dawley rats (200-250 grams, b.w., Sasco-King) were used in this study. Five rats were used for immunocytochemical analysis and five for radioimmunoassay.
Five rats were anesthetized with chloral hydrate and perfused through the left ventricle with I50 ml of a flush solution of 0.1% procaine and 0.1% heparin in 0.01 M phosphatebuffered saline (PBS) followed by 200 ml of 4% paraformaldehyde and 0.17~ glutaraldehyde in 0. I M phosphate buffer (pH 7.5) at 4°C. Individual spinal cord segments, from Cl-S4, were then dissected out through a dorsal laminectomy. using the work of Padmanabhan and Singh [37] as a guide, and post-fixed in fresh fixative for 2 hours. Thirty micron sections of each spinal cord segment were cut on a cryostat after being cryoprotected in a 30% sucrose solution.
‘Requests for reprints should be addressed to Donald H. Harkness. Department of Comparative Medicine, University of Wisconsin, 2015 Linden Drive West, Madison, WI 53706. ‘Completed as partial requirement for the Master of Science degree in the Department of Veterinary “Supported by grants from the University of Wisconsin Graduate School.
Biosciences, Science.
School
of Veterinary
HARKNESS
18
AND BROWNFIELD
TRH IN RAT SPINAL CORD
13
Tissue sections were processed for immunocytochemistry using a modified peroxidase anti-peroxidase method [31,44]. Briefly, tissue sections were washed in 0.01 M PBS and then incubated in 1% hydrogen peroxide in 0.01 M PBS, washed again in 0.01 M PBS and then incubated in a stock buffer of 0.1% gelatin, 2% normal sheep serum, and 0.2% Triton-X 100 (Sigma) in 0.01 M PBS. Sequential incubations in the primary antibody for 16 hours at 4”C, 150 anti-rabbit gamma globulin, and I:200 rabbit peroxidase anti-peroxidase (Miles) were followed by washes in the stock buffer. All antibodies were made up in the stock buffer. After the final antibody incubation, sections were washed in 0.1 M Tris buffer before being reacted in 0.01% diaminobenzidine (Sigma) with 0.01% hydrogen peroxide for seven minutes. After a final wash in 0. I M Tris buffer, tissue sections were mounted permanently on gelatin-coated glass microscope slides. Three TRH antisera were evaluated in this study. Antibody No. 1120 (produced in the laboratory of Dr. I. M. D. Jackson as previously reported [23]) gave the most consistent and intense results. Two other antisera, one provided by Drs. Y. F. Chen and V. Ramirez and one from Arnel Laboratories gave similiar results; however, fiber staining was not as intense, and background staining was higher. Therefore, immunocytochemical data reported in this study were primarily obtained with anti-TRH No. 1120. The TRH antibody was diluted I:2000 in the stock buffer. To test the specificity of the reaction, the TRH antibody was incubated with up to SO pg/ml of luteinizing hormone-releasing hormone (LHRH), TRH-amide (TRH-NH2). TRH-acid (TRHOH), substance P (SP). Leu- and Met-enkephalins (ENK), and serotonin-bovine serum albumin conjugate. Control experiments were also performed following the procedure exactly, but substituting the primary antibody with normal rabbit serum.
The group of five rats for RIA was euthanized, and the spinal columns were removed on ice and stored at -80°C until extraction. At the time of extraction, the spinal cord segments were removed on ice in the same manner as the segments for ICC. TRH was extracted from each segment by sonication in I ml 90% (v:v) menthanol:water. After centrifugation for 5 minutes, the supernatant was decanted and dried by an airstream at 55°C and stored at -80°C until assayed. The same three TRH antisera evaluated for the immunocytochemical studies were also evaluated for use in the radioimmunoassay studies. While each antibody had similiar standard curves and gave similar results in sample tissue tests, the Arnel Laboratories anti-TRH was used due to economy of reagents. Synthetic TRH (Peninsula) was used as the standard and for radioiodination. Sample TRH was measured by an equilibrium double antibody radioimmunoassay. Standards (5 pg to 2000 pg) and samples were done in the same assay and were suspended in 0.01 M phosphate-buffered saline with 0.1% gelatin and 1% normal rabbit serum (PBS-gel-NRS). TRH antibody was used at a dilution of 1:1333. followed by lZZI-TRH with about 10.000
FACING FIG.
cpm. The final antibody dilution in the assay was 1:8000. The assay was incubated for 24 hours at 4°C. After the incubation period, 1: 10 goat anti-rabbit gamma globulin in PBS with 0. I% gelatin and 0.01 M EDTA was added to each tube and incubated for 30 minutes. Polethylene glycol (3.3% in PBS) was added and the tubes were centrifuged at 2500 rpm for 25 minutes at 22°C. The samples were then counted on a gamma counter (1290 Gamma Trac, TM Analytic). Cross reactivity of the anti-TRH with TRH-OH and cycle-His-Pro was less than 0. I%. Coefficient of variation was less than 5%. Assay sensitivity was about I5 pg/tube. RESULTS
Immunocytochemical control experiments showed no staining when the anti-TRH was incubated with TRH-amide as low as IO &ml (Fig. 2B,D) and when the anti-TRH was excluded from the experimental procedure. Incubation with the other substances did not inhibit staining. Results with this antibody showed staining consistent to that previously reported in the hypothalamus and the spinal cord [l&27]. TRH immunoreactivity was seen in all segments of the spinal cord in sensory, autonomic, and somatic motor areas (Fig. I). In the sensory column, TRH immunoreactivity was seen as punctate staining and beaded fibers in lamina II and the superficial portions of lamina III in cervical (Fig. 2A). thoracic (Fig. 3A), and lumbar (Fig. 4A) spinal cord segments. Occasional fibers were seen ascending into lamina I; no immunoreactivity was noted in lamina IV. TRH-containing cell bodies were also noted in many spinal cord segments (Figs. 3A, 4A). The cell bodies were small (6-9 microns) and appeared round with processes extending dorsoventrally and possibly rostrocaudally. The cell bodies were distributed randomly in lamina II and along the lamina II/III border. The greatest number of cell bodies was seen in the cervical segments of the cord. The dorsal horn of the sacral segments had the least amount of TRH immunoreactivity. In the autonomic and intermediate areas of the spinal cord, TRH immunoreactivity was especially dense. TRHcontaining fibers were prominent around the sympathetic preganglionic neurons of the IML (Fig. 3B). The IML appeared to be receiving TRH immunoreactive fibers from the lateral funiculus in the thoracic and lumbar areas (Fig. 4B) of the cord. In spinal segments TI and T4-TlO and Tl3, there were dense bands of staining in the intermediate gray of lamina VII (Fig. 3B) from the central canal to the IML. Intense staining was also noted in lamina X dorsal to the central canal (Fig. 3C). Staining around the central canal was prominent in the thoracic, cervical, and lumbar segments of the cord. Occasional TRH-staining fibers were noted in lamina V and VI. In the somatic motor areas (lamina VIII and lamina IX) of the ventral horn, TRH immunoreactivity was especially noticeable in varicose fibers generally associated with motor neurons (Figs. 2C. 3D, 4C,D). These cell groups appeared to be receiving fibers from the ventrolateral funiculus laterally and the ventral funiculus medially the length of the spinal cord. TRH immunoreactive fibers were evenly distributed among the motor nuclei in the ventral horn. The results of the TRH radioimmunoassay studies sup-
PAGE
I. Schematic
representation
of TRH in selected
segments
of the rat spinal
cord as revealed
by immunocytochemistry
FIG. 2. Transverse sections of rat spinal cord demonstrating TRH immunoreactivity in the dorsal horn of segment C3 (A, x480) and in the ventral horn of segment C7 (C. x420) compared with TRH absorbed controls of the dorsal horn of segment C3 (B, x400) and the ventral horn (D, x420). In the dorsat horn, note the punctate immunoreactivity in lamina II and along the iamina II/III border (A), and the absence of immunoreactivity in the control section (B). In the ventral horn, TRH containing tibers are evenly distributed in lamina VIII(C) mostly around the motor neurons. Again, there is no immunoreactivity in the ventral horn of the control section.
FIG. 3. Transverse sections of the rat thoracic spinal cord demonstrating TRH imrnunoreactivity. A TRH immunoreactive cell body (arrow) is seen in the dorsal horn of segment TlD (A, x399). Punctate fiber staining is also seen just dorsal to the cell body. Very dense immunoreactivity is noted in the intermediate gray oflamina VII (open arrows) and around the ~regau~~i~~i~ neurons of the i~termed~olateral ceil column (IML) of T2 (B, x200) and in lamina X dorsal to the central canal (C) (C, x380). In the ventral horn of segment T6, TRH containing fibers are distributed evenly in all lamina (D, x314). Note how many fibers appear to be closely associated with motor neurons (arrows).
HARKNESS
AND RROWNFIELD
17
TRH IN RAT SPINAL CORD port the immunocytochemical studies but more importantly demonstrate a dramatic pattern of TRH concentration and content that immunocytochemistry alone could not reveal (Fig. 5). Content and concentration are reported to demonstrate the total amount of TRH per segment and to normalize &hedata for changes in segmental tissue weight, respectively. The most obvious feature of TRH spinal cord concentration is that it is highest in the areas of the cervical and lumbar enlargements (segments C6 to Tl and Ll to L6). Intermediate but varying concentrations were found mostly in the thoracic segments, although T13 had the highest concentration of any segment in the spinal cord. The lowest concentrations were found in segments T5 and T6, and in segments C2 through C5. The general pattern of TRH content for individual segments paralleled concentrations (Fig. 5). The content for the ng entire spinal cord of the rat was 11S9rtO.61 (meanrSEM). The average concentration for the entire spinal cord was 30.92 pg/mg tissue wet weight’ 1.9 pgimg.
TRH CONTENT 1100 1000 900 800
P 700 g (II
600
3 3
500
n. 400 300 200 100
OJ
r
1
3 5 7 CERVICAL
I
1
8
3
I
I
1
7 9 5 THORACIC
,’
I
I
1
1
6 2 4 LUMBAR $
1113
TRH CONCENTRATION 80
DISCUSSION 2
These studies demonstrate the segmental distribution of TRH by immunocytochemistry and radioimmunoassay in the rat spinal cord. Evaluation of the results shows (1) a TRH-containing system of cell bodies and fibers present in the sensory column suggesting a role in sensory processing; (2) a variable association of TRH-containing fibers in the autonomic columns suggesting differential involvement of TRH with autonomic functions; and (3) the presence of TRH in the ventral motor column suggesting a role in somatic motor function. The immunocytochemical results of the intrasegmental distribution are in general agreement with previous reports [ 15, 16, 18, 19, 25, 281, except for the additional findings in the dorsal horn. The radioimmunoassay studies complement and extend the immunocytochemical studies, providing a very different perspective of regional concentration of the peptide. They show high concentrations in the segments which contribute to the brachial plexus and lumbar plexus, areas where many of the motor neurons in the spinal cord are located, and where immunocytochemistry shows apparent contacts between TRH containing fibers and motor neurons. RIA studies also show intermediate concentrations of TRH in the thoracic cord, an area densely stained for TRH immunocytochemically. In the dorsal horn, the localization of TRH indicates a role for TRH in sensory processing in the spinai cord. TRH immunoreactivity was found mostly in Iamina II and along the lamina II/III border in both fibers and cell bodies. This finding has not been reported previously in the rat by immunocytochemistry, although a few TRH-containing fibers have been localized in the monkey dorsal horn 1281, and TRH-containing cell bodies have been seen in the dorsal horn of the mouse [8]. While colchicine was not used in this study, another study using colchicine has shown TRHcontaining cell bodies only in the dorsal horn, but not in the ventral horn or the IML [17]. RIA studies have also shown
70
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1
3
,
5 7 3 CERVICAL
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1
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7 9 5 THORACIC
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1113
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,
,
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FIG. 5. Segmental concentration and whole segment content of TRH in the rat spinal cord as determined by radioimmunoassay. Note high concentrations in the segments contributing to the brachial plexus and the lumbar plexus. (N=5; squares with bars denote average +SEM.).
TRH in the dorsal horn [26,291, and receptor studies show high levels of TRH receptors in the rabbit dorsal horn [42]. Since TRH, SP, and 5-hydroxytryptamine (5-HT) have been shown to coexist in the raphe nuclei of the medulla and in the ventral horn and IML of the spinal cord [3, 16,25,29], Lechan [28] postulates that TRH immunoreactivity in the dorsal horn of the monkey may coexist with 5-HT. However, in the rat, TRH-containing cell bodies in the dorsal horn indicate that an intrinsic system separate from the supraspinal serotonergic system may exist. Evidence for this possibility is shown elsewhere. In one study, RIA levels of TRH in the lumbar dorsal horn of rats treated with 5,7dihydroxytryptamine (5,7-DHT), a serotonergic neurotoxin, remained unchanged, while in the ventral horn TRH was lost in parallel with .5-HT [29]. In another study, im-
FACING PAGE FIG. 4. TRH immunoreactiv~ty in the lumbar spinal cord. The arrows again indicate TRH-containing cell bodies in the dorsal horn at segment L1 (A, x400). TRH-containing fibers are seen in the lateral funiculus (LF) apparently passing to the IML at Ll (B, x420). In segment L4, beaded TRH-containing fibers are seen in close association with a motor neuron (C, x600). TRH immunoreactivity is seen in beaded fibers around motor neurons in the motor nuclei of segment Ll (D, x330): note the dense immunoreactivity around the motor neuron indicated by open arrow.
IX
HARKNESS
munocytochemical findings also show no change in dorsal horn TRH immunoreactivity of rats treated with 5,‘7-DHT [ 171. Consequently, it appears that not only does TRH participate in sensory processing, but also that this TRH system is intrinsic to the rat dorsal horn. This does not preclude the possibility that TRH is present in two dorsal horn systems, a raphe-spinal system and an intrinsic spinal system; however, the intrinsic system appears to be the dominant one. Certainly, the exact function of TRH in the dorsal horn remains to be elucidated. The concentration of TRH immunoreactivity in the thoracic spinal cord indicates a role for TRH in the autonomic nervous system. RIA shows inte~ediate levels of TRH in the rostra1 and caudal thoracic spinal cord, but low levels in the middle thoracic region. Immuno~ytochemically, high levels of immunoreactive product are found in the IML. the dorsal lamina X, and the intermediate gray of lamina VII, areas known to contain preganglionic sympathetic neurons in many species 110, 38, 391. Localization of high levels of TRH by ICC and RIA between segments T7 and 119 may indicate preferential autonomic effects on organ systems such as the adrenal as has been suggested for oxytocin and neurophysin 14.51.A combined retrograde transport and immunocytochemical study shows .5-HT, substance P, and enkepahlin around IML preganglionic sympathetic neurons innervating the adrenal [Zl] in the same segments with high TRH levels. Studies have also shown an increased sympathetic outflow to the adrenal following centrally administered TRH f5,43]. RIA and ICC levels of TRH in T2 through T4 also indicate possible autonomic regulation of other functions of the cardiovascular system. Indeed, TRH does increase heart rate and blood pressure [2] and also increases mean arterial pressure in experimental hemorrhagic and endotoxic shock [ZO]. In the ventral horn, the ciose association of TRH with motor neurons as seen with immunocytochemist~ suggests a role in the ~gulation of motor neuron function. The high levels of TRH by RIA in the segments contributing to the lumbar plexus and the brachial plexus also indicate a role in somatic motor function. Because the IML is not prominent in the spinal cord enlargements, and dorsal horn TRH does not contribute substantia1 amounts to the total TRH content of segments, these high levels are a reflection of high levels in the ventral horn, probably due to the increased number of motor neurons. Immunocytochemically, TRH-containing fibers are seen in motor nuclei of the lumbar ventral horn of the rat spinal cord known to provide innervation for various appendicular and axial muscle groups f4, 34, 411. TRHcontaining fibers in the rat ventral horn appear to be evenly distributed throughout the motor nuclei of the ventral horn,
AND BROWNFIELD
unlike the distribution reported for the monkey spinal cord where the heaviest concentrations were in the ventrolateral motor nuclei [ZS]. Neurophysiological studies support the somatic motor function of TRH. TRH appears to enhance membrane conduction and excitability of motor neurons in amphibian spinal cord [33], and, after an initial inhibition, to facilitate the excitabihty of motor neurons to the amino acids glutamate and aspartate in the rat 1461. As well, TRH enhancement of choline acetyltransferase and creatine kinase activities in cultured spinal ventral horn neurons, may indicate a trophic effect on the the-survivai of motor neurons [40]. Finally, TRH stimulation of muscle activity by direct action of peripherally administered TRH on the cat spinal cord [9] and on the rat spinal cord [I] after spinal cord transection suggests that TRH found in the cervical and lumbar enlargements acts on motor neurons in the normal animal. TRH may be involved in the motor control of respiration at the level of the cervical ventral horn. TRH, SP, and 5-HT have been identi~ed as coexisting in the phrenic nucleus of cervical segments four, five, and six of the cat f22], and centrally applied TRH has been reported to increase respiratory rate and minute ventilation [35]. When rats are treated with 5,7-dihydroxyt~p~mine, a supersensitivity to the respiratory stimulatory effect of TRH develops possibly due to the depletion of TRH as well as 5-HT coexisting in nerve fibers of the ventral horn of the cervical spinal cord [32]. The functional role for TRH-containing terminals on motor neurons is also supported by evidence that TRH may be involved in disease processes. In the spinal cords of patients with amyotrophic lateral sclerosis (ALS). a degenerative motor neuron disease, there are lower concentrations of immunorea~tive TRH than in the spinal cords of patients with non-neurological diseases ]30], and ALS patients transientty respond to treatment with TRH [I I]. As well, TRH has been reported to improve neurologic recovery as assessed by improvement in motor function after acute spinal cord injury in the cat even 24 hours post-trauma [12-141. Finally. motor neuron damage induced by sciatic neuroectomy or retrovirus infection in the mouse resulted in an increase in diameter of TRH containing fibers around the affected motor neurons, a change that was reversed in retrovirus infections with TRH treatment 148J. The segmental dist~bution of TRH presented in this report provides an anatomical framework for the function of TRH in the rat spinal cord. Many more investigations will be
necessary
to fufly determine
TRH in the sensory, spinal cord.
the functional
autonomic,
and motor
significance portions
of
of the
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TRH IN RAT SPINAL CORD
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31
R. M. and I. M. D. Jackson. Immunohistochemical localization of thyrotropin-releasing hormone in the rat hypothalamus and pituitary. Endocrinology 11: 55-65, 1982. Lechan, R. M., S. B. Snapper, S. Jacobsen and I. M. D. Jackson. The distribution of thyrotropin-releasing hormone (TRH) in the Rhesus monkey spinal cord. Paptides 5: Suppl 1, 185-194, 1984. Lighton, C., C. A. Marsden and G. W. Bennett. The effects of and p-chlorophenyalanine on 5,7_dihydroxytryptamine thyrotropin-releasing hormone in regions of the brain and spinal cord of the rat. NPuroDhnrrcrcok,.~v 23: 55-60. 1984. Mitsuma. T.. T. Nogimori, K. Adachi, M. Mukoyama and K. Ando. Concentrations of immunoreactive thyrotropin-releasing hormone in spinal cord of patients with amyotrophic lateral sclerosis. At77 J Mcd Sri 287: 34-36, 1984. Moriarty, G. C., C. M. Moriarty and L. A. Stemberger. Ultrastructural immunocytochemistry by unlabelled antibodies and the peroxidase anti-peroxidase complex (PAP). A technique more sensitive than radioimmunoassay. J Hi.sfoc.ho?~ Cytoc,hem 21: 825-833, 1973.
32 Mueller, R. A., A. C. Towle and G. R. Breese. Supersensitivity to the respiratory stimulatory effect of TRH in 5,7dihydroxytryptamine-treated rats. Brctirl Rcs 298: 370-373, 1984. 33. Nicoll, R. A. Excitatory action ofTRH on spinal motoneurones. Ncrttrw 265: 242-243. 1977. 34. Nicolopoulos-Stournaras, S. and J. F. Iles. Motor neuron columns in the lumbar spinal cord of the rat. .f Cottrg Nertrol 217: 75-85, 1983. 35. Niewoehner, D. E., A. S. Levine and J. E. Morley. Central effects of peptides on ventilation in the rat. Pepti&s 4: 277-281, 1983. 36. Oliver, C.. R. L. Eskay, N. Ben-Jonathan and J. C. Porter. Distribution and concentration of TRH in the rat brain. Ozdoc~ririolog~ 95: 540-546, 1974. 37. Padmanabhan, R. and S. Singh. Observations on the topographical relations of spinal nerve roots in the rat. Ac,to Allat (Btrscl) 105: 378-380, 1979. 38. Petras, J. M. and J. F. Cummings. Autonomic neurons in the spinal cord of the rhesus monkey: a correlation of the findings of cytoarchitectonics and sympathectomy with fiber degeneration following dorsal rhizotomy. J Camp Neuml 146: 189-218, 1982. 39. Petras, J. M. and A. I. Faden. The origin of sympathetic preganglionic neurons in the dog. B&rz Rcs 144: 353-357, 1978. 40. Schmidt-Aches, K. M., V. Askanas and W. K. Engel. Thyrotropin-releasing hormone enhances choline acetyltransferase and creatine kinase in cultured spinal ventral horn neurons. I Ncrrrochrw 43: 586589, 1984. 41. Schroder, H. D. Organization of the motoneurons innervating the pelvic muscles of the male rat. .I Con7p NeNroI 192: 567-587, 1980. 42. Sharif. N. A. and D. R. Burt. Receptors for thyrotropinreleasing hormone (TRH) in rabbit spinal cord. BrcJin Rrs 270: 259-263. 1983. 43. Somiya. H. and T. Tonoue. Neuropeptides as central integrators of autonomic nerve activity: effects of TRH, SRIF, VIP, and bombesin on gastric and adrenal nerves. Rcg~d Pcpt 9: 47-52, 1984.
44. Sternberger, L. A. Itnmtrnoc.~tot,~?~r~7i~~tr~,second edition. New York: John Wiley and Sons, 1979. 45. Swanson. L. W. and S. McKellar. The distribution of oxytocin-and neurophysin-stained fibers in the spinal cord of the rat and monkey. J Cornp Neural 188: 87-106, 1979. 46. White, S. R. A comparison of the effects of serotonin, substance P and thyrotropin-releasing hormone on excitability of rat spinal motoneurons in vivo. Bruin Rcs 335: 63-70, 1985. 47. Winokur, A. and R. D. Utiger. Thyrotropin-releasing hormone: regional distribution in the rat brain. Science 185:265-267, 1974. 48. Zimmerman. E. M., J. A. Coffield, V. Miletic, M. J. Hoffert and B. R. Brooks. Localization of substance P, 5HT and TRH-like immunoreactivity in mouse spinal cord following motor neuron damage. .S~IC~ Nfwosci Ahstr 10: 891, 1984.
Bullrtirr.
Vol. 17. pp. I l-19,
1986. ’ Ankho
International
Inc.
Printed
0361.9?30/86
in the U.8.A
$3.00 + .OO
Segmental Distribution of Thyrotropin Releasing Hormone in Rat Spinal Cord DONALD
H. HARKNESS’.’
Received
AND MARK S. BROWNFIELD:’
7 February 1986
HARKNESS. D. H. AND M. S. BROWNFIELD. S~,,~I?IOII~(//cli.strihri,m ~~f’th~rotn~pi~r w/crr.ti~~g /zotwrr~~rc~in ror .\piutr/ cord. BRAIN RES BULL 17(I) I l-19. 1986.-The segmental and laminar distribution of thyrotropin releasing hormone (TRH) was determined in the rat spinal cord using radioimmunoassay (RIA) and immunocytochemistry (ICC). Immunoreactive TRH was found in sensory, autonomic. and motor spinal columns. Dorsal horn TRH-containing fibers and cell bodies in lamina II and along the lamina II/III border were seen by ICC in all spinal cord segments. ICC showed dense TRH immunoreactivity in the sympathetic areas of the thoracic cord. Densely staining TRH-containing fibers were seen in the ventral horn of all spinal segments. RIA of whole segment extracts showed high concentrations in CGTI and Tl2-L6. Low levels were seen in C2-C4 and TS-T6. Other segments were intermediate in concentration. RIA and ICC results were comparatively evaluated. Thyrotropin
releasing
hormone
(TRH)
Spinal cord
THYROTROPIN releasing hormone (TRH) has been localized in several areas of the central nervous system (CNS), including the spinal cord, by both immunocytochemistry [ 15. 18, 19, 24, 27, 281 and radioimmunoassay [7, 23, 26, 36, 471. Immunocytochemical studies of rat spinal cord generally show immunoreactivity in the intermediolateral (IML) cell column and in the ventral horn, but not in the dorsal horn [l5, 16, 18, 191. However, radioimmunoassay studies [26,29] and recent immunocytochemical reports [8.17] do show the presence of TRH in the dorsal horn. Most approaches to neuropeptide spinal cord localization have involved an analysis of laminar distribution at selected segments of the spinal cord rather than a longitudinal segmental distribution. While the laminar approach is useful in predicting the general function a peptide may have, a spinal cord segmental approach to neuropeptide localization provides an anatomical understanding of the importance of a peptide to the innervation of certain peripheral structures. lmmunocytochemistry provides a good qualitative evaluation, while a quantitative method such as radioimmunoassay is useful in determining where a substance is most concentrated in various regions of the spinal cord, and consequently, in what physiological processes it may have a role. Spinal cord segmental localization of TRH by both ICC and RIA is of interest in that TRH appears to elicit effects on the autonomic nervous system [5, 6, 431 and to have an excitatory action on ventral horn motoneurons [33,46]. TRH localization is also of interest in providing a basis for the site
lmmunocytochemistry
Radioimmunoassay
Rat
of action for potential therapeutic uses in spinal cord trauma [ 12-141 and degenerative spinal cord diseases [I I]. To determine if these putative physiological actions have an anatomical correlation, a segmental and laminar study of the distribution of TRH in the rat spinal cord using both immunocytochemistry and radioimmunoassay was undertaken. METHOD
Ten male Sprague-Dawley rats (200-250 grams, b.w., Sasco-King) were used in this study. Five rats were used for immunocytochemical analysis and five for radioimmunoassay.
Five rats were anesthetized with chloral hydrate and perfused through the left ventricle with I50 ml of a flush solution of 0.1% procaine and 0.1% heparin in 0.01 M phosphatebuffered saline (PBS) followed by 200 ml of 4% paraformaldehyde and 0.17~ glutaraldehyde in 0. I M phosphate buffer (pH 7.5) at 4°C. Individual spinal cord segments, from Cl-S4, were then dissected out through a dorsal laminectomy. using the work of Padmanabhan and Singh [37] as a guide, and post-fixed in fresh fixative for 2 hours. Thirty micron sections of each spinal cord segment were cut on a cryostat after being cryoprotected in a 30% sucrose solution.
‘Requests for reprints should be addressed to Donald H. Harkness. Department of Comparative Medicine, University of Wisconsin, 2015 Linden Drive West, Madison, WI 53706. ‘Completed as partial requirement for the Master of Science degree in the Department of Veterinary “Supported by grants from the University of Wisconsin Graduate School.
Biosciences, Science.
School
of Veterinary
HARKNESS
18
AND BROWNFIELD
TRH IN RAT SPINAL CORD
13
Tissue sections were processed for immunocytochemistry using a modified peroxidase anti-peroxidase method [31,44]. Briefly, tissue sections were washed in 0.01 M PBS and then incubated in 1% hydrogen peroxide in 0.01 M PBS, washed again in 0.01 M PBS and then incubated in a stock buffer of 0.1% gelatin, 2% normal sheep serum, and 0.2% Triton-X 100 (Sigma) in 0.01 M PBS. Sequential incubations in the primary antibody for 16 hours at 4”C, 150 anti-rabbit gamma globulin, and I:200 rabbit peroxidase anti-peroxidase (Miles) were followed by washes in the stock buffer. All antibodies were made up in the stock buffer. After the final antibody incubation, sections were washed in 0.1 M Tris buffer before being reacted in 0.01% diaminobenzidine (Sigma) with 0.01% hydrogen peroxide for seven minutes. After a final wash in 0. I M Tris buffer, tissue sections were mounted permanently on gelatin-coated glass microscope slides. Three TRH antisera were evaluated in this study. Antibody No. 1120 (produced in the laboratory of Dr. I. M. D. Jackson as previously reported [23]) gave the most consistent and intense results. Two other antisera, one provided by Drs. Y. F. Chen and V. Ramirez and one from Arnel Laboratories gave similiar results; however, fiber staining was not as intense, and background staining was higher. Therefore, immunocytochemical data reported in this study were primarily obtained with anti-TRH No. 1120. The TRH antibody was diluted I:2000 in the stock buffer. To test the specificity of the reaction, the TRH antibody was incubated with up to SO pg/ml of luteinizing hormone-releasing hormone (LHRH), TRH-amide (TRH-NH2). TRH-acid (TRHOH), substance P (SP). Leu- and Met-enkephalins (ENK), and serotonin-bovine serum albumin conjugate. Control experiments were also performed following the procedure exactly, but substituting the primary antibody with normal rabbit serum.
The group of five rats for RIA was euthanized, and the spinal columns were removed on ice and stored at -80°C until extraction. At the time of extraction, the spinal cord segments were removed on ice in the same manner as the segments for ICC. TRH was extracted from each segment by sonication in I ml 90% (v:v) menthanol:water. After centrifugation for 5 minutes, the supernatant was decanted and dried by an airstream at 55°C and stored at -80°C until assayed. The same three TRH antisera evaluated for the immunocytochemical studies were also evaluated for use in the radioimmunoassay studies. While each antibody had similiar standard curves and gave similar results in sample tissue tests, the Arnel Laboratories anti-TRH was used due to economy of reagents. Synthetic TRH (Peninsula) was used as the standard and for radioiodination. Sample TRH was measured by an equilibrium double antibody radioimmunoassay. Standards (5 pg to 2000 pg) and samples were done in the same assay and were suspended in 0.01 M phosphate-buffered saline with 0.1% gelatin and 1% normal rabbit serum (PBS-gel-NRS). TRH antibody was used at a dilution of 1:1333. followed by lZZI-TRH with about 10.000
FACING FIG.
cpm. The final antibody dilution in the assay was 1:8000. The assay was incubated for 24 hours at 4°C. After the incubation period, 1: 10 goat anti-rabbit gamma globulin in PBS with 0. I% gelatin and 0.01 M EDTA was added to each tube and incubated for 30 minutes. Polethylene glycol (3.3% in PBS) was added and the tubes were centrifuged at 2500 rpm for 25 minutes at 22°C. The samples were then counted on a gamma counter (1290 Gamma Trac, TM Analytic). Cross reactivity of the anti-TRH with TRH-OH and cycle-His-Pro was less than 0. I%. Coefficient of variation was less than 5%. Assay sensitivity was about I5 pg/tube. RESULTS
Immunocytochemical control experiments showed no staining when the anti-TRH was incubated with TRH-amide as low as IO &ml (Fig. 2B,D) and when the anti-TRH was excluded from the experimental procedure. Incubation with the other substances did not inhibit staining. Results with this antibody showed staining consistent to that previously reported in the hypothalamus and the spinal cord [l&27]. TRH immunoreactivity was seen in all segments of the spinal cord in sensory, autonomic, and somatic motor areas (Fig. I). In the sensory column, TRH immunoreactivity was seen as punctate staining and beaded fibers in lamina II and the superficial portions of lamina III in cervical (Fig. 2A). thoracic (Fig. 3A), and lumbar (Fig. 4A) spinal cord segments. Occasional fibers were seen ascending into lamina I; no immunoreactivity was noted in lamina IV. TRH-containing cell bodies were also noted in many spinal cord segments (Figs. 3A, 4A). The cell bodies were small (6-9 microns) and appeared round with processes extending dorsoventrally and possibly rostrocaudally. The cell bodies were distributed randomly in lamina II and along the lamina II/III border. The greatest number of cell bodies was seen in the cervical segments of the cord. The dorsal horn of the sacral segments had the least amount of TRH immunoreactivity. In the autonomic and intermediate areas of the spinal cord, TRH immunoreactivity was especially dense. TRHcontaining fibers were prominent around the sympathetic preganglionic neurons of the IML (Fig. 3B). The IML appeared to be receiving TRH immunoreactive fibers from the lateral funiculus in the thoracic and lumbar areas (Fig. 4B) of the cord. In spinal segments TI and T4-TlO and Tl3, there were dense bands of staining in the intermediate gray of lamina VII (Fig. 3B) from the central canal to the IML. Intense staining was also noted in lamina X dorsal to the central canal (Fig. 3C). Staining around the central canal was prominent in the thoracic, cervical, and lumbar segments of the cord. Occasional TRH-staining fibers were noted in lamina V and VI. In the somatic motor areas (lamina VIII and lamina IX) of the ventral horn, TRH immunoreactivity was especially noticeable in varicose fibers generally associated with motor neurons (Figs. 2C. 3D, 4C,D). These cell groups appeared to be receiving fibers from the ventrolateral funiculus laterally and the ventral funiculus medially the length of the spinal cord. TRH immunoreactive fibers were evenly distributed among the motor nuclei in the ventral horn. The results of the TRH radioimmunoassay studies sup-
PAGE
I. Schematic
representation
of TRH in selected
segments
of the rat spinal
cord as revealed
by immunocytochemistry
FIG. 2. Transverse sections of rat spinal cord demonstrating TRH immunoreactivity in the dorsal horn of segment C3 (A, x480) and in the ventral horn of segment C7 (C. x420) compared with TRH absorbed controls of the dorsal horn of segment C3 (B, x400) and the ventral horn (D, x420). In the dorsat horn, note the punctate immunoreactivity in lamina II and along the iamina II/III border (A), and the absence of immunoreactivity in the control section (B). In the ventral horn, TRH containing tibers are evenly distributed in lamina VIII(C) mostly around the motor neurons. Again, there is no immunoreactivity in the ventral horn of the control section.
FIG. 3. Transverse sections of the rat thoracic spinal cord demonstrating TRH imrnunoreactivity. A TRH immunoreactive cell body (arrow) is seen in the dorsal horn of segment TlD (A, x399). Punctate fiber staining is also seen just dorsal to the cell body. Very dense immunoreactivity is noted in the intermediate gray oflamina VII (open arrows) and around the ~regau~~i~~i~ neurons of the i~termed~olateral ceil column (IML) of T2 (B, x200) and in lamina X dorsal to the central canal (C) (C, x380). In the ventral horn of segment T6, TRH containing fibers are distributed evenly in all lamina (D, x314). Note how many fibers appear to be closely associated with motor neurons (arrows).
HARKNESS
AND RROWNFIELD
17
TRH IN RAT SPINAL CORD port the immunocytochemical studies but more importantly demonstrate a dramatic pattern of TRH concentration and content that immunocytochemistry alone could not reveal (Fig. 5). Content and concentration are reported to demonstrate the total amount of TRH per segment and to normalize &hedata for changes in segmental tissue weight, respectively. The most obvious feature of TRH spinal cord concentration is that it is highest in the areas of the cervical and lumbar enlargements (segments C6 to Tl and Ll to L6). Intermediate but varying concentrations were found mostly in the thoracic segments, although T13 had the highest concentration of any segment in the spinal cord. The lowest concentrations were found in segments T5 and T6, and in segments C2 through C5. The general pattern of TRH content for individual segments paralleled concentrations (Fig. 5). The content for the ng entire spinal cord of the rat was 11S9rtO.61 (meanrSEM). The average concentration for the entire spinal cord was 30.92 pg/mg tissue wet weight’ 1.9 pgimg.
TRH CONTENT 1100 1000 900 800
P 700 g (II
600
3 3
500
n. 400 300 200 100
OJ
r
1
3 5 7 CERVICAL
I
1
8
3
I
I
1
7 9 5 THORACIC
,’
I
I
1
1
6 2 4 LUMBAR $
1113
TRH CONCENTRATION 80
DISCUSSION 2
These studies demonstrate the segmental distribution of TRH by immunocytochemistry and radioimmunoassay in the rat spinal cord. Evaluation of the results shows (1) a TRH-containing system of cell bodies and fibers present in the sensory column suggesting a role in sensory processing; (2) a variable association of TRH-containing fibers in the autonomic columns suggesting differential involvement of TRH with autonomic functions; and (3) the presence of TRH in the ventral motor column suggesting a role in somatic motor function. The immunocytochemical results of the intrasegmental distribution are in general agreement with previous reports [ 15, 16, 18, 19, 25, 281, except for the additional findings in the dorsal horn. The radioimmunoassay studies complement and extend the immunocytochemical studies, providing a very different perspective of regional concentration of the peptide. They show high concentrations in the segments which contribute to the brachial plexus and lumbar plexus, areas where many of the motor neurons in the spinal cord are located, and where immunocytochemistry shows apparent contacts between TRH containing fibers and motor neurons. RIA studies also show intermediate concentrations of TRH in the thoracic cord, an area densely stained for TRH immunocytochemically. In the dorsal horn, the localization of TRH indicates a role for TRH in sensory processing in the spinai cord. TRH immunoreactivity was found mostly in Iamina II and along the lamina II/III border in both fibers and cell bodies. This finding has not been reported previously in the rat by immunocytochemistry, although a few TRH-containing fibers have been localized in the monkey dorsal horn 1281, and TRH-containing cell bodies have been seen in the dorsal horn of the mouse [8]. While colchicine was not used in this study, another study using colchicine has shown TRHcontaining cell bodies only in the dorsal horn, but not in the ventral horn or the IML [17]. RIA studies have also shown
70
0 w 60 iz t; 50 5 gj 40 # E 30 c) r 20
B
‘O- 40, 07
1
3
,
5 7 3 CERVICAL
‘s
1
,
3
,
1
I
7 9 5 THORACIC
,
1113
b
/
,
,
6 2 4 LUMBAR S
FIG. 5. Segmental concentration and whole segment content of TRH in the rat spinal cord as determined by radioimmunoassay. Note high concentrations in the segments contributing to the brachial plexus and the lumbar plexus. (N=5; squares with bars denote average +SEM.).
TRH in the dorsal horn [26,291, and receptor studies show high levels of TRH receptors in the rabbit dorsal horn [42]. Since TRH, SP, and 5-hydroxytryptamine (5-HT) have been shown to coexist in the raphe nuclei of the medulla and in the ventral horn and IML of the spinal cord [3, 16,25,29], Lechan [28] postulates that TRH immunoreactivity in the dorsal horn of the monkey may coexist with 5-HT. However, in the rat, TRH-containing cell bodies in the dorsal horn indicate that an intrinsic system separate from the supraspinal serotonergic system may exist. Evidence for this possibility is shown elsewhere. In one study, RIA levels of TRH in the lumbar dorsal horn of rats treated with 5,7dihydroxytryptamine (5,7-DHT), a serotonergic neurotoxin, remained unchanged, while in the ventral horn TRH was lost in parallel with .5-HT [29]. In another study, im-
FACING PAGE FIG. 4. TRH immunoreactiv~ty in the lumbar spinal cord. The arrows again indicate TRH-containing cell bodies in the dorsal horn at segment L1 (A, x400). TRH-containing fibers are seen in the lateral funiculus (LF) apparently passing to the IML at Ll (B, x420). In segment L4, beaded TRH-containing fibers are seen in close association with a motor neuron (C, x600). TRH immunoreactivity is seen in beaded fibers around motor neurons in the motor nuclei of segment Ll (D, x330): note the dense immunoreactivity around the motor neuron indicated by open arrow.
IX
HARKNESS
munocytochemical findings also show no change in dorsal horn TRH immunoreactivity of rats treated with 5,‘7-DHT [ 171. Consequently, it appears that not only does TRH participate in sensory processing, but also that this TRH system is intrinsic to the rat dorsal horn. This does not preclude the possibility that TRH is present in two dorsal horn systems, a raphe-spinal system and an intrinsic spinal system; however, the intrinsic system appears to be the dominant one. Certainly, the exact function of TRH in the dorsal horn remains to be elucidated. The concentration of TRH immunoreactivity in the thoracic spinal cord indicates a role for TRH in the autonomic nervous system. RIA shows inte~ediate levels of TRH in the rostra1 and caudal thoracic spinal cord, but low levels in the middle thoracic region. Immuno~ytochemically, high levels of immunoreactive product are found in the IML. the dorsal lamina X, and the intermediate gray of lamina VII, areas known to contain preganglionic sympathetic neurons in many species 110, 38, 391. Localization of high levels of TRH by ICC and RIA between segments T7 and 119 may indicate preferential autonomic effects on organ systems such as the adrenal as has been suggested for oxytocin and neurophysin 14.51.A combined retrograde transport and immunocytochemical study shows .5-HT, substance P, and enkepahlin around IML preganglionic sympathetic neurons innervating the adrenal [Zl] in the same segments with high TRH levels. Studies have also shown an increased sympathetic outflow to the adrenal following centrally administered TRH f5,43]. RIA and ICC levels of TRH in T2 through T4 also indicate possible autonomic regulation of other functions of the cardiovascular system. Indeed, TRH does increase heart rate and blood pressure [2] and also increases mean arterial pressure in experimental hemorrhagic and endotoxic shock [ZO]. In the ventral horn, the ciose association of TRH with motor neurons as seen with immunocytochemist~ suggests a role in the ~gulation of motor neuron function. The high levels of TRH by RIA in the segments contributing to the lumbar plexus and the brachial plexus also indicate a role in somatic motor function. Because the IML is not prominent in the spinal cord enlargements, and dorsal horn TRH does not contribute substantia1 amounts to the total TRH content of segments, these high levels are a reflection of high levels in the ventral horn, probably due to the increased number of motor neurons. Immunocytochemically, TRH-containing fibers are seen in motor nuclei of the lumbar ventral horn of the rat spinal cord known to provide innervation for various appendicular and axial muscle groups f4, 34, 411. TRHcontaining fibers in the rat ventral horn appear to be evenly distributed throughout the motor nuclei of the ventral horn,
AND BROWNFIELD
unlike the distribution reported for the monkey spinal cord where the heaviest concentrations were in the ventrolateral motor nuclei [ZS]. Neurophysiological studies support the somatic motor function of TRH. TRH appears to enhance membrane conduction and excitability of motor neurons in amphibian spinal cord [33], and, after an initial inhibition, to facilitate the excitabihty of motor neurons to the amino acids glutamate and aspartate in the rat 1461. As well, TRH enhancement of choline acetyltransferase and creatine kinase activities in cultured spinal ventral horn neurons, may indicate a trophic effect on the the-survivai of motor neurons [40]. Finally, TRH stimulation of muscle activity by direct action of peripherally administered TRH on the cat spinal cord [9] and on the rat spinal cord [I] after spinal cord transection suggests that TRH found in the cervical and lumbar enlargements acts on motor neurons in the normal animal. TRH may be involved in the motor control of respiration at the level of the cervical ventral horn. TRH, SP, and 5-HT have been identi~ed as coexisting in the phrenic nucleus of cervical segments four, five, and six of the cat f22], and centrally applied TRH has been reported to increase respiratory rate and minute ventilation [35]. When rats are treated with 5,7-dihydroxyt~p~mine, a supersensitivity to the respiratory stimulatory effect of TRH develops possibly due to the depletion of TRH as well as 5-HT coexisting in nerve fibers of the ventral horn of the cervical spinal cord [32]. The functional role for TRH-containing terminals on motor neurons is also supported by evidence that TRH may be involved in disease processes. In the spinal cords of patients with amyotrophic lateral sclerosis (ALS). a degenerative motor neuron disease, there are lower concentrations of immunorea~tive TRH than in the spinal cords of patients with non-neurological diseases ]30], and ALS patients transientty respond to treatment with TRH [I I]. As well, TRH has been reported to improve neurologic recovery as assessed by improvement in motor function after acute spinal cord injury in the cat even 24 hours post-trauma [12-141. Finally. motor neuron damage induced by sciatic neuroectomy or retrovirus infection in the mouse resulted in an increase in diameter of TRH containing fibers around the affected motor neurons, a change that was reversed in retrovirus infections with TRH treatment 148J. The segmental dist~bution of TRH presented in this report provides an anatomical framework for the function of TRH in the rat spinal cord. Many more investigations will be
necessary
to fufly determine
TRH in the sensory, spinal cord.
the functional
autonomic,
and motor
significance portions
of
of the
REFERENCES 1. Barbeau, H. and P. Bedard. Similar motor effects of 5-HT and TRH in rats following chronic spinal transection and 5,7dihydroxytryptamine injection. ~e~~f~p~~rmu~~~~~~~~ 20: 477481, 1981. 2. Beale, J. S., R. P. White and S.-P. Huang. EEG and blood pressure effects of TRH in rabbits. Nawopharmncolog~ 16: 499-506, 1977‘ 3. Bowker, R. M., K. N. Westlund, M. C. Suilivan, J. F. Wilber and 1. D. Coulter, Transmitters of the raphe-spinal complex: Immunocytocbemical studies. Feprilirs 3: 291-298, 1982. 4. Brink, E.. E., J. I. Morrell and D. W. Pfaff. Localization of lumbar eoaxiaf motoneurons in the rat. Bruirz Res 170: 23-41, 1979. .
Brown, M. R. Thyrotrop~n releasing factor: a putative CNS renulator of the autonomic nervous system. I_$~~Sri 28: 178917%. 1981. Brown, M. and Y. Tache. ~y~thalamic peptides: central nervous svstem control of visceral functions. Fed Proc $0: 25% 2569,‘1981. Brownstein, M. 1.. M. Palkovits, J. M. Saavedra, R. M. Bassiri and R. D. Utiner. Thvrotrouin-re~easinR hormone in specific nuclei of rat b&in. Scikzw 63% 267-269‘1 1974, Cnffreld. __._._.., .I. A.. V. Mitetic. E. M. Zimmerman. M. S. Hoffert and B. R. Brooks’Demonstration of thyrotropin releasing hormone immunoreactivity in neurons of the mouse dorsal horn. ,I Nc~/trr~sci6: 1194-l 197. 1986.
TRH IN RAT SPINAL CORD
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