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Cytoplasmic vacuole formation in cultured neurons treated with lithium ions
SHORT COMMUNICATIONS
155
Cytoplasmic vacuole formation in cultured neurons treated with lithium ions The development of a reversible dilatation of the Golgi complex in cultured sensory neurons incubated in feeding solutions containing the N a - K - A T P a s e inhibitor, ouabain, has been recently reported 12. In the present study, undertaken to further investigate these neuronal Golgi changes, cultured sensory neurons were incubated in feeding media in which sodium ions had been partially replaced by lithium ions. Our observations are reported here to point out some apparent similarities in the initial neuronal responses in these cultures and the neuronal responses in cultures exposed to ouabain. Experiments in this study were carried out using mature cultures of rat dorsal root ganglion prepared and maintained as described by Bunge et al. 3. The usual maintenance feeding medium for these cultures contains 50 ~ Eagle's Basal Medium, 25 ~ human placental serum, and 25 %ochick embryo extract supplemented with 900 mg ~ glucose. Lithium-containing feeding media (see Table I) were prepared by two TABLE
l
SOME C O N S T I T U E N T S OF C O N T R O L A N D E X P E R I M E N T A L M E D I A
[Li+]
[Na+]
[K+]
( mequiv./ l)
( mequiv./1)
( rnequiv./ I)
Osmolarity (mO s M)
135 130-140 28 76 116
9 7-9 8 9 9.5
315 310-330 330 320 320
Standard Medium 0 Control Media 0 Experimental MediumA 109 Experimental Medium B 55 Experimental Medium C 18
different methods. For replacement of less than half the total sodium concentration by lithium, a calculated amount of LiCI was placed directly into an N a C l - K C l - p o o r Minimal Essential Medium (especially prepared by Microbiological Associates, Bethesda, Md.). Desired amounts of NaC1 and KC1 were then added, and this solution was combined with chick embryo extract and placental serum (50: 25: 25), and glucose (900 m g ~ ) , to make an Li+-containing feeding medium. For replacement of more than half the total sodium concentration, standard feeding medium was dialyzed against an Li +-containing balanced salt solution for 20-30 rain at 4°C. Na+-containing control media were simultaneously prepared by the same methods. In each solution Li-', N a + and K + levels were determined by a D B G spectrophotometer, Beckman model 979, and osmolarity was measured with an Osmette osmometer. Three different groups of cultures were used. Each group was incubated in one of the three Li+-containing feeding solutions (Table I). Cellular responses during incubation in Li~-media were observed and photographed with light microscopic bright field optics using a Bausch and Lomb fluorite oil immersion lens. Then the cultures were fixed (in 2 ~ osmium buffered with veronal acetate), embedded in Epon, and studied with an R,C.A, 3G Electron Microscope. Brain Research, 19 (1970) 155-159
156
SHORT COM MI_JNICATIONS
In all cultures treated with Li +. most neurons developed abnormal cytoplasmic granularity with an appearance suggesting the presence of scattered cytoplasmic vacuoles (Figs. 2 and 31. Neuronal nuclei became progressively obscured, and some increase in cell size seemed to develop. The time required for the occurrence of this response varied inversely with the concentration of Li + and directly with the concentration of Na . Light microscopic observations in living tissue: In cultures incubated in Experimental Medium A (Table I) neurons developed cytoplasmic changes (like Fig. 2) by about 5-6 h; by 10-11 h of incubation, the reaction was more advanced {like Fig, 3). Cultures in Medium B showed abnormal cytoplasmic granularity in neurons by about 18 h of incubation (Fig. 2), and by 24 h. the reaction was pronounced ( Fig. 3). Cultures in Medium C showed little neuronal change until about 45-50 h when the early reaction was observed (like Fig. 2): a distinctly abnormal appearance (like Fig. 3) developed by about 60 h of incubation in this medium. The neuronzd changes reported here appeared similar to those observed in cultures incubated in ouabain ~. but these changes were not reversible upon removal of the cultures from the Li -media. During incubation in Li 4. some unmyelinated nerve fibers showed focal dilatations similar to those described in cultures under other conditions6,.'L These changes are not evaluated in this report. Supporting elements of the tissue seemed to react by cell swelling, but increased cytoplasmic granularity, if il developed, was not visualized in the living Schwann cells or satellite cells. Cultures incubated m control media for up to 96 h (the usual interwash period) showed no abnormalities. Electron microscopic observations: Some cultures incubated in Li~-media were fixed at the first appearance of the granular response in neurons (Fig. 2): others were fixed at more advanced stages (Fig. 3). Electron microscopic study of neurons m these cultures showed that. in affected neurons, clusters of cytoplasmic vacuoles arising in the regions of the Golgi complex appeared to replace the usual Gotgi configuration (Fig. 5); other organelles seemed unaltered. Preliminary evaluation of Schwann cells and satellite ceils in the Li '-treated cultures indicates abnormal dilatation of small clusters of Golgi vesicles with no apparent effect on other organelles. Neurons and supporting tissue in control cultures appeared normal (Fig. 4/. Among monovalent cations other than Na*. Li ions have been found to substitute for Na ions during the production of the action potential in frog nerve 4 and in mammalian non-myelinated nerve fibers 1°. Araki et al. 1 report that ki ions behave exactly as Na ions in the process of generating an action potential in the cat spinal motoneuron but that Li ions are extruded from these%ells at a rate of about half that of N a ions. Keynes and Swan 8 have observed that in resting frog sartorius muscle. the rate of Li + influx is approximately equal to that o f N a +. but the rate of Li-' efflux is about 1/10-t/25 that of Na +. They suggest that though the membrane does not differentiate between Na + and Li ~ during passive influx, there is appreciable discrimiBrain Research, 19 (1970) 155-129
SHORT COMMUNICATIONS
157
Fig. 1 (magn. 900 x ) shows a normal, living neuron from a mature culture of rat dorsal root ganglion. Fig. 2 (magn. 950 > ) illustrates changes developing within the living neuron after incubation in Experimental Medium B (Table 1) for approximately 18 h. Fig. 3 (magn. 900 ×) shows the same neuron after incubation in Experimental Medium B for approximately 25 h. The increasing granularity of the neuron (shown in Figs. 2 and 3), the scattered vacuole-like structures (arrows), and the changes in the cell's dimensions, suggest reaction in the cell during incubation in Medium B. Fig. 4. Cytoplasm of a neuron of rat dorsal root ganglion after incubation in a control medium (Table 1) for approximately 96 h. Cytoplasmic morphology appears to be typical of that of a normal, mature neuron of rat dorsal root ganglion in culture. Magn. 20,000 ×. Fig. 5. Cytoplasmic configuration of a neuron of rat dorsal root ganglion incubated in Experimental Medium B (Table I) for approximately 25 h. Cytoplasmic vacuoles seem to arise by dilatation of components of the Golgi complex+ Magn. 19,000 : .
Brain Research, 19 (1970) 155-159
158
SHORT COMMLNI('ATfONS
nation between N a + and Li + by active t r a n s p o r t m e c h a n i s m s d u r i n g extrusion of these ions. If such a n i o n - d i s c r i m i n a t i n g m e c h a n i s m operates in resting nerve tissue, substituted Li ions (as in our experiments) might help to d e m o n s t r a t e the locations of some N a + - p u m p i n g sites. If intracellular N a + - p u m p i n g sites are present a l o n g m e m branes of the cisternal system in neurons, as previously suggested7,12, Li ions supplied in place of N a ions (see refs. 5. t 1) might tend to accumulate at or near these sites. A c c u m u l a t i o n of Li ions (and water) at such p o s t u l a t e d N a ÷ - p u m p i n g sites a l o n g the Golgi m e m b r a n e might p r o d u c e dilatation of the Golgi cisterns. Other mechanisms, for example, failure of p l a s m a l e m m a l p u m p i n g systems or alterations in c o n c e n t r a t i o n of N a ions or some other c o m p o n e n t ( s ) of the experimental m e d i u m , must also be considered as possible bases for the cellular responses reported here. F u r t h e r evaluation of this m i c r o s o m a l p h e n o m e n o n in cultured n e u r o n s is now underway. The possibility of any relationship between o u r observations and the effects of Li + salts in a n i m a l s and m a n z is also being considered. This study was supported by N a t i o n a l Institute of Health G r a n t s NB,04325, GM-00256 a n d NB-05242 a n d by N a t i o n a l Multiple Sclerosis Society G r a n t 428. We wish to express our gratitude for the guidance a n d interest o f Dr. Richard P. Bunge, our mentor. We also gratefully acknowledge the valuable technical assistance of Mrs. Julia Shen in h a n d l i n g culture material a n d of Miss Ellen Sutter a n d Miss B a r b a r a Jones in m a k i n g cation d e t e r m i n a t i o n s . Department of Anatomy, College of Physicians and Surgeons of Columbia University. New York City, N.Y. 10032 /U.S.A.)
W. O. WHETSELL. JR * J. J. MIRE**
1 ARAKI.T.. ITO. M., KOSTYUK.P. G., OSCARSSON,O., AND OSHIMA,T., The effects of alkaline cations on the responses of cat spinal motoneurons, and their removal from the cells. Proc. Roy. Soc. B. 162 (19653 319-332. 2 BRACELAND.F. J. (Ed.), This month's special section. Lithium carbonate, Amer. J. Psychiat., 125 (1968) 487-549. 3 BUNGE.M. B.. BLrNGE.R. P.. PETERSON,E. R., AND MURRAY.M. R.. A light and electron m~croscope study of long-term organized cultures of rat dorsal root ganglia. J. Cell BioL. 32 (19671 439-466. 4 GALLEGO. A.. AND LORENTEDE Nt3. R.. 0 n the effect of ammonium and lithium ions upon frog nerve deprived of sodium. J. gen. Physiol.. 35 (1951) 227-244. 5 HARTMANN,J. F.. Newer knowledge of the neuron. In O. T. BAILEY(Ed,L International Academy of Pathology. Monograph 9. Central Nervous System. Williams and Witkins, Baltimore, 1968, pp. 1-20. 6 HENDELMAN. W.. AND MIRE. J. J.. Observations on a swelling phase during degeneration of unmyelinated nerve fibers. Anat. Rec.. 160 (1968) 364. 7 J~RNEFELT. J.. Some aspects of the physiological significance of the adenosinetriphosphatase of brain microsomes. Biochim. biophys. Acta /Amst.), 59 (1962} 655-662.
* Present address: Naval Aerospace Medical Institute, Naval Aerospace Medical Center, Pensacola, Fla. 32512, U.S.A. ** Present address: Department of Anatomy, American University of Beirut, Beirut, Lebanon. Brain Research, 19 (1970) 155-159
SHORT COMMUNICATIONS
159
8 KEYNES, R. D., AND SWAN, R. C., The permeability of frog muscle fibres to lithium ions, J. Physiol. (Lond.), 147 (1959)626 638. 9 MIRE, J. J., HENDELMAN W. J., AND BUNGE, R. P., Observations on a transient phase of focal swelling in degenerating unmyelinated nerve fibers, J. Cell Biol., in press. 10 RHCHIE, J. M., AND STRAUB, R. W., The hyperpolarization which follows activity in mammalian non-medullated fibers, J. Physiol. (Lond.), 136 (1957) 80-97. 11 WnETSELL, W. O., JR., Further observations on Golgi complex alterations in ouabain-treated neurons, Anat. Rec., 163 (1969)284. 12 WnETSELL, W. O., JR., AND BUNGE, R. P., Reversible alterations in the Golgi complex of cultured neurons treated with an inhibitor of active Na and K transport, J. Cell Biol., 42 (1969)490-500.
(Accepted January 19th, 1970)
Brain Research, 19 (1970) 155-159
155
Cytoplasmic vacuole formation in cultured neurons treated with lithium ions The development of a reversible dilatation of the Golgi complex in cultured sensory neurons incubated in feeding solutions containing the N a - K - A T P a s e inhibitor, ouabain, has been recently reported 12. In the present study, undertaken to further investigate these neuronal Golgi changes, cultured sensory neurons were incubated in feeding media in which sodium ions had been partially replaced by lithium ions. Our observations are reported here to point out some apparent similarities in the initial neuronal responses in these cultures and the neuronal responses in cultures exposed to ouabain. Experiments in this study were carried out using mature cultures of rat dorsal root ganglion prepared and maintained as described by Bunge et al. 3. The usual maintenance feeding medium for these cultures contains 50 ~ Eagle's Basal Medium, 25 ~ human placental serum, and 25 %ochick embryo extract supplemented with 900 mg ~ glucose. Lithium-containing feeding media (see Table I) were prepared by two TABLE
l
SOME C O N S T I T U E N T S OF C O N T R O L A N D E X P E R I M E N T A L M E D I A
[Li+]
[Na+]
[K+]
( mequiv./ l)
( mequiv./1)
( rnequiv./ I)
Osmolarity (mO s M)
135 130-140 28 76 116
9 7-9 8 9 9.5
315 310-330 330 320 320
Standard Medium 0 Control Media 0 Experimental MediumA 109 Experimental Medium B 55 Experimental Medium C 18
different methods. For replacement of less than half the total sodium concentration by lithium, a calculated amount of LiCI was placed directly into an N a C l - K C l - p o o r Minimal Essential Medium (especially prepared by Microbiological Associates, Bethesda, Md.). Desired amounts of NaC1 and KC1 were then added, and this solution was combined with chick embryo extract and placental serum (50: 25: 25), and glucose (900 m g ~ ) , to make an Li+-containing feeding medium. For replacement of more than half the total sodium concentration, standard feeding medium was dialyzed against an Li +-containing balanced salt solution for 20-30 rain at 4°C. Na+-containing control media were simultaneously prepared by the same methods. In each solution Li-', N a + and K + levels were determined by a D B G spectrophotometer, Beckman model 979, and osmolarity was measured with an Osmette osmometer. Three different groups of cultures were used. Each group was incubated in one of the three Li+-containing feeding solutions (Table I). Cellular responses during incubation in Li~-media were observed and photographed with light microscopic bright field optics using a Bausch and Lomb fluorite oil immersion lens. Then the cultures were fixed (in 2 ~ osmium buffered with veronal acetate), embedded in Epon, and studied with an R,C.A, 3G Electron Microscope. Brain Research, 19 (1970) 155-159
156
SHORT COM MI_JNICATIONS
In all cultures treated with Li +. most neurons developed abnormal cytoplasmic granularity with an appearance suggesting the presence of scattered cytoplasmic vacuoles (Figs. 2 and 31. Neuronal nuclei became progressively obscured, and some increase in cell size seemed to develop. The time required for the occurrence of this response varied inversely with the concentration of Li + and directly with the concentration of Na . Light microscopic observations in living tissue: In cultures incubated in Experimental Medium A (Table I) neurons developed cytoplasmic changes (like Fig. 2) by about 5-6 h; by 10-11 h of incubation, the reaction was more advanced {like Fig, 3). Cultures in Medium B showed abnormal cytoplasmic granularity in neurons by about 18 h of incubation (Fig. 2), and by 24 h. the reaction was pronounced ( Fig. 3). Cultures in Medium C showed little neuronal change until about 45-50 h when the early reaction was observed (like Fig. 2): a distinctly abnormal appearance (like Fig. 3) developed by about 60 h of incubation in this medium. The neuronzd changes reported here appeared similar to those observed in cultures incubated in ouabain ~. but these changes were not reversible upon removal of the cultures from the Li -media. During incubation in Li 4. some unmyelinated nerve fibers showed focal dilatations similar to those described in cultures under other conditions6,.'L These changes are not evaluated in this report. Supporting elements of the tissue seemed to react by cell swelling, but increased cytoplasmic granularity, if il developed, was not visualized in the living Schwann cells or satellite cells. Cultures incubated m control media for up to 96 h (the usual interwash period) showed no abnormalities. Electron microscopic observations: Some cultures incubated in Li~-media were fixed at the first appearance of the granular response in neurons (Fig. 2): others were fixed at more advanced stages (Fig. 3). Electron microscopic study of neurons m these cultures showed that. in affected neurons, clusters of cytoplasmic vacuoles arising in the regions of the Golgi complex appeared to replace the usual Gotgi configuration (Fig. 5); other organelles seemed unaltered. Preliminary evaluation of Schwann cells and satellite ceils in the Li '-treated cultures indicates abnormal dilatation of small clusters of Golgi vesicles with no apparent effect on other organelles. Neurons and supporting tissue in control cultures appeared normal (Fig. 4/. Among monovalent cations other than Na*. Li ions have been found to substitute for Na ions during the production of the action potential in frog nerve 4 and in mammalian non-myelinated nerve fibers 1°. Araki et al. 1 report that ki ions behave exactly as Na ions in the process of generating an action potential in the cat spinal motoneuron but that Li ions are extruded from these%ells at a rate of about half that of N a ions. Keynes and Swan 8 have observed that in resting frog sartorius muscle. the rate of Li + influx is approximately equal to that o f N a +. but the rate of Li-' efflux is about 1/10-t/25 that of Na +. They suggest that though the membrane does not differentiate between Na + and Li ~ during passive influx, there is appreciable discrimiBrain Research, 19 (1970) 155-129
SHORT COMMUNICATIONS
157
Fig. 1 (magn. 900 x ) shows a normal, living neuron from a mature culture of rat dorsal root ganglion. Fig. 2 (magn. 950 > ) illustrates changes developing within the living neuron after incubation in Experimental Medium B (Table 1) for approximately 18 h. Fig. 3 (magn. 900 ×) shows the same neuron after incubation in Experimental Medium B for approximately 25 h. The increasing granularity of the neuron (shown in Figs. 2 and 3), the scattered vacuole-like structures (arrows), and the changes in the cell's dimensions, suggest reaction in the cell during incubation in Medium B. Fig. 4. Cytoplasm of a neuron of rat dorsal root ganglion after incubation in a control medium (Table 1) for approximately 96 h. Cytoplasmic morphology appears to be typical of that of a normal, mature neuron of rat dorsal root ganglion in culture. Magn. 20,000 ×. Fig. 5. Cytoplasmic configuration of a neuron of rat dorsal root ganglion incubated in Experimental Medium B (Table I) for approximately 25 h. Cytoplasmic vacuoles seem to arise by dilatation of components of the Golgi complex+ Magn. 19,000 : .
Brain Research, 19 (1970) 155-159
158
SHORT COMMLNI('ATfONS
nation between N a + and Li + by active t r a n s p o r t m e c h a n i s m s d u r i n g extrusion of these ions. If such a n i o n - d i s c r i m i n a t i n g m e c h a n i s m operates in resting nerve tissue, substituted Li ions (as in our experiments) might help to d e m o n s t r a t e the locations of some N a + - p u m p i n g sites. If intracellular N a + - p u m p i n g sites are present a l o n g m e m branes of the cisternal system in neurons, as previously suggested7,12, Li ions supplied in place of N a ions (see refs. 5. t 1) might tend to accumulate at or near these sites. A c c u m u l a t i o n of Li ions (and water) at such p o s t u l a t e d N a ÷ - p u m p i n g sites a l o n g the Golgi m e m b r a n e might p r o d u c e dilatation of the Golgi cisterns. Other mechanisms, for example, failure of p l a s m a l e m m a l p u m p i n g systems or alterations in c o n c e n t r a t i o n of N a ions or some other c o m p o n e n t ( s ) of the experimental m e d i u m , must also be considered as possible bases for the cellular responses reported here. F u r t h e r evaluation of this m i c r o s o m a l p h e n o m e n o n in cultured n e u r o n s is now underway. The possibility of any relationship between o u r observations and the effects of Li + salts in a n i m a l s and m a n z is also being considered. This study was supported by N a t i o n a l Institute of Health G r a n t s NB,04325, GM-00256 a n d NB-05242 a n d by N a t i o n a l Multiple Sclerosis Society G r a n t 428. We wish to express our gratitude for the guidance a n d interest o f Dr. Richard P. Bunge, our mentor. We also gratefully acknowledge the valuable technical assistance of Mrs. Julia Shen in h a n d l i n g culture material a n d of Miss Ellen Sutter a n d Miss B a r b a r a Jones in m a k i n g cation d e t e r m i n a t i o n s . Department of Anatomy, College of Physicians and Surgeons of Columbia University. New York City, N.Y. 10032 /U.S.A.)
W. O. WHETSELL. JR * J. J. MIRE**
1 ARAKI.T.. ITO. M., KOSTYUK.P. G., OSCARSSON,O., AND OSHIMA,T., The effects of alkaline cations on the responses of cat spinal motoneurons, and their removal from the cells. Proc. Roy. Soc. B. 162 (19653 319-332. 2 BRACELAND.F. J. (Ed.), This month's special section. Lithium carbonate, Amer. J. Psychiat., 125 (1968) 487-549. 3 BUNGE.M. B.. BLrNGE.R. P.. PETERSON,E. R., AND MURRAY.M. R.. A light and electron m~croscope study of long-term organized cultures of rat dorsal root ganglia. J. Cell BioL. 32 (19671 439-466. 4 GALLEGO. A.. AND LORENTEDE Nt3. R.. 0 n the effect of ammonium and lithium ions upon frog nerve deprived of sodium. J. gen. Physiol.. 35 (1951) 227-244. 5 HARTMANN,J. F.. Newer knowledge of the neuron. In O. T. BAILEY(Ed,L International Academy of Pathology. Monograph 9. Central Nervous System. Williams and Witkins, Baltimore, 1968, pp. 1-20. 6 HENDELMAN. W.. AND MIRE. J. J.. Observations on a swelling phase during degeneration of unmyelinated nerve fibers. Anat. Rec.. 160 (1968) 364. 7 J~RNEFELT. J.. Some aspects of the physiological significance of the adenosinetriphosphatase of brain microsomes. Biochim. biophys. Acta /Amst.), 59 (1962} 655-662.
* Present address: Naval Aerospace Medical Institute, Naval Aerospace Medical Center, Pensacola, Fla. 32512, U.S.A. ** Present address: Department of Anatomy, American University of Beirut, Beirut, Lebanon. Brain Research, 19 (1970) 155-159
SHORT COMMUNICATIONS
159
8 KEYNES, R. D., AND SWAN, R. C., The permeability of frog muscle fibres to lithium ions, J. Physiol. (Lond.), 147 (1959)626 638. 9 MIRE, J. J., HENDELMAN W. J., AND BUNGE, R. P., Observations on a transient phase of focal swelling in degenerating unmyelinated nerve fibers, J. Cell Biol., in press. 10 RHCHIE, J. M., AND STRAUB, R. W., The hyperpolarization which follows activity in mammalian non-medullated fibers, J. Physiol. (Lond.), 136 (1957) 80-97. 11 WnETSELL, W. O., JR., Further observations on Golgi complex alterations in ouabain-treated neurons, Anat. Rec., 163 (1969)284. 12 WnETSELL, W. O., JR., AND BUNGE, R. P., Reversible alterations in the Golgi complex of cultured neurons treated with an inhibitor of active Na and K transport, J. Cell Biol., 42 (1969)490-500.
(Accepted January 19th, 1970)
Brain Research, 19 (1970) 155-159