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Chronic stimulation of the peroneal nerve in rats upregulates the pro-opiomelanocortin gene in spinal motoneurones
Brain Research 887 (2000) 191–193 www.elsevier.com / locate / bres
Short communication
Chronic stimulation of the peroneal nerve in rats upregulates the pro-opiomelanocortin gene in spinal motoneurones a b a, Sharon Hughes , Ruth A. Shiner , Margaret E. Smith * a
Division of Medical Sciences, Medical School, University of Birmingham, Birmingham B15 2 TT, UK b School of Health Sciences, University of Wolverhampton, Wolverhampton, WV1 1 SB, UK Accepted 3 October 2000
Abstract Continuous unilateral stimulation of the peroneal nerve in rats for 8 h per day for 2 or 7 days caused significant increases in POMC mRNA and b-endorphin immunoreactivity in both ipsilateral and contralateral motoneurones. Intermittent stimulation, for 10-min periods with 90-min rest periods, for 8 h per day for 2 days also caused upregulation of POMC mRNA. It is suggested that expression of POMC-derived peptides in motoneurones may be important for maintaining muscle contractile function. 2000 Elsevier Science B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters, and receptors Topic: Peptides: anatomy and physiology Keywords: b-Endorphin; Motoneuron; Pro-opiomelanocortin; Nerve stimulation; Exercise
b-Endorphin increases muscle contraction amplitude and decreases fatigue in isolated nerve–muscle preparations stimulated via the nerves [9], and stimulates glucose uptake in contracting muscles [2]. Furthermore b-endorphin receptors are present in normal adult muscles of rodents [7]. This peptide, which is derived from proopiomelanocortin (POMC) is released from the pituitary during exercise, and it may therefore have a role in the control of muscle function during exercise. However it can be released from developing intramuscular motor nerves in vitro by electrical stimulation of the nerves [4], and therefore neuronally released b-endorphin may also be important in muscle function. Although b-endorphin immunoreactivity is normally barely detectable in adult motoneurones [3,5,6], it is expressed in conditions where neuromuscular function may be suboptimum such as congenital muscular dystrophy [3] and diabetes mellitus [7] which are characterised by the presence of secondary motor neuropathy, and in develop-
*Corresponding author. Tel.: 144-121-414-6903; fax: 144-121-4146919. E-mail address: [email protected] (M.E. Smith).
ment [3]. It seems possible therefore that this peptide could be induced during prolonged strenuous exercise when neuromuscular fatigue is present as a consequence of declining release of acetylcholine. In order to investigate this possibility the effect of chronic stimulation of the peroneal motor nerve, on the expression of both b-endorphin immunoreactivity and POMC mRNA in the lumbar spinal cord was studied in adult rats. Part of this work has been published in abstract form [8]. Male Sprague–Dawley rats (|350 g body weight) were implanted with stainless steel, multi-stranded, coiled, teflon-insulated, electrodes near the right lateral popliteal nerve under aseptic conditions and 1–2% halothane (Fluothane ICI) anaesthesia, as described previously [1]. Electrical stimulation (0.3 ms pulse width, 10 Hz and up to 6 V) of the right peroneal nerve was started the day after the operation. The normal discharge frequency for motoneurones innervating slow muscles is 10–20 Hz. The electrodes were connected to Neurotech (Shannon, Ireland) stimulators via light-weight leads. Stimulation was continuous (8 h each day), or intermittent (seven times a day, for periods of 10 min, with 90-min rest periods), for 2 or 7 days. In sham-operated animals the implanted electrodes were not stimulated. Animals were killed by an overdose
0006-8993 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 00 )03055-9
192
S. Hughes et al. / Brain Research 887 (2000) 191 – 193
of sodium pentobarbitone (Sagatal, RMB) |16 h after the first stimulation period. The spinal cord was removed, washed thoroughly in phosphate-buffered saline (0.1 M) containing phenylmethylsulphonyl fluoride (0.1 M) and cyclohexamide (0.1 mM), pH 7.4, and quickly frozen in isopentane cooled in liquid nitrogen. Serial cryostat sections (20 mm thick, 10 to 12 per animal) were prepared from the lumbar segments, and examined for POMC mRNA and b-endorphin immunoreactivity. POMC transcript was detected by in situ hybridisation using a 24 base cDNA oligonucleotide antisense probe complementary to the ACTH 4–11 encoding region of rat POMC (Affiniti Research Products Ltd.). The corresponding sense probe was used in control experiments. The oligoprobes were covalently conjugated to calf intestinal alkaline phosphatase and the presence of POMC transcript was detected using the histochemical colorimetric method of McGadey [11] as described elsewhere [6]. Immunoreactivity was detected in every third section using an antibody to b-endorphin (Immuno-diagnostic Systems Ltd.) and the indirect peroxidase–antiperoxidase method as described previously [5]. Adjacent sections were stained with toluidine blue [10] to enable the total numbers of neurones to be counted. Only cells with a visible nucleus were included. The results were expressed as the proportion of ventral horn motoneurones that expressed the POMC transcript or the peptide immunoreactivity. Statistical significance was determined using ANOVA In unoperated rats faint staining for POMC mRNA was seen in a few cells, the proportion of cells being 4.060.6% (S.E.M., n53). The proportion of stained cells in shamoperated animals at 2 days or 7 days was not significantly different. However in stimulated animals the staining was more intense, and was evident in a greater proportion of motoneurones. Fig. 1A compares the proportions on the stimulated side in intermittently stimulated, continuously stimulated, and sham-operated rats (operated side) at 2 days and 7 days. The proportions were significantly (approximately threefold) higher at 2 days in both groups of stimulated animals compared to sham-operated animals (P,0.02, in each case). At 7 days the proportion was over fourfold higher in continuously stimulated animals than in the sham-operated animals (P,0.01), and over twofold higher than in the intermittently stimulated animals (P, 0.01). The value for the stimulated side at 7 days was significantly higher than that for the stimulated side at 2 days (P,0.05). Fig. 1B shows the effect of intermittent stimulation on the expression of POMC mRNA in the contralateral (left) side of the spinal cord. At 2 days the expression was significantly higher in both groups of stimulated animals than in sham-operated animals. In the continuously stimulated animals the proportion of stained cells on the contralateral side was similar at 2 and 7 days. In the
Fig. 1. (A) Effect of stimulation of the peroneal nerve on POMC mRNA expression in the ipsilateral spinal cord. (B) Effect of stimulation of the peroneal nerve on POMC mRNA expression in the contralateral spinal cord. Black columns, sham operated (n55); hatched columns, intermittent stimulation (n53); grey columns, continuous stimulation (2 days, n54; 7 days, n56). The values are means6S.E.M. (bars). *Significant compared to sham-operated animals.
intermittently stimulated animals however, the proportion was significantly lower at 7 days than in the continuously stimulated animals (P,0.02), and was not significantly different from sham-operated animals. Intense immunostaining for b-endorphin was seen in motoneurones in animals which had been stimulated continuously for 8 h per day for 2 or 7 days. Fig. 2 shows that the proportion of immunostained motoneurones was significantly increased on both sides of the spinal cord at 2 and 7 days. The increases were of similar magnitude at the two time points. The results show that chronic stimulation of the peroneal nerve in rats increases the expression of both POMC mRNA and b-endorphin immunoreactivity in lumbar motoneurones. This effect is unlikely to be a response to damage caused by the surgical procedure as the expression was not significantly increased in sham-operated animals. It is possible therefore that the increases were triggered by action potentials in the motor nerves. Alternatively it could
S. Hughes et al. / Brain Research 887 (2000) 191 – 193
193
tins, also influence muscle function (for a review see Ref. [12]), and these may act in concert with b-endorphin to maintain muscle function during exercise.
Acknowledgements We are grateful to Professor O. Hudlicka for performing the surgical and stimulation procedures, and to the Wellcome Trust for financial support.
References
Fig. 2. Effect of continuous stimulation on the expression of b-endorphin immunoreactivity in the lumbar spinal cord. Black columns, sham operated (n55); open columns, stimulated side (2 days, n54; 7 days, n56); dark grey columns, contralateral side. The values are means6S.E.M. (bars). *Significant compared to sham-operated animals.
be an indirect effect due to activity in the sensory nerves which are also stimulated by the procedure, or a feedback effect from the contracting muscles. At 2 days the proportion of cells expressing POMC mRNA was similar in the intermittently stimulated and chronically stimulated rats. However in the intermittently stimulated animals the proportion was lower at 7 days than at 2 days, although the difference between the two time points was not statistically significant. Moreover the proportion was significantly lower in intermittently stimulated animals at 7 days than in chronically stimulated animals at 7 days. Thus, some adaptation to the less severe stimulation regime may have occurred by 7 days. The increased expression in the contralateral motoneurones may be due to a transneuronal mechanism [5] or the effect of a blood-borne influence from the stimulated muscles. b-Endorphin may be released at the neuromuscular junction during muscle contraction [4] to augment the effect of the circulating peptide during exercise, when it could help to maintain the contractile strength of the muscles [9] and promote glucose utilisation [2]. Interestingly other POMC-derived peptides, notably melanocor-
[1] S. Egginton, O. Hudlicka, M. Glover, Fine structure of capillaries in ischaemic and non-ischaemic rat striated muscle. Effect of torbafylline, Int. J. Microcirc. Clin. Exp. 12 (1993) 33–44. [2] A.A.L. Evans, S. Khan, M.E. Smith, Evidence for a hormonal action of b-endorphin to increase glucose uptake in resting and contracting skeletal muscle, J. Endocrinol. 155 (1997) 387–392. [3] L.W. Haynes, M.E. Smith, Presence of immunoreactive a-melanotropin and b-endorphin in spinal motoneurones of the dystrophic mouse, Neurosci. Lett. 58 (1985) 13–18. [4] L.W. Haynes, M.E. Smith, Release of b-endorphin from the motor nerve of the immature rat, J. Physiol. 351 (1984) 22P. [5] S. Hughes, M.E. Smith, Pro-opiomelanocortin-derived peptides in transected and contralateral motor nerves of the rat, J. Chem. Neuroanat. 2 (1989) 227–237. [6] S. Hughes, M.E. Smith, Upregulation of the pro-opiomelanocortin gene in motoneurones after nerve section in mice, Mol. Brain Res. 25 (1994) 41–49. [7] S. Hughes, M.E. Smith, C.J. Bailey, b-Endorphin and corticotrophin immunoreactivity and specific binding in the neuromuscular system of obese-diabetic mice, Neuroscience 48 (1992) 463–468. [8] S. Hughes, M.E. Smith, O. Hudlicka, Effect of peroneal nerve stimulation in vivo in rats on the expression of the proopiomelanocortin gene in spinal motoneurones, J. Physiol. 481P (1994) 69P. [9] S. Khan, M.E. Smith, Effect of b-endorphin on the contractile response in mouse skeletal muscle, Muscle Nerve 18 (1995) 1250– 1256. [10] K. Kramer, G.M. Windrum, The metachromatic staining reaction, J. Histochem. Cytochem. 3 (1955) 227–237. [11] J. McGadey, A tetrazolium method for non-specific alkaline phosphatase, Histochemie 23 (1970) 180–184. [12] F.L. Strand, K.J. Rose, L.A. Zuccarelli, J. Kume, S.E. Alves, F.J. Antonowich, L.Y. Garrett, Neuropeptide hormones as neurotrophic factors, Physiol. Rev. 71 (1991) 1017–1046.
Short communication
Chronic stimulation of the peroneal nerve in rats upregulates the pro-opiomelanocortin gene in spinal motoneurones a b a, Sharon Hughes , Ruth A. Shiner , Margaret E. Smith * a
Division of Medical Sciences, Medical School, University of Birmingham, Birmingham B15 2 TT, UK b School of Health Sciences, University of Wolverhampton, Wolverhampton, WV1 1 SB, UK Accepted 3 October 2000
Abstract Continuous unilateral stimulation of the peroneal nerve in rats for 8 h per day for 2 or 7 days caused significant increases in POMC mRNA and b-endorphin immunoreactivity in both ipsilateral and contralateral motoneurones. Intermittent stimulation, for 10-min periods with 90-min rest periods, for 8 h per day for 2 days also caused upregulation of POMC mRNA. It is suggested that expression of POMC-derived peptides in motoneurones may be important for maintaining muscle contractile function. 2000 Elsevier Science B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters, and receptors Topic: Peptides: anatomy and physiology Keywords: b-Endorphin; Motoneuron; Pro-opiomelanocortin; Nerve stimulation; Exercise
b-Endorphin increases muscle contraction amplitude and decreases fatigue in isolated nerve–muscle preparations stimulated via the nerves [9], and stimulates glucose uptake in contracting muscles [2]. Furthermore b-endorphin receptors are present in normal adult muscles of rodents [7]. This peptide, which is derived from proopiomelanocortin (POMC) is released from the pituitary during exercise, and it may therefore have a role in the control of muscle function during exercise. However it can be released from developing intramuscular motor nerves in vitro by electrical stimulation of the nerves [4], and therefore neuronally released b-endorphin may also be important in muscle function. Although b-endorphin immunoreactivity is normally barely detectable in adult motoneurones [3,5,6], it is expressed in conditions where neuromuscular function may be suboptimum such as congenital muscular dystrophy [3] and diabetes mellitus [7] which are characterised by the presence of secondary motor neuropathy, and in develop-
*Corresponding author. Tel.: 144-121-414-6903; fax: 144-121-4146919. E-mail address: [email protected] (M.E. Smith).
ment [3]. It seems possible therefore that this peptide could be induced during prolonged strenuous exercise when neuromuscular fatigue is present as a consequence of declining release of acetylcholine. In order to investigate this possibility the effect of chronic stimulation of the peroneal motor nerve, on the expression of both b-endorphin immunoreactivity and POMC mRNA in the lumbar spinal cord was studied in adult rats. Part of this work has been published in abstract form [8]. Male Sprague–Dawley rats (|350 g body weight) were implanted with stainless steel, multi-stranded, coiled, teflon-insulated, electrodes near the right lateral popliteal nerve under aseptic conditions and 1–2% halothane (Fluothane ICI) anaesthesia, as described previously [1]. Electrical stimulation (0.3 ms pulse width, 10 Hz and up to 6 V) of the right peroneal nerve was started the day after the operation. The normal discharge frequency for motoneurones innervating slow muscles is 10–20 Hz. The electrodes were connected to Neurotech (Shannon, Ireland) stimulators via light-weight leads. Stimulation was continuous (8 h each day), or intermittent (seven times a day, for periods of 10 min, with 90-min rest periods), for 2 or 7 days. In sham-operated animals the implanted electrodes were not stimulated. Animals were killed by an overdose
0006-8993 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 00 )03055-9
192
S. Hughes et al. / Brain Research 887 (2000) 191 – 193
of sodium pentobarbitone (Sagatal, RMB) |16 h after the first stimulation period. The spinal cord was removed, washed thoroughly in phosphate-buffered saline (0.1 M) containing phenylmethylsulphonyl fluoride (0.1 M) and cyclohexamide (0.1 mM), pH 7.4, and quickly frozen in isopentane cooled in liquid nitrogen. Serial cryostat sections (20 mm thick, 10 to 12 per animal) were prepared from the lumbar segments, and examined for POMC mRNA and b-endorphin immunoreactivity. POMC transcript was detected by in situ hybridisation using a 24 base cDNA oligonucleotide antisense probe complementary to the ACTH 4–11 encoding region of rat POMC (Affiniti Research Products Ltd.). The corresponding sense probe was used in control experiments. The oligoprobes were covalently conjugated to calf intestinal alkaline phosphatase and the presence of POMC transcript was detected using the histochemical colorimetric method of McGadey [11] as described elsewhere [6]. Immunoreactivity was detected in every third section using an antibody to b-endorphin (Immuno-diagnostic Systems Ltd.) and the indirect peroxidase–antiperoxidase method as described previously [5]. Adjacent sections were stained with toluidine blue [10] to enable the total numbers of neurones to be counted. Only cells with a visible nucleus were included. The results were expressed as the proportion of ventral horn motoneurones that expressed the POMC transcript or the peptide immunoreactivity. Statistical significance was determined using ANOVA In unoperated rats faint staining for POMC mRNA was seen in a few cells, the proportion of cells being 4.060.6% (S.E.M., n53). The proportion of stained cells in shamoperated animals at 2 days or 7 days was not significantly different. However in stimulated animals the staining was more intense, and was evident in a greater proportion of motoneurones. Fig. 1A compares the proportions on the stimulated side in intermittently stimulated, continuously stimulated, and sham-operated rats (operated side) at 2 days and 7 days. The proportions were significantly (approximately threefold) higher at 2 days in both groups of stimulated animals compared to sham-operated animals (P,0.02, in each case). At 7 days the proportion was over fourfold higher in continuously stimulated animals than in the sham-operated animals (P,0.01), and over twofold higher than in the intermittently stimulated animals (P, 0.01). The value for the stimulated side at 7 days was significantly higher than that for the stimulated side at 2 days (P,0.05). Fig. 1B shows the effect of intermittent stimulation on the expression of POMC mRNA in the contralateral (left) side of the spinal cord. At 2 days the expression was significantly higher in both groups of stimulated animals than in sham-operated animals. In the continuously stimulated animals the proportion of stained cells on the contralateral side was similar at 2 and 7 days. In the
Fig. 1. (A) Effect of stimulation of the peroneal nerve on POMC mRNA expression in the ipsilateral spinal cord. (B) Effect of stimulation of the peroneal nerve on POMC mRNA expression in the contralateral spinal cord. Black columns, sham operated (n55); hatched columns, intermittent stimulation (n53); grey columns, continuous stimulation (2 days, n54; 7 days, n56). The values are means6S.E.M. (bars). *Significant compared to sham-operated animals.
intermittently stimulated animals however, the proportion was significantly lower at 7 days than in the continuously stimulated animals (P,0.02), and was not significantly different from sham-operated animals. Intense immunostaining for b-endorphin was seen in motoneurones in animals which had been stimulated continuously for 8 h per day for 2 or 7 days. Fig. 2 shows that the proportion of immunostained motoneurones was significantly increased on both sides of the spinal cord at 2 and 7 days. The increases were of similar magnitude at the two time points. The results show that chronic stimulation of the peroneal nerve in rats increases the expression of both POMC mRNA and b-endorphin immunoreactivity in lumbar motoneurones. This effect is unlikely to be a response to damage caused by the surgical procedure as the expression was not significantly increased in sham-operated animals. It is possible therefore that the increases were triggered by action potentials in the motor nerves. Alternatively it could
S. Hughes et al. / Brain Research 887 (2000) 191 – 193
193
tins, also influence muscle function (for a review see Ref. [12]), and these may act in concert with b-endorphin to maintain muscle function during exercise.
Acknowledgements We are grateful to Professor O. Hudlicka for performing the surgical and stimulation procedures, and to the Wellcome Trust for financial support.
References
Fig. 2. Effect of continuous stimulation on the expression of b-endorphin immunoreactivity in the lumbar spinal cord. Black columns, sham operated (n55); open columns, stimulated side (2 days, n54; 7 days, n56); dark grey columns, contralateral side. The values are means6S.E.M. (bars). *Significant compared to sham-operated animals.
be an indirect effect due to activity in the sensory nerves which are also stimulated by the procedure, or a feedback effect from the contracting muscles. At 2 days the proportion of cells expressing POMC mRNA was similar in the intermittently stimulated and chronically stimulated rats. However in the intermittently stimulated animals the proportion was lower at 7 days than at 2 days, although the difference between the two time points was not statistically significant. Moreover the proportion was significantly lower in intermittently stimulated animals at 7 days than in chronically stimulated animals at 7 days. Thus, some adaptation to the less severe stimulation regime may have occurred by 7 days. The increased expression in the contralateral motoneurones may be due to a transneuronal mechanism [5] or the effect of a blood-borne influence from the stimulated muscles. b-Endorphin may be released at the neuromuscular junction during muscle contraction [4] to augment the effect of the circulating peptide during exercise, when it could help to maintain the contractile strength of the muscles [9] and promote glucose utilisation [2]. Interestingly other POMC-derived peptides, notably melanocor-
[1] S. Egginton, O. Hudlicka, M. Glover, Fine structure of capillaries in ischaemic and non-ischaemic rat striated muscle. Effect of torbafylline, Int. J. Microcirc. Clin. Exp. 12 (1993) 33–44. [2] A.A.L. Evans, S. Khan, M.E. Smith, Evidence for a hormonal action of b-endorphin to increase glucose uptake in resting and contracting skeletal muscle, J. Endocrinol. 155 (1997) 387–392. [3] L.W. Haynes, M.E. Smith, Presence of immunoreactive a-melanotropin and b-endorphin in spinal motoneurones of the dystrophic mouse, Neurosci. Lett. 58 (1985) 13–18. [4] L.W. Haynes, M.E. Smith, Release of b-endorphin from the motor nerve of the immature rat, J. Physiol. 351 (1984) 22P. [5] S. Hughes, M.E. Smith, Pro-opiomelanocortin-derived peptides in transected and contralateral motor nerves of the rat, J. Chem. Neuroanat. 2 (1989) 227–237. [6] S. Hughes, M.E. Smith, Upregulation of the pro-opiomelanocortin gene in motoneurones after nerve section in mice, Mol. Brain Res. 25 (1994) 41–49. [7] S. Hughes, M.E. Smith, C.J. Bailey, b-Endorphin and corticotrophin immunoreactivity and specific binding in the neuromuscular system of obese-diabetic mice, Neuroscience 48 (1992) 463–468. [8] S. Hughes, M.E. Smith, O. Hudlicka, Effect of peroneal nerve stimulation in vivo in rats on the expression of the proopiomelanocortin gene in spinal motoneurones, J. Physiol. 481P (1994) 69P. [9] S. Khan, M.E. Smith, Effect of b-endorphin on the contractile response in mouse skeletal muscle, Muscle Nerve 18 (1995) 1250– 1256. [10] K. Kramer, G.M. Windrum, The metachromatic staining reaction, J. Histochem. Cytochem. 3 (1955) 227–237. [11] J. McGadey, A tetrazolium method for non-specific alkaline phosphatase, Histochemie 23 (1970) 180–184. [12] F.L. Strand, K.J. Rose, L.A. Zuccarelli, J. Kume, S.E. Alves, F.J. Antonowich, L.Y. Garrett, Neuropeptide hormones as neurotrophic factors, Physiol. Rev. 71 (1991) 1017–1046.