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Some effects of magnesium ions upon conduction and synaptic activity in the isolated spinal cord of the mouse
410
Brain Research, 177 (1979) 410-413 © Elsevier/North-Holland Biomedical Press
Some effects of magnesium ions upon conduction and synaptic activity in the isolated spinal cord of the mouse
J. BAGUST and G. A. KERKUT Department of Neurophysiology, University of Southampton, S09 3 TU (England)
(Accepted July 26th, 1979)
The development of in vitro techniques for studying mammalian central nervous system tissue allows the composition of the extracellular fluid to be altered making detailed pharmacological investigations possible. The present communication describes the use of an isolated spinal cord preparation of the mouse, which continued to show good evoked and spontaneous electrical activity for up to 50 h after isolation. The elimination of synaptic responses is sometimes necessary in these experiments to ensure that the observed effects are direct upon the cell under observation, and not indirect via some interneurone. In electrophysiological experiments also, it is often useful when recording extracellularly to be able to separate that part of an evoked response which is due to conduction along nerve fibres directly excited by the stimulus, and that which is due to synaptic activity. The presence of calcium ions has been shown to be an essential requirement for the release of transmitter substances from presynaptic endings 6 and reducing the calcium level in the extracellular fluid is one way of blocking synaptic activity. Magnesium ions antagonize the action of calcium 7, and both lowered calcium and raised magnesium levels have been used to block the synaptic activation of cells in in vitro preparations1,2,4,10,11. High concentrations of magnesium ions have also been used to produce localized synaptic blockade on the cerebral cortex in vivo z. Frankenhaeuser and Meves~ demonstrated that the amplitude of the action potential propagated along single frog nerve fibres could be reduced by high extracellular levels of magnesium, and more recently the excitability of nerve cell membranes has been shown to be lowered by the extraceilular iontophoresis of magnesium ions4,S, ~. We report here some observations on the effects of changing calcium and magnesium levels on both conducted and synaptic activity in a preparation of the mouse spinal cord. Lower spinal cords from the mid-thoracic to the sacral segments were removed from adult mice weighing 30-40 g, under urethane or halothane anaesthesia. The cord was either hemisected with the spinal roots intact, or the surrounding membranes and roots were stripped off under cold Ringer solution (approximately 5 °C) and the cord pinned to the floor of a recording chamber perfused with Ringer solution at 26 °C
411 (NaCI, 118 mM; KC1, 2.0 mM; CaC12, 2.5 mM; NaHCO3, 24 mM; glucose, 12 mM; gassed with 95 % 02/5 % COg; pH 7.4). The fibre tracts of the dorsal funiculus were stimulated in the lower thoracic region using a pair of electrodes made from fine, insulated, stainless steel wires with bared tips. Recordings were made using two independent glass micropipettes, the ends of which had been broken down to give a resistance of I-5 M f l and plugged with 1% Agar in 4 M NaCI to reduce leakage of the electrolyte solution from the tip. One recording electrode was placed in the dorsal white matter in the region of the 3rd or 4th lumbar segment, and the other in the grey matter of the dorsal horn. In this way it was possible to record both the directly propagated activity in the dorsal tracts, (a biphasic wave, although the initial positive deflection was often masked by the stimulus artefact), and simultaneously a combination of propagated and synaptic activity in the dorsal horn (Fig. la). Sixteen successive responses were averaged and measurements were made of the peak amplitude of the propagated action potential in the dorsal tracts, and a suitable wave in the synaptic response (usually having a latency of 3-4 msec). Reducing the calcium content of the perfusing medium caused a reduction in the synaptic activity recorded in the dorsal horn of more than 75 %, whereas the dorsal tract response was either unchanged or increased in amplitude (Figs. 1 and 2). Maximum inhibition of the dorsal horn response was usually achieved after 15 min, and on return to the normal Ringer solution recovery was complete. The addition of MgSOa in concentrations usually used to block synapses (20 mM) ~ produced a complete block of the dorsal horn synaptic response, and a reduction in the amplitude of the conducted action potential by 50-70 %. These effects were reversable provided the cord was not exposed to the raised levels of magnesium for more than 30 min. Addition of 40 mM sucrose to the perfusion medium had little effect upon either the propagated or the synaptic activity, demonstrating that the effects of the magnesium were not due to changes in osmotic pressure.
f Ringer
0 Co++
20raM Mg++
Ringer
+
0.2 mV [
2msec
Fig. 1. Responses recorded in the dorsal funicutus (DF) and dorsal horn (DH) in the mid-lumbar region of the isolated mouse spinal cord to stimulation of the dorsal funiculus in the mid-thoracic region. Each trace is the average of 16 successive responses recorded after 20 min exposure to Ringer solution containing altered levels of calcium and magnesium. The arrow below the first dorsal horn trace indicates a typical negative wave of long latency (3-5 msec) used to quantify the synapticallyevoked response.
412 "E 100" o4~>___c~.,.. p E ~ E
/m~_
lOO-
75"
75-
"--- 50-
50-
25"
25-
,,t
0"~tf
o.1 o'.z
o's
0-
1-b z'.o
o.5
i
Co+'~mM)
2 5 ~o zo3o Mg++ (mM)
Fig. 2. Dose-related effects of CaCI2 and MgSO4 upon the responses evoked from the dorsal funiculus ((3) and dorsal horn (Q) in the lumbar region of the isolated mouse spinal cord by stimulation of the dorsal funiculus in the mind-thoracic region. Each point represents the amplitude of the average of ! 6 successive responses following 20 min exposure to the test solution.
Dose-response curves obtained for calcium and magnesium illustrate the different effects of changing the levels of these ions (Fig. 2). Increasing the concentration of calcium ions reduced the amplitude of the propagated response, but more than 1.5 mM calcium was necessary for efficient synaptic transmission. Magnesium reduced both synaptic and propagated responses. In 4 preparations the amplitude of the ventral horn response to antidromic stimulation of the ventral roots was reduced by 10 mM MgSO4 to an average of 47 % of the control value. This agrees well with the effects of magnesium seen in the dorsal funiculus. The design of these experiments does not permit a distinction to be made between the possible effects of magnesium ions in reducing the amplitude of the action potentials in individual nerve fibres, or increasing the threshold to excitation of the nerve membranes, resulting in fewer fibres being excited by any given stimulus. Fig. 3 shows the results obtained from a hemisected cord preparation in which the dorsal roots were isolated from the bath fluid by a barrier of silicone grease. The maximum "75ol .,.., o >
='50+
23
Z- .25o..
E 0"
2'5
s'o
7's
~60
Stimulus (volts)
Fig. 3. A mouse hemicord preparation showing the effect of varying the stimulus intensity to the 4th lumbar dorsal root (stimulus duration 0.05 msec) upon the amplitude of the response evoked in the dorsal funiculus in the lower thoracic region of the cord. Following 20 rain exposure to Ringer solution containing 20 mM MgSO4 the amplitude of the action potential was reduced (open circles). Each point is the average of 16 responses.
413 a m p l i t u d e o f the c o m p o u n d action p o t e n t i a l r e c o r d e d f r o m t h e d o r s a l white m a t t e r r o s t r a l to the p o i n t o f e n t r y to the c o r d o f the s t i m u l a t e d r o o t was r e d u c e d by raising the m a g n e s i u m c o n c e n t r a t i o n in the p e r f u s i o n m e d i u m . Since the p o i n t o f s t i m u l a t i o n o f the r o o t was n o t e x p o s e d to the c h a n g e d ion level the r e d u c t i o n in the a m p l i t u d e o f ghe c o m p o u n d action p o t e n t i a l i n d i c a t e d t h a t the m a g n e s i u m either caused a r e d u c t i o n in the a m p l i t u d e o f the a c t i o n potentials in i n d i v i d u a l nerve fibres or caused p r o p a g a t i o n to fail c o m p l e t e l y in a p r o p o r t i o n o f the fibres. This p o i n t c a n only be resolved b y single fibre r e c o r d i n g techniques, b u t these experiments show t h a t high c o n c e n t r a t i o n s o f m a g n e s i u m ions n o t only b l o c k synapses b u t also affect nerve conduction.
1 Barker, J. L. and Nicoll, R. A., The pharmacology and ionic dependency of amino acid responses in the frog spinal cord, J. Physiol. (Lond.), 228 (1973) 259-277. 2 Berry, M. S. and Pentreath, V. W., Criteria for distinguishing between monosynaptic and polysynaptic transmission, Brain Research, 105 (1976) 1-20. 3 Bindman, L., Lippold, O. C. J. and Milne, A. R., Prolonged changes in the excitability of pyramidal tract neurones in the cat: a post-synaptic mechanism, J. PhysioL (Lond.), 286 (1979) 457477. 4 Erulkar, S. D., Dambach, G. E. and Mender, D., The effect of magnesium at motoneurones of the isolated spinal cord of the frog, Brain Research, 66 (1974) 413-424. 5 Frankenhaeuser, B. and Meves, H., The effects of magnesium and calcium on the frog myelinated nerve fibre, J. Physiol. (Lond.), 142 (1958) 360-365. 6 Hubbard, J. I., Mechanisms of transmitter release, Progr. Biophys. molec. Biol., 21 (1970) 35-124. 7 Jenkinson, D. H., The nature of the antagonism between calcium and magnesium ions at the neuromuscular junction, J. Physiol. (Lond.), 138 (1957) 434444. 8 Kato, G. and Somjen, G. G., Effects of microiontophoretic administration of magnesium and calcium on neurones in the central nervous system of cats, J. NeurobioL, 2 (1969) 181-195. 9 Kelly, J. S., Krnjevic, K. and Somjen, G., Divalent cation and electrical properties of cortical cells, J. neurobiol., 2 (1969) 197-208. 10 Richards, C. D. and Sercombe, R., Calcium, magnesium and the electrical activity of guinea pig olfactor cortex in-vitro, J. Physiol. (Lond.), 211 (1970) 571-584. 11 Shapovalov, A. I. and Shiriaev, B. I., Electrical coupling between primary afferents and amphibian motor neurones, Exp. Brain Res., 33 (1978) 299-312.
Brain Research, 177 (1979) 410-413 © Elsevier/North-Holland Biomedical Press
Some effects of magnesium ions upon conduction and synaptic activity in the isolated spinal cord of the mouse
J. BAGUST and G. A. KERKUT Department of Neurophysiology, University of Southampton, S09 3 TU (England)
(Accepted July 26th, 1979)
The development of in vitro techniques for studying mammalian central nervous system tissue allows the composition of the extracellular fluid to be altered making detailed pharmacological investigations possible. The present communication describes the use of an isolated spinal cord preparation of the mouse, which continued to show good evoked and spontaneous electrical activity for up to 50 h after isolation. The elimination of synaptic responses is sometimes necessary in these experiments to ensure that the observed effects are direct upon the cell under observation, and not indirect via some interneurone. In electrophysiological experiments also, it is often useful when recording extracellularly to be able to separate that part of an evoked response which is due to conduction along nerve fibres directly excited by the stimulus, and that which is due to synaptic activity. The presence of calcium ions has been shown to be an essential requirement for the release of transmitter substances from presynaptic endings 6 and reducing the calcium level in the extracellular fluid is one way of blocking synaptic activity. Magnesium ions antagonize the action of calcium 7, and both lowered calcium and raised magnesium levels have been used to block the synaptic activation of cells in in vitro preparations1,2,4,10,11. High concentrations of magnesium ions have also been used to produce localized synaptic blockade on the cerebral cortex in vivo z. Frankenhaeuser and Meves~ demonstrated that the amplitude of the action potential propagated along single frog nerve fibres could be reduced by high extracellular levels of magnesium, and more recently the excitability of nerve cell membranes has been shown to be lowered by the extraceilular iontophoresis of magnesium ions4,S, ~. We report here some observations on the effects of changing calcium and magnesium levels on both conducted and synaptic activity in a preparation of the mouse spinal cord. Lower spinal cords from the mid-thoracic to the sacral segments were removed from adult mice weighing 30-40 g, under urethane or halothane anaesthesia. The cord was either hemisected with the spinal roots intact, or the surrounding membranes and roots were stripped off under cold Ringer solution (approximately 5 °C) and the cord pinned to the floor of a recording chamber perfused with Ringer solution at 26 °C
411 (NaCI, 118 mM; KC1, 2.0 mM; CaC12, 2.5 mM; NaHCO3, 24 mM; glucose, 12 mM; gassed with 95 % 02/5 % COg; pH 7.4). The fibre tracts of the dorsal funiculus were stimulated in the lower thoracic region using a pair of electrodes made from fine, insulated, stainless steel wires with bared tips. Recordings were made using two independent glass micropipettes, the ends of which had been broken down to give a resistance of I-5 M f l and plugged with 1% Agar in 4 M NaCI to reduce leakage of the electrolyte solution from the tip. One recording electrode was placed in the dorsal white matter in the region of the 3rd or 4th lumbar segment, and the other in the grey matter of the dorsal horn. In this way it was possible to record both the directly propagated activity in the dorsal tracts, (a biphasic wave, although the initial positive deflection was often masked by the stimulus artefact), and simultaneously a combination of propagated and synaptic activity in the dorsal horn (Fig. la). Sixteen successive responses were averaged and measurements were made of the peak amplitude of the propagated action potential in the dorsal tracts, and a suitable wave in the synaptic response (usually having a latency of 3-4 msec). Reducing the calcium content of the perfusing medium caused a reduction in the synaptic activity recorded in the dorsal horn of more than 75 %, whereas the dorsal tract response was either unchanged or increased in amplitude (Figs. 1 and 2). Maximum inhibition of the dorsal horn response was usually achieved after 15 min, and on return to the normal Ringer solution recovery was complete. The addition of MgSOa in concentrations usually used to block synapses (20 mM) ~ produced a complete block of the dorsal horn synaptic response, and a reduction in the amplitude of the conducted action potential by 50-70 %. These effects were reversable provided the cord was not exposed to the raised levels of magnesium for more than 30 min. Addition of 40 mM sucrose to the perfusion medium had little effect upon either the propagated or the synaptic activity, demonstrating that the effects of the magnesium were not due to changes in osmotic pressure.
f Ringer
0 Co++
20raM Mg++
Ringer
+
0.2 mV [
2msec
Fig. 1. Responses recorded in the dorsal funicutus (DF) and dorsal horn (DH) in the mid-lumbar region of the isolated mouse spinal cord to stimulation of the dorsal funiculus in the mid-thoracic region. Each trace is the average of 16 successive responses recorded after 20 min exposure to Ringer solution containing altered levels of calcium and magnesium. The arrow below the first dorsal horn trace indicates a typical negative wave of long latency (3-5 msec) used to quantify the synapticallyevoked response.
412 "E 100" o4~>___c~.,.. p E ~ E
/m~_
lOO-
75"
75-
"--- 50-
50-
25"
25-
,,t
0"~tf
o.1 o'.z
o's
0-
1-b z'.o
o.5
i
Co+'~mM)
2 5 ~o zo3o Mg++ (mM)
Fig. 2. Dose-related effects of CaCI2 and MgSO4 upon the responses evoked from the dorsal funiculus ((3) and dorsal horn (Q) in the lumbar region of the isolated mouse spinal cord by stimulation of the dorsal funiculus in the mind-thoracic region. Each point represents the amplitude of the average of ! 6 successive responses following 20 min exposure to the test solution.
Dose-response curves obtained for calcium and magnesium illustrate the different effects of changing the levels of these ions (Fig. 2). Increasing the concentration of calcium ions reduced the amplitude of the propagated response, but more than 1.5 mM calcium was necessary for efficient synaptic transmission. Magnesium reduced both synaptic and propagated responses. In 4 preparations the amplitude of the ventral horn response to antidromic stimulation of the ventral roots was reduced by 10 mM MgSO4 to an average of 47 % of the control value. This agrees well with the effects of magnesium seen in the dorsal funiculus. The design of these experiments does not permit a distinction to be made between the possible effects of magnesium ions in reducing the amplitude of the action potentials in individual nerve fibres, or increasing the threshold to excitation of the nerve membranes, resulting in fewer fibres being excited by any given stimulus. Fig. 3 shows the results obtained from a hemisected cord preparation in which the dorsal roots were isolated from the bath fluid by a barrier of silicone grease. The maximum "75ol .,.., o >
='50+
23
Z- .25o..
E 0"
2'5
s'o
7's
~60
Stimulus (volts)
Fig. 3. A mouse hemicord preparation showing the effect of varying the stimulus intensity to the 4th lumbar dorsal root (stimulus duration 0.05 msec) upon the amplitude of the response evoked in the dorsal funiculus in the lower thoracic region of the cord. Following 20 rain exposure to Ringer solution containing 20 mM MgSO4 the amplitude of the action potential was reduced (open circles). Each point is the average of 16 responses.
413 a m p l i t u d e o f the c o m p o u n d action p o t e n t i a l r e c o r d e d f r o m t h e d o r s a l white m a t t e r r o s t r a l to the p o i n t o f e n t r y to the c o r d o f the s t i m u l a t e d r o o t was r e d u c e d by raising the m a g n e s i u m c o n c e n t r a t i o n in the p e r f u s i o n m e d i u m . Since the p o i n t o f s t i m u l a t i o n o f the r o o t was n o t e x p o s e d to the c h a n g e d ion level the r e d u c t i o n in the a m p l i t u d e o f ghe c o m p o u n d action p o t e n t i a l i n d i c a t e d t h a t the m a g n e s i u m either caused a r e d u c t i o n in the a m p l i t u d e o f the a c t i o n potentials in i n d i v i d u a l nerve fibres or caused p r o p a g a t i o n to fail c o m p l e t e l y in a p r o p o r t i o n o f the fibres. This p o i n t c a n only be resolved b y single fibre r e c o r d i n g techniques, b u t these experiments show t h a t high c o n c e n t r a t i o n s o f m a g n e s i u m ions n o t only b l o c k synapses b u t also affect nerve conduction.
1 Barker, J. L. and Nicoll, R. A., The pharmacology and ionic dependency of amino acid responses in the frog spinal cord, J. Physiol. (Lond.), 228 (1973) 259-277. 2 Berry, M. S. and Pentreath, V. W., Criteria for distinguishing between monosynaptic and polysynaptic transmission, Brain Research, 105 (1976) 1-20. 3 Bindman, L., Lippold, O. C. J. and Milne, A. R., Prolonged changes in the excitability of pyramidal tract neurones in the cat: a post-synaptic mechanism, J. PhysioL (Lond.), 286 (1979) 457477. 4 Erulkar, S. D., Dambach, G. E. and Mender, D., The effect of magnesium at motoneurones of the isolated spinal cord of the frog, Brain Research, 66 (1974) 413-424. 5 Frankenhaeuser, B. and Meves, H., The effects of magnesium and calcium on the frog myelinated nerve fibre, J. Physiol. (Lond.), 142 (1958) 360-365. 6 Hubbard, J. I., Mechanisms of transmitter release, Progr. Biophys. molec. Biol., 21 (1970) 35-124. 7 Jenkinson, D. H., The nature of the antagonism between calcium and magnesium ions at the neuromuscular junction, J. Physiol. (Lond.), 138 (1957) 434444. 8 Kato, G. and Somjen, G. G., Effects of microiontophoretic administration of magnesium and calcium on neurones in the central nervous system of cats, J. NeurobioL, 2 (1969) 181-195. 9 Kelly, J. S., Krnjevic, K. and Somjen, G., Divalent cation and electrical properties of cortical cells, J. neurobiol., 2 (1969) 197-208. 10 Richards, C. D. and Sercombe, R., Calcium, magnesium and the electrical activity of guinea pig olfactor cortex in-vitro, J. Physiol. (Lond.), 211 (1970) 571-584. 11 Shapovalov, A. I. and Shiriaev, B. I., Electrical coupling between primary afferents and amphibian motor neurones, Exp. Brain Res., 33 (1978) 299-312.