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Effects of glycine on spinal neurones grown in tissue culture
399
SHORT COMMUNICATIONS
Effects of glycine on spinal neurones grown in tissue culture Glycine has been proposed to be an inhibitory transmitter substance in the spinal cord of the catS,11; the amino acid hyperpolarizes and increases the membrane conductance of spinal motoneurones and interneuronesl,S, 11. In the present investigation we were interested to study whether glycine has a similar action on the membrane potential and conductance of spinal neurones grown in tissue culture. The cultures were prepared from spinal cord of newborn rats and grown on collagen-coated coverslips in Maximov assemblies 8. For electrophysiological investigations the coverslip with the culture was mounted as a 'lying-drop' preparation in a Falcon petri dish (diameter 50 mm) filled with Gey's balanced salt solution. The temperature of the solution (34-36°C) was regulated by an automatic heating system, the pH was kept at 7.0--7.2 by passing 5 ~ COz--95~ 02 over the surface of the bathing solution. The chamber was fixed to the mechanical stage of a reverse Zeiss microscope. The microelectrodes were introduced from above into the culture at an angle of 15-20 ° by means of two Leitz micromanipulators. For intracellular recordings 3 M KCl-containing glass microelectrodes (tip diameter < 1 #m) were used, most of which were bent at an angle of 50 °, 3--4 mm back from the tip. Using the two types of electrodes (straight and bent) we observed that impalements with bent electrodes were more successful. The recording microelectrode was connected through an Ag-AgC1 wire to a cathode follower and potentials were displayed on an oscilloscope. To determine the resistance of the cell membrane square pulses (depolarizing and hyperpolarizing) of 30--40 msec duration were passed through the recording electrode by means of a device similar to that described by Fein 6. Amino acids were administered microelectrophoretically into the external environment of neurones using a 4-barrel micropipette (tip diameter 3~,/~m) placed close to the cell membrane (Fig. 1). Glycine (Fluka, 0.5 M) was adjusted to pH 3.4 and ejected as cation (qcurrent). Sodium DL-homocysteate (DLH, 0.2 M) and monosodium L-glutamate (Fluka, 1.0 M) were adjusted to pH 8.0 and ejected as anions (-- currents)L Microelectrode impalements were made of neurones lying in the dense zones of the explant as well as in the zones of migration of 13-33-day-old spinal cord cultures. Although most of the recordings were obtained from large cells (30-50/~m in diameter) some smaller neurones have also been successfully impaled. The neurones were identified by their morphological appearance using phase-contrast optics (shape, Nissl substance, nuclear morphology). Some of the cultures were also stained (Nissl, methylene blue, Bodian) after recordings have been made. As was observed by Hild and Tasaki 7 and Wardell 1° it was often difficult to maintain membrane potentials over a long period of time. In a large proportion of cells the membrane potential decayed rapidly (within several seconds) after impalement. However, in some cells it was possible to record reasonably stable potentials for some minutes (up to 20 min). The fall in membrane potential was often accompanied by morphological changes of the cells (increased granularity, vacuoles, shrinkage, Fig. 1C). The action of glycine was tested on 18 neurones with reasonably stable membrane potentials ranging from --40 to --65 mV. When ejected with currents of Brain Research, 34 (1971) 399-402
400
SHORT COMMUNICATIONS
80-100 nA onto these neurones, glycine often caused a hyperpolarization with a time course similar to that observed in spinal motoneurones of the cat 5 (Fig. 2A). The considerable varieties of the effectiveness of glycine between individual neurones could be due to differences in the distance of the tip of the drug-administering electrode from the cell membrane, as well as to differences in the amino acid sensitivity of the cells tested. Two small neurones were depolarized by glycine, presumably because of the spontaneous diffusion of chloride ions from the recording microelectrode 5,11.
Brain Research, 34 (1971) 399-402
401
SHORT COMMUNICATIONS
Fig. I. Phase-contrast picture of a neurone of a 20-day-old spinal cord culture. A, Before impalement with the recording microelectrode (R). The drug-containing micropipette (M) is placed close to the cell membrane. B, During impalement with the recording electrode. C, After withdrawal of the recording electrode from the cell. Notice marked morphological changes of the cell. Bar: 20 #m.
A GLY
80nA
m
3 see
B GLY 100 nA
I
5nA
I 30mY
m
20 rnsec
Fig. 2. Actions of glycine on the membrane potential and membrane conductance of two different neurones. A, Spinal neurone, 21 days in vitro (resting potential ---42 mV). Glycine was ejected with a current of 80 nA. Duration of the glycine application is indicated by horizontal bar above the trace. B, Spinal neurone of a 15-day-old culture, resting potential --40 mV (dotted line). Glycine (100 nA) reversibly reduces the amplitude of the intracellular hyperpolarizing pulse (lower trace). Current (nA) and duration of intracellular pulse are indicated in upper trace.
402
SHORT COMMUNICATIONS
The hyperpolarization by glycine was accompanied by an increase in m e m b r a n e conductance. This effect is illustrated in Fig. 2B. Glycine (100 nA) reduced the membrane resistance o f this neurone from 12 Mr2 to 7.5 M ~ and caused a hyperpolarization o f approximately 5 mV. After termination o f the glycine ejection, the m e m b r a n e resistance increased to 14 M ~ . Preliminary observations on two neurones indicate that D L H and L-glutamate (80 nA) depolarize cultured nerve cells. Electrophysiological investigations by Crain and Peterson 4 have shown that the formation o f synapses in spinal cord cultures2, 3 is correlated with the onset o f bioelectric activity. They also found that strychnine enhanced bioelectric responses in these cultures suggesting that 'an unusually high ratio o f inhibitory to excitatory synapses may develop under conditions o f neuronal isolation in vitro '4. O u r observations indicate that cultured spinal neurones have receptors which are influenced by glycine in a similar fashion to those o f spinal motoneurones in vivo 5,11. It has yet to be demonstrated that inhibitory postsynaptic potentials can be evoked in these cells by selective stimulation and that strychnine affects postsynaptic inhibition as well as the action o f glycine. This work was supported in part by the Sandoz-Stiftung zur FSrderung der medizinisch-biologischen Wissenschaften, Basel. Department of Neurophysiology, Neurological Clinic of the University of Basle, 4051 Basle (Switzerland)
L. HOSLI P. F. ANDRI~S E. HOSLI
1 BRUGGENCATE,G. TEN,AND ENGBERG, I., Analysis of glycine actions on spinal interneurones by intracellular recording, Brain Research, I 1 (1968) 446-450. 2 BUNGE, M. B., BUNGE, R. P., AND PETERSON, E. R., The onset of synapse formation in spinal cord cultures as studied by electron microscopy, Brain Research, 6 (1967) 728-749. 3 BUNGE,R. P., BUNGE,M. B., ANDPETERSON,E. R., An electron microscope study of cultured rat spinal cord, J. Cell Biol., 24 (1965) 163-191. 4 CRAIN, S. M., AND PETERSON, E. R., Onset and development of functional interneuronal connections in explants of rat spinal cord-ganglia during maturation in culture, Brain Research, 6 (1967) 750-762. 5 CURTIS,I). R., HOSLI, L., JOHNSTON,G. A. R., AND JOHNSTON,I. H., The hyperpolarization of spinal motoneurones by glycine and related amino acids, Exp. Brain Res., 5 (1968) 235-258. 6 FEIN, H., Passing current through recording glass micropipette electrodes, IEEE Trans. Bio-Med. Engin., BME-13 (1966) 211-212. 7 HILD, W., ANDTASAKr,I., Morphological and physiological properties of neurons and glial cells in tissue culture, J. Neurophysiol., 25 (1962) 277-304. 8 H6SLI, E., AND H/)SLI, L., Acetylcholinesterase in cultured rat spinal cord, Brain Research, 30 (1971) 193-197. 9 H6SLI, L., AND TEBECIS, A. K., Actions of amino acids and convulsants on bulbar reticular neurones, Exp. Brain Res., 11 (1970) 111-127. 10 WARDELL, W. M., Electrical and pharmacological properties of mammalian neuroglial cells in tissue-culture, Proc. roy. Soc. B, 165 (1966) 326-361. ll WERMAN,R., DAVIDOFF,R. A., AND APRISON, M. H.. Inhibitory acition of glycine on spinal neurons in the cat, J. Neurophysiol., 31 (1968) 81-95. (Accepted August 28th, 1971)
Brain Research, 34 (1971) 399402
SHORT COMMUNICATIONS
Effects of glycine on spinal neurones grown in tissue culture Glycine has been proposed to be an inhibitory transmitter substance in the spinal cord of the catS,11; the amino acid hyperpolarizes and increases the membrane conductance of spinal motoneurones and interneuronesl,S, 11. In the present investigation we were interested to study whether glycine has a similar action on the membrane potential and conductance of spinal neurones grown in tissue culture. The cultures were prepared from spinal cord of newborn rats and grown on collagen-coated coverslips in Maximov assemblies 8. For electrophysiological investigations the coverslip with the culture was mounted as a 'lying-drop' preparation in a Falcon petri dish (diameter 50 mm) filled with Gey's balanced salt solution. The temperature of the solution (34-36°C) was regulated by an automatic heating system, the pH was kept at 7.0--7.2 by passing 5 ~ COz--95~ 02 over the surface of the bathing solution. The chamber was fixed to the mechanical stage of a reverse Zeiss microscope. The microelectrodes were introduced from above into the culture at an angle of 15-20 ° by means of two Leitz micromanipulators. For intracellular recordings 3 M KCl-containing glass microelectrodes (tip diameter < 1 #m) were used, most of which were bent at an angle of 50 °, 3--4 mm back from the tip. Using the two types of electrodes (straight and bent) we observed that impalements with bent electrodes were more successful. The recording microelectrode was connected through an Ag-AgC1 wire to a cathode follower and potentials were displayed on an oscilloscope. To determine the resistance of the cell membrane square pulses (depolarizing and hyperpolarizing) of 30--40 msec duration were passed through the recording electrode by means of a device similar to that described by Fein 6. Amino acids were administered microelectrophoretically into the external environment of neurones using a 4-barrel micropipette (tip diameter 3~,/~m) placed close to the cell membrane (Fig. 1). Glycine (Fluka, 0.5 M) was adjusted to pH 3.4 and ejected as cation (qcurrent). Sodium DL-homocysteate (DLH, 0.2 M) and monosodium L-glutamate (Fluka, 1.0 M) were adjusted to pH 8.0 and ejected as anions (-- currents)L Microelectrode impalements were made of neurones lying in the dense zones of the explant as well as in the zones of migration of 13-33-day-old spinal cord cultures. Although most of the recordings were obtained from large cells (30-50/~m in diameter) some smaller neurones have also been successfully impaled. The neurones were identified by their morphological appearance using phase-contrast optics (shape, Nissl substance, nuclear morphology). Some of the cultures were also stained (Nissl, methylene blue, Bodian) after recordings have been made. As was observed by Hild and Tasaki 7 and Wardell 1° it was often difficult to maintain membrane potentials over a long period of time. In a large proportion of cells the membrane potential decayed rapidly (within several seconds) after impalement. However, in some cells it was possible to record reasonably stable potentials for some minutes (up to 20 min). The fall in membrane potential was often accompanied by morphological changes of the cells (increased granularity, vacuoles, shrinkage, Fig. 1C). The action of glycine was tested on 18 neurones with reasonably stable membrane potentials ranging from --40 to --65 mV. When ejected with currents of Brain Research, 34 (1971) 399-402
400
SHORT COMMUNICATIONS
80-100 nA onto these neurones, glycine often caused a hyperpolarization with a time course similar to that observed in spinal motoneurones of the cat 5 (Fig. 2A). The considerable varieties of the effectiveness of glycine between individual neurones could be due to differences in the distance of the tip of the drug-administering electrode from the cell membrane, as well as to differences in the amino acid sensitivity of the cells tested. Two small neurones were depolarized by glycine, presumably because of the spontaneous diffusion of chloride ions from the recording microelectrode 5,11.
Brain Research, 34 (1971) 399-402
401
SHORT COMMUNICATIONS
Fig. I. Phase-contrast picture of a neurone of a 20-day-old spinal cord culture. A, Before impalement with the recording microelectrode (R). The drug-containing micropipette (M) is placed close to the cell membrane. B, During impalement with the recording electrode. C, After withdrawal of the recording electrode from the cell. Notice marked morphological changes of the cell. Bar: 20 #m.
A GLY
80nA
m
3 see
B GLY 100 nA
I
5nA
I 30mY
m
20 rnsec
Fig. 2. Actions of glycine on the membrane potential and membrane conductance of two different neurones. A, Spinal neurone, 21 days in vitro (resting potential ---42 mV). Glycine was ejected with a current of 80 nA. Duration of the glycine application is indicated by horizontal bar above the trace. B, Spinal neurone of a 15-day-old culture, resting potential --40 mV (dotted line). Glycine (100 nA) reversibly reduces the amplitude of the intracellular hyperpolarizing pulse (lower trace). Current (nA) and duration of intracellular pulse are indicated in upper trace.
402
SHORT COMMUNICATIONS
The hyperpolarization by glycine was accompanied by an increase in m e m b r a n e conductance. This effect is illustrated in Fig. 2B. Glycine (100 nA) reduced the membrane resistance o f this neurone from 12 Mr2 to 7.5 M ~ and caused a hyperpolarization o f approximately 5 mV. After termination o f the glycine ejection, the m e m b r a n e resistance increased to 14 M ~ . Preliminary observations on two neurones indicate that D L H and L-glutamate (80 nA) depolarize cultured nerve cells. Electrophysiological investigations by Crain and Peterson 4 have shown that the formation o f synapses in spinal cord cultures2, 3 is correlated with the onset o f bioelectric activity. They also found that strychnine enhanced bioelectric responses in these cultures suggesting that 'an unusually high ratio o f inhibitory to excitatory synapses may develop under conditions o f neuronal isolation in vitro '4. O u r observations indicate that cultured spinal neurones have receptors which are influenced by glycine in a similar fashion to those o f spinal motoneurones in vivo 5,11. It has yet to be demonstrated that inhibitory postsynaptic potentials can be evoked in these cells by selective stimulation and that strychnine affects postsynaptic inhibition as well as the action o f glycine. This work was supported in part by the Sandoz-Stiftung zur FSrderung der medizinisch-biologischen Wissenschaften, Basel. Department of Neurophysiology, Neurological Clinic of the University of Basle, 4051 Basle (Switzerland)
L. HOSLI P. F. ANDRI~S E. HOSLI
1 BRUGGENCATE,G. TEN,AND ENGBERG, I., Analysis of glycine actions on spinal interneurones by intracellular recording, Brain Research, I 1 (1968) 446-450. 2 BUNGE, M. B., BUNGE, R. P., AND PETERSON, E. R., The onset of synapse formation in spinal cord cultures as studied by electron microscopy, Brain Research, 6 (1967) 728-749. 3 BUNGE,R. P., BUNGE,M. B., ANDPETERSON,E. R., An electron microscope study of cultured rat spinal cord, J. Cell Biol., 24 (1965) 163-191. 4 CRAIN, S. M., AND PETERSON, E. R., Onset and development of functional interneuronal connections in explants of rat spinal cord-ganglia during maturation in culture, Brain Research, 6 (1967) 750-762. 5 CURTIS,I). R., HOSLI, L., JOHNSTON,G. A. R., AND JOHNSTON,I. H., The hyperpolarization of spinal motoneurones by glycine and related amino acids, Exp. Brain Res., 5 (1968) 235-258. 6 FEIN, H., Passing current through recording glass micropipette electrodes, IEEE Trans. Bio-Med. Engin., BME-13 (1966) 211-212. 7 HILD, W., ANDTASAKr,I., Morphological and physiological properties of neurons and glial cells in tissue culture, J. Neurophysiol., 25 (1962) 277-304. 8 H6SLI, E., AND H/)SLI, L., Acetylcholinesterase in cultured rat spinal cord, Brain Research, 30 (1971) 193-197. 9 H6SLI, L., AND TEBECIS, A. K., Actions of amino acids and convulsants on bulbar reticular neurones, Exp. Brain Res., 11 (1970) 111-127. 10 WARDELL, W. M., Electrical and pharmacological properties of mammalian neuroglial cells in tissue-culture, Proc. roy. Soc. B, 165 (1966) 326-361. ll WERMAN,R., DAVIDOFF,R. A., AND APRISON, M. H.. Inhibitory acition of glycine on spinal neurons in the cat, J. Neurophysiol., 31 (1968) 81-95. (Accepted August 28th, 1971)
Brain Research, 34 (1971) 399402