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Thyrotropin-releasing hormone (TRH) neurons sprout in cervical spinal cord of Wobbler mouse
Brain Research, 626 (1993) 83-89
83
© 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00 BRES 19329
Thyrotropin-releasing hormone (TRH) neurons sprout in cervical spinal cord of Wobbler mouse J. S h i
a
and L.L. Vacca-Galloway
b
aDepartment of Neurobiology, TongjiMedical University, Wuhan (China)and 6Department of Anatomy, Universityof Hong Kong, HongKong (HongKong) (Accepted 4 May 1993)
Key words: Motoneuron disease; Thyrotropin-releasinghormone; Immunocytochemistry;Spinal cord; Computerized morphometry
The present study was undertaken to quantify the immunocytochemicalchanges for thyrotropin-releasing hormone (TRH) within the ventral horn of the cervical spinal cord from Wobbler (wr/wr) mice selected at postnatal ages 3 weeks to 5 months compared with the normal phenotype (NFR/wr) littermates as well as mice from two related normal mouse strains: the NFR/N parent strain, and the closely related C57BI/6N mouse strain. The immunoreactive(IR) neuronal processes containing TRH appeared in all specimens within Rexed's laminae VIII, IX, and X. Compared with the normal (C57B1/6N, NFR/N) specimens, the pair-matched normal phenotype (NFR/wr) and Wobbler (wr/wr) specimens possessed significantlygreater numbers of IR-TRH containing processes at every age studied. Compared with the normal phenotype (NFR/wr) specimens, greater numbers of IR-TRH containing processes appeared in the ventral horn region studied from the Wobbler (wr/wr) specimens taken early (Stage 1) as well as later (Stages 3 and 4) in the motoneuron disease. An age-related decline in the number of IR-TRH processes was apparent among the specimens from the Wobbler mouse strain (NFR/wr, wr/wr), but not the normal (NFR/N, C57BI/6N) mouse strains. The data suggest that TRH may play a significant role in the Wobbler disease, possibly even before the symptoms become apparent. In addition strain-related differences exist which may be important to the etiologyof the Wobbler disorder.
INTRODUCTION The Wobbler mouse ( w r / w r ) shows progressive weakness and atrophy primarily of the forelimb musculature, secondary to cervical spinal cord motoneuron degeneration, and therefore has been considered as a model for human motoneuron diseases (amyotrophic lateral sclerosis, ALS; and infantile spinal muscular atrophy, ISMA) 6'9. Our early immunocytochemical (ICC) study in the cervical spinal cord showed that the amount of immunostaining for thyrotropin-releasing hormone (TRH) in immunoreactive (IR) neuronal processes seemed to be greater in the ventral horn of the Wobbler ( w r / w r ) mouse compared with the normal phenotype ( N F R / w r ) littermate control specimens 29. The increase of I R - T R H within neuronal processes was clearly manifested by the time the symptoms were well defined (Stages 2 and 3), with a slight increase noted earlier (Stage 1) 29. It was hypothesized that the increased amount of immunoreactivity, detected by optical density readings, might represent a change in
the synthesis or degradation of the neurotransmitters, a n d / o r the sprouting of preterminal neurons z9. Previous ICC studies verified increased amounts of immunoreactivity for substance P-(SP) 28 and serotonin(5-HT) 29 containing neuronal processes within the ventral horn region of Wobbler ( w r / w r ) mice. Recent quantitative ICC studies from our laboratory indicate that the number of IR-neuronal processes containing SP, 5-HT, leucine and methionine enkephalin (LE, ME), cholecystokinin (CCK), and galanin are greater in the cervical ventral horn of the Wobbler ( w r / w r ) mouse studied at different stages (either early a n d / o r late) in the motoneuron disease compared with the control littermates 3°,34. However IR-neuronal processes as well as perikarya motoneuron containing calcitonin gene-related peptide (CGRP) are lower 3°'34. Studies by radioimmunoassay (RIA) have shown that higher concentrations of I R - T R H occur within the spinal cord, pons and medulla taken from two to three-month-old Wobbler mice (C57B1/6N mouse strain) 4A6'26. Our recent R I A studies indicate that IR-
Correspondence: L.L. Vacca-Galloway,Department of Anatomy, University of Hong Kong, Faculty of Medicine, Li Shu Fan Building, 5 Sassoon Road, Hong Kong. Fax: (852) 817-0857.
~4 T R H c o n t e n t s in the W o b b l e r ( N F R / w r
m o u s e strain)
tions report no c h a n g e for the n u m b e r of I R - n e u r o n a i
cervical spinal cord are significantly g r e a t e r early in the
processes
disease (Stage l) at postnatal age 22 days and later
e n k e p h a l i n ( E N K ) and T R H , w h e r e a s o t h e r s reporl
in
the
ventral
horn
that
contain
CCK.
(Stage 3) at postnatal age 2 months, and a p p r o a c h e d
that
significance at postnatal ages 24 days (Stage 2) and 5.5
C G R P , and o t h e r n e u r o p e p t i d e s are lost lO. R I A s have
m o n t h s (Stage 4), c o m p a r e d with the p a i r - m a t c h e d
m e a s u r e d no d i f f e r e n c e s in T R H
normal
phenotype
the
IR-processes
containing
ENK,
TRH,
SP.
c o n c e n t r a t i o n s per
l i t t e r m a t e s 33. D e s p i t e
clear bio-
milligram protein, a l t h o u g h evalutions m a d e per wet
chemical indications for c h a n g e s in T R H
concentra-
weight of tissue indicate that T R H was lower in the
tions d u r i n g the W o b b l e r disease process, no t h r o u g h
chronically diseased h u m a n tissues ~4
q u a n t i t a t i v e 1CC study has b e e n d o n e t h r o u g h o u t the
T R H is an excitatory n e u r o t r a n s m i t t e r that reportedly causes m o t o n e u r o n excitation in the rat 3'22'32. In
m o t o n e u r o n disease for c o m p a r i s o n . studied
c u l t u r e d rat ventral spinal neurons, T R H e n h a n c e s the
p o s t m o r t e m f r o m c h r o n i c cases, s o m e ICC investiga-
Concerning
human
motoneuron
diseases
activities of choline acetyltransferase and c r e a t i n e ki-
Fig. 1. lmmunocytochemical localization of thyrotropin-releasing hormone (TRH) in the ventrolateral ventral horn region of the cervical spinal cord (ABC technique combined with the glucose oxidase-DAB-nickel method). Arrows point to presumed motoneurons. A: immunoreactive (IR) puncta and varicose neuronal processes appear in a normal phenotype (NFR/wr) littermate specimen (3 weeks old) of the Wobbler mouse strain. B: a pair-matched Wobbler (wr/wr) mouse specimen selected at Stage 1 (3 weeks old) of the motoneuron disease shows more numerous processes IR-puncta and varicose neuronal compared with the NFR/wr specimen shown in A. C: the distribution of IR puncta and varicose neuronal processes is shown in a normal phenotype (NFR/wr) specimen (1 month old) from the Wobbler mouse strain. D: a pair-matched Wobbler (wr/wr) mouse specimen at Stage 2 (1 month old) of the motoneuron disease shows IR puncta and varicose processes around several presumed motoneurons (arrows). Although the number of IR-processes appears to be greater than in the normal phenotype NFR/wr specimen shown in C, actual counts lack statistical significance (see Table I). E: IR puncta and varicose neuronal processes are shown in a normal phenotype (NFR/wr) specimen (2 months old) from the Wobbler mouse strain. F: a pair-matched Wobbler (wr/wr) mouse specimen at Stage 3 (2 months old) of the motoneuron disease shows more numerous IR-puncta and varicose neuronal processes compared with the NFR/wr specimen shown in E. G: IR puncta and varicose neuronal processes appear in a normal phenotype (NFR/wr) specimen (5 months old) from the Wobbler mouse strain. H: the number of IR puncta and varicose neuronal processes appear to be greater in a pair-matched Wobbler mouse at Stage 4 (5 months old), than in the NFR/wr specimen shown in G. Bar = 70/xm.
85
Fig. 1 (continued).
naseY
and benefits neuronal
g r o w t h 23. T h e r e f o r e ,
has been suggested that TRH
has a r o l e in n e u r o n a l
g r o w t h as w e l l as n e u r o m o d u l a t i o n . use of TRH cal
it
Accordingly, the
has b e e n a d v o c a t e d for s e v e r a l n e u r o l o g i -
conditions
including
acute
spinal
cord
injury 8,
s p i n o - c e r e b e l l a r d e g e n e r a t i o n 25, a n d A L S 7. T h e p r e s e n t I C C s t u d y was u n d e r t a k e n t h e c h a n g e s for I R - T R H
to q u a n t i f y
neuronal processes within the
ventral horn of the cervical spinal cord of the Wobbler mouse and
(NFR/wr
late
mouse strain) throughout
stages of the
motoneuron
the early
disease
(Stages
1-4). For normal controls, the pair-matched
normal
p h e n o t y p e l i t t e r m a t e s w e r e u s e d , as w e l l as m i c e f r o m two n o r m a l m o u s e strains: t h e N F R / N and the C57BL/6N
p a r e n t strain,
m o u s e s t r a i n r e l a t e d to t h e origi-
nal C 5 7 B I / 6 N m u t a n t s f r o m w h i c h t h e o r i g i n a l W o b b l e r m o u s e s t r a i n d e r i v e s 9.
MATERIALS AND METHODS Wobbler mice (NFR/wr strain, NIH, Bethesda, MD) were staged 2s during the motoneuron disease according to behavioral tests 11A7. Behavioral tests included the evaluation of front leg power
by the ability to pick up a small cardboard box, to climb a vertical grating, to walk 17, and to perform the clasp-knife reflex 11. After anesthetizing with an intraperitoneal injection of sodium pentobarbital (60 mg/kg), the mice were perfused through the left ventricle with Tyrode's buffer-free Ca 2+ (35°C) followed by 4% paraformaldehyde, 0.2% glutaraldehyde and 0.2% picric acid in phosphate buffer (0.1 M pH 7.2, 4°C) 27. Spinal cord segments C3 to T1 were quickly removed and immersed in the same fixative for 2 h at 4°C. Then the specimens were placed overnight in phosphate buffer (0.1 M pH 7.2, 4°C) containing 30% sucrose. Ten um transverse sections were cut with a cryostat (SLEE Instruments) and mounted on poly-L-lysine coated slides. The tissue sections were immunostained using rabbit anti-TRH (1:4000, 24 h, RT)visualized with the avidin-biotin complex (ABC) technique 12 combined with the glucose oxidase-DAB-nickel method 24. Antibodies to synthetic TRH (Hoechst Ag Frankfurt, FRG) coupled to human serum albumin were generated in rabbits 27,31, and generously given to us (J. Visser) for this study. Antiserum specificity was identified by: (1) incubation with normal rabbit serum in place of TRH antiserum in the first layer; (2) specific absorption of first layer antiserum with antigen TRH (Peninsula Laboratories, Inc.). Both types of controls showed that non-specific staining could not account for the immunocytochemical results. Quantitative studies were performed using computerized morphometry (Vidas, Zeiss) in order to determine the fiber density for the immunostained neuronal processes visualized in the ventral horn of the cervical spinal cord between segments C5 and C8~ In view of the fact that the Wobbler disease expresses itself most severely in the forelimbs 5'6'9, we selected the motoneuron pool of the triceps muscle, using the results from a previous studing of horseradish peroxidase uptake from the periphery 2, in order to limit the scope of the
86
I1
immunocytochemical quantifications. Therefore the region selected for study was the ventrolateral quadrant of the ventral horn 3°'34, known from the H R P tracing studies to contain the motoneurons that innervate the triceps muscle ~-. For this part the work, Wobbler mice were examined at Stages 1 to 4 of the motoneuron disease and compared with the pair-matched normal phenotype littermates as well as the age- and sex-matched mice from the two normal mouse strains ( N F R / N and C57BI/6N). For each specimen, every fifth tissue section was counted, after first capturing an image on the Vidas screen using a microscope (Zeiss Optiphot, 20×objective) fitted with a black-and-white video camera (JVC). A program was developed to overlay the captured image with a grid of 1 4 × 1 4 squares, each measuring 2 0 x 2 0 /zm (total area 78400 /xm-~). Point counts were made of the IR-processes crossing the vertical and horizontal grid lines using a cursor. The number of hits were counted for the IR-neuronal processes, and the data used to determine the results were compared statistically using Student's t-tesl.
wr/WF
NFR/wr C57BI/6N NFR/N
~-] [~ d-
4
.Of .Of *0~
o o o
5
O<
.Q(
*0(
*0(
o o0J
2
o, o
O(
2
L ~
O<
o
~ o
Z
RESULTS 22 d a y s (Stage 1)
I R-TRH puncta and varicose neuronal processes were observed in the ventrolateral motoneurons studied from the Wobbler (wr/wr) and normal phenotype (NFR/wr) littermate mice as well as the mice from the two normal mouse strains: N F R / N and C57B1/6N (Fig. 1). IR-TRH fibers could be visualized around the somata of the ventrolateral motoneurons, normal and diseased, thought to innervate the triceps muscle 2 (Fig. 1). Often the accumulation of immunoreactive sites for TRH seemed to be greater around the degenerating motoneurons in the Wobbler specimens taken at all stages of the disease (Fig. 1B, D, F, H arrows) compared with the normal motoneurons observed in the pair-matched littermate controls (Fig. 1A, C, E, G arrows). However, normal motoneurons observed in the normal phenotype (NFR/wr) specimens were sometimes heavily innervated as well (Fig. 1E arrow). Interestingly, IR-TRH containing fibers, but not perikarya, were visualized in the dorsal horn of the C57BI/6N specimens (not shown), but they were barely detectable in this region of the normal N F R / N , the
E
1 month (Stage 2)
months (Stage 3)
°
5 months (Stage 4)
Fig. 2. Bar graph showing the number (No.) of hits for I R - T R H neuronal processes counted per 1000 ~ m 2 in the ventrolateral quadrant of the ventral horn from the cervical spinal cord (C5-C8) taken from Wobbler ( w r / w r ) , and pair-matched normal phenotype ( N F R / w r ) littermates as well as normal ( N F R / N , C57BI/6N) mice. ** P < 0.05 and * * * P < 0.005 compared with pair-matched normal phenotype ( N F R / w r ) littermates. ~o P<^0.005 compared with pairmatched normal N F R / N specimens. P < 0.005 compared with pair-matched normal C57BI/6N specimens.
normal phenotype (NFR/wr), and the Wobbler (wr/
wr) specimens. Quantitatively, significantly greater numbers of IRneuronal processes were counted for TRH in the ventrolateral ventral horn of the Wobbler (wr/wr) mice at Stage 1 (P < 0.05), Stage 3 (P < 0.005), and Stage 4 (P<0.005), but not Stage 2, compared with pairmatched normal phenotype (NFR/wr) littermates (Table I, Fig. 2). Compared with the normal (NFR/N, C57BI/6N) mice, the number of IR-TRH containing processes in the ventrolateral quadrant of the ventral
TABLE I
Number of immunoreactive processes counted per 1000 ixm 2 area in the lateral ventral horn of the cervical spinal cord (C5- C8) taken from normal parent strain (C57Bl / 6N, NFR / N) mice and the mice from the Wobbler strain (NFR / N, normal phenotype; wr / wr, Wobbler phenotype at Stages 1 to 4 of the motoneuron disease) Statistical comparisons were accomplished using Student's t-test ± S.D. N u m b e r of animals is in parentheses.
Stage / age
Mouse strain wr / wr
NFR / wr
Stage Stage Stage Stage
2.75 ± 0.89 (6) * * 1.43 ± 0.29 (6) 2.17 ± 0.38 (6) * * * 1.58 ± 0.43 (6) * * *
1.88 ± 1.36 ± 1.35 ± 0.99 ±
1/22 days 2 / 1 month 3 / 2 months 4 / 5 months
0.30 0.20 0.32 0.30
(6) (6) (6) (6)
NFR / N
C57Bl / 6N
0.55 + 0.09 (6) 0.56 ± 0.12 (6) 0.62 ± 0.17 (6) 0.54 ± 0.11 (6)
0.98 ± 0.14 1.08 ± 0.13 1.04 ± 0.17 1.24 ± 0.29
* * * P < 0.005; * * P < 0.05 compared with pair-matched normal phenotype ( N F R / w r ) littermate specimens. °°° P < 0.005 compared with pair-matched normal N F R / N mouse specimens.
(6) (6) (6) (6)
°°° °°° °°° °°°
87
n
22 days ~ - ~ 1 month 2 months [ ~ 5 months
.0 .0 *0
d~
(Stage (Stage (Stage (Stage
1) 2) 3) 4)
0 0 0
0
.o<
nor the normal phenotype (NFR/wr) littermate specimens (r = - 0 . 8 3 P > 0.05) (Fig. 4). However, regression analyses from our previous R I A data showed a significant age-related decrease indeed exists for T R H concentrations measured in the cervical spinal cord from both Wobbler (wr/wr) and normal phenotype (NFR/wr) control mice 33. Normal ( N F R / N , C57B1/ 6N) mice were not examined 33. DISCUSSION
d
Z
0 wr/wr
NFR/wr
C57B1/6N NFR/N
Fig. 3. Bar graph showing the number (No.) of hits for IR-TRH neuronal processes counted per 1000 ~m 2 in the ventrolateral quadrant of the ventral horn of the cervical spinal cord (C5-C8) taken from young and old Wobbler (wr/wr)mice, pair-matched normal phenotype (NFR/wr) littermates, and normal (NFR/N, C57BI/6N) mice at postnatal ages 22 days, 1 month, 2 months and 5 months. °°°P <0.005 comparedwith age 1 month. *** P < 0.005 compared with age 5 months. P < 0.005 compared with age 2 months.
horn region studied from the Wobbler (wr/wr) mouse specimens was significantly greater at Stage 1 ( P < 0.005), Stage 2 ( P < 0.005), Stage 3 ( P < 0.005) and Stage 4 ( P < 0.005) (Fig. 2). The normal phenotype (NFR/wr) specimens possessed greater numbers than the normal N F R / N specimens at all ages and the C 5 7 B I / 6 N specimens except at age 5 months (Fig. 2). Comparing the two normal ( N F R / N , C 5 7 B I / 6 N ) mouse specimens, the number of I R - T R H neuronal processes was significantly greater in the C 5 7 B I / 6 N specimens compared with the age-matched N F R / N mouse specimens at all ages, young and old (Table I, Fig. 2). Comparing the Wobbler (wr/wr) specimens taken at the earliest stage (Stage 1, postnatal age 22 days) in the motoneuron disease with those taken at the latest stage (Stage 4, postnatal age 5 months), a significant ( P < 0.005) age-related numerical decrease could be detected for the I R - T R H containing processes in the ventrolateral ventral horn (Table I, Fig. 3). The same significant trend ( P < 0.005) was established for the normal phenotype (NFR/wr) littermates, but not for the normal N F R / N and C 5 7 B I / 6 N specimens (Fig. 3). Unfortunately regression analyses did not reveal a consistently significant trend for the age-related decrease in the number of the I R - T R H containing processes in the Wobbler (wr/wr) specimens (r = - 0 . 4 6 , P > 0.05)
The data verify that the number of I R - T R H containing processes is greater around the cervical spinal ventrolateral motoneurons thought to innervate the triceps muscle 2 of the Wobbler mouse (wr/wr) at early (Stage 1) and late (Stages 3, 4) stages of the motoneuron disease compared with the pair-matched normal phenotype (NFR/wr) littermate controls. These results partly confirm and expand our earlier ICC results which measured by photometry an increased amount of T R H immunoreactivity at Stages 2 and 3 within neuronal processes afferent to Wobbler motoneurons 29. The present data also correlate well with our recent R I A findings, showing that higher concentrations of I R - T R H exist in the cervical spinal cord of the Wobbler mouse taken early (Stage 1) and late (Stage 3) in
•
--
•
wr/wr
• •
--
•
--
•
NFR/wr NFR/N
•
--
•
CS,TBI/6N regression
line
o o o
o
6
Z
. . . . . . . . . i " i
0
-
0
•
................
I
I
I
I
I
30
60
90
120
150
Age
(days)
Fig. 4. Regression analyses for the number of IR-TRH neuronal processes in the ventrolateral quadrant of the ventral horn from Wobbler (wr/wr) and normal phenotype (NFR/wr) littermate specimens. The data show r and P values are respectively: wr/wr, r = 0.46, P > 0.05; NFR/wr, r = -0.8351, P > 0.05; C57BI/6N, r = +0.924, P > 0.05; NFR/N, r = -0.2506, P > 0.05.
~8 the motoneuron disease 3-~. Despite descrepant findings from the previous ICC and R I A data at Stages 2 and 4 respectively, there seem to be two phases for the presumed sprouting of the I R - T R H processes, one early and one late in the motoneuron disease. Why no increase could be found by R I A at Stage 2 is not known. However it has been reported that Stages 2 and 3 contain greater variations in neurotransmitter concentrations than Stages 1 and 433. Observer variation in staging the mice may also be a factor. The increased concentrations of I R - T R H 29'33 within greater numbers of neuronal processes counted in the Wobbler ventral horn may derive from the decreased release or the increased biosynthesis of T R H , or both. Although the mechanism remains unclear, the increased numbers of I R - T R H neuronal processes and concentration detected in the Wobbler mouse (wr/wr) spinal cord at cervical levels, the region of greatest pathological degeneration, hypothetically may be secondary to the alteration of postsynaptic T R H receptors on degenerating motoneurons. This hypothesis is supported the increased numbers of TRH-containing processes observed around the diseased motoneurons (Fig 1B, D, F, H arrows). Alternatively an altered metabolism of T R H may occur. The early increase of T R H suggests that this peptide may be important in the etiology the motoneuron disease. Without data on the T R H receptors (work in progress), the decrease of T R H concentrations with age detected previously by R I A 33 and herein by ICC comparing ages 22 days and 5 months (Fig. 3) in the normal phenotype (NFR/wr) and Wobbler specimens, but not in the normal C 5 7 B I / 6 N and N F R / N specimens, leads us to suspect that the postnatal development of T R H neurons may be abnormal in the Wobbler mouse strain. These data underscore the possibility that the TRH-containing neurons in the Wobbler (NFR/wr) mouse strain may differ from normal. Perhaps such a defect underlies the Wobbler abnormality. Studies in presymptomatic mice are warrented, but they are limited by difficulties in the detection of diseased littermates prior to 3 weeks of age 5'6'9. The finding that the number of I R - T R H containing processes in the ventrolaterat ventral horn region of the cervical spinal cord from the C57B1/6N mouse specimens is greater than in the pair-matched N F R / N specimens may indicate that other strain-related differences exist for T R H neurons, but for which no behavioral abnormality can be found. The early increase of TRH-containing processes suggests that T R H may play a significant role in the disease process, even before the symptoms of the disease become apparent. The presumed sprouting of
T R H containing processes around diseased motoneurons suggests that the motoneurons themselves may solicit the exhuberrant growth. ,4cknowledgements. The work is supported by award from UPGC (388/031/0006, LLV-G), Croucher Foundation (360/031/0814, LLV-G) and CRCG of the University of Hong Kong (372/162/6396). The authors would also like to thank Miss A. Li for her excellent technical assistance. We are grateful to Dr. T.J. Visser for his generous gift of TRH antiserum, and to Dr. T. H6kfelt for his beneficial advice. We also appreciate the kind support of Professor B. Weatherhead and the Department of Anatomy (University of Hong Kong) throughout the course of our work.
REFERENCES l Bradley, W.G., Munsat, T.L., Pelham, R.W., Rasool, L.G., Baruah, J.K., Chanerjee, A., Silber, D. and Kugelmann, K., In Kidman, A.D. and Tomkin, J.K. (Eds.), Muscle Nerve and Brain Degeneration, pp. 67-84, Excerpta Medica, Amsterdam, 1979. 2 Cheng, R., Tay, D.K.C. and Vacca-Galloway, L.L., The motoneuron pools of the triceps and flexor muscles of the Wobbler mouse, a model for motoneuron disease, according to the retrograde transport of horseradish peroxidase, Neurosci. Lett., Suppl. 40 (1990) $53. 3 Clarke, K.A., and Stirk, G.C., Motoneuron excitability after adminstration of a thyrotrophin releasing hormone analogue, Br. J. Pharmacol., 80 (1983) 561-565. 4 Court, J.A., Mcdermott, J.R., Gibonson, A.M., Marshall, E., Bloxham, C.A., Perry, R.H. and Edwardson, J.A., Raised thyrotrophin-releasing hormone, pyroglutamylamino peptidase, and proline endopeptidase are present in the spinal cord of Wobbler mice but not in human motor neurone disease, J. Neurochem., 49 (1987) 1084-1090. 5 Duchen, L.W., Falconer, D.S. and Strich, J.S., Hereditary progressive neurogenic muscular atrophy in the mouse, J. Physiol., 183 (1966) 53-55. 6 Duchen, L.W. and Strich, S.J., A hereditary motor neuron disease with progressive denervation of muscle in the mouse: the mutant 'Wobbler', J. Neurol. Neurosurg. Psychiatry, 31 (1968) 535-542. 7 Engel, W.K., Siddique and Nicoloff, J.T., Thyrotropin-releasing hormone (TRH) acutely in amyotrophic lateral sclerosis (ALS) patients causes increased mobility and strenth, lessened spasticity, shivering and tachypnea, Neurology, 33 (Suppl. 2) (1983) 120. 8 Faden, A.I., Jacobs, T.P. and Holaday, J.W., Thyrotropin releasing hormone improves neurologic recovery after spinal trauma in cat, N. Engl. J. Med., 305 (1981) 1063-1067. 9 Falconer, D.S., Wobbler, Mouse News Lett., 15 (1956) 23. 10 Gibson, S.J., Polak, J.M., Katagiri, T., Su, H., Weller, R.O., Brownell, D.B., Holland, S., Hughes, J.T., Kikuyama, S., Ball, J., Bloom S.R., Steiner, T.J., de Belleroche, J. and Rose, F.C., A comparison of the distributions of eight peptides in spinal cord from normal controls and cases of motor neuron disease with special reference to Onuf's nucleus, Brain Res., 474 (1988) 255278. 11 Hanson, P.A., Research strategies in infantile spinal muscular atrophy. In Gamstorp, I. and Sarnat, H.B. (Eds.), Progressive Spinal Muscular Atrophies Vol. 37, 1984, pp. 209-224. 12 Hsu, S.M., Raine, L. and Fanger, H., Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques. A comparison between ABC and unlabelled antibody (PAP) procedure, J. Histochem. Cytochem., 29 (1981) 1349-1353. 13 Huttinlocher, R.P. and Cohen, R.B., Oxidative enzymes in spinal neurons in Werding-Hoffman disease, Neurology, 16 (1966) 390. 14 Jackson, I.M.D., Adelman, L.S., Munsat, T.L., Forte, S. and Lechan, R.M., Amyotrophic lateral sclerosis: thyrotropin releasing hormone and histidyl proline diketopiperazine in the spinal cord and cerebrospinal fluid, Neurology, 36 (1986) 1218-1223.
89 15 Kardon, F.C., Winokur, A. and Utiger, R.D., Thyrotropin releasing hormone (TRH) in rat spinal cord, Brain Res., 122 (1977) 578-581. 16 Kozachuk, W.E., Mitsumoto, H., Salanga, V.D., Beck, G.J. and Wilber, J.F., Thyrotropin-releasing hormone (TRH) in murine motor neuron disease (the wobbler mouse), J. Neurol. Sci., 78 (1987) 253-260. 17 Lange, D.J., Good, P.F. and Bradley, W.G., A therapeutic trial of gangliosides and thymosin in the wobbler mouse model of motor neuron disease, J. NeuroL Sci., 61 (1983) 211-216 18 Mitsumoto, H. and Bradley, W.G., Murine motor neuron disease (the Wobbler mouse): degeneration and regeneration of the lower motor neuron, Brain, 105 (1982) 811-834. 19 Mitsumoto, H., Salgado, E.D., Negroski, D., Hanson, M.R., Saalariga, V.D. and Wibourn, A., Double-blind crossover trial with acute intravenous thyrotropin-releasing hormone infusion in patients with amyotrophic lateral sclerosis negative studies, Ann. Neurol., 16 (1984) 109. 20 Murakami, T., Mann, D.M.A. and Bradley, W.G., Abnormal RNA metabolism in spinal motor neurons in the Wobbler mouse, Muscle Nerve, 4 (1981) 407-412. 21 Naftchi, N.E., Abrahams, S.J., St. Paul, H.M. and Vacca, L.L., Substance P and leucine-enkphain changes in spinal cord of paraplegic rats and cats, Peptides, 2, (Suppl. 1) (1981) pp85-101. 22 Nicoll, R.A., Excitatory action of TRH on spinal motoneurons, Nature, 265 (1977) 242-243. 23 Schmidt-Achert, K.M., Askanas, V. and Engel, W.K., Thyrotropin-releasing hormone enhances choline acetyltransferase and creatine kinase in cultured spinal ventral neurons, J. Neurochem., 43 (1984) 586-589. 24 Shu, S.Y., Ju, G. and Lingzhi, F., The glucose oxidase-DAB-nickel method un peroxidase histochemitry of the nervous system, Neurosci. Lett., 85 (1988) 169-171. 25 Sobue, I., Yamanoto, H., Konagay, M., Iida, M. and Takayanagi, T., Effects of thyrotropin-releasing hormone on ataxia of spinocerebellar degeneration, Lancet, i (1980) 418-419. 26 Tang, F., Cheung, A. and Vacca-Galloway, L.L., Measurement of neuropeptides in the brain and spinal cord of Wobbler mouse: a model for motoneuron disease, Brain Res., 518 (1990) 329-333.
27 Ulfhake, B., Arvidsson, U., Cullheim, S., HSkfelt, T. and Visser, T.J., Thyrotropin-releasing hormone (TRH)-immunoreactive boutons and nerve cell bodies in the dorsal horn of the cat L7 spinal cord, Neurosci. Lett., 73 (1987) 3-8. 28 Vacca-Galloway, L.L. and Steinberg, C.C., Substance P neurons sprout in the cervical spinal cord of the wobbler mouse: a model for motoneuron disease, J. Neurosci. Res., 16 (1986) 657-670. 29 Vacca-Galloway, L.L., Steinberger, C., Pool, E. and Menon, C.D., Substance P, thyrotropin-releasing hormone and serotonin neurons sprout in cervical ventral horn of the wobbler mouse a model for motoneuron disease. In Henry, J.L. et al. (Eds.), Substance P and Neurokinins, 1987, pp. 359-362. 30 Vacca-Galloway, L.L. and Zhang, Y.Q., Decreased immunoreactive (IR) calcitonin gene-related peptide correlates with sprouting of IR-peptidergic and serotonergic neurona processes in spinal cord and during motoneuron disease, Neurosci. Lett., Suppl. 41 (1991) S19 31 Visser, T.J., Klootwilk, W., Doctor, R. and Henneman, G., A different approach to the radioimmunoassay of thyrotropin-releasing hormone. In Radioimmunoassay and Related Procedures in Medicine, National Atomic Energy Agency Vienna, 1977, pp. 469-477 32 White, S.R., A comparison of the effects of serotonin, substance P and thyrotropin-releasing hormone on excitability of rat spinal motoneurons in vivo, Brain Res., 335 (1985) 63-70. 33 Yung, K.K.L, Tang, F., Fielding, R., Du, Y.H. and Vacca-Galloway, L.L., Alteration in the levels of thyrotropin releasing hormone, substance P and enkephalins in the spinal cord, brainstem, hypothalamus and midbrain of the Wobbler mouse at different stages of the motoneuron disease, Neuroscience, 50 (1992) 209222. 34 Zhang, Y.Q. and Vacca-Galloway, L.L., Decreased immunoreactive (IR) calcitonin gene-related peptide correlates with sprouting of IR-peptidergic and serotonergic neuronal processes in spinal cord and brain nuclei from the Wobbler mouse during motoneuron disease, Brain Res., 587 (1992) 169-177.
83
© 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00 BRES 19329
Thyrotropin-releasing hormone (TRH) neurons sprout in cervical spinal cord of Wobbler mouse J. S h i
a
and L.L. Vacca-Galloway
b
aDepartment of Neurobiology, TongjiMedical University, Wuhan (China)and 6Department of Anatomy, Universityof Hong Kong, HongKong (HongKong) (Accepted 4 May 1993)
Key words: Motoneuron disease; Thyrotropin-releasinghormone; Immunocytochemistry;Spinal cord; Computerized morphometry
The present study was undertaken to quantify the immunocytochemicalchanges for thyrotropin-releasing hormone (TRH) within the ventral horn of the cervical spinal cord from Wobbler (wr/wr) mice selected at postnatal ages 3 weeks to 5 months compared with the normal phenotype (NFR/wr) littermates as well as mice from two related normal mouse strains: the NFR/N parent strain, and the closely related C57BI/6N mouse strain. The immunoreactive(IR) neuronal processes containing TRH appeared in all specimens within Rexed's laminae VIII, IX, and X. Compared with the normal (C57B1/6N, NFR/N) specimens, the pair-matched normal phenotype (NFR/wr) and Wobbler (wr/wr) specimens possessed significantlygreater numbers of IR-TRH containing processes at every age studied. Compared with the normal phenotype (NFR/wr) specimens, greater numbers of IR-TRH containing processes appeared in the ventral horn region studied from the Wobbler (wr/wr) specimens taken early (Stage 1) as well as later (Stages 3 and 4) in the motoneuron disease. An age-related decline in the number of IR-TRH processes was apparent among the specimens from the Wobbler mouse strain (NFR/wr, wr/wr), but not the normal (NFR/N, C57BI/6N) mouse strains. The data suggest that TRH may play a significant role in the Wobbler disease, possibly even before the symptoms become apparent. In addition strain-related differences exist which may be important to the etiologyof the Wobbler disorder.
INTRODUCTION The Wobbler mouse ( w r / w r ) shows progressive weakness and atrophy primarily of the forelimb musculature, secondary to cervical spinal cord motoneuron degeneration, and therefore has been considered as a model for human motoneuron diseases (amyotrophic lateral sclerosis, ALS; and infantile spinal muscular atrophy, ISMA) 6'9. Our early immunocytochemical (ICC) study in the cervical spinal cord showed that the amount of immunostaining for thyrotropin-releasing hormone (TRH) in immunoreactive (IR) neuronal processes seemed to be greater in the ventral horn of the Wobbler ( w r / w r ) mouse compared with the normal phenotype ( N F R / w r ) littermate control specimens 29. The increase of I R - T R H within neuronal processes was clearly manifested by the time the symptoms were well defined (Stages 2 and 3), with a slight increase noted earlier (Stage 1) 29. It was hypothesized that the increased amount of immunoreactivity, detected by optical density readings, might represent a change in
the synthesis or degradation of the neurotransmitters, a n d / o r the sprouting of preterminal neurons z9. Previous ICC studies verified increased amounts of immunoreactivity for substance P-(SP) 28 and serotonin(5-HT) 29 containing neuronal processes within the ventral horn region of Wobbler ( w r / w r ) mice. Recent quantitative ICC studies from our laboratory indicate that the number of IR-neuronal processes containing SP, 5-HT, leucine and methionine enkephalin (LE, ME), cholecystokinin (CCK), and galanin are greater in the cervical ventral horn of the Wobbler ( w r / w r ) mouse studied at different stages (either early a n d / o r late) in the motoneuron disease compared with the control littermates 3°,34. However IR-neuronal processes as well as perikarya motoneuron containing calcitonin gene-related peptide (CGRP) are lower 3°'34. Studies by radioimmunoassay (RIA) have shown that higher concentrations of I R - T R H occur within the spinal cord, pons and medulla taken from two to three-month-old Wobbler mice (C57B1/6N mouse strain) 4A6'26. Our recent R I A studies indicate that IR-
Correspondence: L.L. Vacca-Galloway,Department of Anatomy, University of Hong Kong, Faculty of Medicine, Li Shu Fan Building, 5 Sassoon Road, Hong Kong. Fax: (852) 817-0857.
~4 T R H c o n t e n t s in the W o b b l e r ( N F R / w r
m o u s e strain)
tions report no c h a n g e for the n u m b e r of I R - n e u r o n a i
cervical spinal cord are significantly g r e a t e r early in the
processes
disease (Stage l) at postnatal age 22 days and later
e n k e p h a l i n ( E N K ) and T R H , w h e r e a s o t h e r s reporl
in
the
ventral
horn
that
contain
CCK.
(Stage 3) at postnatal age 2 months, and a p p r o a c h e d
that
significance at postnatal ages 24 days (Stage 2) and 5.5
C G R P , and o t h e r n e u r o p e p t i d e s are lost lO. R I A s have
m o n t h s (Stage 4), c o m p a r e d with the p a i r - m a t c h e d
m e a s u r e d no d i f f e r e n c e s in T R H
normal
phenotype
the
IR-processes
containing
ENK,
TRH,
SP.
c o n c e n t r a t i o n s per
l i t t e r m a t e s 33. D e s p i t e
clear bio-
milligram protein, a l t h o u g h evalutions m a d e per wet
chemical indications for c h a n g e s in T R H
concentra-
weight of tissue indicate that T R H was lower in the
tions d u r i n g the W o b b l e r disease process, no t h r o u g h
chronically diseased h u m a n tissues ~4
q u a n t i t a t i v e 1CC study has b e e n d o n e t h r o u g h o u t the
T R H is an excitatory n e u r o t r a n s m i t t e r that reportedly causes m o t o n e u r o n excitation in the rat 3'22'32. In
m o t o n e u r o n disease for c o m p a r i s o n . studied
c u l t u r e d rat ventral spinal neurons, T R H e n h a n c e s the
p o s t m o r t e m f r o m c h r o n i c cases, s o m e ICC investiga-
Concerning
human
motoneuron
diseases
activities of choline acetyltransferase and c r e a t i n e ki-
Fig. 1. lmmunocytochemical localization of thyrotropin-releasing hormone (TRH) in the ventrolateral ventral horn region of the cervical spinal cord (ABC technique combined with the glucose oxidase-DAB-nickel method). Arrows point to presumed motoneurons. A: immunoreactive (IR) puncta and varicose neuronal processes appear in a normal phenotype (NFR/wr) littermate specimen (3 weeks old) of the Wobbler mouse strain. B: a pair-matched Wobbler (wr/wr) mouse specimen selected at Stage 1 (3 weeks old) of the motoneuron disease shows more numerous processes IR-puncta and varicose neuronal compared with the NFR/wr specimen shown in A. C: the distribution of IR puncta and varicose neuronal processes is shown in a normal phenotype (NFR/wr) specimen (1 month old) from the Wobbler mouse strain. D: a pair-matched Wobbler (wr/wr) mouse specimen at Stage 2 (1 month old) of the motoneuron disease shows IR puncta and varicose processes around several presumed motoneurons (arrows). Although the number of IR-processes appears to be greater than in the normal phenotype NFR/wr specimen shown in C, actual counts lack statistical significance (see Table I). E: IR puncta and varicose neuronal processes are shown in a normal phenotype (NFR/wr) specimen (2 months old) from the Wobbler mouse strain. F: a pair-matched Wobbler (wr/wr) mouse specimen at Stage 3 (2 months old) of the motoneuron disease shows more numerous IR-puncta and varicose neuronal processes compared with the NFR/wr specimen shown in E. G: IR puncta and varicose neuronal processes appear in a normal phenotype (NFR/wr) specimen (5 months old) from the Wobbler mouse strain. H: the number of IR puncta and varicose neuronal processes appear to be greater in a pair-matched Wobbler mouse at Stage 4 (5 months old), than in the NFR/wr specimen shown in G. Bar = 70/xm.
85
Fig. 1 (continued).
naseY
and benefits neuronal
g r o w t h 23. T h e r e f o r e ,
has been suggested that TRH
has a r o l e in n e u r o n a l
g r o w t h as w e l l as n e u r o m o d u l a t i o n . use of TRH cal
it
Accordingly, the
has b e e n a d v o c a t e d for s e v e r a l n e u r o l o g i -
conditions
including
acute
spinal
cord
injury 8,
s p i n o - c e r e b e l l a r d e g e n e r a t i o n 25, a n d A L S 7. T h e p r e s e n t I C C s t u d y was u n d e r t a k e n t h e c h a n g e s for I R - T R H
to q u a n t i f y
neuronal processes within the
ventral horn of the cervical spinal cord of the Wobbler mouse and
(NFR/wr
late
mouse strain) throughout
stages of the
motoneuron
the early
disease
(Stages
1-4). For normal controls, the pair-matched
normal
p h e n o t y p e l i t t e r m a t e s w e r e u s e d , as w e l l as m i c e f r o m two n o r m a l m o u s e strains: t h e N F R / N and the C57BL/6N
p a r e n t strain,
m o u s e s t r a i n r e l a t e d to t h e origi-
nal C 5 7 B I / 6 N m u t a n t s f r o m w h i c h t h e o r i g i n a l W o b b l e r m o u s e s t r a i n d e r i v e s 9.
MATERIALS AND METHODS Wobbler mice (NFR/wr strain, NIH, Bethesda, MD) were staged 2s during the motoneuron disease according to behavioral tests 11A7. Behavioral tests included the evaluation of front leg power
by the ability to pick up a small cardboard box, to climb a vertical grating, to walk 17, and to perform the clasp-knife reflex 11. After anesthetizing with an intraperitoneal injection of sodium pentobarbital (60 mg/kg), the mice were perfused through the left ventricle with Tyrode's buffer-free Ca 2+ (35°C) followed by 4% paraformaldehyde, 0.2% glutaraldehyde and 0.2% picric acid in phosphate buffer (0.1 M pH 7.2, 4°C) 27. Spinal cord segments C3 to T1 were quickly removed and immersed in the same fixative for 2 h at 4°C. Then the specimens were placed overnight in phosphate buffer (0.1 M pH 7.2, 4°C) containing 30% sucrose. Ten um transverse sections were cut with a cryostat (SLEE Instruments) and mounted on poly-L-lysine coated slides. The tissue sections were immunostained using rabbit anti-TRH (1:4000, 24 h, RT)visualized with the avidin-biotin complex (ABC) technique 12 combined with the glucose oxidase-DAB-nickel method 24. Antibodies to synthetic TRH (Hoechst Ag Frankfurt, FRG) coupled to human serum albumin were generated in rabbits 27,31, and generously given to us (J. Visser) for this study. Antiserum specificity was identified by: (1) incubation with normal rabbit serum in place of TRH antiserum in the first layer; (2) specific absorption of first layer antiserum with antigen TRH (Peninsula Laboratories, Inc.). Both types of controls showed that non-specific staining could not account for the immunocytochemical results. Quantitative studies were performed using computerized morphometry (Vidas, Zeiss) in order to determine the fiber density for the immunostained neuronal processes visualized in the ventral horn of the cervical spinal cord between segments C5 and C8~ In view of the fact that the Wobbler disease expresses itself most severely in the forelimbs 5'6'9, we selected the motoneuron pool of the triceps muscle, using the results from a previous studing of horseradish peroxidase uptake from the periphery 2, in order to limit the scope of the
86
I1
immunocytochemical quantifications. Therefore the region selected for study was the ventrolateral quadrant of the ventral horn 3°'34, known from the H R P tracing studies to contain the motoneurons that innervate the triceps muscle ~-. For this part the work, Wobbler mice were examined at Stages 1 to 4 of the motoneuron disease and compared with the pair-matched normal phenotype littermates as well as the age- and sex-matched mice from the two normal mouse strains ( N F R / N and C57BI/6N). For each specimen, every fifth tissue section was counted, after first capturing an image on the Vidas screen using a microscope (Zeiss Optiphot, 20×objective) fitted with a black-and-white video camera (JVC). A program was developed to overlay the captured image with a grid of 1 4 × 1 4 squares, each measuring 2 0 x 2 0 /zm (total area 78400 /xm-~). Point counts were made of the IR-processes crossing the vertical and horizontal grid lines using a cursor. The number of hits were counted for the IR-neuronal processes, and the data used to determine the results were compared statistically using Student's t-tesl.
wr/WF
NFR/wr C57BI/6N NFR/N
~-] [~ d-
4
.Of .Of *0~
o o o
5
O<
.Q(
*0(
*0(
o o0J
2
o, o
O(
2
L ~
O<
o
~ o
Z
RESULTS 22 d a y s (Stage 1)
I R-TRH puncta and varicose neuronal processes were observed in the ventrolateral motoneurons studied from the Wobbler (wr/wr) and normal phenotype (NFR/wr) littermate mice as well as the mice from the two normal mouse strains: N F R / N and C57B1/6N (Fig. 1). IR-TRH fibers could be visualized around the somata of the ventrolateral motoneurons, normal and diseased, thought to innervate the triceps muscle 2 (Fig. 1). Often the accumulation of immunoreactive sites for TRH seemed to be greater around the degenerating motoneurons in the Wobbler specimens taken at all stages of the disease (Fig. 1B, D, F, H arrows) compared with the normal motoneurons observed in the pair-matched littermate controls (Fig. 1A, C, E, G arrows). However, normal motoneurons observed in the normal phenotype (NFR/wr) specimens were sometimes heavily innervated as well (Fig. 1E arrow). Interestingly, IR-TRH containing fibers, but not perikarya, were visualized in the dorsal horn of the C57BI/6N specimens (not shown), but they were barely detectable in this region of the normal N F R / N , the
E
1 month (Stage 2)
months (Stage 3)
°
5 months (Stage 4)
Fig. 2. Bar graph showing the number (No.) of hits for I R - T R H neuronal processes counted per 1000 ~ m 2 in the ventrolateral quadrant of the ventral horn from the cervical spinal cord (C5-C8) taken from Wobbler ( w r / w r ) , and pair-matched normal phenotype ( N F R / w r ) littermates as well as normal ( N F R / N , C57BI/6N) mice. ** P < 0.05 and * * * P < 0.005 compared with pair-matched normal phenotype ( N F R / w r ) littermates. ~o P<^0.005 compared with pairmatched normal N F R / N specimens. P < 0.005 compared with pair-matched normal C57BI/6N specimens.
normal phenotype (NFR/wr), and the Wobbler (wr/
wr) specimens. Quantitatively, significantly greater numbers of IRneuronal processes were counted for TRH in the ventrolateral ventral horn of the Wobbler (wr/wr) mice at Stage 1 (P < 0.05), Stage 3 (P < 0.005), and Stage 4 (P<0.005), but not Stage 2, compared with pairmatched normal phenotype (NFR/wr) littermates (Table I, Fig. 2). Compared with the normal (NFR/N, C57BI/6N) mice, the number of IR-TRH containing processes in the ventrolateral quadrant of the ventral
TABLE I
Number of immunoreactive processes counted per 1000 ixm 2 area in the lateral ventral horn of the cervical spinal cord (C5- C8) taken from normal parent strain (C57Bl / 6N, NFR / N) mice and the mice from the Wobbler strain (NFR / N, normal phenotype; wr / wr, Wobbler phenotype at Stages 1 to 4 of the motoneuron disease) Statistical comparisons were accomplished using Student's t-test ± S.D. N u m b e r of animals is in parentheses.
Stage / age
Mouse strain wr / wr
NFR / wr
Stage Stage Stage Stage
2.75 ± 0.89 (6) * * 1.43 ± 0.29 (6) 2.17 ± 0.38 (6) * * * 1.58 ± 0.43 (6) * * *
1.88 ± 1.36 ± 1.35 ± 0.99 ±
1/22 days 2 / 1 month 3 / 2 months 4 / 5 months
0.30 0.20 0.32 0.30
(6) (6) (6) (6)
NFR / N
C57Bl / 6N
0.55 + 0.09 (6) 0.56 ± 0.12 (6) 0.62 ± 0.17 (6) 0.54 ± 0.11 (6)
0.98 ± 0.14 1.08 ± 0.13 1.04 ± 0.17 1.24 ± 0.29
* * * P < 0.005; * * P < 0.05 compared with pair-matched normal phenotype ( N F R / w r ) littermate specimens. °°° P < 0.005 compared with pair-matched normal N F R / N mouse specimens.
(6) (6) (6) (6)
°°° °°° °°° °°°
87
n
22 days ~ - ~ 1 month 2 months [ ~ 5 months
.0 .0 *0
d~
(Stage (Stage (Stage (Stage
1) 2) 3) 4)
0 0 0
0
.o<
nor the normal phenotype (NFR/wr) littermate specimens (r = - 0 . 8 3 P > 0.05) (Fig. 4). However, regression analyses from our previous R I A data showed a significant age-related decrease indeed exists for T R H concentrations measured in the cervical spinal cord from both Wobbler (wr/wr) and normal phenotype (NFR/wr) control mice 33. Normal ( N F R / N , C57B1/ 6N) mice were not examined 33. DISCUSSION
d
Z
0 wr/wr
NFR/wr
C57B1/6N NFR/N
Fig. 3. Bar graph showing the number (No.) of hits for IR-TRH neuronal processes counted per 1000 ~m 2 in the ventrolateral quadrant of the ventral horn of the cervical spinal cord (C5-C8) taken from young and old Wobbler (wr/wr)mice, pair-matched normal phenotype (NFR/wr) littermates, and normal (NFR/N, C57BI/6N) mice at postnatal ages 22 days, 1 month, 2 months and 5 months. °°°P <0.005 comparedwith age 1 month. *** P < 0.005 compared with age 5 months. P < 0.005 compared with age 2 months.
horn region studied from the Wobbler (wr/wr) mouse specimens was significantly greater at Stage 1 ( P < 0.005), Stage 2 ( P < 0.005), Stage 3 ( P < 0.005) and Stage 4 ( P < 0.005) (Fig. 2). The normal phenotype (NFR/wr) specimens possessed greater numbers than the normal N F R / N specimens at all ages and the C 5 7 B I / 6 N specimens except at age 5 months (Fig. 2). Comparing the two normal ( N F R / N , C 5 7 B I / 6 N ) mouse specimens, the number of I R - T R H neuronal processes was significantly greater in the C 5 7 B I / 6 N specimens compared with the age-matched N F R / N mouse specimens at all ages, young and old (Table I, Fig. 2). Comparing the Wobbler (wr/wr) specimens taken at the earliest stage (Stage 1, postnatal age 22 days) in the motoneuron disease with those taken at the latest stage (Stage 4, postnatal age 5 months), a significant ( P < 0.005) age-related numerical decrease could be detected for the I R - T R H containing processes in the ventrolateral ventral horn (Table I, Fig. 3). The same significant trend ( P < 0.005) was established for the normal phenotype (NFR/wr) littermates, but not for the normal N F R / N and C 5 7 B I / 6 N specimens (Fig. 3). Unfortunately regression analyses did not reveal a consistently significant trend for the age-related decrease in the number of the I R - T R H containing processes in the Wobbler (wr/wr) specimens (r = - 0 . 4 6 , P > 0.05)
The data verify that the number of I R - T R H containing processes is greater around the cervical spinal ventrolateral motoneurons thought to innervate the triceps muscle 2 of the Wobbler mouse (wr/wr) at early (Stage 1) and late (Stages 3, 4) stages of the motoneuron disease compared with the pair-matched normal phenotype (NFR/wr) littermate controls. These results partly confirm and expand our earlier ICC results which measured by photometry an increased amount of T R H immunoreactivity at Stages 2 and 3 within neuronal processes afferent to Wobbler motoneurons 29. The present data also correlate well with our recent R I A findings, showing that higher concentrations of I R - T R H exist in the cervical spinal cord of the Wobbler mouse taken early (Stage 1) and late (Stage 3) in
•
--
•
wr/wr
• •
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Fig. 4. Regression analyses for the number of IR-TRH neuronal processes in the ventrolateral quadrant of the ventral horn from Wobbler (wr/wr) and normal phenotype (NFR/wr) littermate specimens. The data show r and P values are respectively: wr/wr, r = 0.46, P > 0.05; NFR/wr, r = -0.8351, P > 0.05; C57BI/6N, r = +0.924, P > 0.05; NFR/N, r = -0.2506, P > 0.05.
~8 the motoneuron disease 3-~. Despite descrepant findings from the previous ICC and R I A data at Stages 2 and 4 respectively, there seem to be two phases for the presumed sprouting of the I R - T R H processes, one early and one late in the motoneuron disease. Why no increase could be found by R I A at Stage 2 is not known. However it has been reported that Stages 2 and 3 contain greater variations in neurotransmitter concentrations than Stages 1 and 433. Observer variation in staging the mice may also be a factor. The increased concentrations of I R - T R H 29'33 within greater numbers of neuronal processes counted in the Wobbler ventral horn may derive from the decreased release or the increased biosynthesis of T R H , or both. Although the mechanism remains unclear, the increased numbers of I R - T R H neuronal processes and concentration detected in the Wobbler mouse (wr/wr) spinal cord at cervical levels, the region of greatest pathological degeneration, hypothetically may be secondary to the alteration of postsynaptic T R H receptors on degenerating motoneurons. This hypothesis is supported the increased numbers of TRH-containing processes observed around the diseased motoneurons (Fig 1B, D, F, H arrows). Alternatively an altered metabolism of T R H may occur. The early increase of T R H suggests that this peptide may be important in the etiology the motoneuron disease. Without data on the T R H receptors (work in progress), the decrease of T R H concentrations with age detected previously by R I A 33 and herein by ICC comparing ages 22 days and 5 months (Fig. 3) in the normal phenotype (NFR/wr) and Wobbler specimens, but not in the normal C 5 7 B I / 6 N and N F R / N specimens, leads us to suspect that the postnatal development of T R H neurons may be abnormal in the Wobbler mouse strain. These data underscore the possibility that the TRH-containing neurons in the Wobbler (NFR/wr) mouse strain may differ from normal. Perhaps such a defect underlies the Wobbler abnormality. Studies in presymptomatic mice are warrented, but they are limited by difficulties in the detection of diseased littermates prior to 3 weeks of age 5'6'9. The finding that the number of I R - T R H containing processes in the ventrolaterat ventral horn region of the cervical spinal cord from the C57B1/6N mouse specimens is greater than in the pair-matched N F R / N specimens may indicate that other strain-related differences exist for T R H neurons, but for which no behavioral abnormality can be found. The early increase of TRH-containing processes suggests that T R H may play a significant role in the disease process, even before the symptoms of the disease become apparent. The presumed sprouting of
T R H containing processes around diseased motoneurons suggests that the motoneurons themselves may solicit the exhuberrant growth. ,4cknowledgements. The work is supported by award from UPGC (388/031/0006, LLV-G), Croucher Foundation (360/031/0814, LLV-G) and CRCG of the University of Hong Kong (372/162/6396). The authors would also like to thank Miss A. Li for her excellent technical assistance. We are grateful to Dr. T.J. Visser for his generous gift of TRH antiserum, and to Dr. T. H6kfelt for his beneficial advice. We also appreciate the kind support of Professor B. Weatherhead and the Department of Anatomy (University of Hong Kong) throughout the course of our work.
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