132
Brain Research, 572 (1~92) I32-13~ © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.0(i
BRES 17439
Release of neurite outgrowth promoting factors by Helisoma central ganglia depends on neural activity Robert C. Berdan and Richard L. Ridgway* Department of Physiology, University of Alberta, Edmonton, Alta. (Canada)
(Accepted 1 October 1991) Key words: Neural activity; Molluscan neuron; Nerve regeneration; Growth factor; Cell culture
Identified buccal neurons B5 and B19 from the mollusc, Hetisoma trivolvis, were plated into cell culture in order to assay for neurite outgrowth promoting factors released from central ring ganglia. The release and attachment of neurite promoting factors to the substratum of poly-lysine coated dishes could be inhibited by blocking spontaneous bioelectric activity in central ring ganglia used to condition the medium and dishes. Bioelectric activity within neurons in central ring ganglia was assayed by intracellular recording and found to be inhibited by exposure to the sodium channel blocker, tetrodotoxin (TTX; 2 x 10-5 M), or CoCl 2 (10 raM). Neither of these agents appeared to be toxic over a three day period since activity within neurons in central ring ganglia was restored following superfnsion with safine. To examine the effect of blocking neural activity on the ability of central ring ganglia to release neurite outgrowth promoting factors, we compared the percentage of neurons that extended processes under 5 different conditions: (1) dishes containing conditioned medium and substrate attached growth factors (Super SAM); (2) dishes with substrate attached growth factors only and defined medium (SAM); (3) dishes containing substrate attached growth factors prepared in the presence of TTX; or (4) COC12; and (5) dishes containing unconditioned defined medium. The percentage of neurons extending processes under the 5 conditions were: (1) 71% (n = 32); (2) 51% (n = 33); (3) 14% (n -- 37); (4) 15% (n = 47); (5) 0% (n = 40), respectively. The addition of TTX to dishes containing neurons in conditioned medium (super SAM) did not inhibit neurite outgrowth, but even brief applications of CoC12 to neurons under the same conditions inhibited neurite outgrowth in a non-reversible manner. Analysis of concentrated conditioned medium by sodium dodecyl-sulphate polyacrylamide gel electrophoresis and silver staining revealed that a number of proteins having apparent molecular weights of 44, 29, 26, 23, 17, 15, and 14.5 kDa were significantly reduced in quantity in conditioned medium produced in the presence of TI3( or CoC12. Taken together, these observations indicate that neurite outgrowth promoting factors were released from central ring ganglia in a manner dependent on neural activity. INTRODUCTION
rite outgrowth, sprouting and the specificity of neuronal connections in some species 14'19'23'46. Cohan 14 d e m o n -
Cultured cells from a variety of tissues have b e e n found to release growth factors into culture m e d i u m which enhance the survival of neurons in vitro, p r o m o t e neurite outgrowth or induce differentiation 2'3'12A5'17'29'
strated that direct electrical stimulation of molluscan neurons in culture can inhibit neurite outgrowth in a reversible manner. O t h e r neurons can be induced to sprout following the elimination of neural activity with tetrodotoxin s . O n e mechanism by which neural activity may m o d u l a t e neurite outgrowth is by regulating the release of neurite growth factors controlling neurite outgrowth. Many neurotransmitters and n e u r o p e p t i d e s in the molluscan nervous system are structurally similar to those in vertebrates, and this conservation m a y also extend to nerve growth factors 4°;43'49. Studies on isolated neurons from molluscs and several other invertebrates have b e e n valuable in examining the role of intrinsic and extrinsic influences on neurite outgrowth 1'7'1°'25'37.
36. A single growth factor m a y exert several functions or alternatively different molecules m a y be responsible for inducing neurite outgrowth o r neuronal survival. N e r v e growth factors have b e e n shown to be released in vivo during d e v e l o p m e n t and following injury to certain tissues 20'27. Despite the purification, identification and characterization of several nerve growth factors, their exact m o d e of action is still u n k n o w n (for reviews see refs. 4,16,42,45,47,48). Nevertheless, the potential clinical value these molecules hold for the t r e a t m e n t of various n e u r o d e g e n e r a t i v e diseases or neurons injured following t r a u m a currently underlies an extensive effort towards their identification and characterization. Neural activity has also b e e n shown to influence neu-
In o r d e r for isolated, identified neurons from t h e p o n d snail, Helisoma trivolvis, to extend neurites in culture, a defined m e d i u m must be ' c o n d i t i o n e d ' for three days with central ring ganglia 51. The central ring ganglia ap-
* Present address: Department of Biology, Seattle Pacific University, Seattle, WA 98119, U.S.A. Correspondence: R.C. Berdan, Department of Physiology, University of Alberta. Edmonton, AIta., T6G 2H7 Canada.
133 pears to be the only tissue capable of releasing growth factors into the m e d i u m r e q u i r e d for the outgrowth of Helisoma neurons. F u r t h e r m o r e , the release of nerve growth factors from central ring ganglia into culture med i u m is d e p e n d e n t u p o n new p r o t e i n synthesis and the molecules responsible for p r o m o t i n g neurite outgrowth are trypsin and heat sensitive, and bind to the surface of poly-lysine c o a t e d Petri dishes 5°'51. A l t h o u g h central ring ganglia constitute a source of the growth factors in Helisoma, it is not known w h e t h e r the cellular source is neurons o r n o n - n e u r o n a l cells, or w h e t h e r the release of growth factors is d e p e n d e n t on neural activity. T h e present study was u n d e r t a k e n in o r d e r to d e t e r m i n e w h e t h e r the release of neurite outgrowth p r o m o t i n g factors from central ring ganglia occurred in a m a n n e r dep e n d e n t on neural activity. T h e results indicate that the release o f neurite outgrowth p r o m o t i n g factors from central ring ganglia was b l o c k e d by agents that inhibit neural activity in Helisoma neurons. MATERIALS AND METHODS
Cell culture Identified neurons B5 and B19 were isolated from Helisoma buccal ganglia under sterile conditions and placed into culture in halfstrength Leibovitz~ medium (Gibco special order) containing inorganic salts at the concentration of: NaCI 51.3 raM, KC1 1.7 mM, CaCI2 4.1 raM, MgCI2 1.5 raM, and HEPES buffer 5 or 20 mM, pH 7.3, as previously describeds. Since both B5 and B19 neurons exhibit about the same growth potential in culture34, no attempt to analyze differences between them was undertaken in this study. Petri dishes (Falcon 3001) were coated with 0.1% poly-L-lysine (Mr 4-15 kDa, Sigma) in 0.15 M Tris buffer, pH 8.1. Isolated neurons were plated into dishes containing defined culture medium (DM); dishes containing medium conditioned with central ring ganglia for 3 days (Super SAM); dishes which were conditioned with central ring ganglia for 3 days, rinsed twice with defined medium, and to which defined medium was added. Since these dishes contained neurite promoting factors attached only to their surface, they are referred to as SAM (surface attached material) dishes. SAM dishes were also prepared with central ring ganglia in the presence of tetrodotoxin (T/'X; 2 × 10-5 M), and 10 mM CoCI v A drop in pH of the medium after the addition of CoCI2 was circumvented by increasing the HEPES buffer to 20 mM instead of 5 mM; this increase in HEPES had no apparent effect on neurite outgrowth. Neurons were cultured at room temperature (22-24 *C) in a humidified chamber. Neurite outgrowth from neurons was assessed by phase contrast microscopy 24-72 h after plating. Neurite outgrowth was considered to have occurred if two or more neurites extended at least two cell diameters from the soma or preexisting axon segment. The viability of neurons in culture was assessed by intracellular recording of their membrane potential and ability to elicit action potentials. Standard electrophysiological recording equipment was used (for details see ref. 5). Statistical analysis between sample groups was determined by Fisher's 2 x 2 exact test, and results were considered significant if P < 0.05. Biochemical analysis In preparation for sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE), defined medium was conditioned for 3 days with central ring ganglia (2/ml) and concentrated to approximately 30-50 x by passage through a Centricon-10 membrane ill-
tration unit (10 kDa nominal molecular weight cut-off; Amicon). The retentate was rinsed twice with sterile Super Q (Millipore) water and then diluted to give a 20 x sample having a 3:1 ratio with SDS sample buffer (40% glycerol, 8% Bromophenol blue, 8% SDS, 200 mM dithiothreitol, 0.25 M Tris buffer, pH 6.8). Samples of medium conditioned in the presence of T I X (2 x 10-s M) or CoC12 (10 mM) were prepared in the same manner. Samples were boiled for 3 rain and while still warm, 3 #1 of 6.75 M iodoacetamide (Sigma) was added to each 100 gl of the samples. Identical amounts (usually 15-30 #1) of each sample were loaded. Combined high and low molecular weight standards (Sigma) were diluted to 1% of their recommended concentration and loaded. Electrophoresis was carried out as described by Laemmli2s with 5% stacking and 12.5% resolving gels. Following eleetrophoresis, gels were stained for 30 rain in 0.25% Coomassie blue in 40% methanol/7% acetic acid and destained overnight in the same solvent. The gels were then fixed in 10% giutaraldehyde for 30 rain, rinsed thoroughly with Super Q water and silver stained3s. RESULTS To d e t e r m i n e if neural activity affects the release of growth factors from central ring ganglia, it was first essential to d e t e r m i n e w h e t h e r activity could be b l o c k e d in neurons from the central ring ganglia. We superfused T T X (2 x 10 -5 M) o r COC12 (10 m M ) onto isolated central ring ganglia and r e c o r d e d intracellularly from a variety of unidentified neurons chosen r a n d o m l y . In all instances, action potentials were absent (n = 10-15 neurons/ganglia; n = 3 ganglionic rings). To d e t e r m i n e if incubation in the presence of T I X or CoC12 for 3 days would effectively block action potentials over this time p e r i o d and not be toxic, intracellular recordings were p e r f o r m e d on whole ganglia cultured in defined m e d i u m containing T T X or CoC12 for three days. A f t e r 3 days in organ culture, intracellular recording s from r a n d o m l y chosen neurons r e v e a l e d stable m e m b r a n e potentials (-30 to - 6 5 mV) and an absence of spontaneous activity which was r e s t o r e d following superfusion with normal saline. The effects of T I X on neural activity were reversed rapidly (often within seconds), whereas the effects of CoC12 required several minutes superfusion with saline before spontaneous activity returned. These results indicate that activity could be suppressed in neurons m a i n t a i n e d in organ culture for 3 days and that the reagents were not acutely toxic to neurons. Before we could test w h e t h e r activity was essential for the release of neurite outgrowth p r o m o t i n g factors, we n e e d e d to establish what percentage of neurons grow in conditioned m e d i u m and, as a negative control, what percentage of neurons grow in defined m e d i u m . Isolated neurons B5 and B19 p l a t e d into defined m e d i u m adh e r e d to poly-lysine coated dishes within a few minutes, but did not extend processes even after several days (Fig. l a ) . These neurons had resting m e m b r a n e potentials b e t w e e n -30 to -50 m V and spontaneously fired action potentials. A significant a m o u n t of m e m b r a n e move-
134 ment called 'veiling' could be seen around the soma and
gentle shaking than were neurons plated onto poly-lysine coated dishes containing conditioned medium (super
processes of the neurons, but no new processes formed. Interestingly, axons associated with n e u r o n s plated onto poly-lysine dishes in defined m e d i u m adhered tightly to the substratum, and the axons appeared to retract into
factors to the bottom of the Petri dish appears to reduce the adhesiveness of the substratum (polyqysine coated
the soma less frequently than those plated on poly-lysine
plastic). Evidently, adhesion to the substratum is clearly
coated surfaces adsorbed with conditioning factors (compare Fig. l a with Fig. l c and d). F u r t h e r m o r e , neurons
not sufficient to promote neurite outgrowth. Almost three-quarters of the neurons plated onto Petri
plated onto polyqysine coated dishes adhered more rap-
dishes containing both conditioned m e d i u m and surface
idly and, in general, were more difficult to dislodge by
associated growth factors (i.e. Super SAM dishes) ad-
SAM dishes). Therefore, the attachment of conditioning
Fig. 1. Phase-contrast photomicrographs of isolated identified neurons from Helisoma. A: neurons plated in defined medium onto poly-lysine coated dishes. Note long axons and flattened membranes around the soma and processes. Adhesion of the axons to the poly-lysine appears to have prevented axon retraction. B: identified neurons B5 and B19 exhibited extensive outgrowth in defined medium if the substratum of the Petri dish had been exposed to medium conditioned by central ring ganglia (SAM dishes). C: neurons plated into SAM dishes prepared in the presence of CoC12 did not support neurite outgrowth, note blebbing around some of the neurons. D: neurite outgrowth was also absent in SAM dishes exposed to medium conditioned in the presence of T r x . E: neurite outgrowth from two neurons B5 in conditioned medium with TFX added. Bar = 100 pm.
135
Neurite Outgrowth from Isolated Neurons In Vitro
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Fig. 2. Histogram summarizing the culture experiments. Super SAM, Petri dishes containing conditioned medium; SAM, Petri dishes originally containing medium conditioned with central ring ganglia for three days and then replaced with defined medium (DM). TTX and CoC12 refer to Petri dishes with substrate-bound material deposited from central ring ganglia in the presence of either of these agents. Neurite outgrowth in qq"X dishes was significantly reduced (P < 0.02) in comparison to outgrowth in SAM dishes. Similarly, neurite outgrowth was significantly reduced (P < 0.001) in dishes exposed to medium conditioned in the presence of CoC12. DM refers to Petri dishes plated with neurons containing only defined medium.
hered within a few hours and sent out numerous neurites within 24-36 h (not shown). About half of the neurons plated into dishes containing defined medium in
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B19
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Fig. 3. IntraceUular recordings from neurons B5 and B19 which did not extend neurites after plating in dishes conditioned in the presence of COC12 show that, even though neurons failed to extend processes, they exhibited good membrane potentials and fired action potentials, in this case, in response to brief hyperpolarizing current pulses (arrows) or depolarizing current pulses (asterisk). B19 would also fire in a bursting pattern in response to several hyperpolarizing current pulses (arrows) reminiscent of its endogenous firing pattern observed in isolated ganglia.
-1
o m
o
Fig. 4. Photograph of a polyacrylmide gel loaded with equal volumes of concentrated conditioned medium (CM), CM produced in the presence of "I'TX, CM produced in the presence of CoC12, and defined medium (DM). Arrows point to protein staining bands that appear reduced in staining intensity in CM produced in the presence of qTX or CoCI2. Proteins having apparent molecular weights of 23 and 15 kDa exhibited the greatest reduction.
which only the substratum possessed neurite promoting factors (SAM dishes) exhibited neurite outgrowth (Fig. lb). There was, however, a significant reduction in the percentage of neurons that extended neurites (in defined medium) when plated into dishes in which the substrate was conditioned with central ring ganglia (SAM) in the presence of C o C I 2 (Fig. lc) or TFX (Fig. ld). The results are summarized in Fig. 2. Neurons plated onto SAM dishes prepared in the presence of "I~X o r CoC12 exhibited spontaneous action potentials and membrane potentials similar to those observed in super SAM dishes (Fig. 3). Membrane blebbing from the soma was occasionally seen around some neurons plated into SAM dishes prepared in the presence of CoC12 (Fig. lc). In the experiments described above, T-FX and C o C I 2 were presumably removed from SAM culture dishes after several rinses with defined medium before neurons were plated onto their surface. Intracellular recordings from neurons plated into SAM dishes prepared in the presence of TTX o r C o C l 2 did not reveal any evidence for the suppression of activity; nevertheless, it is possi-
136 ble that small amounts of TTX or CoC12 may have remained attached to the substratum. To test whether neurite outgrowth could occur in the presence of either of the agents, we plated neurons into conditioned medium (super SAM dishes) to which we added TTX (2 x 10 -5 M) or CoCI 2 (10 mM). Neurite outgrowth failed to occur in the presence 10 mM CoCI 2 and this concentration effectively blocked action potentials in the isolated neurons. The effect of even a brief exposure to CoC12 (several minutes) was not readily reversible, as neurons did not extend processes when the CoCI 2 containing medium was replaced with fresh conditioned medium even after several changes of conditioned medium. The addition of T]?X to conditioned medium inhibited spontaneous action potentials in neurons B5 and B19 in vitro; however, TFX had no apparent effect on neurite outgrowth (7/9 neurons sprouted; e.g. Fig. le). Adjacent neurons extending neurites in the presence of TFX also formed electrical synaptic connections that were normally observed between these neurons when cultured in conditioned medium. In an attempt to identify putative neurite promoting proteins we examined the relative abundance of proteins in conditioned medium by SDS-PAGE and compared them with the protein profiles in the presence of T-FX or CoCI 2. Conditioned medium was concentrated about 30-50-fold and equal volumes were loaded onto polyacrylamide gels and separated by electrophoresis and stained with silver. The results are shown in Fig. 4. The overall protein content in conditioned medium produced in the presence of TTX or CoC12 appeared to be reduced. Proteins that stained less intensely in both CoCI 2 and TTX conditioned medium had apparent molecular weights of 44, 29, 26, 24, 23, 17, 15 and 14.5 kDa. Proteins having apparent molecular weights of 23 and 15 kDa exhibited the greatest reduction in the presence of T I ' X or CoC12. DISCUSSION The experiments described in the present study were concerned with identifying whether neural activity was required for central ring ganglia to release neurite outgrowth promoting factors during organ culture. Our findings indicate that the release of neurite outgrowth promoting factors into culture medium was dependent on spontaneous electrical activity normally associated with neurons in central ring ganglia. It is unlikely that TTX would inhibit the secretion of growth factors from non-neuronal cells (glia, hemocytes and sheath cells) since secretion is primarily a calcium-regulated process 21. The possibility that neural activity may indirectly modulate the release of growth factors from non-neuronal
cells, however, cannot be excluded in these studies. A previous study that examined Aplysia neurons in culture found that hemolymph contained neurite outgrowth promoting factors 4l, but a more recent study found that the periesophageal ganglion also released neurite outgrowth promoting factors that may be different TM. In contrast, hemolymph from Helisoma contains substances that inhibit neurite outgrowth 24. Phagocytic cells, also called hemocytes or ameobocytes, in the hemolymph of a related species, Lymnaea stagnalis, have also been implicated as a putative source of neurite growth factors a2. Highly motile hemocytes were present in our cultures arising primarily from the surface of the central ring ganglia (see Fig. 4d, ref. 7). If hemocytes contribute neurite outgrowth promoting factors in the present study, their contribution would appear to be small since outgrowth was significantly reduced by TTX, which is not known to directly inhibit secretion from non-neuronal cells. However, a small percentage of neurons did exhibit outgrowth in dishes conditioned in the presence of TTX or CoC12. It is possible that the release of a small amount of growth factor may not be linked to activity (e.g. from non-neuronal cells), or alternatively, that not all neural activity was blocked by TTX or CoCl2, since some neurons may have predominantly sodium- or calcium-dependent action potentials. Fibronectin-like molecules have been found in association with hemocytes from Helisoma 33, but fihronectin alone is insufficient to promote neurite outgrowth 33"51. Interestingly, however, if the Petri dish is coated with fibronectin and calcium channel blockers (lanthanum, cobalt or cadmium) are added to defined medium, neurite outgrowth from Helisoma neurons can be induced in the absence of exogenous growth factors 34. The growth promoting properties of these calcium channel blockers operate only in a narrow concentration range well below that required to block action potentials. Our studies used a high concentration of cobalt sufficient to block calcium-dependent action potentials or calcium-dependent secretion. Under these conditions, no neurite outgrowth was observed even after the cobalt was rinsed from the isolated neurons with defined medium. It is possible that in our experiments some of the cobalt remained associated with the neurons and inhibited outgrowth by binding to and blocking the receptors for the growth factors. Taken together, these studies suggest that a fibronectinlike molecule secreted by hemocytes in culture has a permissive role in neurite outgrowth of Helisoma neurons and also suggests the possibility that neurite promoting factors may act by modulating the influx of extracellular calcium 34. Grimm-Jorgensen 22 has suggested that the ability of somatostatin and calcitonin (released from hemocytes)
137 to promote neurite outgrowth of some molluscan neurons also correlates with their ability to modulate intracellular calcium. However, recent studies on mammalian neurons in culture suggest that calcium signals are not necessary for NGF-dependent neurite outgrowth 13'44. We have been unable to detect changes in the levels of intracellular calcium (using Fura-2 imaging) or changes in whole cell patch clamp currents of Helisoma neuron B5 superfused with conditioned medium (Berdan and Wang, unpublished results). These preliminary results suggest that the neurite outgrowth promoting factors from Helisoma central ring ganglia do not appear to act on receptor-mediated ion channels or by inducing rapid changes in the levels of intracellular calcium. TTX added to conditioned medium did not suppress neurite outgrowth of Helisoma neurons B5 and B19, and these results are similar to those we reported previously in which axotomized neurons B5 extended processes equally well within buccal ganglia maintained in organ culture in the presence or absence of q'TX 5. In contrast, other investigators have reported that neurite outgrowth from rat cerebral cortex cells in culture is enhanced in the presence of q'TX 46. Our findings suggest that voltage-activated sodium channels do not play a crucial role in neurite outgrowth of Helisoma neurons B5 and B19 in culture, but species and tissue differences may exist. Neural activity is thought to play an important role during the development, and wiring of the nervous system and it has been suggested that trophic support is somehow linked to neural activity39. In one investigation, blocking neural activity of retinal ganglion cells in culture with TTX resulted in their death and prevented them from conditioning medium that would otherwise rescue the cells 31. On the basis of these results, Lipton 31 suggested that survival of ganglion cells was dependent on a chemical factor whose presence was dependent on neural activity. Our results are similar in that the release
of neurite promoting factors from central ganglia was dependent on neural activity, but differ in that neuronal survival over 5-6 days was not. Recently, neural activity has been found to regulate N G F and B D N F (brain derived neurotrophic factor) gene expression in hippocampal neurons 32'52. These results raise the interesting possibility that trophic factor synthesis and release modified by neural activity could modulate synaptogenesis and thereby have important implications for learning and memory. Furthermore, an increase in impulse activity associated with seizures could elevate neurotrophic factors and perpetuate epileptic activity through synaptic strengthening 32. The putative neurite promoting factors released by Helisoma central ring ganglia have not yet been identified. Our biochemical results indicated that the staining of a number of proteins was reduced in conditioned medium produced in the presence of q T X or CoCl 2. Further biochemical and immunological studies will be required to identify whether any of these proteins represent bonafide neurite promoting factors. Thus far, an insulinlike peptide 26, glutamate 9 and nerve growth factor have been shown to stimulate neurite outgrowth of some molluscan neurons 4°. Recently, a laminin-like protein has been purified from Helisoma conditioned medium and this protein appears to exhibit a small amount of neurite promoting activity35. It is likely that medium conditioned by Helisoma central ganglia will contain several neurite promoting factors as appears to be the case in vertebrates brains.
Acknowledgments: This research was supported by the Alberta Heritage Foundation for Medical Research. We thank Drs. N.I. Syed, Sanders, and Mr. P. Nguyen-Ho for their comments on the manuscript. We thank Dr. A.G.M Bulloch for supplying some of the reagents and equipment used in this study.
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fibronectin-like molecule promotes neurite outgrowth, J. Neurobiol., 19 (1988) 239-256. Mattson, M.P., Guthrie, P.B. and Kater, S.B., Components of neurite outgrowth that determine neuronal cytoarchitecture: influence of calcium and the growth substrate, J. Neurosci. Res., 20 (1988) 331-345. Miller, J.D. and Hadley, R.D., Laminin-like immunoreactivity in snail Helisoma: involvement of a 300 kDa extracellular matrix protein in promoting neurite outgrowth, J. Neurobiol., 22 (1991) 431-442. Muller, H.W., Bockh, S. and Seifert, W., Neurotrophic factor for central neurons, Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 1241-1252. Nicholts, J.G., The Search for Connections: Studies of Regeneration in the Nervous System of the Leech, Sinauer Associates, Sunderland, MA, 1987. Oakley, B.R., Kirsh, D.R. and Morris, N.R., A simplified ultrasensitive silver stain for detecting proteins in polyaerylamide gels, Anal Biochem., 105 (1980) 361-363. Purves, D. and Lichtman, J., Principles of Neural Development, Sinauer Associates, Sunderland, MA, 1985, pp. 290. Ridgway, R.L., Syed, N.I., Lukowiak, K. and Bulloch, A.G.M., Nerve growth factor (NGF) induces sprouting of specific neurons of the snail, Lymnaea stagnalis, J. Neurobiol., (in press. Schacher, S.M. and Proshansky, E., Neurite regeneration by Aplysia neurons in dissociated cell culture: modulation by Aplysia hemolymph and the presence of the initial axon segment, J. Neurosci., 3 (1983) 2403-2413. Snider, W.D. and Johnson, E.M., Neurotrophic molecules, Ann. Neurol., 26 (1989) 489-506. S-Roza, K., The pharmacology of molluscan neurons, Prog. Neurobiol., 23 (1984) 79-150. Tolkvosky, A.M., Walker, A.E., Murrel, R.D. and Suidan, H.S., Ca z+ transients are not required as signals for long-term neurite outgrowth from cultured sympathetic neurons, J. Cell Biol., 110 (1990) 1295-1306. Thoenen, H. The changing scene of neurotrophic factors, Trends Neurosci., 14 (1991) 165-170. Van Huizen, F., Romijn, H.J., Tetrodotoxin enhances initial neurite outgrowth from fetal rat cerebral cortex cells in vitro, Brain Research, 408 (1987) 271-274. Varon, S., Manthorpe, M. and Williams, L.R., Neuronotrophic and neurite-promoting factors and their clinical potentials, Dev. Neurosci., 6 (1983/84) 73-100. Walicke, P.A., Novel neurotrophic factors, receptors, and oncogenes, Annu. Rev. Neurosci., 12 (1989) 103-126. Wendelaar Bonga, S., Lafeber, EP.J.G., Flik, G., Kaneko, T. and Pang, P.K.T., Immunocytochemical demonstration of a novel system of neuroendocrine peptidergic neurons in the pond snail, Lymnaea stagnalis, with antisera to the teloestean hormone hypocalcin and mammalian parathyroid hormone, Gen. Comp. Endocr., 75 (1989) 29-38. Wong, R.G., Martel, E.C. and Kater, S.B., Conditioning factor produced by several molluscan species promote neurite outgrowth in cell culture, J. Exp. Biol., 105 (1983) 389-393. Wong, R.G., Barker, D.L., Kater, S.B. and Bodnar D.A., Nerve-growth promoting factor produced in culture media conditioned by specific CNS tissues of the snail Helisoma, Brain Research, 292 (1984) 81-91. Zafra, E, Hengerer, B., Leibrock, J. , Thoenen, H. and Lindholm, D., Activity dependent regulation of BDNF and NGF mRNAs in the rat hippocampus is mediated by non-NMDA glutamate receptors, EMBO J., 9 (1991) 3545-3550.
Brain Research, 572 (1~92) I32-13~ © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.0(i
BRES 17439
Release of neurite outgrowth promoting factors by Helisoma central ganglia depends on neural activity Robert C. Berdan and Richard L. Ridgway* Department of Physiology, University of Alberta, Edmonton, Alta. (Canada)
(Accepted 1 October 1991) Key words: Neural activity; Molluscan neuron; Nerve regeneration; Growth factor; Cell culture
Identified buccal neurons B5 and B19 from the mollusc, Hetisoma trivolvis, were plated into cell culture in order to assay for neurite outgrowth promoting factors released from central ring ganglia. The release and attachment of neurite promoting factors to the substratum of poly-lysine coated dishes could be inhibited by blocking spontaneous bioelectric activity in central ring ganglia used to condition the medium and dishes. Bioelectric activity within neurons in central ring ganglia was assayed by intracellular recording and found to be inhibited by exposure to the sodium channel blocker, tetrodotoxin (TTX; 2 x 10-5 M), or CoCl 2 (10 raM). Neither of these agents appeared to be toxic over a three day period since activity within neurons in central ring ganglia was restored following superfnsion with safine. To examine the effect of blocking neural activity on the ability of central ring ganglia to release neurite outgrowth promoting factors, we compared the percentage of neurons that extended processes under 5 different conditions: (1) dishes containing conditioned medium and substrate attached growth factors (Super SAM); (2) dishes with substrate attached growth factors only and defined medium (SAM); (3) dishes containing substrate attached growth factors prepared in the presence of TTX; or (4) COC12; and (5) dishes containing unconditioned defined medium. The percentage of neurons extending processes under the 5 conditions were: (1) 71% (n = 32); (2) 51% (n = 33); (3) 14% (n -- 37); (4) 15% (n = 47); (5) 0% (n = 40), respectively. The addition of TTX to dishes containing neurons in conditioned medium (super SAM) did not inhibit neurite outgrowth, but even brief applications of CoC12 to neurons under the same conditions inhibited neurite outgrowth in a non-reversible manner. Analysis of concentrated conditioned medium by sodium dodecyl-sulphate polyacrylamide gel electrophoresis and silver staining revealed that a number of proteins having apparent molecular weights of 44, 29, 26, 23, 17, 15, and 14.5 kDa were significantly reduced in quantity in conditioned medium produced in the presence of TI3( or CoC12. Taken together, these observations indicate that neurite outgrowth promoting factors were released from central ring ganglia in a manner dependent on neural activity. INTRODUCTION
rite outgrowth, sprouting and the specificity of neuronal connections in some species 14'19'23'46. Cohan 14 d e m o n -
Cultured cells from a variety of tissues have b e e n found to release growth factors into culture m e d i u m which enhance the survival of neurons in vitro, p r o m o t e neurite outgrowth or induce differentiation 2'3'12A5'17'29'
strated that direct electrical stimulation of molluscan neurons in culture can inhibit neurite outgrowth in a reversible manner. O t h e r neurons can be induced to sprout following the elimination of neural activity with tetrodotoxin s . O n e mechanism by which neural activity may m o d u l a t e neurite outgrowth is by regulating the release of neurite growth factors controlling neurite outgrowth. Many neurotransmitters and n e u r o p e p t i d e s in the molluscan nervous system are structurally similar to those in vertebrates, and this conservation m a y also extend to nerve growth factors 4°;43'49. Studies on isolated neurons from molluscs and several other invertebrates have b e e n valuable in examining the role of intrinsic and extrinsic influences on neurite outgrowth 1'7'1°'25'37.
36. A single growth factor m a y exert several functions or alternatively different molecules m a y be responsible for inducing neurite outgrowth o r neuronal survival. N e r v e growth factors have b e e n shown to be released in vivo during d e v e l o p m e n t and following injury to certain tissues 20'27. Despite the purification, identification and characterization of several nerve growth factors, their exact m o d e of action is still u n k n o w n (for reviews see refs. 4,16,42,45,47,48). Nevertheless, the potential clinical value these molecules hold for the t r e a t m e n t of various n e u r o d e g e n e r a t i v e diseases or neurons injured following t r a u m a currently underlies an extensive effort towards their identification and characterization. Neural activity has also b e e n shown to influence neu-
In o r d e r for isolated, identified neurons from t h e p o n d snail, Helisoma trivolvis, to extend neurites in culture, a defined m e d i u m must be ' c o n d i t i o n e d ' for three days with central ring ganglia 51. The central ring ganglia ap-
* Present address: Department of Biology, Seattle Pacific University, Seattle, WA 98119, U.S.A. Correspondence: R.C. Berdan, Department of Physiology, University of Alberta. Edmonton, AIta., T6G 2H7 Canada.
133 pears to be the only tissue capable of releasing growth factors into the m e d i u m r e q u i r e d for the outgrowth of Helisoma neurons. F u r t h e r m o r e , the release of nerve growth factors from central ring ganglia into culture med i u m is d e p e n d e n t u p o n new p r o t e i n synthesis and the molecules responsible for p r o m o t i n g neurite outgrowth are trypsin and heat sensitive, and bind to the surface of poly-lysine c o a t e d Petri dishes 5°'51. A l t h o u g h central ring ganglia constitute a source of the growth factors in Helisoma, it is not known w h e t h e r the cellular source is neurons o r n o n - n e u r o n a l cells, or w h e t h e r the release of growth factors is d e p e n d e n t on neural activity. T h e present study was u n d e r t a k e n in o r d e r to d e t e r m i n e w h e t h e r the release of neurite outgrowth p r o m o t i n g factors from central ring ganglia occurred in a m a n n e r dep e n d e n t on neural activity. T h e results indicate that the release o f neurite outgrowth p r o m o t i n g factors from central ring ganglia was b l o c k e d by agents that inhibit neural activity in Helisoma neurons. MATERIALS AND METHODS
Cell culture Identified neurons B5 and B19 were isolated from Helisoma buccal ganglia under sterile conditions and placed into culture in halfstrength Leibovitz~ medium (Gibco special order) containing inorganic salts at the concentration of: NaCI 51.3 raM, KC1 1.7 mM, CaCI2 4.1 raM, MgCI2 1.5 raM, and HEPES buffer 5 or 20 mM, pH 7.3, as previously describeds. Since both B5 and B19 neurons exhibit about the same growth potential in culture34, no attempt to analyze differences between them was undertaken in this study. Petri dishes (Falcon 3001) were coated with 0.1% poly-L-lysine (Mr 4-15 kDa, Sigma) in 0.15 M Tris buffer, pH 8.1. Isolated neurons were plated into dishes containing defined culture medium (DM); dishes containing medium conditioned with central ring ganglia for 3 days (Super SAM); dishes which were conditioned with central ring ganglia for 3 days, rinsed twice with defined medium, and to which defined medium was added. Since these dishes contained neurite promoting factors attached only to their surface, they are referred to as SAM (surface attached material) dishes. SAM dishes were also prepared with central ring ganglia in the presence of tetrodotoxin (T/'X; 2 × 10-5 M), and 10 mM CoCI v A drop in pH of the medium after the addition of CoCI2 was circumvented by increasing the HEPES buffer to 20 mM instead of 5 mM; this increase in HEPES had no apparent effect on neurite outgrowth. Neurons were cultured at room temperature (22-24 *C) in a humidified chamber. Neurite outgrowth from neurons was assessed by phase contrast microscopy 24-72 h after plating. Neurite outgrowth was considered to have occurred if two or more neurites extended at least two cell diameters from the soma or preexisting axon segment. The viability of neurons in culture was assessed by intracellular recording of their membrane potential and ability to elicit action potentials. Standard electrophysiological recording equipment was used (for details see ref. 5). Statistical analysis between sample groups was determined by Fisher's 2 x 2 exact test, and results were considered significant if P < 0.05. Biochemical analysis In preparation for sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE), defined medium was conditioned for 3 days with central ring ganglia (2/ml) and concentrated to approximately 30-50 x by passage through a Centricon-10 membrane ill-
tration unit (10 kDa nominal molecular weight cut-off; Amicon). The retentate was rinsed twice with sterile Super Q (Millipore) water and then diluted to give a 20 x sample having a 3:1 ratio with SDS sample buffer (40% glycerol, 8% Bromophenol blue, 8% SDS, 200 mM dithiothreitol, 0.25 M Tris buffer, pH 6.8). Samples of medium conditioned in the presence of T I X (2 x 10-s M) or CoC12 (10 mM) were prepared in the same manner. Samples were boiled for 3 rain and while still warm, 3 #1 of 6.75 M iodoacetamide (Sigma) was added to each 100 gl of the samples. Identical amounts (usually 15-30 #1) of each sample were loaded. Combined high and low molecular weight standards (Sigma) were diluted to 1% of their recommended concentration and loaded. Electrophoresis was carried out as described by Laemmli2s with 5% stacking and 12.5% resolving gels. Following eleetrophoresis, gels were stained for 30 rain in 0.25% Coomassie blue in 40% methanol/7% acetic acid and destained overnight in the same solvent. The gels were then fixed in 10% giutaraldehyde for 30 rain, rinsed thoroughly with Super Q water and silver stained3s. RESULTS To d e t e r m i n e if neural activity affects the release of growth factors from central ring ganglia, it was first essential to d e t e r m i n e w h e t h e r activity could be b l o c k e d in neurons from the central ring ganglia. We superfused T T X (2 x 10 -5 M) o r COC12 (10 m M ) onto isolated central ring ganglia and r e c o r d e d intracellularly from a variety of unidentified neurons chosen r a n d o m l y . In all instances, action potentials were absent (n = 10-15 neurons/ganglia; n = 3 ganglionic rings). To d e t e r m i n e if incubation in the presence of T I X or CoC12 for 3 days would effectively block action potentials over this time p e r i o d and not be toxic, intracellular recordings were p e r f o r m e d on whole ganglia cultured in defined m e d i u m containing T T X or CoC12 for three days. A f t e r 3 days in organ culture, intracellular recording s from r a n d o m l y chosen neurons r e v e a l e d stable m e m b r a n e potentials (-30 to - 6 5 mV) and an absence of spontaneous activity which was r e s t o r e d following superfusion with normal saline. The effects of T I X on neural activity were reversed rapidly (often within seconds), whereas the effects of CoC12 required several minutes superfusion with saline before spontaneous activity returned. These results indicate that activity could be suppressed in neurons m a i n t a i n e d in organ culture for 3 days and that the reagents were not acutely toxic to neurons. Before we could test w h e t h e r activity was essential for the release of neurite outgrowth p r o m o t i n g factors, we n e e d e d to establish what percentage of neurons grow in conditioned m e d i u m and, as a negative control, what percentage of neurons grow in defined m e d i u m . Isolated neurons B5 and B19 p l a t e d into defined m e d i u m adh e r e d to poly-lysine coated dishes within a few minutes, but did not extend processes even after several days (Fig. l a ) . These neurons had resting m e m b r a n e potentials b e t w e e n -30 to -50 m V and spontaneously fired action potentials. A significant a m o u n t of m e m b r a n e move-
134 ment called 'veiling' could be seen around the soma and
gentle shaking than were neurons plated onto poly-lysine coated dishes containing conditioned medium (super
processes of the neurons, but no new processes formed. Interestingly, axons associated with n e u r o n s plated onto poly-lysine dishes in defined m e d i u m adhered tightly to the substratum, and the axons appeared to retract into
factors to the bottom of the Petri dish appears to reduce the adhesiveness of the substratum (polyqysine coated
the soma less frequently than those plated on poly-lysine
plastic). Evidently, adhesion to the substratum is clearly
coated surfaces adsorbed with conditioning factors (compare Fig. l a with Fig. l c and d). F u r t h e r m o r e , neurons
not sufficient to promote neurite outgrowth. Almost three-quarters of the neurons plated onto Petri
plated onto polyqysine coated dishes adhered more rap-
dishes containing both conditioned m e d i u m and surface
idly and, in general, were more difficult to dislodge by
associated growth factors (i.e. Super SAM dishes) ad-
SAM dishes). Therefore, the attachment of conditioning
Fig. 1. Phase-contrast photomicrographs of isolated identified neurons from Helisoma. A: neurons plated in defined medium onto poly-lysine coated dishes. Note long axons and flattened membranes around the soma and processes. Adhesion of the axons to the poly-lysine appears to have prevented axon retraction. B: identified neurons B5 and B19 exhibited extensive outgrowth in defined medium if the substratum of the Petri dish had been exposed to medium conditioned by central ring ganglia (SAM dishes). C: neurons plated into SAM dishes prepared in the presence of CoC12 did not support neurite outgrowth, note blebbing around some of the neurons. D: neurite outgrowth was also absent in SAM dishes exposed to medium conditioned in the presence of T r x . E: neurite outgrowth from two neurons B5 in conditioned medium with TFX added. Bar = 100 pm.
135
Neurite Outgrowth from Isolated Neurons In Vitro
÷ t~
80 N=32
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o t-
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o
64 ' NR,~
~4s, uJ 32 16
• NI~37
.
N~47
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SAM TTX Conditioned Culture
CO++ Ivled~um
Fig. 2. Histogram summarizing the culture experiments. Super SAM, Petri dishes containing conditioned medium; SAM, Petri dishes originally containing medium conditioned with central ring ganglia for three days and then replaced with defined medium (DM). TTX and CoC12 refer to Petri dishes with substrate-bound material deposited from central ring ganglia in the presence of either of these agents. Neurite outgrowth in qq"X dishes was significantly reduced (P < 0.02) in comparison to outgrowth in SAM dishes. Similarly, neurite outgrowth was significantly reduced (P < 0.001) in dishes exposed to medium conditioned in the presence of CoC12. DM refers to Petri dishes plated with neurons containing only defined medium.
hered within a few hours and sent out numerous neurites within 24-36 h (not shown). About half of the neurons plated into dishes containing defined medium in
*
B19
1664t~664b
t tt • tt~-
'~
25 s
Fig. 3. IntraceUular recordings from neurons B5 and B19 which did not extend neurites after plating in dishes conditioned in the presence of COC12 show that, even though neurons failed to extend processes, they exhibited good membrane potentials and fired action potentials, in this case, in response to brief hyperpolarizing current pulses (arrows) or depolarizing current pulses (asterisk). B19 would also fire in a bursting pattern in response to several hyperpolarizing current pulses (arrows) reminiscent of its endogenous firing pattern observed in isolated ganglia.
-1
o m
o
Fig. 4. Photograph of a polyacrylmide gel loaded with equal volumes of concentrated conditioned medium (CM), CM produced in the presence of "I'TX, CM produced in the presence of CoC12, and defined medium (DM). Arrows point to protein staining bands that appear reduced in staining intensity in CM produced in the presence of qTX or CoCI2. Proteins having apparent molecular weights of 23 and 15 kDa exhibited the greatest reduction.
which only the substratum possessed neurite promoting factors (SAM dishes) exhibited neurite outgrowth (Fig. lb). There was, however, a significant reduction in the percentage of neurons that extended neurites (in defined medium) when plated into dishes in which the substrate was conditioned with central ring ganglia (SAM) in the presence of C o C I 2 (Fig. lc) or TFX (Fig. ld). The results are summarized in Fig. 2. Neurons plated onto SAM dishes prepared in the presence of "I~X o r CoC12 exhibited spontaneous action potentials and membrane potentials similar to those observed in super SAM dishes (Fig. 3). Membrane blebbing from the soma was occasionally seen around some neurons plated into SAM dishes prepared in the presence of CoC12 (Fig. lc). In the experiments described above, T-FX and C o C I 2 were presumably removed from SAM culture dishes after several rinses with defined medium before neurons were plated onto their surface. Intracellular recordings from neurons plated into SAM dishes prepared in the presence of TTX o r C o C l 2 did not reveal any evidence for the suppression of activity; nevertheless, it is possi-
136 ble that small amounts of TTX or CoC12 may have remained attached to the substratum. To test whether neurite outgrowth could occur in the presence of either of the agents, we plated neurons into conditioned medium (super SAM dishes) to which we added TTX (2 x 10 -5 M) or CoCI 2 (10 mM). Neurite outgrowth failed to occur in the presence 10 mM CoCI 2 and this concentration effectively blocked action potentials in the isolated neurons. The effect of even a brief exposure to CoC12 (several minutes) was not readily reversible, as neurons did not extend processes when the CoCI 2 containing medium was replaced with fresh conditioned medium even after several changes of conditioned medium. The addition of T]?X to conditioned medium inhibited spontaneous action potentials in neurons B5 and B19 in vitro; however, TFX had no apparent effect on neurite outgrowth (7/9 neurons sprouted; e.g. Fig. le). Adjacent neurons extending neurites in the presence of TFX also formed electrical synaptic connections that were normally observed between these neurons when cultured in conditioned medium. In an attempt to identify putative neurite promoting proteins we examined the relative abundance of proteins in conditioned medium by SDS-PAGE and compared them with the protein profiles in the presence of T-FX or CoCI 2. Conditioned medium was concentrated about 30-50-fold and equal volumes were loaded onto polyacrylamide gels and separated by electrophoresis and stained with silver. The results are shown in Fig. 4. The overall protein content in conditioned medium produced in the presence of TTX or CoC12 appeared to be reduced. Proteins that stained less intensely in both CoCI 2 and TTX conditioned medium had apparent molecular weights of 44, 29, 26, 24, 23, 17, 15 and 14.5 kDa. Proteins having apparent molecular weights of 23 and 15 kDa exhibited the greatest reduction in the presence of T I ' X or CoC12. DISCUSSION The experiments described in the present study were concerned with identifying whether neural activity was required for central ring ganglia to release neurite outgrowth promoting factors during organ culture. Our findings indicate that the release of neurite outgrowth promoting factors into culture medium was dependent on spontaneous electrical activity normally associated with neurons in central ring ganglia. It is unlikely that TTX would inhibit the secretion of growth factors from non-neuronal cells (glia, hemocytes and sheath cells) since secretion is primarily a calcium-regulated process 21. The possibility that neural activity may indirectly modulate the release of growth factors from non-neuronal
cells, however, cannot be excluded in these studies. A previous study that examined Aplysia neurons in culture found that hemolymph contained neurite outgrowth promoting factors 4l, but a more recent study found that the periesophageal ganglion also released neurite outgrowth promoting factors that may be different TM. In contrast, hemolymph from Helisoma contains substances that inhibit neurite outgrowth 24. Phagocytic cells, also called hemocytes or ameobocytes, in the hemolymph of a related species, Lymnaea stagnalis, have also been implicated as a putative source of neurite growth factors a2. Highly motile hemocytes were present in our cultures arising primarily from the surface of the central ring ganglia (see Fig. 4d, ref. 7). If hemocytes contribute neurite outgrowth promoting factors in the present study, their contribution would appear to be small since outgrowth was significantly reduced by TTX, which is not known to directly inhibit secretion from non-neuronal cells. However, a small percentage of neurons did exhibit outgrowth in dishes conditioned in the presence of TTX or CoC12. It is possible that the release of a small amount of growth factor may not be linked to activity (e.g. from non-neuronal cells), or alternatively, that not all neural activity was blocked by TTX or CoCl2, since some neurons may have predominantly sodium- or calcium-dependent action potentials. Fibronectin-like molecules have been found in association with hemocytes from Helisoma 33, but fihronectin alone is insufficient to promote neurite outgrowth 33"51. Interestingly, however, if the Petri dish is coated with fibronectin and calcium channel blockers (lanthanum, cobalt or cadmium) are added to defined medium, neurite outgrowth from Helisoma neurons can be induced in the absence of exogenous growth factors 34. The growth promoting properties of these calcium channel blockers operate only in a narrow concentration range well below that required to block action potentials. Our studies used a high concentration of cobalt sufficient to block calcium-dependent action potentials or calcium-dependent secretion. Under these conditions, no neurite outgrowth was observed even after the cobalt was rinsed from the isolated neurons with defined medium. It is possible that in our experiments some of the cobalt remained associated with the neurons and inhibited outgrowth by binding to and blocking the receptors for the growth factors. Taken together, these studies suggest that a fibronectinlike molecule secreted by hemocytes in culture has a permissive role in neurite outgrowth of Helisoma neurons and also suggests the possibility that neurite promoting factors may act by modulating the influx of extracellular calcium 34. Grimm-Jorgensen 22 has suggested that the ability of somatostatin and calcitonin (released from hemocytes)
137 to promote neurite outgrowth of some molluscan neurons also correlates with their ability to modulate intracellular calcium. However, recent studies on mammalian neurons in culture suggest that calcium signals are not necessary for NGF-dependent neurite outgrowth 13'44. We have been unable to detect changes in the levels of intracellular calcium (using Fura-2 imaging) or changes in whole cell patch clamp currents of Helisoma neuron B5 superfused with conditioned medium (Berdan and Wang, unpublished results). These preliminary results suggest that the neurite outgrowth promoting factors from Helisoma central ring ganglia do not appear to act on receptor-mediated ion channels or by inducing rapid changes in the levels of intracellular calcium. TTX added to conditioned medium did not suppress neurite outgrowth of Helisoma neurons B5 and B19, and these results are similar to those we reported previously in which axotomized neurons B5 extended processes equally well within buccal ganglia maintained in organ culture in the presence or absence of q'TX 5. In contrast, other investigators have reported that neurite outgrowth from rat cerebral cortex cells in culture is enhanced in the presence of q'TX 46. Our findings suggest that voltage-activated sodium channels do not play a crucial role in neurite outgrowth of Helisoma neurons B5 and B19 in culture, but species and tissue differences may exist. Neural activity is thought to play an important role during the development, and wiring of the nervous system and it has been suggested that trophic support is somehow linked to neural activity39. In one investigation, blocking neural activity of retinal ganglion cells in culture with TTX resulted in their death and prevented them from conditioning medium that would otherwise rescue the cells 31. On the basis of these results, Lipton 31 suggested that survival of ganglion cells was dependent on a chemical factor whose presence was dependent on neural activity. Our results are similar in that the release
of neurite promoting factors from central ganglia was dependent on neural activity, but differ in that neuronal survival over 5-6 days was not. Recently, neural activity has been found to regulate N G F and B D N F (brain derived neurotrophic factor) gene expression in hippocampal neurons 32'52. These results raise the interesting possibility that trophic factor synthesis and release modified by neural activity could modulate synaptogenesis and thereby have important implications for learning and memory. Furthermore, an increase in impulse activity associated with seizures could elevate neurotrophic factors and perpetuate epileptic activity through synaptic strengthening 32. The putative neurite promoting factors released by Helisoma central ring ganglia have not yet been identified. Our biochemical results indicated that the staining of a number of proteins was reduced in conditioned medium produced in the presence of q T X or CoCl 2. Further biochemical and immunological studies will be required to identify whether any of these proteins represent bonafide neurite promoting factors. Thus far, an insulinlike peptide 26, glutamate 9 and nerve growth factor have been shown to stimulate neurite outgrowth of some molluscan neurons 4°. Recently, a laminin-like protein has been purified from Helisoma conditioned medium and this protein appears to exhibit a small amount of neurite promoting activity35. It is likely that medium conditioned by Helisoma central ganglia will contain several neurite promoting factors as appears to be the case in vertebrates brains.
Acknowledgments: This research was supported by the Alberta Heritage Foundation for Medical Research. We thank Drs. N.I. Syed, Sanders, and Mr. P. Nguyen-Ho for their comments on the manuscript. We thank Dr. A.G.M Bulloch for supplying some of the reagents and equipment used in this study.
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