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Inhibition of nerve fiber regeneration in cultured sympathetic neurons by local, high potassium
BrainResearch, 293(1984)159--163 Elsevier
159
BRE20015
Inhibition of nerve fiber regeneration in cultured sympathetic neurons by local, high potassium ROBERT B. CAMPENOT Section of Neurobiology and Behavior, Cornell University, Ithaca, NY14853 (U.S.A.) (Accepted October llth, 1983) Key words: sympathetic neurons - - potassium - - nerve regeneration - - nerve growth - - depolarization
Distal regeneration of neurites from cultured sympathetic neurons of newborn rats was stunted by exposure to local, high K ÷ (20 mM), only when more proximal neurite regions and the cell bodies were exposed to a normal K + concentration (5 mM). Neurites grew luxuriantly and extensively when exposure to high K ÷ was uniform over the entirety of the neurons. Thus, neurite growth can be strongly influenced by regional variations in K ÷ which are probably within the naturally occurring range, especially during development and regeneration. A recent report by Lowl0 indicates that regeneration of the sciatic nerve occurs in an environment of high extracellular potassium ([K+]e). Following transection of adult rat sciatic nerve, [K+]e was found to reach 15 m M in the e n d o n e u r i u m distal to the cut, about a 4-fold increase from an average of 4 m M in intact nerves. Conners et al. 4 r e p o r t that [K÷]e in newborn rat optic nerve can increase m o r e than 4fold, to 20 m M during electrical stimulation at 20 Hz, whereas levels in optic nerves of adults stimulated at 400 Hz increased to only 11 mM. These experiments suggest that [K+]e in the neuronal environment may vary substantially, especially during develo p m e n t and regeneration in the m a m m a l i a n peripheral and central nervous systems. In o r d e r to investigate the possibility that variations in [K+]e might influence nerve fiber growth, I observed the regeneration of neurites in principal neurons cultured from the superior cervical ganglia of newborn rats in normal (5 mM) and elevated (20 mM) K ÷. The neurons were cultured on multiple collagen channels in 3 - c o m p a r t m e n t dishes (Fig. 1) such that neurites originating from cell bodies in a center c o m p a r t m e n t p e n e t r a t e d silicone grease barriers, emerging into s e p a r a t e fluid environments within left and right c o m p a r t m e n t s 1. In this way neurons were exposed to a uniform increase in K ÷ in all 3 c o m p a r t m e n t s , or a local increase in K +, confined to the neurites in one side c o m p a r t m e n t only. F o r m u l a 0006-8993/84/$03.00 © 1984 Elsevier Science Publishers B.V.
tions and general culturing p r o c e d u r e s were as described by H a w r o t and Patterson5 except that 7S nerve growth factor was further purified by gel filtration of the D E A E - c e l l u l o s e fractions through Sephadex G-150. E l e v a t e d K ÷ was achieved by adding one part of 150 m M KC1 to 9 parts of culture medium. Control m e d i u m was m a d e by adding 150 m M NaC1 to 9 parts of culture m e d i u m from the same batch. High K + m e d i u m contained a final concentration of 20 m M K ÷, and control m e d i u m contained 5 m M K ÷. Except where noted, 7S nerve growth factor was provided in all c o m p a r t m e n t s at 1/~g/ml. Neurons in 9 sister cultures plated under 3 different regimes of K ÷ were examined 1.8 days and 3.8 days later. T h r e e dishes contained control m e d i u m in all c o m p a r t m e n t s , 3 contained high K ÷ m e d i u m in all compartments, and 3 contained high K ÷ m e d i u m in one side c o m p a r t m e n t , with control m e d i u m elsewhere. Many channels in side c o m p a r t m e n t s of all of the cultures contained neurites on day 1.8. On day 3.8, neurites on these channels had elongated to a similar extent and with c o m p a r a b l e density in cultures containing only control m e d i u m or only high K ÷ medium. Mean neurite extension reached about 3 mm, with little difference between left and right compartments. Thus, uniformly high K ÷ had no observed effect on neurite growth. In the 3 cultures with high K ÷ m e d i u m locally, in one side c o m p a r t m e n t only, c o m p a r a b l e neurite ex-
160
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0.5
0
0
0.5
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1
mm
mm
Fig. 1. A schematic diagram of neurons from newborn rats in a 3-compartment culture. A Teflon divider partitioned the culture dish (dish not shown) into 3 compartments, a narrow (1.5 × 5 mm) center compartment (a) open to the bulk of the culture dish, and left (b) and right (c) side compartments. The collagen-coated floor of the dish was scored with 21 parallel scratches that spanned the center compartment and extended into the side compartments as shown. Twenty collagen channels, each 200-300 #m wide and bordered by the scratches, served to orient the outgrowth of neurites from sympathetic neurons in the center compartment to the left and right, as indicated by the enlargement of a single channel. Although the silicone grease formed an effective seal against fluid movement between the compartments, neurites readily penetrated beneath it, crossing into the separate fluid environments of the side compartments where their extension along the channels could be measured (note scale). Three-compartment dishes permit exposure of different regions of the neurons to different media constituents. Except for manipulations of the ionic environment and nerve growth factor described in the text, media formulations and general culturing procedures were as previously described 3,5, with ascorbate and rat serum provided in the center compartments only. Compartmentalized cultures permit the ready mechanical removal of neurites in the side compartments by means of a jet of distilled water delivered with a syringe2.3. This in vitro neuritotomy is without effect on the healthy appearance or number (unpublished) of neurons in the center compartment, and neurites promptly regenerate into the side compartments.
tension (mean = 3.14 m m , S.E. = 0.13, n = 20) was
density differences would have escaped notice, but
observed on day 3.8 in side c o m p a r t m e n t s containing
large ones were easily d o c u m e n t e d by this m et h o d .
control medium, but extension into high K + compart-
Local, high K ÷ caused a reduction in neurite density
ments was about 30% less (mean = 1.88 + 0.10, n =
in the 3 cultures described above and in two addition-
20). The ad v an cem en t of neurites along the channels
al sister cultures raised under identical conditions.
between days 1.8 and 3.8 was r e d u c e d 30% in high,
Nine days after plating, a reduced neurite density in
local K +, indicating a reduced rate of extension oc-
compartments given high K ÷ was observed on 54 of
curred within the compartments containing local,
the 64 channels e x a m i n e d in these cultures. Eight
high K +. Qualitative, visual comparisons were periodically
channels showed equal density in high K ÷ and con-
made of the density of neurites in the left and right compartments of the cultures. Each channel was ex-
high K+.
amined individually. The initial 1 m m in the left and right compartments was examined under phase contrast at 2 0 0 x , and it was noted if the density of neurites was clearly greater on one side than on the other. If no obvious density difference was observed, the channel was noted to have an approximately equal density in the left and right compartments. Small
trol m ed i u m , and two channels were m o r e dense in The effect of local, high K ÷ was subjected to some further tests by means of in vitro neuritotomy2, 3 of neurons ranging 14-33 days in culture. Six cultures were used, all grown under symmetrical conditions, 3 with normal K ÷ in all c o m p a r t m e n t s and 3 with high K+ in all compartments (2 of the high K ÷ cultures were participants in the previous experiment). Prior to neuritotomy, nerve growth factor was withdrawn
161 from the center c o m p a r t m e n t s of 2 of the cultures in
right compartments extending beyond the scale
order to limit neurite regeneration into the center compartment and thus confine it largely to the side
( > 5 mm) on all channels, and with noteworthy differences in density on only 9 of 70 channels.
compartments3. Before neuritotomy, all cultures
Immediately after removal of neurites from the left and right compartments, the center c o m p a r t m e n t
contained luxuriant neurite outgrowth in left and
Fig. 2. Photomicrographs showing neurons on a single collagen channel, 40 days in culture and 7 days after neuritotomy in both side compartments. The channel is 210/~m wide, and the scratches bordering it are visible at the top and bottom of each panel. The 5 panels show a continuous stretch of the channel (see Fig. 1). The top two panels show neurites regenerated in the left compartment given high K÷ medium (20 mM), the middle panel shows the center compartment containing the clusters of cell bodies and given control medium (5 mM K+), and the bottom two panels show neurites regenerated in the right compartment given control medium. Note the drastic reduction of extension and density in the neurites regenerated for 7 days in high local K÷ compared with 7 days regeneration in normal K+. This culture had previously been given high K÷ medium in all compartments continuously since plating and showed a luxuriant and comparable neurite growth in the left and right compartment when neuritotomy was performed.
162 and one side compartment of each culture were given control medium and the remaining side compartment was given high K ÷ medium. A clear inhibitory effect of local, high K ÷ on neurite regeneration was apparent within a few days. Between 1 and 3 days after neuritotomy, 59 of 70 channels showed a higher neurite density in control than in high K + medium. The density differences persisted, becoming more apparent with time, and at the termination of observations neurites on 68 of the 70 channels were more dense in normal than in high K +. While no attempt was made to quantify the magnitude of the density differences, typically the outgrowth in side compartments given normal K ÷ was luxuriant on every channel, and the relative reduction in compartments supplied with high K ÷ was so striking as to leave no doubt of the veracity of the effect (see Fig. 2). The distance that neurites extended into side compartments was initially similar in high and normal K ÷ on many channels, but within 4 days an inhibition of neurite advancement by high K ÷ was apparent, and was well developed in all 6 cultures by days 7-8 after neuritotomy. Mean distances of neurite extension in local, high K + were variable between cultures at this time, ranging 0.95-2.86 mm (S.E. ranged 0.14-0.40). But all cultures showed mean neurite extension exceeding 3.78 mm in opposite side compartments containing control medium. The reduction in neurite extension and density by high, local K ÷ was exhibited by all cultures tested with no apparent differences attributable to nerve growth factor withdrawal from the center compartment or to previous high K + exposure. Clearly, neurons react to regional differences in K + with a stunting of the growth of neurites which encounter levels elevated with respect to that of the soma and more proximal neurite regions. Two of the cultures with local, high K + were maintained for 11 and 16 days after neuritotomy without altering the culture conditions. Neurites on all of the channels in control medium, side compartments had extended beyond the scale, but neurites in high K + compartments never did. They remained shorter and, in fact, showed definite signs of degeneration. Several additional points may be made about the reaction of neurites to local, high K +. In experiments where the initial outgrowth of neurons in culture cross under the barrier and encounter local, high K +,
the reduction in outgrowth results from the exposure of intact and otherwise undamaged neurites. Exposure of damaged neurites to local, high K + as in the in vitro neuritotomy experiments was not necessary for the observed effect. Also, since the elongation of neurites within side compartments containing local, high K + was reduced, and since the effect was observed in cultures where neurite growth back into control medium in the center compartment was severely limited by withdrawal of nerve growth factor3~ a mechanism involving taxis of growth cones along a gradient of decreasing K ÷ is not indicated. Rather, a remote mechanism whereby a local elevation in K* along the neurite has a stunting effect on all neurite outgrowth distal to it seems to be involved. Interference with axonal transport may be indicated. The mechanism of the effect of local, high K ÷ is unknown, but one possibility is that local depolarization of the membrane potential by K ÷ is responsible. If so, it is interesting that a local depolarization distal from the cell body would produce within the neurite a flow of electric current towards the cell body. This is the opposite current direction that would result from an external electric field produced by a proximal anode and distal cathode, an arrangement shown to enhance neurite growth7. 8. A common mechanism may be indicated, and the possibility is raised that the effects of experimentally applied extracellular electric fields might be mediated by intracetlular electric fields in vivo set up by natural, regional variations in [K+]e . Also, reports indicate that nerve growth factor may enhance neurite growth by means of stimulation of a hyperpolarizing, electrogenic pump 11. This hints at a possible connection between the influence of high K ÷ and the mechanism of nerve growth factor action. Similarities in the effects of high K ÷ and nerve growth factor on neuronal survival 12 and tyrosine hydroxylase induction 6 have also been reported. Unlike the present experiments, these involve effects of exposure of the entirety of the neurons to uniformly high K + . Extracellular K + is known to increase locally in the central nervous system during normal physiological function 9. The present work along with the recent report of an increase in [K+]e after nerve damage I0 raises the distinct possibility that natural variations in [K+]e may have important consequences for nerve fiber growth and maintenance, especially during de-
163 v e l o p m e n t 4 and r e g e n e r a t i o n . T h e possibility that in-
National
j u r e d n e r v e s m a y b e e x p o s e d in v i v o to local K ÷ con-
T h a n k s are due to M a r y E. C o r d e r m a n for assisting
Institutes
of
Health
Grant
NS15559.
c e n t r a t i o n s that are s u b o p t i m a l for r e g e n e r a t i o n is intriguing, especially with r e g a r d to the p r o b l e m of re-
in the l a b o r a t o r y , to B a r b a r a Seely for typing the
c o v e r y f r o m spinal c o r d injury.
for supplying a p r o t o c o l for t h e n e r v e g r o w t h factor
m a n u s c r i p t , and to E r i c S h o o t e r and J o h n B r a m h a l l purification p r o c e d u r e .
F i n a n c i a l s u p p o r t for this w o r k was p r o v i d e d by
1 Campenot, R. B., Independent control of the local environment of somas and neurites. In W. B. Jacoby and I. H. Pastan (Eds.), Methods in Enzymology, Vol. 28, Academic Press, New York, 1979, pp. 302-307. 2 Campenot, R. B., Regeneration of neurites in long-term cultures of sympathetic neurons deprived of nerve growth factor, Science, 214 (1981) 579-581. 3 Campenot, R. B., Development of sympathetic neurons in compartmentalized cultures. I. Local control of neurite growth by nerve growth factor, Develop. Biol., 93 (1982) 1-12. 4 Conners, B. W., Ransom, B. R., Kunis, D. M. and Gutnick, M. J., Activity-dependent K ÷ accumulation in the developing rat optic nerve, Science, 216 (1982) 1341-1343. 5 Hawrot, E. and Patterson, P. H., Long-term culture of dissociated sympathetic neurons. In W. B. Jacoby and I. H. Pastan (Eds.), Methods in Enzymology, Vol. 28, Academic Press, New York, 1979, pp. 574-584. 6 Hefti, F., Gnahn, H., Schwab, M. E. and Thoenen, H., Induction of tyrosine hydroxylase by nerve growth factor and by elevated K ÷ concentrations in cultures of dissociated sympathetic neurons, J. Neurosci., 2 (1982) 1554-1566.
7 Hinkle, L., McCaig, C. D. and Robinson, K. R., The direction of growth of differentiating neurones and myoblasts from frog embryos in an applied electric field, J. Physiol. (Lond.), 314 (1981) 121-135. 8 Jaffee, L. F. and Poo, M.-M., Neurites grow faster toward the cathode than the anode in a steady field, J. exp. Zool., 209 (1979) 115-128. 9 Kelly, J. P. and Van Essen, D. C., Cell structure and function in the visual cortex of the cat, J. Physiol. (Lond.), 238 (1974) 515-547. 10 Low, P. A., Damaged peripheral nerve axons regenerate in a milieu of increased [K+]e. Physiologic implications for neuropathic symptoms, Soc. Neurosci. Abstr., 8 (1982) 72.1. 11 Skaper, S. D. and Varon, S., Nerve growth factor influences potassium movements in chick embryo dorsal root ganglionic cells, Exp. Cell Res., 131 (1981) 353-361. 12 Wakade, A. R., Edgar, D. and Thoenen, H., Both nerve growth factor and high K ÷ concentrations support the survival of chick embryo sympathetic neurons, Exp. Cell Res., 144 (1983) 377-384.
159
BRE20015
Inhibition of nerve fiber regeneration in cultured sympathetic neurons by local, high potassium ROBERT B. CAMPENOT Section of Neurobiology and Behavior, Cornell University, Ithaca, NY14853 (U.S.A.) (Accepted October llth, 1983) Key words: sympathetic neurons - - potassium - - nerve regeneration - - nerve growth - - depolarization
Distal regeneration of neurites from cultured sympathetic neurons of newborn rats was stunted by exposure to local, high K ÷ (20 mM), only when more proximal neurite regions and the cell bodies were exposed to a normal K + concentration (5 mM). Neurites grew luxuriantly and extensively when exposure to high K ÷ was uniform over the entirety of the neurons. Thus, neurite growth can be strongly influenced by regional variations in K ÷ which are probably within the naturally occurring range, especially during development and regeneration. A recent report by Lowl0 indicates that regeneration of the sciatic nerve occurs in an environment of high extracellular potassium ([K+]e). Following transection of adult rat sciatic nerve, [K+]e was found to reach 15 m M in the e n d o n e u r i u m distal to the cut, about a 4-fold increase from an average of 4 m M in intact nerves. Conners et al. 4 r e p o r t that [K÷]e in newborn rat optic nerve can increase m o r e than 4fold, to 20 m M during electrical stimulation at 20 Hz, whereas levels in optic nerves of adults stimulated at 400 Hz increased to only 11 mM. These experiments suggest that [K+]e in the neuronal environment may vary substantially, especially during develo p m e n t and regeneration in the m a m m a l i a n peripheral and central nervous systems. In o r d e r to investigate the possibility that variations in [K+]e might influence nerve fiber growth, I observed the regeneration of neurites in principal neurons cultured from the superior cervical ganglia of newborn rats in normal (5 mM) and elevated (20 mM) K ÷. The neurons were cultured on multiple collagen channels in 3 - c o m p a r t m e n t dishes (Fig. 1) such that neurites originating from cell bodies in a center c o m p a r t m e n t p e n e t r a t e d silicone grease barriers, emerging into s e p a r a t e fluid environments within left and right c o m p a r t m e n t s 1. In this way neurons were exposed to a uniform increase in K ÷ in all 3 c o m p a r t m e n t s , or a local increase in K +, confined to the neurites in one side c o m p a r t m e n t only. F o r m u l a 0006-8993/84/$03.00 © 1984 Elsevier Science Publishers B.V.
tions and general culturing p r o c e d u r e s were as described by H a w r o t and Patterson5 except that 7S nerve growth factor was further purified by gel filtration of the D E A E - c e l l u l o s e fractions through Sephadex G-150. E l e v a t e d K ÷ was achieved by adding one part of 150 m M KC1 to 9 parts of culture medium. Control m e d i u m was m a d e by adding 150 m M NaC1 to 9 parts of culture m e d i u m from the same batch. High K + m e d i u m contained a final concentration of 20 m M K ÷, and control m e d i u m contained 5 m M K ÷. Except where noted, 7S nerve growth factor was provided in all c o m p a r t m e n t s at 1/~g/ml. Neurons in 9 sister cultures plated under 3 different regimes of K ÷ were examined 1.8 days and 3.8 days later. T h r e e dishes contained control m e d i u m in all c o m p a r t m e n t s , 3 contained high K ÷ m e d i u m in all compartments, and 3 contained high K ÷ m e d i u m in one side c o m p a r t m e n t , with control m e d i u m elsewhere. Many channels in side c o m p a r t m e n t s of all of the cultures contained neurites on day 1.8. On day 3.8, neurites on these channels had elongated to a similar extent and with c o m p a r a b l e density in cultures containing only control m e d i u m or only high K ÷ medium. Mean neurite extension reached about 3 mm, with little difference between left and right compartments. Thus, uniformly high K ÷ had no observed effect on neurite growth. In the 3 cultures with high K ÷ m e d i u m locally, in one side c o m p a r t m e n t only, c o m p a r a b l e neurite ex-
160
j
j
/
/j
j
___1
1
I
I
I
1
0.5
0
0
0.5
1___
1
mm
mm
Fig. 1. A schematic diagram of neurons from newborn rats in a 3-compartment culture. A Teflon divider partitioned the culture dish (dish not shown) into 3 compartments, a narrow (1.5 × 5 mm) center compartment (a) open to the bulk of the culture dish, and left (b) and right (c) side compartments. The collagen-coated floor of the dish was scored with 21 parallel scratches that spanned the center compartment and extended into the side compartments as shown. Twenty collagen channels, each 200-300 #m wide and bordered by the scratches, served to orient the outgrowth of neurites from sympathetic neurons in the center compartment to the left and right, as indicated by the enlargement of a single channel. Although the silicone grease formed an effective seal against fluid movement between the compartments, neurites readily penetrated beneath it, crossing into the separate fluid environments of the side compartments where their extension along the channels could be measured (note scale). Three-compartment dishes permit exposure of different regions of the neurons to different media constituents. Except for manipulations of the ionic environment and nerve growth factor described in the text, media formulations and general culturing procedures were as previously described 3,5, with ascorbate and rat serum provided in the center compartments only. Compartmentalized cultures permit the ready mechanical removal of neurites in the side compartments by means of a jet of distilled water delivered with a syringe2.3. This in vitro neuritotomy is without effect on the healthy appearance or number (unpublished) of neurons in the center compartment, and neurites promptly regenerate into the side compartments.
tension (mean = 3.14 m m , S.E. = 0.13, n = 20) was
density differences would have escaped notice, but
observed on day 3.8 in side c o m p a r t m e n t s containing
large ones were easily d o c u m e n t e d by this m et h o d .
control medium, but extension into high K + compart-
Local, high K ÷ caused a reduction in neurite density
ments was about 30% less (mean = 1.88 + 0.10, n =
in the 3 cultures described above and in two addition-
20). The ad v an cem en t of neurites along the channels
al sister cultures raised under identical conditions.
between days 1.8 and 3.8 was r e d u c e d 30% in high,
Nine days after plating, a reduced neurite density in
local K +, indicating a reduced rate of extension oc-
compartments given high K ÷ was observed on 54 of
curred within the compartments containing local,
the 64 channels e x a m i n e d in these cultures. Eight
high K +. Qualitative, visual comparisons were periodically
channels showed equal density in high K ÷ and con-
made of the density of neurites in the left and right compartments of the cultures. Each channel was ex-
high K+.
amined individually. The initial 1 m m in the left and right compartments was examined under phase contrast at 2 0 0 x , and it was noted if the density of neurites was clearly greater on one side than on the other. If no obvious density difference was observed, the channel was noted to have an approximately equal density in the left and right compartments. Small
trol m ed i u m , and two channels were m o r e dense in The effect of local, high K ÷ was subjected to some further tests by means of in vitro neuritotomy2, 3 of neurons ranging 14-33 days in culture. Six cultures were used, all grown under symmetrical conditions, 3 with normal K ÷ in all c o m p a r t m e n t s and 3 with high K+ in all compartments (2 of the high K ÷ cultures were participants in the previous experiment). Prior to neuritotomy, nerve growth factor was withdrawn
161 from the center c o m p a r t m e n t s of 2 of the cultures in
right compartments extending beyond the scale
order to limit neurite regeneration into the center compartment and thus confine it largely to the side
( > 5 mm) on all channels, and with noteworthy differences in density on only 9 of 70 channels.
compartments3. Before neuritotomy, all cultures
Immediately after removal of neurites from the left and right compartments, the center c o m p a r t m e n t
contained luxuriant neurite outgrowth in left and
Fig. 2. Photomicrographs showing neurons on a single collagen channel, 40 days in culture and 7 days after neuritotomy in both side compartments. The channel is 210/~m wide, and the scratches bordering it are visible at the top and bottom of each panel. The 5 panels show a continuous stretch of the channel (see Fig. 1). The top two panels show neurites regenerated in the left compartment given high K÷ medium (20 mM), the middle panel shows the center compartment containing the clusters of cell bodies and given control medium (5 mM K+), and the bottom two panels show neurites regenerated in the right compartment given control medium. Note the drastic reduction of extension and density in the neurites regenerated for 7 days in high local K÷ compared with 7 days regeneration in normal K+. This culture had previously been given high K÷ medium in all compartments continuously since plating and showed a luxuriant and comparable neurite growth in the left and right compartment when neuritotomy was performed.
162 and one side compartment of each culture were given control medium and the remaining side compartment was given high K ÷ medium. A clear inhibitory effect of local, high K ÷ on neurite regeneration was apparent within a few days. Between 1 and 3 days after neuritotomy, 59 of 70 channels showed a higher neurite density in control than in high K + medium. The density differences persisted, becoming more apparent with time, and at the termination of observations neurites on 68 of the 70 channels were more dense in normal than in high K +. While no attempt was made to quantify the magnitude of the density differences, typically the outgrowth in side compartments given normal K ÷ was luxuriant on every channel, and the relative reduction in compartments supplied with high K ÷ was so striking as to leave no doubt of the veracity of the effect (see Fig. 2). The distance that neurites extended into side compartments was initially similar in high and normal K ÷ on many channels, but within 4 days an inhibition of neurite advancement by high K ÷ was apparent, and was well developed in all 6 cultures by days 7-8 after neuritotomy. Mean distances of neurite extension in local, high K + were variable between cultures at this time, ranging 0.95-2.86 mm (S.E. ranged 0.14-0.40). But all cultures showed mean neurite extension exceeding 3.78 mm in opposite side compartments containing control medium. The reduction in neurite extension and density by high, local K ÷ was exhibited by all cultures tested with no apparent differences attributable to nerve growth factor withdrawal from the center compartment or to previous high K + exposure. Clearly, neurons react to regional differences in K + with a stunting of the growth of neurites which encounter levels elevated with respect to that of the soma and more proximal neurite regions. Two of the cultures with local, high K + were maintained for 11 and 16 days after neuritotomy without altering the culture conditions. Neurites on all of the channels in control medium, side compartments had extended beyond the scale, but neurites in high K + compartments never did. They remained shorter and, in fact, showed definite signs of degeneration. Several additional points may be made about the reaction of neurites to local, high K +. In experiments where the initial outgrowth of neurons in culture cross under the barrier and encounter local, high K +,
the reduction in outgrowth results from the exposure of intact and otherwise undamaged neurites. Exposure of damaged neurites to local, high K + as in the in vitro neuritotomy experiments was not necessary for the observed effect. Also, since the elongation of neurites within side compartments containing local, high K + was reduced, and since the effect was observed in cultures where neurite growth back into control medium in the center compartment was severely limited by withdrawal of nerve growth factor3~ a mechanism involving taxis of growth cones along a gradient of decreasing K ÷ is not indicated. Rather, a remote mechanism whereby a local elevation in K* along the neurite has a stunting effect on all neurite outgrowth distal to it seems to be involved. Interference with axonal transport may be indicated. The mechanism of the effect of local, high K ÷ is unknown, but one possibility is that local depolarization of the membrane potential by K ÷ is responsible. If so, it is interesting that a local depolarization distal from the cell body would produce within the neurite a flow of electric current towards the cell body. This is the opposite current direction that would result from an external electric field produced by a proximal anode and distal cathode, an arrangement shown to enhance neurite growth7. 8. A common mechanism may be indicated, and the possibility is raised that the effects of experimentally applied extracellular electric fields might be mediated by intracetlular electric fields in vivo set up by natural, regional variations in [K+]e . Also, reports indicate that nerve growth factor may enhance neurite growth by means of stimulation of a hyperpolarizing, electrogenic pump 11. This hints at a possible connection between the influence of high K ÷ and the mechanism of nerve growth factor action. Similarities in the effects of high K ÷ and nerve growth factor on neuronal survival 12 and tyrosine hydroxylase induction 6 have also been reported. Unlike the present experiments, these involve effects of exposure of the entirety of the neurons to uniformly high K + . Extracellular K + is known to increase locally in the central nervous system during normal physiological function 9. The present work along with the recent report of an increase in [K+]e after nerve damage I0 raises the distinct possibility that natural variations in [K+]e may have important consequences for nerve fiber growth and maintenance, especially during de-
163 v e l o p m e n t 4 and r e g e n e r a t i o n . T h e possibility that in-
National
j u r e d n e r v e s m a y b e e x p o s e d in v i v o to local K ÷ con-
T h a n k s are due to M a r y E. C o r d e r m a n for assisting
Institutes
of
Health
Grant
NS15559.
c e n t r a t i o n s that are s u b o p t i m a l for r e g e n e r a t i o n is intriguing, especially with r e g a r d to the p r o b l e m of re-
in the l a b o r a t o r y , to B a r b a r a Seely for typing the
c o v e r y f r o m spinal c o r d injury.
for supplying a p r o t o c o l for t h e n e r v e g r o w t h factor
m a n u s c r i p t , and to E r i c S h o o t e r and J o h n B r a m h a l l purification p r o c e d u r e .
F i n a n c i a l s u p p o r t for this w o r k was p r o v i d e d by
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