- Email: [email protected]
Alterations in acetylcholinesterase and choline acetyltransferase activities and neuropeptide levels in the ventral spinal cord of the Wobbler mouse during inherited motoneuron disease
BRAIN RESEARCH ELSEVIER
Brain Research 638 (1994) 337-342
Short Communication
Alterations in acetylcholinesterase and choline acetyltransferase activities and neuropeptide levels in the ventral spinal cord of the Wobbler mouse during inherited motoneuron disease K.K.L. Yung a,b,**, F. Tang b, L.L. Vacca-Galloway a,. Departments of ~Anatomy and b Physiology, Faculty of Medicine, University of Hong Kong, Hong Kong (Accepted 23 November 1993)
Abstract
Enymatic assays for acetylcholine esterase (ACHE) and choline acetyltransferase (CHAT) were applied to dorsal and ventral cervical spinal cord regions taken from the Wobbler mouse, a model for inherited motoneuron disease. Early in the disease, CHAT (but not ACHE) activity is significantly greater compared with the control littermate specimens. The high ChAT activity correlates with the high thyrotropin releasing hormone (also leucine-enkephalin) concentrations measured in the Wobbler ventral horn early in the disease. Late in the motoneuron disease, both AChE and ChAT activities are significantly lower than in the control littermate specimens. These data correlate with the high substance P, methionine and leucine enkephalin concentrations measured in the Wobbler ventral horn late in the motoneuron disease.
Key words: Radioimmunoassay; Radioenzymatic assay; Wobbler mouse motoneuron disease; Cervical spinal cord; Acetyicholinesterase; Choline acetyitransferase; Thyrotropin-releasing hormone; Substance P; Methionine-enkephalin; Leucine-enkephalin
The Wobbler mouse [9] has been used as a model of human motoneuron diseases (e.g., amyotrophic lateral sclerosis, ALS and infantile spinal muscular atrophy, ISMA). The most characteristic abnormality is the loss of motoneurons, in the spinal cord and the brainstem [1,5,8,27,40]. The early symptoms, identified at about 3 weeks of age [8,9,15,30,41], include the reduction of weight, shakiness, unsteady gait, hypoactivity, upper limb weakness, an upward-pointing snout [8,27] and the 'clasp-knife' reflex response [15]. Since the progress of the motoneuron disease differs among individual Wobbler mice [8], it is important to categorize the stages of the disease according to the motor deficits [41,43]. The pathogenesis of the motoneuron loss in Wobbler is unknown [27,36]. Alterations in the levels of neuropeptides, neurotransmitters and neurotransmit-
* Corresponding author. Fax: (852) 1-817-0857. ** Present address: MRC Anatomical Neuropharmacology Unit, University Department of Pharmacology, Oxford OX1 3TH, UK.
0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 3 ) E 1 5 5 5 - J
ter-related enzymes have been noted in the spinal cord, brainstem and other brain regions by immunocytochemistry [24,41,42,45] and radioimmunoassays (RIAs) [7,23,39,43]. It remains unknown whether the changes in neurotransmitters may be the primary cause of, or the secondary response to, the degeneration of motoneurons. No studies relate the biochemical aspects of the motoneuron degeneration and the changes in neurotransmitter levels. In the present study, the activities of two enzymes, acetylcholinesterase (ACHE) and choline acetyltransferase (CHAT), related to the neurotransmitter acetylcholine, are used as markers of cholinergic motoneurons in the Wobbler cervical spinal cord according to radioenzymatic assays. Neuropeptides (thyrotropin releasing hormone [TRH], substance P [SP], methionine enkephalin [ME] and leucine enkephalin [LE]) are quantified by RIAs. Wobbler mice (NFR/wr strain, NIH, Animal Resources, Bethesda, MD) were maintained in the Laboratory Animal Unit, University of Hong Kong (23°C; 65% humidity; 12 h:12 h light-dark cycle). Males and females were used since no obvious sex-related differ-
K.K.L. Yung et al. /Brain Research 638 (1994) 337-342
338
ences have been detected. The Wobbler mice (wr/wr) and their pair-matched normal phenotype control littermates (NFR/wr)were identified, weighed, and classified into different stages of the motoneuron disease by applying a set of behavioral tests [15,25,42]. Then they were decapitated; the cervical spinal cord was dissected out, and transversely sectioned by a razor blade on a glass slide cooled on dry ice in a petri dish. Under a dissection microscope (Zeiss), the dorsal and ventral halves were bissected using the central canal as the marking point (see [32]). Neuropeptide contents were measured by RIAs (TRH [2], SP, ME and LE [20]). The sources of antisera and cross-reactivities were reported previously [43]. For the radioenzymatic assays, the bissected spinal cord samples were homogenized (5% w/v) in buffered EDTA solution (0.01 M, pH 7.4) using a motorized homogenizer (Lightning) fitted with a teflon pestle [10,11]. The homogenates were treated with Triton X-100 (0.05 v/v in final concentration) to insure total release of enzyme activity [10,11]. The AChE assay [22] used a premixed incubation mixture of labelled substrate [3H]acetylcholine chloride (approximately 10,000 cpm, spec. act. 2 Ci/mmol) diluted in potassium phosphate buffer (0.05 M, pH 7.0, Amersham, Buckinghamshire, UK). The extracted enzyme homogenates were diluted 100-fold in EDTA solution (0.01 M, pH 7.4). The diluted enzyme samples or enzyme standards (electric eel ACHE, Sigma, St Louis, MO, USA) in appropriate activities were added to 10 ml scintillation vials. Then the incubation mixture (80/zl) was added, mixed, and vortexed. The reaction mixture was incubated (15 min at 25°C); the reaction was stopped by the 'standard stopping mixture' (1 M monochloro-acetic acid, 0.5 M NaOH, 2.0 M NAC1, 100 /~1 [22]); then 4 ml of scintillation mixture (0.5%
PPO, 0.03% POPOP in 90% toluene, and 10% v/v isoamyl alcohol) was added immediately. The reaction product was selectively extracted into the scintillation mixture. The ChAT assay [10,11] used an incubation mixture containing (in final concentration) labelled acetyl donor ([3H]acetyl-CoA, approximately 10,000 cpm, spec. act. 2.5 Ci/mmol; Amersham, Buckinghamshire, UK), 0.05 M sodium phosphate buffer (pH 7.4), 0.3 M NaCI, 8 mM choline chloride, 0.02 M EDTA, and 0.1 mM physostigmine. The extracted enzyme homogenates (20 /zl) or the enzyme standards (Bovine Brain CHAT, Sigma, St. Louis, MO, USA)were added in appropriate activities to 10 ml scintillation vials. The incubation mixture (100 /xl) was added and the contents mixed, vortexed, and incubated (15 min at 25°C). The reaction was stopped by adding 1 ml sodium phosphate buffer (0.01 M, pH 7.4), followed by 4 ml scintillation mixture (0.5% PPO, 0.03% POPOP in 80% v/v toluene, and 20% v/v acetonitrile containing 0.5% w/v Kalignost; Sigma, St. Louis, MO, USA). The vial was shaken slightly and the organic and aqueous layers were allowed to separate for 10 min. The reaction product was selectively extracted into the scintillation mixture. All vials were counted using a liquid scintillation counter (Parkard, Tri-Carb 2000CA). The enzyme activity in each sample was found by reading from a standard curve. The protein content of the tissues was determined [27]. The activities of the AChE and ChAT were expressed in units (of activities) per mg protein. The concentrations of the neuropeptides were expressed in pg or ng per mg protein. Statistical comparisons of the neuropeptide contents and enzyme activities in Wobbler mice and their ageand sex-matched normal phenotype control littermates
Table 1 Neuropeptide contents in bissected cervical spinal cord in Wobbler mice (Stage 1 and Stage 4) compared with the pair-matched control littermates Peptides
TRH (pg/mg SP (ng/mg ME (ng/mg LE (pg/mg
Ventral
protein) protein) protein) protein)
Dorsal
Ventral
Dorsal
NFR/wr
wr/wr
NFR/wr
wr/wr
NFR/wr
wr/wr
NFR/wr
wr/wr
Stage 0
Stage 1
Stage 0
Stage 1
Stage 0
Stage 4
Stage 0
Stage 4
298.5 5:_48.01 (8) 1.09 5:0.13 (8) 1.35 +0.28 (8) 92.72 5:26.62 (8)
]"413.05 5:13.31 (8) * 1.11 ___0.66 (8) 1.64 +0.23 (8) $158.88 + 19.28 (8) *
292.34 5:73.39 (8) 2.81 + 0.29 (8) 2.06 +0.26 (8) 265.66 5:35.80 (8)
321.72 + 79.60 (8) 2.54 _+0.60 (8) 2.05 +0.54 (8) 240.34 + 32.99 (8)
99.52 5:11.62 (9) 0.67 _ 0.13 (9) 0.94 +0.07 (9) 57.57 5:7.51 (9)
126.92 5:19.59 (9) 1' 1.46 5:0.23 (9) * * 1' 1.30 +0.16 (9) * $137.14 5:24.55 (9) * *
370.64 + 98.41 (8) 3.93 5:0.54 (9) 2.39 5:0.27 (9) 139.89 5:18.13 (9)
273.74 ± 61.64 3.12 _+0.39 2.15 5:0.31 3"236.88 5:31.27
(8) (9) (9) (9) *
Values are mean 5: S.E.M. $ Higher than the control values. TRH, thyrotropin releasing hormone; SP, substance P; ME, methionine-enkephalin; LE, leucine-enkephalin; NFR/wr, control littermates; wr/wr, Wobbler mouse. No. of animals in parentheses. * P < 0.05; * * P < 0.01 compared with control values by Student's unpaired t-test.
K~K.L. Yung et al. / Brain Research 638 (1994) 337-342
were performed using individual pair-matched values. The mean values + standard error (S.E.M.) were compared using two-tailed Student's t-test [35]. Table 1 shows the results of the immunoreactive (IR) neuropeptides measured by RIAs in the bissected cervical spinal cord (ventral and dorsal portions) from the Wobbler mice (Stage 1, age 19-21 days and Stage 4, age 3-4 months) compared with the pair-matched control littermates. The TRH values were significantly higher in the ventral (but not the dorsal) half of the spinal cord taken from Wobbler mice at Stage 1 compared with the pair-matched control mice. At Stage 4, there were no differences for the TRH contents in the ventral and dorsal halves of the Wobbler spinal cord specimens compared with the littermate controls (Table 1). For SP and ME levels, there was no significant difference in the ventral spinal cord taken from the Wobbler mice at Stage 1 compared with the littermate controls. However, the SP and ME contents were significantly greater in the Wobbler ventral spinal cord at Stage 4. In the dorsal half, no significant difference was found for SP contents (Table 1). LE contents were also significantly increased in the ventral spinal cord of the Wobbler mice at Stage 1, and in both the ventral and dorsal halves of the spinal cord taken at Stage 4 (Table 1). In the ventral spinal cord, a significantly lower level of AChE activity was detected at Stage 4 in the Wobbler specimens compared with the control littermates (Fig. la). There was no significant difference in the AChE activity of the dorsal half of the spinal cord throughout all four stages of the Wobbler disorder compared with the littermate controls (Fig. lb). Significantly greater ChAT activities were detected in the ventral spinal cord taken from Stage 1 Wobbler specimens compared with the control specimens, whereas significantly lower ChAT activities occurred at Stage 4 (Fig. 2a). There were no significant differences in ChAT activities measured for Wobbler and control littermate specimens of dorsal spinal cord (Fig. 2b). In the control littermate specimens, there was a progressive increase with age in the ChAT activity of the ventral spinal cord. The ChAT activity in the Wobbler specimens tended to decline with age and stage of the motoneuron disease (Fig. 2a). In the dorsal spinal cord, the ChAT activities in both groups of mice were similar (Fig. 2b). AChE and ChAT enzymes are used as markers for motoneurons in the ventral spinal cord [31], wherein only the motoneurons are cholinergic [3,16]. However, AChE is ubiquitous and may not be localized exclusively to motoneurons. Relatively few AChE-positive non-motoneuron cells, with unknown functions, are scattered in the intermediate gray and ventral dorsal horn regions [3]. Therefore in vitro assays may be influenced by AChE activities associated with other
339
la. AChE (ventral) 1412-
10"
t it
8"
~
Wobbler
--'e--
Control
/I
6" 4" 2" 0
Stage
lb. AChE (dorsal) 8-
6"
4'
---0---
Wobbl~
---e--
Control
2'
0
Stage
Fig. 1. Acetylcholinesterase (ACHE) activity in the cervical spinal cord of Wobbler mice at different stages (Stages 1 to 4, classified by behaviourial tests, see text) of the motoneuron disease compared with the normal phenotype control littermates. A shows the AChE activities in the ventral half of the spinal cord from the Wobbler and control littermate specimens. B shows the AChE activities in the dorsal half of the spinal cord from the specimens. Number of animals ( n ) = 8. Statistical comparisons were made by Student's t-test (** P < 0.01). mg, milligram.
cell types besides motoneurons. ChAT is regarded as the most valid marker for cholinergic (moto-) neurons because it is selective [37]. Still, both AChE and ChAT are best studied together in order to evaluate the motoneurons. The decreased activities for AChE and ChAT in the Wobbler ventral horn at Stage 4 could conceivably result from the severe degeneration and loss of motoneurons known to occur in the chronically diseased mice [1,8,9,27,30]. From the results shown for AChE and ChAT activities at Stage 1, it would seem that only a negligeable population of motoneurons undergo degeneration during Stage 1 of the disease. A recent retrograde tracer study showed that the loss of motoneurons is progressively greater in Wobbler (C57 B1/Fa) mice studied at ages 4 and 6 weeks old, but was not detected at 3 weeks of age [33]. Unfortunately, the ages of the mice were not related to the stages of the disease.
340
K.ICL. Yung et al. /Brain Research 638 (1994) 337-342 2a. ChAT (ventral) 20-
15-
Wobbler 10-- t---
Control
5-
i
E ¢I,
1
2
i
i
3
4
Stage
2b. ChAT
(dorsal)
15
~ - - ~
10
Wobbler -
Control
5-
0
Stage
Fig. 2. Choline acetyltransferase (CHAT) activity in the cervical spinal cord of Wobbler mice at different stages (Stages 1 to 4) of the motoneuron disease compared with their normal phenotype control littermates, A shows the ChAT activities in the ventral half of the spinal cord taken from the Wobbler and control littermate specimens. B shows the ChAT activities in the dorsal half of the spinal cord from the same specimens. Number of animals (n)= 8. Statistical comparisons were made by Student's t-test (* P < 0.05; * * * * P < 0.001). mg, milligram.
A recent retrograde tracing study from our laboratory showed that significant motoneuron loss (38%) occurs in young Stage 1 (age 18 to 21 days) Wobbler ( N F R / w r strain) mice [5]. These results are difficult to explain based on the present findings showing a significant increase in C h A T activity in the Stage 1 Wobbler ventral horn specimens. Perhaps the surviving motoneurons compensate for other cell losses by expressing increased C h A T activity. Alternatively the early degeneration of motoneurons [5] could result in the leakage of C h A T enzyme into the surrounding neuropil or another cell c o m p a r t m e n t that was then measured in the ventral horn. It is tempting to speculate that the high levels of C h A T at Stage 1 of the motoneuron disease represent an abnormally high number of motoneurons, destined for an accelerated process of cell death (see [15]). Considering cell counts based on previous retrograde tracing studies [5,33] the excessive numbers of motoneurons could go undetected if they
were aberrant a n d / o r not connected with the periphery. Reportedly, T R H enhances C h A T activity in ventral horn neurons in vitro [34]. Therefore perhaps the early increase of C h A T activity detected in the Wobbler ventral spinal cord at Stage 1 relates to the early increase of T R H . The C h A T activity in the Stage 1 Wobbler ventral horn is almost 4 times the control littermate levels. Alternatively, this high C h A T activity might reflect an abnormality in cholinergic neurotransmission during the disease process, since C h A T is the synthesizing enzyme for acetylcholine. There was a trend towards decreasing activities for C h A T in the Wobbler ventral spinal cord from Stage 1 to Stage 4. H u m a n postmortem tissues taken from terminal ALS patients also possess a significant decrease in C h A T activity in the lateral ventral horn [14]. A previous study in Wobbler (C57BI/6J) mutants, ages 2 to 2 3 / 4 months (equivalent to our Stage 3 mice), showed no significant difference for the ChAT activity measured in whole spinal cord compared with the pair-matched control littermates [24]. The trend towards decreasing A C h E activities was only significant at Stage 4. Enzyme histochemistry studies showed that the intensity of A C h E staining in degenerating motoneurons appeared decreased at Stages 2 and 3 [41]; a more extensive decrease was noted at Stage 4 [42]. A C h E is capable of hydrolysing SP in vitro [6,28]. If this occurs in vivo, then the late increase of SP levels in the Wobbler ventral spinal cord correlates well with the low level of A C h E activity detected at Stage 4 in the ventral horn. Hypothetically, the hydrolysis of SP by A C h E could keep the SP level from increasing during the early stages of the Wobbler syndrome. Similarly, the low A C h E activity detected in the Stage 4 Wobbler specimens could increase the SP levels. Thus SP may increase secondarily in response to the degeneration of motoneurons. Unfortunately, if SP concentrations are held in check by motoneuronal A C h E activity, the hypothesis cannot be proven since we cannot detect SP alterations at Stage 1. We have shown that T R H and LE (also serotonin [45]) increase early in the motoneuron disease (Stage 1) whereas SP and ME (also LE) increase late (Stage 4) in the cervical ventral horn. These data verify our previous R I A results in the whole cervical spinal cord [43]. The early increases of T R H , LE and serotonin lead us to speculate that they may play a role in the etiology of the disease. The late increases of SP and ME suggest that they may occur secondarily. How these peptides are involved in the Wobbler disease is unknown [39,43]. It has been reported that T R H in the cultured spinal cord [34], enkephalins in the developing cerebellum [44], and serotonin in the intact spinal cord [26], may be neurotropic, and may
K.K.L. Yung et al. / Brain Research 638 (1994) 337-342
interact with each other in functions yet unknown [13]. TR H can protect spinal motoneurons from the toxic effects of an SP antagonist [12]. TRH, SP and serotonin, which coexist within neuronal processes in the ventral horn of the spinal cord [4,13,17,18,19,21] may act as a self-repair system [18]. There is no convincing evidence suggesting that these transmitters may have detrimental effects on motoneurons. In conclusion, the significantly lower enzyme activities for AChE and ChAT detected in the Wobbler ventral horn late in the disease (Stage 4) probably reflect the prominent degeneration of motoneurons. The lack of change for ACHE, and the greater activity in CHAT, detected at Stage 1 are more difficult to interpret, and imply that massive motoneuron degeneration does not occur at Stage 1, despite our recent anatomical findings to the contrary [5]. The early motoneuron loss [5] is small in view of the total loss. Thus indeed the early increases in neurotransmitters (TRH, LE and serotonin) might not occur in response to massive motoneuron degeneration. Coincident with the massive motoneuron loss occurring later in the Wobbler disease [5,33], there is a late increase in the ventral horn contents of SP and ME. The late increases are more likely to represent secondary responses to the huge accumulation of motoneuron deaths. Studies in younger Wobblers are needed, but remain hindered by difficulties in identifying the diseased mice prior to the expression of symptoms. Additional studies in genetically normal mice are in progress in order to evaluate the extent of wr gene penetrance into the phenotypically normal heterozygote ( N F R / w r ) littermates. The authors like to thank Dr. J. Hong (TRH) and Dr. J.S. Kizer (SP) for their gifts of antisera. KKLY would like to thank Dr. J.P. Bolam (Oxford) for his valuable advice on the manuscript. We are also grateful to the support given to us by Prof. Brian Weatherhead and the Department of Anatomy, University of Hong Kong. This research is supported by the following grants: UPGC (338/031/0006), Croucher Foundation (360/031/0814), Run Run Shaw Foundation (372/162/6396), and CRCG (372/162/6994). [1] Andrews, J.M. and Andrews, R.L., The comparative neuropathology of motor neuron disease. In J.M. Andrews, R.T. Johnson and M.A.B. Brazier (Eds.), UCLA Forum in Medical Sciences, No, 19, Amyotrophic Lateral Sclerosis, Recent Research Trends, Academic Press, New York, 1976, pp. 181-216. [2] Bassiri, R.M. and Utiger, R.D., The preparation and specificity of antibody to thyrotropin releasing hormone, Endocrinology, 90 (1972) 722-727. [3] Butcher, L.L. and Woolf, N.J., Histochemical distribution of acetylcholinesterase in the central nervous system: clues to the localization of cholinergic neurons. In T. Bj6rklund, T. H6kfelt and M.J. Kuhar (Eds.), Handbook of Chemical Neuroanatomy, VoL 3: Classical Transmitters and Transmitter Receptors in the CNS, Part H Elsevier, Amsterdam, 1984, pp. 1-50. [4] Chan-Palay, V., Jonsson, G. and Palay, S.L., Serotonin and substance P co-exist in neurons of the rat's central nervous system, Proc. Natl. Acad. Sci. USA, 5 (1978) 1582-1586.
341
[5] Cheng, R.W.K., Tay, D. and Vacca-Galloway, L.L., Quantatitive changes in gamma- and alpha-motoneurons innervating the forelimb flexor and extensor muscles of the Wobbler mouse after intramucular injection of HRP tracer, Neurosci. Lett., Suppl. 41 (1991) $21. [6] Chubb, I.W., Hodgson, A.J. and White, G.H., Acetylcholinesterase hydrolyzes substance P, Neuroscience, 5 (1980) 2065 -2072. [7] Court, J.A., Mcdermott, J.R., Gibson, 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. [8] Duchen, L.W., Strich, S.J. and Falconer, D.S., An hereditary motoneuron disease with progressive denervation of muscle in the mouse, the mutant 'wobbler', J. Neurol. Neurosurg. Psychiat., 31 (1968) 535-542. [9] Falconer, D.S., Wobbler (wr), Mouse News Lett., 15 (1956) 22. [10] Fonnum, F., Radiochemical micro assays for the determination of choline acetyltransferase and acetylcholinesterase activities, Biochem. J., 115 (1969) 465-472. [11] Fonnum, F., A rapid radiochemical method for the determination of choline acetyltransferase, J. Neurochem., 24 (1975) 407409. [12] Freedman, J., H6kfelt, T., Jonsson, G. and Post, C., Thyrotropin-releasing hormone (TRH) counteracts neuronal damage induced by a substance P antagonist, Exp. Brain Res., 62 (1986) 175-178. [13] Gilbert, R.F.T., Emson, P.C., Hunt, S.P., Bennett, G.W. and Marsden, C.A., Neuronal coexistence of putative transmitters in the spinal cord and brainstem of the rat. In A.C. Cuello (Ed.), Co-transmission, MacMillan, New York, 1982, [~p. 51-75. [14] Gillberg, P-G., Aquilonius, S-T., Eckern~is, S-A., Lundqvist, G. and Winblad, B., Choline acetyltransferase and substance P-like immunoreactivity in the human spinal cord: changes in amyotrophic lateral sclerosis, Brain Res., 250 (1982) 394-397. [15] Hanson, P.A., Research strategies in infantile spinal muscular atrophy. In J. Gamstoid and H.B. Sarnat (Eds.), Progressive Spinal Muscular Atrophies, Raven, New York, 1984, pp. 209-224. [16] Heimer, L., The Human Brain and Spinal Cord, Springer, New York, 1983. [17] H6kfelt, T., Ljungdahl, A., Steinbusch, H., Verhofstad, A., Nilsson, G., Broden, E., Pernow, B. and Goldstein, M, Immunohistochemical evidence of substance P-like immunoreactivity in some 5-hydroxytryptamine-containing neurons in the rat central nervous system, Neuroscience, 3 (1978) 517-538. [18] H6kfelt, T., Johansson, O., Ljungdahl, ,~., Lundberg, J.M. and Schultzberg, M., Peptidergic neurones. Nature, 284 (1980) 515521. [19] H6kfelt, T., Lundberg, J.M., Schultzberg, M., Johansson, O. and Ljungdahl, A., Coexistence of peptides and putative transmitters in neurons, Ad~,. Biochem. Psychopharmacol., 22 (1980) 1-23. [20] Hong, J.S., Yoshikawa, K. and Hendren, R.W., Measurement of /3-endorphin and enkephalins in biological tissues and fluids. In P.M. Conn (Ed.), Methods in Enzymology, Vol. 103, Academic Press, New York, 1983, pp. 547-564. [21] Johansson, O., H6kfelt, T., Pernow, B., Jeffcoate, S.L., White, N., Steinberger, H.W.M., Verhofstad, A.A.J., Emson, P.C. and Spindel, E., Immunohistochemical support for three putative transmitters in one neuron: coexistence of 5-hydroxytryptamine, substance P and thyrotropin-releasing hormone-like activity in medullary neurones projecting to the spinal cord, Neuroscience, 6 (1981) 1857-1881. [22] Johnson, C.D. and Russell, R.L., A rapid, simple radiometric assay for cholinesterase, suitable for multiple determination, Anal. Biochem., 64 (1975) 229-238.
342
K.KL. Yung et al. /Brain Research 638 (1994) 337-342
[23] Kozachuk, W.E., Mitsumoto, H., Salanga, V.D., Beck, G.J. and Wilber, J.F., Thyrotrophin-releasing hormone (TRH) in murine motor neuron disease (the wobbler mouse), J. Neurol. Sci., 78 (1987) 253-260. [24] Laage, S., Zobel, G. and Jockusch, H., Astrocyte overgrowth in the brain stem and spinal cord of mice affected by spinal atrophy, Wobbler, De~. Neurosci., 10 (1988) 190-198. [25] 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. [26] Lauder, J.M., Wallace, J.A., Wilkie, M.B., DiNome, A. and Krebs, H., Roles for serotonin in neurogenesis, Monogr Neural Sci., 9 (1983) 3-10. [27] Leestma, J.E., Animal model: motor neuron disease in Wobbler mouse (wr/wr), Am. J. Pathol., 100 (1980) 821-824. [28] Lockridge, O., Substance P hydrolysis by human serum cholinesterase, ,L Neurochem., 39 (1982) 106-110. [29] Lowry, O.H., Rosebrough, W.J., Farr, A.L. and Randall, R.J., Protein measurement with the folic phenol reagent, J. Biol. Chem., 193 (1951) 265-275. [30] 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. [31] Paxinos, G. and Butcher, L.L., Organizational principles of the brain revealed by choline acetyltransferase and acetylcholinesterase distribution and projections. In G. Paxinos (Ed.), The Rat NerlJous System, Part 1, Academic Press, Australia, 1985, pp. 487-515. [32] Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, 2nd edn., Academic Press, Australia, 1986. [33] Pollin, M.M., McHanwell, S. and Slater, C.R., Loss of motor neurons from the median nerve motor nucleus of the mutant mouse 'wobbler', J. Neurocytol., 19 (1990) 29-38. [34] Schmidt-Achert, K., Askanas, V. and Engel, W.K., Thyrotropin-releasing hormone enhances choline acetyltransferase and creative kinase in cultured spinal ventral horn neurons, J. Neuroehem., 43 (1984) 586-589. [35] Siegel, S., Statistical errors. In: Nonparametric Statistics for the Behacioral Sciences, MacGraw-Hill, New York, 1956, pp. 55-70. [36] Sillevis Smitt, P.A, and de Jong, J.M., Animal models of amyotrophic lateral sclerosis and the spinal muscular atrophies, J. Neurol. Sci., 91 (1989) 231-258.
[37] Sofroniew, M.V., Campbell, P.E., Cuello, A.C. and Eckenstein, F., Central cholinergic neurons visualized by immunohistochemical detection of choline acetyltransferase. In G. Paxinos (Ed.), The Rat Nercous System, Part l, Academic Press, Australia, 1985, pp. 471-485. [38] Tang, F., Change in met-enkephalin and Beta-endorphin contents in the hypothalamus and the pituitary in diabetic erat: effect of insulin therapy, Clin. Exp. Pharmacol. Physiol., 16 (1989) 65-75. [39] 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. [40] Papapetropoulos, A. and Bradley, W.G., Spinal motor neuones in murine muscular dystrophy and spinal muscular atrophy, J. Neurol. Neurosurg. Psychiatry, 35 (1972) 60-65. [41] Vacca-Galloway, L.L. and Steinberger, C.C., Substance P neurons sprout in the cervical spinal cord of the wobbler mouse: a model for motoneuron disease, Z Neurosei. Res., 16 (1986) 657-670. [42] Vacca-Galloway, L.L., Steinberger, C., Poole, E. and Menon, C.D., Substance P, thyrotropin releasing hormone, and serotonin neurons sprout in cervical spinal cord of Wobbler mouse, a model for motoneuron disease. In J.L. Henry, R. Couture, A.C. Cuello, G.R. Pelletier and R. Quirion (Eds.), Substance P and Neurokinins, Springer, New York, 1987, pp. 359-362. [43] 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) 2(/9-222. [44] Zagon, I.S. and McLaughlin, P.J., Ultrastructural localization of enkephalin-like immunoreactivity in developing rat cerebellum, Neuroscience, 34 (1990) 479-489. [45] 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.
Brain Research 638 (1994) 337-342
Short Communication
Alterations in acetylcholinesterase and choline acetyltransferase activities and neuropeptide levels in the ventral spinal cord of the Wobbler mouse during inherited motoneuron disease K.K.L. Yung a,b,**, F. Tang b, L.L. Vacca-Galloway a,. Departments of ~Anatomy and b Physiology, Faculty of Medicine, University of Hong Kong, Hong Kong (Accepted 23 November 1993)
Abstract
Enymatic assays for acetylcholine esterase (ACHE) and choline acetyltransferase (CHAT) were applied to dorsal and ventral cervical spinal cord regions taken from the Wobbler mouse, a model for inherited motoneuron disease. Early in the disease, CHAT (but not ACHE) activity is significantly greater compared with the control littermate specimens. The high ChAT activity correlates with the high thyrotropin releasing hormone (also leucine-enkephalin) concentrations measured in the Wobbler ventral horn early in the disease. Late in the motoneuron disease, both AChE and ChAT activities are significantly lower than in the control littermate specimens. These data correlate with the high substance P, methionine and leucine enkephalin concentrations measured in the Wobbler ventral horn late in the motoneuron disease.
Key words: Radioimmunoassay; Radioenzymatic assay; Wobbler mouse motoneuron disease; Cervical spinal cord; Acetyicholinesterase; Choline acetyitransferase; Thyrotropin-releasing hormone; Substance P; Methionine-enkephalin; Leucine-enkephalin
The Wobbler mouse [9] has been used as a model of human motoneuron diseases (e.g., amyotrophic lateral sclerosis, ALS and infantile spinal muscular atrophy, ISMA). The most characteristic abnormality is the loss of motoneurons, in the spinal cord and the brainstem [1,5,8,27,40]. The early symptoms, identified at about 3 weeks of age [8,9,15,30,41], include the reduction of weight, shakiness, unsteady gait, hypoactivity, upper limb weakness, an upward-pointing snout [8,27] and the 'clasp-knife' reflex response [15]. Since the progress of the motoneuron disease differs among individual Wobbler mice [8], it is important to categorize the stages of the disease according to the motor deficits [41,43]. The pathogenesis of the motoneuron loss in Wobbler is unknown [27,36]. Alterations in the levels of neuropeptides, neurotransmitters and neurotransmit-
* Corresponding author. Fax: (852) 1-817-0857. ** Present address: MRC Anatomical Neuropharmacology Unit, University Department of Pharmacology, Oxford OX1 3TH, UK.
0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 3 ) E 1 5 5 5 - J
ter-related enzymes have been noted in the spinal cord, brainstem and other brain regions by immunocytochemistry [24,41,42,45] and radioimmunoassays (RIAs) [7,23,39,43]. It remains unknown whether the changes in neurotransmitters may be the primary cause of, or the secondary response to, the degeneration of motoneurons. No studies relate the biochemical aspects of the motoneuron degeneration and the changes in neurotransmitter levels. In the present study, the activities of two enzymes, acetylcholinesterase (ACHE) and choline acetyltransferase (CHAT), related to the neurotransmitter acetylcholine, are used as markers of cholinergic motoneurons in the Wobbler cervical spinal cord according to radioenzymatic assays. Neuropeptides (thyrotropin releasing hormone [TRH], substance P [SP], methionine enkephalin [ME] and leucine enkephalin [LE]) are quantified by RIAs. Wobbler mice (NFR/wr strain, NIH, Animal Resources, Bethesda, MD) were maintained in the Laboratory Animal Unit, University of Hong Kong (23°C; 65% humidity; 12 h:12 h light-dark cycle). Males and females were used since no obvious sex-related differ-
K.K.L. Yung et al. /Brain Research 638 (1994) 337-342
338
ences have been detected. The Wobbler mice (wr/wr) and their pair-matched normal phenotype control littermates (NFR/wr)were identified, weighed, and classified into different stages of the motoneuron disease by applying a set of behavioral tests [15,25,42]. Then they were decapitated; the cervical spinal cord was dissected out, and transversely sectioned by a razor blade on a glass slide cooled on dry ice in a petri dish. Under a dissection microscope (Zeiss), the dorsal and ventral halves were bissected using the central canal as the marking point (see [32]). Neuropeptide contents were measured by RIAs (TRH [2], SP, ME and LE [20]). The sources of antisera and cross-reactivities were reported previously [43]. For the radioenzymatic assays, the bissected spinal cord samples were homogenized (5% w/v) in buffered EDTA solution (0.01 M, pH 7.4) using a motorized homogenizer (Lightning) fitted with a teflon pestle [10,11]. The homogenates were treated with Triton X-100 (0.05 v/v in final concentration) to insure total release of enzyme activity [10,11]. The AChE assay [22] used a premixed incubation mixture of labelled substrate [3H]acetylcholine chloride (approximately 10,000 cpm, spec. act. 2 Ci/mmol) diluted in potassium phosphate buffer (0.05 M, pH 7.0, Amersham, Buckinghamshire, UK). The extracted enzyme homogenates were diluted 100-fold in EDTA solution (0.01 M, pH 7.4). The diluted enzyme samples or enzyme standards (electric eel ACHE, Sigma, St Louis, MO, USA) in appropriate activities were added to 10 ml scintillation vials. Then the incubation mixture (80/zl) was added, mixed, and vortexed. The reaction mixture was incubated (15 min at 25°C); the reaction was stopped by the 'standard stopping mixture' (1 M monochloro-acetic acid, 0.5 M NaOH, 2.0 M NAC1, 100 /~1 [22]); then 4 ml of scintillation mixture (0.5%
PPO, 0.03% POPOP in 90% toluene, and 10% v/v isoamyl alcohol) was added immediately. The reaction product was selectively extracted into the scintillation mixture. The ChAT assay [10,11] used an incubation mixture containing (in final concentration) labelled acetyl donor ([3H]acetyl-CoA, approximately 10,000 cpm, spec. act. 2.5 Ci/mmol; Amersham, Buckinghamshire, UK), 0.05 M sodium phosphate buffer (pH 7.4), 0.3 M NaCI, 8 mM choline chloride, 0.02 M EDTA, and 0.1 mM physostigmine. The extracted enzyme homogenates (20 /zl) or the enzyme standards (Bovine Brain CHAT, Sigma, St. Louis, MO, USA)were added in appropriate activities to 10 ml scintillation vials. The incubation mixture (100 /xl) was added and the contents mixed, vortexed, and incubated (15 min at 25°C). The reaction was stopped by adding 1 ml sodium phosphate buffer (0.01 M, pH 7.4), followed by 4 ml scintillation mixture (0.5% PPO, 0.03% POPOP in 80% v/v toluene, and 20% v/v acetonitrile containing 0.5% w/v Kalignost; Sigma, St. Louis, MO, USA). The vial was shaken slightly and the organic and aqueous layers were allowed to separate for 10 min. The reaction product was selectively extracted into the scintillation mixture. All vials were counted using a liquid scintillation counter (Parkard, Tri-Carb 2000CA). The enzyme activity in each sample was found by reading from a standard curve. The protein content of the tissues was determined [27]. The activities of the AChE and ChAT were expressed in units (of activities) per mg protein. The concentrations of the neuropeptides were expressed in pg or ng per mg protein. Statistical comparisons of the neuropeptide contents and enzyme activities in Wobbler mice and their ageand sex-matched normal phenotype control littermates
Table 1 Neuropeptide contents in bissected cervical spinal cord in Wobbler mice (Stage 1 and Stage 4) compared with the pair-matched control littermates Peptides
TRH (pg/mg SP (ng/mg ME (ng/mg LE (pg/mg
Ventral
protein) protein) protein) protein)
Dorsal
Ventral
Dorsal
NFR/wr
wr/wr
NFR/wr
wr/wr
NFR/wr
wr/wr
NFR/wr
wr/wr
Stage 0
Stage 1
Stage 0
Stage 1
Stage 0
Stage 4
Stage 0
Stage 4
298.5 5:_48.01 (8) 1.09 5:0.13 (8) 1.35 +0.28 (8) 92.72 5:26.62 (8)
]"413.05 5:13.31 (8) * 1.11 ___0.66 (8) 1.64 +0.23 (8) $158.88 + 19.28 (8) *
292.34 5:73.39 (8) 2.81 + 0.29 (8) 2.06 +0.26 (8) 265.66 5:35.80 (8)
321.72 + 79.60 (8) 2.54 _+0.60 (8) 2.05 +0.54 (8) 240.34 + 32.99 (8)
99.52 5:11.62 (9) 0.67 _ 0.13 (9) 0.94 +0.07 (9) 57.57 5:7.51 (9)
126.92 5:19.59 (9) 1' 1.46 5:0.23 (9) * * 1' 1.30 +0.16 (9) * $137.14 5:24.55 (9) * *
370.64 + 98.41 (8) 3.93 5:0.54 (9) 2.39 5:0.27 (9) 139.89 5:18.13 (9)
273.74 ± 61.64 3.12 _+0.39 2.15 5:0.31 3"236.88 5:31.27
(8) (9) (9) (9) *
Values are mean 5: S.E.M. $ Higher than the control values. TRH, thyrotropin releasing hormone; SP, substance P; ME, methionine-enkephalin; LE, leucine-enkephalin; NFR/wr, control littermates; wr/wr, Wobbler mouse. No. of animals in parentheses. * P < 0.05; * * P < 0.01 compared with control values by Student's unpaired t-test.
K~K.L. Yung et al. / Brain Research 638 (1994) 337-342
were performed using individual pair-matched values. The mean values + standard error (S.E.M.) were compared using two-tailed Student's t-test [35]. Table 1 shows the results of the immunoreactive (IR) neuropeptides measured by RIAs in the bissected cervical spinal cord (ventral and dorsal portions) from the Wobbler mice (Stage 1, age 19-21 days and Stage 4, age 3-4 months) compared with the pair-matched control littermates. The TRH values were significantly higher in the ventral (but not the dorsal) half of the spinal cord taken from Wobbler mice at Stage 1 compared with the pair-matched control mice. At Stage 4, there were no differences for the TRH contents in the ventral and dorsal halves of the Wobbler spinal cord specimens compared with the littermate controls (Table 1). For SP and ME levels, there was no significant difference in the ventral spinal cord taken from the Wobbler mice at Stage 1 compared with the littermate controls. However, the SP and ME contents were significantly greater in the Wobbler ventral spinal cord at Stage 4. In the dorsal half, no significant difference was found for SP contents (Table 1). LE contents were also significantly increased in the ventral spinal cord of the Wobbler mice at Stage 1, and in both the ventral and dorsal halves of the spinal cord taken at Stage 4 (Table 1). In the ventral spinal cord, a significantly lower level of AChE activity was detected at Stage 4 in the Wobbler specimens compared with the control littermates (Fig. la). There was no significant difference in the AChE activity of the dorsal half of the spinal cord throughout all four stages of the Wobbler disorder compared with the littermate controls (Fig. lb). Significantly greater ChAT activities were detected in the ventral spinal cord taken from Stage 1 Wobbler specimens compared with the control specimens, whereas significantly lower ChAT activities occurred at Stage 4 (Fig. 2a). There were no significant differences in ChAT activities measured for Wobbler and control littermate specimens of dorsal spinal cord (Fig. 2b). In the control littermate specimens, there was a progressive increase with age in the ChAT activity of the ventral spinal cord. The ChAT activity in the Wobbler specimens tended to decline with age and stage of the motoneuron disease (Fig. 2a). In the dorsal spinal cord, the ChAT activities in both groups of mice were similar (Fig. 2b). AChE and ChAT enzymes are used as markers for motoneurons in the ventral spinal cord [31], wherein only the motoneurons are cholinergic [3,16]. However, AChE is ubiquitous and may not be localized exclusively to motoneurons. Relatively few AChE-positive non-motoneuron cells, with unknown functions, are scattered in the intermediate gray and ventral dorsal horn regions [3]. Therefore in vitro assays may be influenced by AChE activities associated with other
339
la. AChE (ventral) 1412-
10"
t it
8"
~
Wobbler
--'e--
Control
/I
6" 4" 2" 0
Stage
lb. AChE (dorsal) 8-
6"
4'
---0---
Wobbl~
---e--
Control
2'
0
Stage
Fig. 1. Acetylcholinesterase (ACHE) activity in the cervical spinal cord of Wobbler mice at different stages (Stages 1 to 4, classified by behaviourial tests, see text) of the motoneuron disease compared with the normal phenotype control littermates. A shows the AChE activities in the ventral half of the spinal cord from the Wobbler and control littermate specimens. B shows the AChE activities in the dorsal half of the spinal cord from the specimens. Number of animals ( n ) = 8. Statistical comparisons were made by Student's t-test (** P < 0.01). mg, milligram.
cell types besides motoneurons. ChAT is regarded as the most valid marker for cholinergic (moto-) neurons because it is selective [37]. Still, both AChE and ChAT are best studied together in order to evaluate the motoneurons. The decreased activities for AChE and ChAT in the Wobbler ventral horn at Stage 4 could conceivably result from the severe degeneration and loss of motoneurons known to occur in the chronically diseased mice [1,8,9,27,30]. From the results shown for AChE and ChAT activities at Stage 1, it would seem that only a negligeable population of motoneurons undergo degeneration during Stage 1 of the disease. A recent retrograde tracer study showed that the loss of motoneurons is progressively greater in Wobbler (C57 B1/Fa) mice studied at ages 4 and 6 weeks old, but was not detected at 3 weeks of age [33]. Unfortunately, the ages of the mice were not related to the stages of the disease.
340
K.ICL. Yung et al. /Brain Research 638 (1994) 337-342 2a. ChAT (ventral) 20-
15-
Wobbler 10-- t---
Control
5-
i
E ¢I,
1
2
i
i
3
4
Stage
2b. ChAT
(dorsal)
15
~ - - ~
10
Wobbler -
Control
5-
0
Stage
Fig. 2. Choline acetyltransferase (CHAT) activity in the cervical spinal cord of Wobbler mice at different stages (Stages 1 to 4) of the motoneuron disease compared with their normal phenotype control littermates, A shows the ChAT activities in the ventral half of the spinal cord taken from the Wobbler and control littermate specimens. B shows the ChAT activities in the dorsal half of the spinal cord from the same specimens. Number of animals (n)= 8. Statistical comparisons were made by Student's t-test (* P < 0.05; * * * * P < 0.001). mg, milligram.
A recent retrograde tracing study from our laboratory showed that significant motoneuron loss (38%) occurs in young Stage 1 (age 18 to 21 days) Wobbler ( N F R / w r strain) mice [5]. These results are difficult to explain based on the present findings showing a significant increase in C h A T activity in the Stage 1 Wobbler ventral horn specimens. Perhaps the surviving motoneurons compensate for other cell losses by expressing increased C h A T activity. Alternatively the early degeneration of motoneurons [5] could result in the leakage of C h A T enzyme into the surrounding neuropil or another cell c o m p a r t m e n t that was then measured in the ventral horn. It is tempting to speculate that the high levels of C h A T at Stage 1 of the motoneuron disease represent an abnormally high number of motoneurons, destined for an accelerated process of cell death (see [15]). Considering cell counts based on previous retrograde tracing studies [5,33] the excessive numbers of motoneurons could go undetected if they
were aberrant a n d / o r not connected with the periphery. Reportedly, T R H enhances C h A T activity in ventral horn neurons in vitro [34]. Therefore perhaps the early increase of C h A T activity detected in the Wobbler ventral spinal cord at Stage 1 relates to the early increase of T R H . The C h A T activity in the Stage 1 Wobbler ventral horn is almost 4 times the control littermate levels. Alternatively, this high C h A T activity might reflect an abnormality in cholinergic neurotransmission during the disease process, since C h A T is the synthesizing enzyme for acetylcholine. There was a trend towards decreasing activities for C h A T in the Wobbler ventral spinal cord from Stage 1 to Stage 4. H u m a n postmortem tissues taken from terminal ALS patients also possess a significant decrease in C h A T activity in the lateral ventral horn [14]. A previous study in Wobbler (C57BI/6J) mutants, ages 2 to 2 3 / 4 months (equivalent to our Stage 3 mice), showed no significant difference for the ChAT activity measured in whole spinal cord compared with the pair-matched control littermates [24]. The trend towards decreasing A C h E activities was only significant at Stage 4. Enzyme histochemistry studies showed that the intensity of A C h E staining in degenerating motoneurons appeared decreased at Stages 2 and 3 [41]; a more extensive decrease was noted at Stage 4 [42]. A C h E is capable of hydrolysing SP in vitro [6,28]. If this occurs in vivo, then the late increase of SP levels in the Wobbler ventral spinal cord correlates well with the low level of A C h E activity detected at Stage 4 in the ventral horn. Hypothetically, the hydrolysis of SP by A C h E could keep the SP level from increasing during the early stages of the Wobbler syndrome. Similarly, the low A C h E activity detected in the Stage 4 Wobbler specimens could increase the SP levels. Thus SP may increase secondarily in response to the degeneration of motoneurons. Unfortunately, if SP concentrations are held in check by motoneuronal A C h E activity, the hypothesis cannot be proven since we cannot detect SP alterations at Stage 1. We have shown that T R H and LE (also serotonin [45]) increase early in the motoneuron disease (Stage 1) whereas SP and ME (also LE) increase late (Stage 4) in the cervical ventral horn. These data verify our previous R I A results in the whole cervical spinal cord [43]. The early increases of T R H , LE and serotonin lead us to speculate that they may play a role in the etiology of the disease. The late increases of SP and ME suggest that they may occur secondarily. How these peptides are involved in the Wobbler disease is unknown [39,43]. It has been reported that T R H in the cultured spinal cord [34], enkephalins in the developing cerebellum [44], and serotonin in the intact spinal cord [26], may be neurotropic, and may
K.K.L. Yung et al. / Brain Research 638 (1994) 337-342
interact with each other in functions yet unknown [13]. TR H can protect spinal motoneurons from the toxic effects of an SP antagonist [12]. TRH, SP and serotonin, which coexist within neuronal processes in the ventral horn of the spinal cord [4,13,17,18,19,21] may act as a self-repair system [18]. There is no convincing evidence suggesting that these transmitters may have detrimental effects on motoneurons. In conclusion, the significantly lower enzyme activities for AChE and ChAT detected in the Wobbler ventral horn late in the disease (Stage 4) probably reflect the prominent degeneration of motoneurons. The lack of change for ACHE, and the greater activity in CHAT, detected at Stage 1 are more difficult to interpret, and imply that massive motoneuron degeneration does not occur at Stage 1, despite our recent anatomical findings to the contrary [5]. The early motoneuron loss [5] is small in view of the total loss. Thus indeed the early increases in neurotransmitters (TRH, LE and serotonin) might not occur in response to massive motoneuron degeneration. Coincident with the massive motoneuron loss occurring later in the Wobbler disease [5,33], there is a late increase in the ventral horn contents of SP and ME. The late increases are more likely to represent secondary responses to the huge accumulation of motoneuron deaths. Studies in younger Wobblers are needed, but remain hindered by difficulties in identifying the diseased mice prior to the expression of symptoms. Additional studies in genetically normal mice are in progress in order to evaluate the extent of wr gene penetrance into the phenotypically normal heterozygote ( N F R / w r ) littermates. The authors like to thank Dr. J. Hong (TRH) and Dr. J.S. Kizer (SP) for their gifts of antisera. KKLY would like to thank Dr. J.P. Bolam (Oxford) for his valuable advice on the manuscript. We are also grateful to the support given to us by Prof. Brian Weatherhead and the Department of Anatomy, University of Hong Kong. This research is supported by the following grants: UPGC (338/031/0006), Croucher Foundation (360/031/0814), Run Run Shaw Foundation (372/162/6396), and CRCG (372/162/6994). [1] Andrews, J.M. and Andrews, R.L., The comparative neuropathology of motor neuron disease. In J.M. Andrews, R.T. Johnson and M.A.B. Brazier (Eds.), UCLA Forum in Medical Sciences, No, 19, Amyotrophic Lateral Sclerosis, Recent Research Trends, Academic Press, New York, 1976, pp. 181-216. [2] Bassiri, R.M. and Utiger, R.D., The preparation and specificity of antibody to thyrotropin releasing hormone, Endocrinology, 90 (1972) 722-727. [3] Butcher, L.L. and Woolf, N.J., Histochemical distribution of acetylcholinesterase in the central nervous system: clues to the localization of cholinergic neurons. In T. Bj6rklund, T. H6kfelt and M.J. Kuhar (Eds.), Handbook of Chemical Neuroanatomy, VoL 3: Classical Transmitters and Transmitter Receptors in the CNS, Part H Elsevier, Amsterdam, 1984, pp. 1-50. [4] Chan-Palay, V., Jonsson, G. and Palay, S.L., Serotonin and substance P co-exist in neurons of the rat's central nervous system, Proc. Natl. Acad. Sci. USA, 5 (1978) 1582-1586.
341
[5] Cheng, R.W.K., Tay, D. and Vacca-Galloway, L.L., Quantatitive changes in gamma- and alpha-motoneurons innervating the forelimb flexor and extensor muscles of the Wobbler mouse after intramucular injection of HRP tracer, Neurosci. Lett., Suppl. 41 (1991) $21. [6] Chubb, I.W., Hodgson, A.J. and White, G.H., Acetylcholinesterase hydrolyzes substance P, Neuroscience, 5 (1980) 2065 -2072. [7] Court, J.A., Mcdermott, J.R., Gibson, 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. [8] Duchen, L.W., Strich, S.J. and Falconer, D.S., An hereditary motoneuron disease with progressive denervation of muscle in the mouse, the mutant 'wobbler', J. Neurol. Neurosurg. Psychiat., 31 (1968) 535-542. [9] Falconer, D.S., Wobbler (wr), Mouse News Lett., 15 (1956) 22. [10] Fonnum, F., Radiochemical micro assays for the determination of choline acetyltransferase and acetylcholinesterase activities, Biochem. J., 115 (1969) 465-472. [11] Fonnum, F., A rapid radiochemical method for the determination of choline acetyltransferase, J. Neurochem., 24 (1975) 407409. [12] Freedman, J., H6kfelt, T., Jonsson, G. and Post, C., Thyrotropin-releasing hormone (TRH) counteracts neuronal damage induced by a substance P antagonist, Exp. Brain Res., 62 (1986) 175-178. [13] Gilbert, R.F.T., Emson, P.C., Hunt, S.P., Bennett, G.W. and Marsden, C.A., Neuronal coexistence of putative transmitters in the spinal cord and brainstem of the rat. In A.C. Cuello (Ed.), Co-transmission, MacMillan, New York, 1982, [~p. 51-75. [14] Gillberg, P-G., Aquilonius, S-T., Eckern~is, S-A., Lundqvist, G. and Winblad, B., Choline acetyltransferase and substance P-like immunoreactivity in the human spinal cord: changes in amyotrophic lateral sclerosis, Brain Res., 250 (1982) 394-397. [15] Hanson, P.A., Research strategies in infantile spinal muscular atrophy. In J. Gamstoid and H.B. Sarnat (Eds.), Progressive Spinal Muscular Atrophies, Raven, New York, 1984, pp. 209-224. [16] Heimer, L., The Human Brain and Spinal Cord, Springer, New York, 1983. [17] H6kfelt, T., Ljungdahl, A., Steinbusch, H., Verhofstad, A., Nilsson, G., Broden, E., Pernow, B. and Goldstein, M, Immunohistochemical evidence of substance P-like immunoreactivity in some 5-hydroxytryptamine-containing neurons in the rat central nervous system, Neuroscience, 3 (1978) 517-538. [18] H6kfelt, T., Johansson, O., Ljungdahl, ,~., Lundberg, J.M. and Schultzberg, M., Peptidergic neurones. Nature, 284 (1980) 515521. [19] H6kfelt, T., Lundberg, J.M., Schultzberg, M., Johansson, O. and Ljungdahl, A., Coexistence of peptides and putative transmitters in neurons, Ad~,. Biochem. Psychopharmacol., 22 (1980) 1-23. [20] Hong, J.S., Yoshikawa, K. and Hendren, R.W., Measurement of /3-endorphin and enkephalins in biological tissues and fluids. In P.M. Conn (Ed.), Methods in Enzymology, Vol. 103, Academic Press, New York, 1983, pp. 547-564. [21] Johansson, O., H6kfelt, T., Pernow, B., Jeffcoate, S.L., White, N., Steinberger, H.W.M., Verhofstad, A.A.J., Emson, P.C. and Spindel, E., Immunohistochemical support for three putative transmitters in one neuron: coexistence of 5-hydroxytryptamine, substance P and thyrotropin-releasing hormone-like activity in medullary neurones projecting to the spinal cord, Neuroscience, 6 (1981) 1857-1881. [22] Johnson, C.D. and Russell, R.L., A rapid, simple radiometric assay for cholinesterase, suitable for multiple determination, Anal. Biochem., 64 (1975) 229-238.
342
K.KL. Yung et al. /Brain Research 638 (1994) 337-342
[23] Kozachuk, W.E., Mitsumoto, H., Salanga, V.D., Beck, G.J. and Wilber, J.F., Thyrotrophin-releasing hormone (TRH) in murine motor neuron disease (the wobbler mouse), J. Neurol. Sci., 78 (1987) 253-260. [24] Laage, S., Zobel, G. and Jockusch, H., Astrocyte overgrowth in the brain stem and spinal cord of mice affected by spinal atrophy, Wobbler, De~. Neurosci., 10 (1988) 190-198. [25] 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. [26] Lauder, J.M., Wallace, J.A., Wilkie, M.B., DiNome, A. and Krebs, H., Roles for serotonin in neurogenesis, Monogr Neural Sci., 9 (1983) 3-10. [27] Leestma, J.E., Animal model: motor neuron disease in Wobbler mouse (wr/wr), Am. J. Pathol., 100 (1980) 821-824. [28] Lockridge, O., Substance P hydrolysis by human serum cholinesterase, ,L Neurochem., 39 (1982) 106-110. [29] Lowry, O.H., Rosebrough, W.J., Farr, A.L. and Randall, R.J., Protein measurement with the folic phenol reagent, J. Biol. Chem., 193 (1951) 265-275. [30] 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. [31] Paxinos, G. and Butcher, L.L., Organizational principles of the brain revealed by choline acetyltransferase and acetylcholinesterase distribution and projections. In G. Paxinos (Ed.), The Rat NerlJous System, Part 1, Academic Press, Australia, 1985, pp. 487-515. [32] Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, 2nd edn., Academic Press, Australia, 1986. [33] Pollin, M.M., McHanwell, S. and Slater, C.R., Loss of motor neurons from the median nerve motor nucleus of the mutant mouse 'wobbler', J. Neurocytol., 19 (1990) 29-38. [34] Schmidt-Achert, K., Askanas, V. and Engel, W.K., Thyrotropin-releasing hormone enhances choline acetyltransferase and creative kinase in cultured spinal ventral horn neurons, J. Neuroehem., 43 (1984) 586-589. [35] Siegel, S., Statistical errors. In: Nonparametric Statistics for the Behacioral Sciences, MacGraw-Hill, New York, 1956, pp. 55-70. [36] Sillevis Smitt, P.A, and de Jong, J.M., Animal models of amyotrophic lateral sclerosis and the spinal muscular atrophies, J. Neurol. Sci., 91 (1989) 231-258.
[37] Sofroniew, M.V., Campbell, P.E., Cuello, A.C. and Eckenstein, F., Central cholinergic neurons visualized by immunohistochemical detection of choline acetyltransferase. In G. Paxinos (Ed.), The Rat Nercous System, Part l, Academic Press, Australia, 1985, pp. 471-485. [38] Tang, F., Change in met-enkephalin and Beta-endorphin contents in the hypothalamus and the pituitary in diabetic erat: effect of insulin therapy, Clin. Exp. Pharmacol. Physiol., 16 (1989) 65-75. [39] 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. [40] Papapetropoulos, A. and Bradley, W.G., Spinal motor neuones in murine muscular dystrophy and spinal muscular atrophy, J. Neurol. Neurosurg. Psychiatry, 35 (1972) 60-65. [41] Vacca-Galloway, L.L. and Steinberger, C.C., Substance P neurons sprout in the cervical spinal cord of the wobbler mouse: a model for motoneuron disease, Z Neurosei. Res., 16 (1986) 657-670. [42] Vacca-Galloway, L.L., Steinberger, C., Poole, E. and Menon, C.D., Substance P, thyrotropin releasing hormone, and serotonin neurons sprout in cervical spinal cord of Wobbler mouse, a model for motoneuron disease. In J.L. Henry, R. Couture, A.C. Cuello, G.R. Pelletier and R. Quirion (Eds.), Substance P and Neurokinins, Springer, New York, 1987, pp. 359-362. [43] 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) 2(/9-222. [44] Zagon, I.S. and McLaughlin, P.J., Ultrastructural localization of enkephalin-like immunoreactivity in developing rat cerebellum, Neuroscience, 34 (1990) 479-489. [45] 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.