Anabolic-androgenic steroid induced alterations in choline acetyltransferase messenger RNA levels of spinal cord motoneurons in the male rat

doi:10.1016/S0306-4522(96)00597-0

Neuroscience

Volume 78, Issue 3, 27 March 1997, Pages 873-882

https://doi.org/10.1016/S0306-4522(96)00597-0 Get rights and content

Abstract

The effect of chronic supraphysiological doses of anabolic-androgenic steroids, such as those illegally used by recreational, amateur and professional athletes to increase muscle mass and strength, on motoneurons has not been established. The choline acetyltransferase activity levels of perineal muscles in the male rat are modulated by plasma testosterone levels. These muscles are innervated by the sexually dimorphic motoneurons of the spinal nucleus of the bulbocavernosus. Since the primary source of choline acetyltransferase in muscle is motoneuronal, testosterone may modulate perineal muscle choline acetyltransferase activity by regulating choline acetyltransferase messenger RNA levels in motoneurons. The purpose of this study was to determine if choline acetyltransferase messenger RNA levels in cervical and lumbar spinal motoneurons are affected by chronic (four weeks) changes of plasma testosterone levels in the adult male rat. Using in situ hybridization, choline acetyltransferase messenger RNA levels were analysed in four motor columns: the spinal nucleus of the bulbocavernosus, the retrodorsal lateral nucleus of the lumbar spinal cord, and the lumbar lateral motor columns of the cervical and lumbar spinal cords. Chronic exposure to supraphysiological levels of testosterone (five- to ten-times physiologic levels) significantly increased choline acetyltransferase messenger RNA in all four motor columns. Subsequent to castration, choline acetyltransferase messenger RNA levels decreased in motoneurons of the spinal nucleus of the bulbocavernosus and the retrodorsal lateral nucleus. This observation suggests that the decrease in choline acetyltransferase activity levels of muscles innervated by spinal nucleus of the bulbocavernosus motoneurons may be due to changes in choline acetyltransferase protein levels. Indeed, testosterone replacement therapy of castrated males prevented the decline of choline acetyltransferase messenger RNA levels in motoneurons.

The results of this study demonstrate that anabolic-androgenic steroids can affect the levels of specific messenger RNAs in motoneuron populations throughout the spinal cord suggesting that motoneuronal characteristics are modulated by circulating anabolic-androgenic steroid levels regardless of the purported “androgen sensitivity” of the specific neuromuscular system.

Section snippets

Animals

Plasma testosterone levels were altered for a 28 day period in age-matched, adult male Long–Evans rats (45–50-days-old). All animals were purchased from Charles Rivers (Wilmington, MA). Animal care and killing were consistent with the established guidelines of the American Veterinary Medical Association. Experimental protocols were approved by the University of California at Los Angeles Chancellor's Animal Research Committee. In Experiment I, plasma testosterone levels were chronically

Gonadally-intact male rats

As illustrated for the SNB, RDLN, dorsal lateral nuclei and the ventral motor pool of spinal cord segment L₅ all motoneurons in spinal cord segment levels L₄–L₅ and C₃–C₅ express ChAT mRNA (Fig. 1). In addition, cell bodies in area X also express ChAT mRNA. These results are consistent with the results of previous in situ hybridization histochemical studies.26, 28, 47However, previous immunohistochemical studies have shown that there are some strongly ChAT immunoreactive cell bodies in lamina

Discussion

The present study demonstrates that in the adult male rat, circulating testosterone levels modulate the steady state levels of ChAT mRNA in somatic motoneurons of the lumbar and cervical spinal cord. Chronic elevation of plasma testosterone levels for 28 days, to approximately 5- and 10-times physiological levels, in gonadally-intact male rats significantly increased motoneuron ChAT mRNA levels in the SNB, RDLN, L_Lat and C_Lat motor columns. Decreasing plasma testosterone levels below

Conclusions

In summary, motoneuronal ChAT mRNA levels varied significantly between different motor columns of the cervical and lumbar spinal cord in adult male rats. Motoneuron ChAT mRNA levels in cervical and lumbar motor columns are dramatically modulated by circulating testosterone levels regardless of the purported “androgen sensitivity” of the muscles that they innervate. These data suggest that the cellular properties of the motoneuronal component of the neuromuscular system can be modulated by

Acknowledgements

The authors would like to thank Dr John Lu for assaying plasma testosterone levels. This research was supported by HD-07228 and NS-21220.

References (75)

M Beato et al.
Gene regulation by steroid hormones
J. Ster. Biochem.
(1987)
D.A Damassa et al.
The relationship between circulating testosterone levels and male sexual behavior in rats
Horm. Behav.
(1977)
R.B Fishman et al.
The androgenic induction of spinal sexual dimorphism is independent of supraspinal afferents
Devl Brain Res.
(1985)
R.B Fishman et al.
Local perineal implants of anti-androgen block masculinization of the spinal nucleus of the bulbocavernosus
Devl Brain Res.
(1992)
M Guzman et al.
Treatment with anabolic steroids increases the activity of the mitochondrial outer carnitine palmitoyltransferase in rat liver and fast-twitch muscle
Biochem. Pharmac.
(1991)
M Kengaku et al.
Multiple mRNA species of choline acetyltransferase from rat spinal cord
Molec. Brain Res.
(1993)
M Krieg
Characterization of the androgen receptor in the skeletal muscle of the rat
Steroids
(1976)
E.M Kurz et al.
Testosterone fails to save androgen-sensitive rat motonuerons following early target removal
Devl Brain Res.
(1992)
M Leslie et al.
Sexual dimorphism and androgen effects on spinal motoneurons innervating the rat flexor digitorum brevis
Brain Res.
(1991)
V Luine et al.
Androgen affects cholinergic enzymes in syringeal motor neurons and muscle
Brain Res.
(1980)

A Matsumoto et al.

Androgen regulates gap junction mRNA expression in androgen-sensitive motoneurons in the rat spinal cord

Neurosci. Lett.

(1991)

A Matsumoto et al.

Effect of androgen on the expression of gap junction and β-actin mRNAs in adult rat motoneurons

J. Neurosci. Res.

(1992)

J.L McManaman et al.

Purification of a skeletal muscle polypeptide which stimulates choline acetyltransferase activity in cultured spinal cord neurons

J. biol. Chem.

(1988)

H Misawa et al.

Gene expression of mouse choline acetyl transferase: alternative splicing and identification of a highly active promoter region

J. biol. Chem.

(1992)

N Mori et al.

Partial cloning of the rat choline acetyltransferase gene and in situ localization of its transcripts in the cell body of cholinergic neurons in the brain stem and spinal cord

Molec. Brain Res.

(1993)

P Popper et al.

Differential regulation of α-calcitonin gene-related peptide and preprocholecystokinin messenger RNA expression in α-motoneurons: effects of testosterone and inactivity induced factors

Neuroscience

(1992)

P Popper et al.

The role of target muscles in the expression of calcitonin gene-related mRNA in the spinal nucleus of the bulbocavernosus

Molec. Brain Res

(1992)

C.A Priest et al.

Estrogen regulates preproenkephalin-A mRNA levels in the rat ventromedial nucleus: temporal and cellular events

Molec. Brain Res.

(1995)

C.A Priest et al.

Temporal regulation of β-preprotachykinin mRNA expression in the rat ventromedial nucleus of the hypothalamus

Molec. Brain. Res.

(1995)

J.E Swett et al.

Motoneurons of the rat sciatic nerve

Expl Neurol.

(1986)

J.-A Tan et al.

Response elements of the androgen-regulated C3 gene

J. biol. Chem.

(1992)

S Tucek et al.

Testosterone-induced changes of choline acetyltransferase and cholinesterase activities in rat levator ani muscle

J. Neurol. Sci.

(1976)

S Tucek et al.

Effects of castration, tesosterone and immobilization of activities of choline acetyltransferase and cholinesterase in rat hindlimb muscles

J. Neurol. Sci.

(1976)

A.J Adler et al.

The stringency and magnitude of androgen-specific gene activation are combinatorial functions of receptor and nonreceptor binding site sequences

Molec. Cell Biol.

(1993)

M Alen et al.

Changes in neuromuscular performance and muscle fiber characteristics of elite power athletes self-administering androgenic and anabolic steroids

Acta physiol. scand.

(1984)

A.P Arnold et al.

Neural plasticity in a hormone-sensitive spinal nucleus

Bull. TMIN

(1988)

A.P Arnold et al.

Hormone concentrating cells in vocal control and other areas of the brain of the zebra finch (Poephila guttata)

Science

(1979)

R.P Barber et al.

The morphology and distribution of neurons containing choline acetyltransferase in the adult rat spinal cord: an immunohistochemical study

J. comp. Neurol.

(1984)

C.E Blanco et al.

Anabolic-androgenic steroid regulation of choline acetyltransferase (ChAT) mRNA in spinal motoneurons

Anat. Rec.

(1993)

S.M Breedlove et al.

Hormone accumulation in a sexually dimorphic motor nucleus of the rat

Science

(1980)

S.M Breedlove et al.

Sex differences in the pattern of steroid accumulation by motoneurons of the rat lumbar spinal cord

J. comp. Neurol.

(1983)

A Brice et al.

Complete sequence of a cDNA encoding an active rat choline acetyltransferase: a tool to investigate the plasticity of cholinergic phenotype expression

J. Neurosci. Res.

(1989)

F Celotti et al.

Anabolic steroids: A review of their effects on the muscles, of their possible mechanisms of action and of their use in athletics

J. Ster. Biochem. Molec. Biol.

(1992)

H.P Clamann et al.

A comparison of electromyographic and mechanical fatigue properties in motor units of the cat hindlimb

Brain Res

(1985)

S Eggington

Effects of an anabolic hormone on aerobic capacity of rat striated muscle

Pflügers Arch.

(1987)

S Eggington

Effects of an anabolic hormone on striated muscle growth and performance

Pflügers Arch.

(1987)

J.D Elashoff et al.

Effects of anabolic-androgenic steroids on muscular strength

Ann. Int. Med.

(1991)

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¹: Present Address: Department of Biokinesiology and Physical Therapy, University of Southern California, 1540 E. Alcazar Street, CHP 155, Los Angeles, CA 90033, U.S.A.

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Anabolic-androgenic steroid induced alterations in choline acetyltransferase messenger RNA levels of spinal cord motoneurons in the male rat

Abstract

Section snippets

Animals

Gonadally-intact male rats

Discussion

Conclusions

Acknowledgements

J. Ster. Biochem.

Horm. Behav.

Devl Brain Res.

Devl Brain Res.

Biochem. Pharmac.

Molec. Brain Res.

Steroids

Devl Brain Res.

Brain Res.

Brain Res.

Neurosci. Lett.

J. Neurosci. Res.

J. biol. Chem.

J. biol. Chem.

Molec. Brain Res.

Neuroscience

Molec. Brain Res

Molec. Brain Res.

Molec. Brain. Res.

Expl Neurol.

J. biol. Chem.

J. Neurol. Sci.

J. Neurol. Sci.

The stringency and magnitude of androgen-specific gene activation are combinatorial functions of receptor and nonreceptor binding site sequences

Molec. Cell Biol.

Changes in neuromuscular performance and muscle fiber characteristics of elite power athletes self-administering androgenic and anabolic steroids

Acta physiol. scand.

Neural plasticity in a hormone-sensitive spinal nucleus

Bull. TMIN

Hormone concentrating cells in vocal control and other areas of the brain of the zebra finch (Poephila guttata)

Science

The morphology and distribution of neurons containing choline acetyltransferase in the adult rat spinal cord: an immunohistochemical study

J. comp. Neurol.

Anabolic-androgenic steroid regulation of choline acetyltransferase (ChAT) mRNA in spinal motoneurons

Anat. Rec.

Hormone accumulation in a sexually dimorphic motor nucleus of the rat

Science

Sex differences in the pattern of steroid accumulation by motoneurons of the rat lumbar spinal cord

J. comp. Neurol.

Complete sequence of a cDNA encoding an active rat choline acetyltransferase: a tool to investigate the plasticity of cholinergic phenotype expression

J. Neurosci. Res.

Anabolic steroids: A review of their effects on the muscles, of their possible mechanisms of action and of their use in athletics

J. Ster. Biochem. Molec. Biol.

A comparison of electromyographic and mechanical fatigue properties in motor units of the cat hindlimb

Brain Res

Effects of an anabolic hormone on aerobic capacity of rat striated muscle

Pflügers Arch.

Effects of an anabolic hormone on striated muscle growth and performance

Pflügers Arch.

Effects of anabolic-androgenic steroids on muscular strength

Ann. Int. Med.