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Selective enhancement of axonal branching of cultured dentate gyrus neurons by neurotrophic factors
~
Pergamon
Neuroscience Vol. 69, No. 3, pp. 763-770, 1995 Elsevier Science Ltd
0306-4522(95)00281-2
IBRO
Printed in Great Britain
SELECTIVE E N H A N C E M E N T OF A X O N A L B R A N C H I N G OF C U L T U R E D D E N T A T E G Y R U S N E U R O N S BY NEUROTROPHIC FACTORS M. N. PATEL*~" and J. O. M c N A M A R A t : ~ Departments of ?Medicine (Neurology), :~Neurology and Pharmacology, Duke University Medical Center, Durham, NC 27710, U.S.A. Abstract--Epileptic seizures in the mature nervous system are associated with axonal sprouting of the hippocampal dentate granule cells and pathological synapse formation. The molecular basis of this morphological rearrangement is obscure. Since epileptic seizures induce the transcriptional activation of genes encoding diverse neurotrophic and growth factors in the dentate granule cells and their targets, morphoregulatory effects of these proteins may contribute to this morphological rearrangement. To determine whether neurotrophins or growth factors exert morphoregulatory effects on dentate gyrus neurons, quite homogeneous preparations of these neurons from postnatal rats were established in primary culture at low density in defined media. Dendrites were distinguished from axons by phase contrast appearance together with microtubule-associated protein-2 immunocytochemistry. Multiple factors enhanced branching of axons but not dendrites of these neurons. The rank order of effectiveness was: basic fibroblast growth factor > brain-derived growth factor > neurotrophin-4 > neurotrophin-3; nerve growth factor was ineffective. No additives of synergistic effects were detected. These results are consistent with the idea that activity-driven expression of these genes contributes to the axonal sprouting and pathological synapse formation evident in diverse forms of epilepsy. Key words: neurotrophins, basic fibroblast growth factor, axon, dendrite, sprouting.
Neuronal growth and synaptogenesis, processes that normally occur during nervous system development, can be recapitulated in adult plasticities. One striking example of a synaptic reorganization in the mature m a m m a l i a n brain is the sprouting of mossy fiber axons of the hippocampal dentate granule cells, first identified following seizures induced by intracerebroventricular kainate in rats. 32 Sprouting of mossy fiber axons has subsequently been identified in many forms of experimental epilepsy, 2j'41 as well as human epilepsy. 18'42 In contrast to many forms of axonal sprouting in the mature brain, mossy fibers form synapses with targets not normally innervated by granule cells. 33'35'41 At least some of these targets are other granule cells, 33'35'41 thereby producing a recurrent excitatory circuit that is not present in the normal brain. 43 Although the functional consequences of mossy fiber sprouting are incompletely understood, 3s electrophysiological analyses disclose increased excitability of the granule cells in the absence 43 or presence of a G A B A A receptor antagon*To whom correspondence should be addressed. Abbreviations: BDNF, brain-derived neurotrophic factor; bFGF, basic fibroblast growth factor; FGF, fibroblast growth factor; FGFRI orflg, FGF receptor-I; GAP-43, growth-associated protein 43; GFAP, glial fabrillary acidic protein; MAP-2, microtubule-associated protein 2; NGF, nerve growth factor; NT-3, neurotrophin 3; NT-4, neurotrophin 4. 763
ist, 1° findings consistent with functional recurrent excitatory synapses. This projection could thus contribute to the increased excitability of an epileptic brain. The molecular mechanisms underlying formation of this anomalous projection are obscure. Seizures have been shown to produce dramatic and fleeting transcriptional activation of neurotrophic factors and their receptors. These include brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin receptors T r k B and TrkC, 3'11'2° as well as members of the fibroblast growth factor ( F G F ) family: basic F G F ( b F G F or FGF-2), F G F - 5 and the F G H receptor ( F G F R - 1 or j~g).13,14,36 Additionally, seizures increase the protein levels of at least some of these neurotrophic factors and their receptors. 3'3°'45 As seen with other brain areas, the seizure-induced expression of these diverse neurotrophic and growth factors may subserve neuroprotective effects on dentate gyrus neurons. 8'34'37'44'5°Alternatively, in the light of the morphoregulatory effects of neurotrophic factors on other neurons, 28,31,46'47 these findings suggest that seizure-induced expression of these neurotrophic and growth factors may contribute to mossy fiber sprouting in these diverse forms of epilepsy. To initiate investigations of whether neurotrophic and growth factors might contribute to mossy fiber sprouting in vivo, we first asked whether these factors were capable of producing morphoregulatory effects
764
M . N . Patel and J. O. McNamara
o n n e u r o n s f r o m the d e n t a t e gyrus & vitro. T o begin to a d d r e s s this q u e s t i o n , we first e s t a b l i s h e d q u i t e h o m o g e n e o u s p r e p a r a t i o n s o f d e n t a t e gyrus n e u r o n s in d e f i n e d m e d i a in p r i m a r y culture. A p a r t i c u l a r l y striking s t r u c t u r a l f e a t u r e o f m o s s y fiber s p r o u t i n g e v i d e n t in G o l g i 35 a n d b i o c y t i n 33 analyses is i n c r e a s e d b r a n c h i n g o f the a x o n s b u t n o t the d e n d r i t e s o f these n e u r o n s . W e t h e r e f o r e s o u g h t to s e p a r a t e l y e x a m i n e the effects o f these f a c t o r s o n the b r a n c h i n g o f a x o n s a n d d e n d r i t e s o f these n e u r o n s . EXPERIMENTAL PROCEDURES
chemical staining with antisera to GFAP, represented 10-15% of the total cells in control cultures. Immunocytochemistry
lmmunocytochemical staining for MAP-2 and GAP-43 were conducted according to the method of Caceres et al. 7 Cells were fixed with 4% paraformaldehyde and incubated for 1 h with polyclonal anti-MAP-2 at 1:200 dilution in blocking solution. The cells were washed and incubated with secondary antibody (fluorescein isothiocyanate-conjugated anti-rabbit at 1:400 dilution). For GAP-43 immunostaining, cells were treated for 15 min with 1:4000 monoclonal anti-GAP-43 followed by incubation with secondary antibody (biotinylated anti-mouse immunoglobulin G). lmmunostaining for G F A P was conducted according to Lerea and McCarthy. 2z
Materials
Quantification of axonal and dendritic branching
Antiserum to glial fibrillary acidic protein (GFAP) was obtained from Dako Corporation (Santa Barbara, CA). Antisera to microtubule-associated protein 2 (MAP-2) and growth-associated protein 43 (GAP-43) were kindly provided by Howard Schulman (Palo Alto, CA) and Pate Skene (Durham, NC), respectively. N G F and b F G F were obtained from Collaborative Sciences (Bedford, MA). Human recombinant BDNF, neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4) were kindly provided by Dr Nancy Ip (Regeneron Pharmaceuticals, Tarrytown, NY).
Cells were evaluated for axonal and dendritic branches using a digital image analyser. Phase-contrast images of live neurons were digitally enhanced such that even minor processes could be clearly visualized. Neurons were chosen for measurements of neurite branching based on the following criteria. (1) Neurons were grown in a "sandwich" area created by two coverslips. Since the coverslips did not overlap perfectly, only neurons growing within the "sandwich" area were selected. (2) Individual neurons whose entire axonal and dendritic domains were distinguishable from those of neighboring neurons were selected. (3) Cells growing in aggregates whose axonal or dendritic processes contacted those of neighboring cells were excluded from measurements. (4) Neurons that met criteria 1 3 were selected if their entire axonal and dendritic domains, including finer processes, were clearly visible when projected on the video monitor screen. (5) Pyramidal-like neurons with a single long branching neurite of uniform diameter (axon) and several short tapering neurites (dendrites) were selected for measurements. Measurements were made on day 8 in vitro (the day of plating was considered day 1) with the exception of time course experiments that were conducted on days 3-8. Branch points per individual axon and dendrite were counted and data were expressed as branch points per neurite.
Animals
Four-day-old Sprague-Dawley rats were obtained from Zivic Miller. Rats were killed by decapitation, which was accomplished quickly so as to minimize suffering. Adult rats were killed by CO 2 anesthesia followed by cervical dislocation. Care was taken that the animals experienced only that discomfort which is unavoidable. Tissue culture
Dentate gyrus neurons were prepared from four-day-old rat pups and grown at low density in serum-free defined medium. Briefly, each hippocampus was dissected and sectioned into 600-800-mm-thick transverse sections using a McIlwain tissue chopper. The fascia dentata was microdissected according to the method described previously by Lerea et al. 23 Efforts were made to obtain the granule cell layer without the adjacent hilar cells. Previous studies characterizing these cultures have identified 95% of the neuronal population as glutamate decarboxylase-negative,23 suggesting that our nei~ronal population most likely consists o f excitatory granule cells. The tissue was enzymatically dissociated with 0.25% trypsin for 30 min at 37°C followed by mechanical dissociation with fire-polished Pasteur pipettes. The cell suspension was centrifuged for 10 min at 100g, and the pellet resuspended in Dulbecco's modified Eagle medium supplemented with I0% fetal calf serum, 1 m M pyruvate, 0.6% glucose and 1 mM N a H C O 3. The cells were plated on poly-D-lysine coated 12 mm round glass coverslips at densities of 7500-8500 cells/cm 2 in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum. After 1 h, the medium was replaced with B-18 defined medium 5 containing the appropriate growth factor and an additional glass coverslip was placed over the cells so as to create a sandwich configuration. The sandwich configuration has been shown to minimize oxygen toxicity and promote long-term survival of low-density cultures? Cells were treated with growth factors 1 h after plating and thereafter on alternate days. In order to avoid the effects of endogenously released glutamate on neurite outgrowth, 27 the excitatory amino acid antagonists 6-cyano-7nitroquinoxaline-2,3-dione ( 1 0 p M ) and D-2-amino-4phosphonovaleric acid (100#M) were routinely included in culture media. Control cultures were treated with culture media or appropriately diluted bovine serum albumin. Astrocytes, identified morphologically and by immunocyto-
Statistical analyses
Treatments were compared by one-way followed by the Tukey-Kramer post hoc test.
ANOVA
RESULTS Characterization o f neurites
T h e d e n d r i t e s a n d a x o n o f n e u r o n s were dist i n g u i s h e d m o r p h o l o g i c a l l y using p h a s e - c o n t r a s t microscopy and MAP-2 and GAP-43 immunocytoc h e m i s t r y . T h e m a j o r i t y o f n e u r o n s in culture c o u l d be classified as p y r a m i d - l i k e n e u r o n s I with a single long b r a n c h i n g n e u r i t e o f u n i f o r m d i a m e t e r a n d several s h o r t t a p e r i n g neurites (Fig. 1A). In e m b r y onic h i p p o c a m p a l n e u r o n s m a i n t a i n e d in p r i m a r y culture, the single, long n e u r i t e has been identified as the a x o n due to the lack o f M A P - 2 a n d p r e s e n c e o f tau i m m u n o r e a c t i v i t y ; the s h o r t t a p e r i n g processes have been identified as d e n d r i t e s d u e to the p r e s e n c e o f M A P - 2 a n d a b s e n c e o f tau i m m u n o r e a c t i v i t y . 6'7 T h e p r e s e n c e o f M A P - 2 i m m u n o r e a c t i v i t y selectively in the s h o r t t a p e r i n g p r o c e s s e s a n d n o t in the single long b r a n c h i n g neurite c o n f i r m e d the findings o f C a c e r e s et al. 7 a n d r e i n f o r c e d the a c c u r a c y o f the m o r p h o l o g i c a l d i s t i n c t i o n o f a x o n s f r o m d e n d r i t e s (Fig. IB, C). G A P - 4 3 i m m u n o r e a c t i v i t y is
Neurotrophic factors and axonal branching
765
Fig. 1. Characterization of dentate gyrus neurons. (A) Phase-contrast image of a representative eight-day-old dentate gyrus neuron used for quantification of axonal and dendritic branch points. The axon can be identified as the single long branched process. Arrows show three examples of the numerous axonal branch points used for measurements. The dendrites can be identified as short tapering processes emerging from the cell soma. (B, C) MAP-2 immunocytochemistry to distinguish axons from dendrites Eight-day-old dentate gyrus neurons were processed for MAP-2 immunocytochemistry as described in Experimental Procedures. B shows the positive staining in the soma and dendrites of a representative neuron. C shows the phase-contrast image of the same neuron. Arrowheads indicate the axon which shows the absence of MAP-2 immunoreactivity. (A) x 250; (B, C) x 400.
preferentially expressed in the soma and axons of hippocampal neurons in primary culture 15 and has been used to characterize subsets o f neurites as axons. By contrast, GAP-43 immunocytochemistry in our cultures of dentate gyrus neurons revealed a pattern of expression similar to that found in the developing rat dentate granule cells in v i v o 29 with immunoreactivity found in the soma, throughout the length of the axon and in the dendrites (not shown). Although GAP-43 immunoreactivity did not help distinguish axons from dendrites, the phase-contrast appearance, together with M A P - 2 immunoreactivity, provided the basis for identifying subsets o f neurites as axons or dendrites.
Effect of basic fibroblast growth factor on neurite branching b F G F produced a concentration-dependent increase in the number of axonal branch points on day 8 (Fig. 2). b F G F (10-100 ng/ml) produced a 1.6- to 2-fold increase in the number of axonal branch points. In contrast, at the concentrations tested b F G F did not change the number of branch points on individual dendrites. The onset of b F G F induction of axonal branching occurred approximately on day 5 in culture (Fig. 3). In addition to increasing the number of branch points per individual axon, b F G F also induced a more rounded appearance of the soma and a small (20%) but statistically significant increase (P < 0.05, one-way A N O V A ) in the number of dendrites per cell.
Effect of the neurotrophins on neurite branching We also examined the morphoregulatory effects of N G F , B D N F , NT-3 and N T - 4 on dentate gyrus neurons, Cells were treated with varying concentrations of neurotrophic factors for eight days. While
testing each neurotrophic factor, b F G F (10 ng/ml) was included in a sister well on the same plate as the positive control. B D N F produced a concentration-dependent increase in the number of axonal branch points (Fig. 4A). At concentrations of 10 and 100 ng/ml, B D N F increased the number of axonal branch points by 36% and 48%, respectively. In contrast with its effect on axonal branching, B D N F did not change the number of branch points per individual dendrite. NT-4 also produced a small but concentrationdependent increase in the number of axonal branch points (28% and 34% increase by 10 and 100 ng/ml, respectively; (Fig. 4B). Like B D N F , NT-4 did not influence the branching of individual dendrites. NT-3 had only a modest effect (20% increase at 100 ng/ml) on axonal branching, detectable only at 20
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Fig. 2. Concentration dependence of bFGF effect on neurite branching. Cells were grown in the presence of 0 (control), 0.1, 1, 10, 30 or 100ng/ml of bFGF for eight days and axonal and dendritic branches counted. Bars represent mean + S.E.M. of 19-49 neurons. Asterisk indicates significant difference from controls (P <0.05, one-way ANOVA).
766
M . N . Patel and J. O. McNamara
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Fig. 3. Time course of bFGF induction of neurite branching. Cells were grown in the presence of control media or 10 ng/ml bFGF and branch points counted on axons (A) and dendrites (B) on days 4-8. Bars represent mean _+ S.E.M. of 11~20 neurons.
the highest c o n c e n t r a t i o n used (Fig. 4C). NT-3 h a d no effect o n the n u m b e r o f dendritic b r a n c h points. In c o n t r a s t to b F G F , B D N F , NT-3 a n d NT-4, N G F h a d no effect o n axonal or dendritic b r a n c h i n g at any o f the c o n c e n t r a t i o n s tested (Fig. 4D). In order to ensure t h a t the N G F p r e p a r a t i o n was biologically active, we tested its ability to induce neurite outg r o w t h in undifferentiated PC 12 cells. In undifferentiated PC12 cells, N G F t r e a t m e n t (50 ng/ml) for five
days p r o d u c e d r o b u s t neurite extension c o m p a r e d to vehicle-treated cells (data not shown). The e n h a n c e d axonal b r a n c h i n g produced by multiple n e u r o t r o p h i c factors led us to query w h e t h e r these actions were additive a n d / o r synergistic. T o test this idea, a single s u b m a x i m a l conc e n t r a t i o n (10 ng/ml) o f each n e u r o t r o p h i n was first examined in sister cultures (Table 1). The most p r o m i n e n t effect was f o u n d with b F G F (73%, P<0.05), B D N F (34%, P < 0 . 0 5 ) and NT-4
~51 A
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Fig. 4. Concentration dependence of neurotrophins on neurite branching. Cells were grown in the presence of 0 (control), 1, 10 or 100 ng/ml of BDNF (a), NT04 (B), NT-3 (C) and NGF (D) for eight days and axonal and dendritic branches counted. Bars represent mean + S.E.M. of 12-37 neurons. Closed bars indicate axonal branch points and open bars indicate dendritic branch points. Asterisk indicates significant difference from controls (P < 0.05, one-way ANOVA).
Neurotrophic factors and axonal branching DISCUSSION
Table 1. Comparison of neurotrophic factor efficacies in enhancing axonal branching Treatment
Branch points/axon
Control bFGF NT-4 BDNF NT-3 NGF
767
7.3 __+0.4 12.6 + 0.7* 10.3 _+ 0.6* 9.8 __+0.6* 8.8 _+ 0.6 6.8 __+0.7
Cells were grown in the presence of 10ng/ml bFGF, NT-4, BDNF, NT-3 or N G F for eight days and axonal branch points counted. Values represent mean+S.E.M, of 1636 neurons. Asterisk indicates significant difference from controls (P < 0.05, one-way ANOVA).
(41%, P < 0.05), confirming the results o f concent r a t i o n - r e s p o n s e studies. A l t h o u g h small additive effects m a y be found, n o evidence of synergy was f o u n d with b F G F a n d B D N F or b F G F a n d NT-4, respectively (Table 2).
The effect of basic fibroblast growth factor on neuronal survival T o ensure t h a t b F G F induced neurite b r a n c h i n g r a t h e r t h a n s u p p o r t the survival of a highly b r a n c h e d s u b p o p u l a t i o n o f neurons, we m e a s u r e d the effect o f b F G F o n n e u r o n a l survival in o u r cultures. Neurons in selected fields were c o u n t e d 24 h after plating (day 2), w h e n the n e u r o n s could be distinguished from n o n - n e u r o n a l cells. T h e same fields were relocated a n d c o u n t e d o n day 8. U n d e r control conditions n e u r o n a l n u m b e r s decreased by 4 5 % on day 8 (12 _+ 2 a n d 7 _+ 1 n e u r o n s per field o n days 2 a n d 8, respectively; n = 4). b F G F p r o d u c e d a small increase in n e u r o n a l survival o n day 8 (13_+ 2 a n d 10 _+ 2 n e u r o n s per field o n days 2 a n d 8, respectively; n = 6 ) , which was n o t statistically significant. A l t h o u g h b F G F showed some i m p r o v e m e n t in neuronal survival, we were u n a b l e to detect statistical significance o f its effect in this limited study. Imm u n o s t a i n i n g for G F A P revealed a m a r k e d increase in the n u m b e r a n d size o f glial cells in the presence o f b F G F (data n o t shown).
T h r e e principal findings emerge from this work. (1) Multiple n e u r o t r o p h i c factors e n h a n c e d axonal b u t n o t dendritic b r a n c h i n g o f d e n t a t e gyrus neurons. (2) The r a n k order of effectiveness was: b F G F > B D N F > N T - 4 > NT-3; N G F was ineffective. (3) N o additive or synergistic effets o f these n e u r o t r o p h i c factors were detected. R o b u s t sprouting o f granule cell axons is observed in m a n y a n i m a l models a n d in h u m a n epilepsy itself, yet the underlying molecular m e c h a n i s m s are unknown. Since n e u r o t r o p h i c factors exert m o r p h o r e g ulatory effects o n neurons, 2 the fact t h a t limbic seizures produce dramatic, transient increases in the expression o f genes e n c o d i n g n e u r o t r o p h i c factors a n d their receptors 3'4'u'~3'~4'2°suggests t h a t one consequence o f these factors m i g h t be i n d u c t i o n axonal sprouting. Since n e u r o t r o p h i c factors evoke striking effects in some b u t n o t all n e u r o n a l populations, the question arose as to whether, a n d which, neurotrophic factors m i g h t exert m o r p h o r e g u l a t o r y effects o n dentate gyrus neurons. A m o n g the diverse morphological properties one m i g h t analyse, axonal b r a n c h i n g seemed of particular relevance since the granule cell axons in the s p r o u t e d c o n d i t i o n exhibit r o b u s t branching. 33,35 The present findings provide direct evidence t h a t some of the n e u r o t r o p h i c factors u n d e r g o i n g increased expression in the dentate gyrus following seizures exert m o r p h o r e g u l a t o r y effects o n dentate gyrus neurons, the most r o b u s t effects being observed with b F G F a n d B D N F . O u r results are consistent with the idea t h a t seizure activity-driven n e u r o t r o p h i c factor gene expression c o n t r i b u t e s to the axonal b r a n c h i n g a n d s p r o u t i n g o f the dentate granule cells evident in m a n y of the epilepsies. The expression o f distinct n e u r o t r o p h i c factor receptors in d e n t a t e gyrus n e u r o n s likely c o n t r i b u t e s to the specificity of the m o r p h o r e g u l a t o r y effects of these diverse n e u r o t r o p h i c factors. B D N F a n d N T - 4 showed a striking similarity in terms of the magnitude a n d c o n c e n t r a t i o n dependence o f their effect on axonal branching. This is consistent with their preference for the same receptor, TrkB. U n l i k e B D N F a n d NT-4, NT-3 was effective at increasing axonal b r a n c h i n g only at the highest c o n c e n t r a t i o n tested
Table 2. The combined effect of basic fibroblast growth factor and brain-derived neurotrophic factor or neurotrophin-4 on neurite branching Experiment 1 Treatment Control bFGF NT bFGF + NT
Experiment 2
Axon
Dendrite
Axon
Dendrite
6.0 -t- 0.4 8.4 + 0.4* 8.3 + 0.8* 9.6 __+0.7*
1.6 + 0.1 1.1 __+0.1 1.2 + 0.1 1.6 + 0.2
7.3 __+0.5 11.0 + 0.8* 9.8 __+0.6* 12.0 + 0.9*
1.6 + 0.I 1.9 -I- 0.1 1.4 + 0.1 1.8 _ 0.2
Cells were grown in the presence of 10ng/ml bFGF alone or in combination with neurotrophin (NT). "NT" in experiments 1 and 2 represents 10 ng/ml BDNF and NT-4, respectively. Values represent axonal and dendritic branch points (mean + S.E.M. of I[~17 neurons in each experiment). Asterisk indicates significant difference from controls (P < 0.05, one-way ANOVA).
768
M.N. Patel and J. O. McNamara
(100 ng/ml). This finding is consistent with the ability of NT-3 to increase Fos-like immunostaining in the dentate gyrus of hippocampal explant cultures at the same concentration.9 While NT-3 is the preferred ligand for TrkC receptors, NT-3 also binds and activates TrkB receptors, although less potently and effectively than BDNF. 4° It seems plausible that axonal branching in our cultures is mediated by TrkB and not TrkC receptors, and the effect of NT-3 was mediated by its action on TrkB receptors. The effects of b F G F could be mediated by the direct interaction of b F G F with F G F receptor (e.g., FGFR1 or f i g ) expressed on dentate gyrus neurons.4s Alternatively, the morphoregulatory effects of b F G F observed here could be mediated indirectly through an action on astrocytes, since b F G F enhanced both size and numbers of astrocytes in these experiments. The absence of N G F responses in dentate gyrus neurons is consistent with previous reports of the lack of N G F effect on neurite outgrowth in cultured hippocampal neurons28 and the absence of TrkA receptors in dentate granule cells in vivo 1~.30 or in hippocampal neurons in v i t r o . 19
The absence of morphoregulatory effects of N G F on dentate dyrus neurons raises the question as to whether the seizure-induced expression of N G F in the dentate granule cells might exert morphoregulatory effects on other neuronal populations in t)il)o. 39 Importantly, seizures not only increase N G F mRNA but also increase N G F protein-like neurotrophic activity in hippocampal extracts isolated from rats following kainate-induced seizures,25 these extracts enhance survival of sympathetic neurons maintained in primary culture, an effect that can be inhibited by anti-NGF antibodies.25 Although it was not certain that the anti-NGF antibodies exerted their effects on N G F p e r se, the results of Lowenstein et al. 25 support the idea that N G F protein is expressed in the hippocampus following limbic seizures. One possibility is that N G F expressed in dentate granule cells exerts its effect on neuronal populations other than the granule cells themselves. The survival-promoting effects of N G F on cholinergic basal forebrain neurons39 have predicted the role of N G F as a target-derived neurotrophic factor for innervating cholinergic neurons of the basal forebrain. The trophic effects of hippocampal infusions of N G F on septal cholinergic neurons ~7 are consistent with this idea. The presence of sprouting of septal cholinergic afferents after kainate-evoked seizures may be one consequence of seizure-evoked expression of N G F mRNA. H'~2 Among the diversity of neurotrophic factors whose expression is enhanced by seizures, our findings suggest that b F G F and BDNF in particular may contribute to the axonal sprouting of dentate granule cells in the epileptic brain. This idea raises the interesting question as to the route of access of the growth factors to the receptors on the neurons. That is, the most prominent site of increased expression of b F G F immunoreactivity following seizures is in as-
trocytes in the molecular leyer of the dentate gyrus and to a lesser extent in the granule cells themselves. 14'45 Seizure-induction of b F G F immunoreactivity is paralleled in both distribution and time course by the induction of immunoreactivity of an F G F receptor, FGFR1:5 If b F G F contributes to the axonal sprouting of the granule cells in vivo, it will be interesting to determine whether b F G F expression in the granule cells exerts autocrine and/or paracrine effects or whether b F G F derived from astrocytes contributes to these effects. With respect to BDNF, kainate-induced seizures are associated with a redistribution of BDNF immunoreactivity from cell soma and dendrites of CA3 and dentate granule cells to the neuropil in the mossy fiber zone: 9 Together with the expression of full length TrkB receptor mRNA in the dentate granule cells, this led Wetmore et al. 49 to suggest that release of BDNF from soma and dendrites into axon terminal zones may promote axonal sprouting in a paracrine or autocrine fashion after kainate-induced seizures. The selective effects of the neurotrophic factors on branching of axons but not on dendrites in dentate gyrus neurons is curious. Although morphoregulatory effects of neurotrophic factors have been identified in diverse populations of neurons maintained in primary culture by numerous investigators, 16'3134'26'46'47 few studies28 have distinguished axons from dendrites. It seems likely that the morphoregulatory effects of neurotrophic factors in vivo will impact on the structure of both axons and dendrites, culminating in synapse formation. This raises the interesting possibility that the initial actions of these growth factors are exerted on the axon of dentate gyrus neurons; for example, the proteins synthesized in response to the neurotrophic factors may be preferentially targeted to the axons. Whether the axon specificity of these effects is specific to dentate gyrus neurons is currently under investigation. Although the absence of effects on dendritic branching is curious, the presence of morphoregulatory effects of b F G F and these neurotrophic factors on the axons of dentate hyrus neurons strengthens the likelihood that these factors contribute to the axonal sprouting of the granule cell axons in vivo. CONCLUSIONS
Two major findings emerge from this work. (1) Multiple neurotrophic factors selectively increased axonal but not dendritic branching of dentate gyrus neurons. (2) The rank order of effectiveness was: b F G F > BDNF > NT-4 > NT-3; N G R was ineffective. These in vitro findings parallel mossy fiber sprouting in t, ivo and are consistent with the idea that activity-driven gene expression contributes to axonal sprouting in diverse forms of epilepsy. Acknowledgements--This work was supported by a grant
from the National Institutes of Health, NS 32334.
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(1981) Lesion induced sprouting of hippocampal mossy fiber collaterals to the fascia dentata in developing and adult rats. J. comp. NeuroL 200, 433-459. 22. Lerea L. S. and McCarthy K. D. (1990) Neuron-associated astroglial cells express b- and al-adrenergic receptors in vitro. Brain Res. 521, 7 14. 23. Lerea L. S., Butler L. S. and McNamara J. O. (1992) NMDA and non-NMDA receptor-mediated increase of c-fos mRNA in dentate gyrus neurons involves calcium influx via different routes. J. Neurosci. 12, 2973-2961. 24. Lindsay R. M. (1988) Nerve growth factors (NGF, BDNF) enhance axonal regeneration but are not required for survival of adult sensory neurons. J. Neurosci. 8, 2394-2405. 25. Lowenstein D. H., Seren M. S. and Longo F. M. (1993) Prolonged increases in neurotrophic activity associated with kainate-induced hippocampal synaptic reorganization. Neuroscience 56, 597~504. 26. Maisonpierre P. C., Belluscio L., Squinto S., Ip N. Y., Furth M. E., Lindsay R. M. and Yancopoulos G. D. (1990) Neurotrophin-3: a neurotrophic factor related to NGF and BDNF. Seience 247, 1446-1451. 27. Mattson M. P., Dou P. and Kater S. B. (1988) Outgrowth-regulating actions of glutamate in isolated hippocampal pyramidal neurons. J. Neurosci. 8, 2087 2100. 28. Mattson M. P., Murrain M., Guthrie P. B. and Kater S. B. (1989) Fibroblast growth factor and glutamate: opposing roles in the generation and degeneration of hippocampal neuroarchitecture. J. Neurosci. 9, 3728-3740. 29. Meberg P. J. and Routtenberg A. (1991) Selective expression of protein F1/(GAP43) mRNA in pyramidal but not granule cells of the hippocampus. Neuroscience 45, 721-733. 30. Merlio J. P., Emfors P., Kokaia Z., Middlemas D. S., Bengzon J., Kokaia M., Smith M. L., Siesjo B. K., Hunter T., Lindvall O. and Persson H. (1993) Increased production of the TrkB protein tyrosine kinase receptor after brain insults. Neuron I0, 151 164. 31. Morrison R. S., Sharma A., De Vellis J. and Bradshaw R. A. (1986) Basic fibroblast growth factor supports the survival of cerebral cortical neurons in primary culture. Proc. natn. Acad. Sci. U.S.A. 83, 7537-7541. 32. Nadler J. V., Perry B. W. and Cotman C. W. (1980) Selective reinnervation of hippocampal area CAI and the fascia dentata after destruction of CA3~CA4 afferents with kainic acid. Brain Res. 182, 14. 33. Okazaki M. M., Evenson D. A. and Nadler J. V. (1995) Hippocampal mossy fiber sprouting and synapse formation after retrograde transport of biocytin. J. comp. Neurol. 352, 515-534. 34. Otto D., Unsicker K. and Grothe C. (1987) Pharmacological effects of nerve growth factor applied to the transectioned sciatic nerve on neuron death in adult rat dorsal root ganglia. Neurosci. Lett. 83, 15(~160.
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35. Represa A., Jorquera I., Le Gal La Salle G. and Ben-Ari Y. (1993) Epilepsy induced collateral sprouting of hippocampal mossy fibers: does it induce the development of ectopic synapses with granule cell dendrites? Hippocarnpus 3, 257~68. 36. Riva M. A., Gale K. and Mocchetti I. (1992) Basic fibroblast growth factor m R N A increases in specific brain regions following convulsive seizures. Molec. Brain Res. 15, 311-318. 37. Sendtner M., Holtman B., Kolbeck R., Thoenen H. and Barde Y. A. (1992) Brain-derived neurotrophic factor prevents the death of motoneurons in newborn rats after nerve section. Nature 360, 757-759. 38. Slovitor R. S. (1992) Possible functional consequences of synaptic reorganization in the dentate gyrus of kainate-treated rats. Neurosci. Lett. 137, 91-96. 39. Snider W. D. and Johnson E. M. (1989) Neurotrophic molecules. Ann. Neurol. 26, 489 506. 40. Squinto S. P,, Stitt T. N., Aldrich T. H., Davis S., Bianco S. M,, Radziejewski C., Glass D. J., Masiakowski P., Furth M. E., Valenzuela D. M., DiStefano P. S. and Yancopoulos G. D. (1991) TrkB encodes a functional receptor for brain-derived neurotropic factor and neurotrophin-3 but not nerve growth factor. Cell 65, 885 893. 41. Sutula T., He X. X., Cavazos J. and Scott G. (1988) Synaptic reorganization induced in the hippocampus by abnormal functional activity. Science 239, 1147-1150. 42. Sutula T., Cascino M. D., Cavazos J., Parada I. and Ramirez L. (1989) Mossy fiber synaptic reorganization in the epileptic human temporal lobe. Ann. Neurol. 26, 321 330. 43. Tauck D. L. and Nadler J. V. (1985) Evidence of functional mossy fiber sprouting in hippocampal formation of kainic acid-treated rats. J. Neurosci. 5, 1016-1022. 44. Unsciker K., Reichert-Preibsch H., Schmidt R., Pettmann B., Labourdette G. and Sensenbrenner M. (1987) Astroglial and fibroblast growth factors have neurotropic functions for cultured peripheral and central nervous system neurons. Proc. natn. Acad. Sci. U.S.A. 84, 5459 5463. 45. van der Wal E. A., Gomez-Pinilla F. and Cotman C. W. (1994) Seizure-associated induction of basic fibroblast growth factor and its receptor in the rat brain. Neuroscience 60, 311 323. 46. Walicke P. A., Cowan W. M., Ueno N., Baird A. and Guillemin R. (1986) Fibroblast growth factor promotes survival of dissociated hippocampal neurons and enhances neurite extension. Proc. natn. Acad. Sci. U.S.A. 83, 3012--3016. 47. Walicke P. A. (1988) Basic and acidic fibroblast growth factors have trophic effects on neurons from multiple CNS regions. J. Neurosci. 8, 2618 2627. 48. Wanaka A., Johnson E. M. and Millbrandt J. (1990) Localization of F G F receptor m R N A in the adult rat central nervous system by in situ hybridization. Neuron 5, 267-281. 49. Wetmore C., Olson L. and Bean A. J. (1994) Regulation of brain-derived neurotrophic factor (BDNF) expression and release from hippocampal neurons is mediated by n o n - N M D A type glutamate receptors. J. Neurosci. 14, 1688 1700. 50. Yan Q., Elliot J. and Snider W. D. (1992) Brain-derived neurotrophic factor rescues spinal motor neurons from axotomy-induced cell death. Nature 360, 753 755. (Accepted 7 June 1995)
Pergamon
Neuroscience Vol. 69, No. 3, pp. 763-770, 1995 Elsevier Science Ltd
0306-4522(95)00281-2
IBRO
Printed in Great Britain
SELECTIVE E N H A N C E M E N T OF A X O N A L B R A N C H I N G OF C U L T U R E D D E N T A T E G Y R U S N E U R O N S BY NEUROTROPHIC FACTORS M. N. PATEL*~" and J. O. M c N A M A R A t : ~ Departments of ?Medicine (Neurology), :~Neurology and Pharmacology, Duke University Medical Center, Durham, NC 27710, U.S.A. Abstract--Epileptic seizures in the mature nervous system are associated with axonal sprouting of the hippocampal dentate granule cells and pathological synapse formation. The molecular basis of this morphological rearrangement is obscure. Since epileptic seizures induce the transcriptional activation of genes encoding diverse neurotrophic and growth factors in the dentate granule cells and their targets, morphoregulatory effects of these proteins may contribute to this morphological rearrangement. To determine whether neurotrophins or growth factors exert morphoregulatory effects on dentate gyrus neurons, quite homogeneous preparations of these neurons from postnatal rats were established in primary culture at low density in defined media. Dendrites were distinguished from axons by phase contrast appearance together with microtubule-associated protein-2 immunocytochemistry. Multiple factors enhanced branching of axons but not dendrites of these neurons. The rank order of effectiveness was: basic fibroblast growth factor > brain-derived growth factor > neurotrophin-4 > neurotrophin-3; nerve growth factor was ineffective. No additives of synergistic effects were detected. These results are consistent with the idea that activity-driven expression of these genes contributes to the axonal sprouting and pathological synapse formation evident in diverse forms of epilepsy. Key words: neurotrophins, basic fibroblast growth factor, axon, dendrite, sprouting.
Neuronal growth and synaptogenesis, processes that normally occur during nervous system development, can be recapitulated in adult plasticities. One striking example of a synaptic reorganization in the mature m a m m a l i a n brain is the sprouting of mossy fiber axons of the hippocampal dentate granule cells, first identified following seizures induced by intracerebroventricular kainate in rats. 32 Sprouting of mossy fiber axons has subsequently been identified in many forms of experimental epilepsy, 2j'41 as well as human epilepsy. 18'42 In contrast to many forms of axonal sprouting in the mature brain, mossy fibers form synapses with targets not normally innervated by granule cells. 33'35'41 At least some of these targets are other granule cells, 33'35'41 thereby producing a recurrent excitatory circuit that is not present in the normal brain. 43 Although the functional consequences of mossy fiber sprouting are incompletely understood, 3s electrophysiological analyses disclose increased excitability of the granule cells in the absence 43 or presence of a G A B A A receptor antagon*To whom correspondence should be addressed. Abbreviations: BDNF, brain-derived neurotrophic factor; bFGF, basic fibroblast growth factor; FGF, fibroblast growth factor; FGFRI orflg, FGF receptor-I; GAP-43, growth-associated protein 43; GFAP, glial fabrillary acidic protein; MAP-2, microtubule-associated protein 2; NGF, nerve growth factor; NT-3, neurotrophin 3; NT-4, neurotrophin 4. 763
ist, 1° findings consistent with functional recurrent excitatory synapses. This projection could thus contribute to the increased excitability of an epileptic brain. The molecular mechanisms underlying formation of this anomalous projection are obscure. Seizures have been shown to produce dramatic and fleeting transcriptional activation of neurotrophic factors and their receptors. These include brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin receptors T r k B and TrkC, 3'11'2° as well as members of the fibroblast growth factor ( F G F ) family: basic F G F ( b F G F or FGF-2), F G F - 5 and the F G H receptor ( F G F R - 1 or j~g).13,14,36 Additionally, seizures increase the protein levels of at least some of these neurotrophic factors and their receptors. 3'3°'45 As seen with other brain areas, the seizure-induced expression of these diverse neurotrophic and growth factors may subserve neuroprotective effects on dentate gyrus neurons. 8'34'37'44'5°Alternatively, in the light of the morphoregulatory effects of neurotrophic factors on other neurons, 28,31,46'47 these findings suggest that seizure-induced expression of these neurotrophic and growth factors may contribute to mossy fiber sprouting in these diverse forms of epilepsy. To initiate investigations of whether neurotrophic and growth factors might contribute to mossy fiber sprouting in vivo, we first asked whether these factors were capable of producing morphoregulatory effects
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o n n e u r o n s f r o m the d e n t a t e gyrus & vitro. T o begin to a d d r e s s this q u e s t i o n , we first e s t a b l i s h e d q u i t e h o m o g e n e o u s p r e p a r a t i o n s o f d e n t a t e gyrus n e u r o n s in d e f i n e d m e d i a in p r i m a r y culture. A p a r t i c u l a r l y striking s t r u c t u r a l f e a t u r e o f m o s s y fiber s p r o u t i n g e v i d e n t in G o l g i 35 a n d b i o c y t i n 33 analyses is i n c r e a s e d b r a n c h i n g o f the a x o n s b u t n o t the d e n d r i t e s o f these n e u r o n s . W e t h e r e f o r e s o u g h t to s e p a r a t e l y e x a m i n e the effects o f these f a c t o r s o n the b r a n c h i n g o f a x o n s a n d d e n d r i t e s o f these n e u r o n s . EXPERIMENTAL PROCEDURES
chemical staining with antisera to GFAP, represented 10-15% of the total cells in control cultures. Immunocytochemistry
lmmunocytochemical staining for MAP-2 and GAP-43 were conducted according to the method of Caceres et al. 7 Cells were fixed with 4% paraformaldehyde and incubated for 1 h with polyclonal anti-MAP-2 at 1:200 dilution in blocking solution. The cells were washed and incubated with secondary antibody (fluorescein isothiocyanate-conjugated anti-rabbit at 1:400 dilution). For GAP-43 immunostaining, cells were treated for 15 min with 1:4000 monoclonal anti-GAP-43 followed by incubation with secondary antibody (biotinylated anti-mouse immunoglobulin G). lmmunostaining for G F A P was conducted according to Lerea and McCarthy. 2z
Materials
Quantification of axonal and dendritic branching
Antiserum to glial fibrillary acidic protein (GFAP) was obtained from Dako Corporation (Santa Barbara, CA). Antisera to microtubule-associated protein 2 (MAP-2) and growth-associated protein 43 (GAP-43) were kindly provided by Howard Schulman (Palo Alto, CA) and Pate Skene (Durham, NC), respectively. N G F and b F G F were obtained from Collaborative Sciences (Bedford, MA). Human recombinant BDNF, neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4) were kindly provided by Dr Nancy Ip (Regeneron Pharmaceuticals, Tarrytown, NY).
Cells were evaluated for axonal and dendritic branches using a digital image analyser. Phase-contrast images of live neurons were digitally enhanced such that even minor processes could be clearly visualized. Neurons were chosen for measurements of neurite branching based on the following criteria. (1) Neurons were grown in a "sandwich" area created by two coverslips. Since the coverslips did not overlap perfectly, only neurons growing within the "sandwich" area were selected. (2) Individual neurons whose entire axonal and dendritic domains were distinguishable from those of neighboring neurons were selected. (3) Cells growing in aggregates whose axonal or dendritic processes contacted those of neighboring cells were excluded from measurements. (4) Neurons that met criteria 1 3 were selected if their entire axonal and dendritic domains, including finer processes, were clearly visible when projected on the video monitor screen. (5) Pyramidal-like neurons with a single long branching neurite of uniform diameter (axon) and several short tapering neurites (dendrites) were selected for measurements. Measurements were made on day 8 in vitro (the day of plating was considered day 1) with the exception of time course experiments that were conducted on days 3-8. Branch points per individual axon and dendrite were counted and data were expressed as branch points per neurite.
Animals
Four-day-old Sprague-Dawley rats were obtained from Zivic Miller. Rats were killed by decapitation, which was accomplished quickly so as to minimize suffering. Adult rats were killed by CO 2 anesthesia followed by cervical dislocation. Care was taken that the animals experienced only that discomfort which is unavoidable. Tissue culture
Dentate gyrus neurons were prepared from four-day-old rat pups and grown at low density in serum-free defined medium. Briefly, each hippocampus was dissected and sectioned into 600-800-mm-thick transverse sections using a McIlwain tissue chopper. The fascia dentata was microdissected according to the method described previously by Lerea et al. 23 Efforts were made to obtain the granule cell layer without the adjacent hilar cells. Previous studies characterizing these cultures have identified 95% of the neuronal population as glutamate decarboxylase-negative,23 suggesting that our nei~ronal population most likely consists o f excitatory granule cells. The tissue was enzymatically dissociated with 0.25% trypsin for 30 min at 37°C followed by mechanical dissociation with fire-polished Pasteur pipettes. The cell suspension was centrifuged for 10 min at 100g, and the pellet resuspended in Dulbecco's modified Eagle medium supplemented with I0% fetal calf serum, 1 m M pyruvate, 0.6% glucose and 1 mM N a H C O 3. The cells were plated on poly-D-lysine coated 12 mm round glass coverslips at densities of 7500-8500 cells/cm 2 in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum. After 1 h, the medium was replaced with B-18 defined medium 5 containing the appropriate growth factor and an additional glass coverslip was placed over the cells so as to create a sandwich configuration. The sandwich configuration has been shown to minimize oxygen toxicity and promote long-term survival of low-density cultures? Cells were treated with growth factors 1 h after plating and thereafter on alternate days. In order to avoid the effects of endogenously released glutamate on neurite outgrowth, 27 the excitatory amino acid antagonists 6-cyano-7nitroquinoxaline-2,3-dione ( 1 0 p M ) and D-2-amino-4phosphonovaleric acid (100#M) were routinely included in culture media. Control cultures were treated with culture media or appropriately diluted bovine serum albumin. Astrocytes, identified morphologically and by immunocyto-
Statistical analyses
Treatments were compared by one-way followed by the Tukey-Kramer post hoc test.
ANOVA
RESULTS Characterization o f neurites
T h e d e n d r i t e s a n d a x o n o f n e u r o n s were dist i n g u i s h e d m o r p h o l o g i c a l l y using p h a s e - c o n t r a s t microscopy and MAP-2 and GAP-43 immunocytoc h e m i s t r y . T h e m a j o r i t y o f n e u r o n s in culture c o u l d be classified as p y r a m i d - l i k e n e u r o n s I with a single long b r a n c h i n g n e u r i t e o f u n i f o r m d i a m e t e r a n d several s h o r t t a p e r i n g neurites (Fig. 1A). In e m b r y onic h i p p o c a m p a l n e u r o n s m a i n t a i n e d in p r i m a r y culture, the single, long n e u r i t e has been identified as the a x o n due to the lack o f M A P - 2 a n d p r e s e n c e o f tau i m m u n o r e a c t i v i t y ; the s h o r t t a p e r i n g processes have been identified as d e n d r i t e s d u e to the p r e s e n c e o f M A P - 2 a n d a b s e n c e o f tau i m m u n o r e a c t i v i t y . 6'7 T h e p r e s e n c e o f M A P - 2 i m m u n o r e a c t i v i t y selectively in the s h o r t t a p e r i n g p r o c e s s e s a n d n o t in the single long b r a n c h i n g neurite c o n f i r m e d the findings o f C a c e r e s et al. 7 a n d r e i n f o r c e d the a c c u r a c y o f the m o r p h o l o g i c a l d i s t i n c t i o n o f a x o n s f r o m d e n d r i t e s (Fig. IB, C). G A P - 4 3 i m m u n o r e a c t i v i t y is
Neurotrophic factors and axonal branching
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Fig. 1. Characterization of dentate gyrus neurons. (A) Phase-contrast image of a representative eight-day-old dentate gyrus neuron used for quantification of axonal and dendritic branch points. The axon can be identified as the single long branched process. Arrows show three examples of the numerous axonal branch points used for measurements. The dendrites can be identified as short tapering processes emerging from the cell soma. (B, C) MAP-2 immunocytochemistry to distinguish axons from dendrites Eight-day-old dentate gyrus neurons were processed for MAP-2 immunocytochemistry as described in Experimental Procedures. B shows the positive staining in the soma and dendrites of a representative neuron. C shows the phase-contrast image of the same neuron. Arrowheads indicate the axon which shows the absence of MAP-2 immunoreactivity. (A) x 250; (B, C) x 400.
preferentially expressed in the soma and axons of hippocampal neurons in primary culture 15 and has been used to characterize subsets o f neurites as axons. By contrast, GAP-43 immunocytochemistry in our cultures of dentate gyrus neurons revealed a pattern of expression similar to that found in the developing rat dentate granule cells in v i v o 29 with immunoreactivity found in the soma, throughout the length of the axon and in the dendrites (not shown). Although GAP-43 immunoreactivity did not help distinguish axons from dendrites, the phase-contrast appearance, together with M A P - 2 immunoreactivity, provided the basis for identifying subsets o f neurites as axons or dendrites.
Effect of basic fibroblast growth factor on neurite branching b F G F produced a concentration-dependent increase in the number of axonal branch points on day 8 (Fig. 2). b F G F (10-100 ng/ml) produced a 1.6- to 2-fold increase in the number of axonal branch points. In contrast, at the concentrations tested b F G F did not change the number of branch points on individual dendrites. The onset of b F G F induction of axonal branching occurred approximately on day 5 in culture (Fig. 3). In addition to increasing the number of branch points per individual axon, b F G F also induced a more rounded appearance of the soma and a small (20%) but statistically significant increase (P < 0.05, one-way A N O V A ) in the number of dendrites per cell.
Effect of the neurotrophins on neurite branching We also examined the morphoregulatory effects of N G F , B D N F , NT-3 and N T - 4 on dentate gyrus neurons, Cells were treated with varying concentrations of neurotrophic factors for eight days. While
testing each neurotrophic factor, b F G F (10 ng/ml) was included in a sister well on the same plate as the positive control. B D N F produced a concentration-dependent increase in the number of axonal branch points (Fig. 4A). At concentrations of 10 and 100 ng/ml, B D N F increased the number of axonal branch points by 36% and 48%, respectively. In contrast with its effect on axonal branching, B D N F did not change the number of branch points per individual dendrite. NT-4 also produced a small but concentrationdependent increase in the number of axonal branch points (28% and 34% increase by 10 and 100 ng/ml, respectively; (Fig. 4B). Like B D N F , NT-4 did not influence the branching of individual dendrites. NT-3 had only a modest effect (20% increase at 100 ng/ml) on axonal branching, detectable only at 20
-I
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m
*
AXON
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15-
Z
010tlJddd 150 z 0-
0
0.1
1
10
30
100
bFGF (nglml)
Fig. 2. Concentration dependence of bFGF effect on neurite branching. Cells were grown in the presence of 0 (control), 0.1, 1, 10, 30 or 100ng/ml of bFGF for eight days and axonal and dendritic branches counted. Bars represent mean + S.E.M. of 19-49 neurons. Asterisk indicates significant difference from controls (P <0.05, one-way ANOVA).
766
M . N . Patel and J. O. McNamara
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Fig. 3. Time course of bFGF induction of neurite branching. Cells were grown in the presence of control media or 10 ng/ml bFGF and branch points counted on axons (A) and dendrites (B) on days 4-8. Bars represent mean _+ S.E.M. of 11~20 neurons.
the highest c o n c e n t r a t i o n used (Fig. 4C). NT-3 h a d no effect o n the n u m b e r o f dendritic b r a n c h points. In c o n t r a s t to b F G F , B D N F , NT-3 a n d NT-4, N G F h a d no effect o n axonal or dendritic b r a n c h i n g at any o f the c o n c e n t r a t i o n s tested (Fig. 4D). In order to ensure t h a t the N G F p r e p a r a t i o n was biologically active, we tested its ability to induce neurite outg r o w t h in undifferentiated PC 12 cells. In undifferentiated PC12 cells, N G F t r e a t m e n t (50 ng/ml) for five
days p r o d u c e d r o b u s t neurite extension c o m p a r e d to vehicle-treated cells (data not shown). The e n h a n c e d axonal b r a n c h i n g produced by multiple n e u r o t r o p h i c factors led us to query w h e t h e r these actions were additive a n d / o r synergistic. T o test this idea, a single s u b m a x i m a l conc e n t r a t i o n (10 ng/ml) o f each n e u r o t r o p h i n was first examined in sister cultures (Table 1). The most p r o m i n e n t effect was f o u n d with b F G F (73%, P<0.05), B D N F (34%, P < 0 . 0 5 ) and NT-4
~51 A
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n 0
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Fig. 4. Concentration dependence of neurotrophins on neurite branching. Cells were grown in the presence of 0 (control), 1, 10 or 100 ng/ml of BDNF (a), NT04 (B), NT-3 (C) and NGF (D) for eight days and axonal and dendritic branches counted. Bars represent mean + S.E.M. of 12-37 neurons. Closed bars indicate axonal branch points and open bars indicate dendritic branch points. Asterisk indicates significant difference from controls (P < 0.05, one-way ANOVA).
Neurotrophic factors and axonal branching DISCUSSION
Table 1. Comparison of neurotrophic factor efficacies in enhancing axonal branching Treatment
Branch points/axon
Control bFGF NT-4 BDNF NT-3 NGF
767
7.3 __+0.4 12.6 + 0.7* 10.3 _+ 0.6* 9.8 __+0.6* 8.8 _+ 0.6 6.8 __+0.7
Cells were grown in the presence of 10ng/ml bFGF, NT-4, BDNF, NT-3 or N G F for eight days and axonal branch points counted. Values represent mean+S.E.M, of 1636 neurons. Asterisk indicates significant difference from controls (P < 0.05, one-way ANOVA).
(41%, P < 0.05), confirming the results o f concent r a t i o n - r e s p o n s e studies. A l t h o u g h small additive effects m a y be found, n o evidence of synergy was f o u n d with b F G F a n d B D N F or b F G F a n d NT-4, respectively (Table 2).
The effect of basic fibroblast growth factor on neuronal survival T o ensure t h a t b F G F induced neurite b r a n c h i n g r a t h e r t h a n s u p p o r t the survival of a highly b r a n c h e d s u b p o p u l a t i o n o f neurons, we m e a s u r e d the effect o f b F G F o n n e u r o n a l survival in o u r cultures. Neurons in selected fields were c o u n t e d 24 h after plating (day 2), w h e n the n e u r o n s could be distinguished from n o n - n e u r o n a l cells. T h e same fields were relocated a n d c o u n t e d o n day 8. U n d e r control conditions n e u r o n a l n u m b e r s decreased by 4 5 % on day 8 (12 _+ 2 a n d 7 _+ 1 n e u r o n s per field o n days 2 a n d 8, respectively; n = 4). b F G F p r o d u c e d a small increase in n e u r o n a l survival o n day 8 (13_+ 2 a n d 10 _+ 2 n e u r o n s per field o n days 2 a n d 8, respectively; n = 6 ) , which was n o t statistically significant. A l t h o u g h b F G F showed some i m p r o v e m e n t in neuronal survival, we were u n a b l e to detect statistical significance o f its effect in this limited study. Imm u n o s t a i n i n g for G F A P revealed a m a r k e d increase in the n u m b e r a n d size o f glial cells in the presence o f b F G F (data n o t shown).
T h r e e principal findings emerge from this work. (1) Multiple n e u r o t r o p h i c factors e n h a n c e d axonal b u t n o t dendritic b r a n c h i n g o f d e n t a t e gyrus neurons. (2) The r a n k order of effectiveness was: b F G F > B D N F > N T - 4 > NT-3; N G F was ineffective. (3) N o additive or synergistic effets o f these n e u r o t r o p h i c factors were detected. R o b u s t sprouting o f granule cell axons is observed in m a n y a n i m a l models a n d in h u m a n epilepsy itself, yet the underlying molecular m e c h a n i s m s are unknown. Since n e u r o t r o p h i c factors exert m o r p h o r e g ulatory effects o n neurons, 2 the fact t h a t limbic seizures produce dramatic, transient increases in the expression o f genes e n c o d i n g n e u r o t r o p h i c factors a n d their receptors 3'4'u'~3'~4'2°suggests t h a t one consequence o f these factors m i g h t be i n d u c t i o n axonal sprouting. Since n e u r o t r o p h i c factors evoke striking effects in some b u t n o t all n e u r o n a l populations, the question arose as to whether, a n d which, neurotrophic factors m i g h t exert m o r p h o r e g u l a t o r y effects o n dentate gyrus neurons. A m o n g the diverse morphological properties one m i g h t analyse, axonal b r a n c h i n g seemed of particular relevance since the granule cell axons in the s p r o u t e d c o n d i t i o n exhibit r o b u s t branching. 33,35 The present findings provide direct evidence t h a t some of the n e u r o t r o p h i c factors u n d e r g o i n g increased expression in the dentate gyrus following seizures exert m o r p h o r e g u l a t o r y effects o n dentate gyrus neurons, the most r o b u s t effects being observed with b F G F a n d B D N F . O u r results are consistent with the idea t h a t seizure activity-driven n e u r o t r o p h i c factor gene expression c o n t r i b u t e s to the axonal b r a n c h i n g a n d s p r o u t i n g o f the dentate granule cells evident in m a n y of the epilepsies. The expression o f distinct n e u r o t r o p h i c factor receptors in d e n t a t e gyrus n e u r o n s likely c o n t r i b u t e s to the specificity of the m o r p h o r e g u l a t o r y effects of these diverse n e u r o t r o p h i c factors. B D N F a n d N T - 4 showed a striking similarity in terms of the magnitude a n d c o n c e n t r a t i o n dependence o f their effect on axonal branching. This is consistent with their preference for the same receptor, TrkB. U n l i k e B D N F a n d NT-4, NT-3 was effective at increasing axonal b r a n c h i n g only at the highest c o n c e n t r a t i o n tested
Table 2. The combined effect of basic fibroblast growth factor and brain-derived neurotrophic factor or neurotrophin-4 on neurite branching Experiment 1 Treatment Control bFGF NT bFGF + NT
Experiment 2
Axon
Dendrite
Axon
Dendrite
6.0 -t- 0.4 8.4 + 0.4* 8.3 + 0.8* 9.6 __+0.7*
1.6 + 0.1 1.1 __+0.1 1.2 + 0.1 1.6 + 0.2
7.3 __+0.5 11.0 + 0.8* 9.8 __+0.6* 12.0 + 0.9*
1.6 + 0.I 1.9 -I- 0.1 1.4 + 0.1 1.8 _ 0.2
Cells were grown in the presence of 10ng/ml bFGF alone or in combination with neurotrophin (NT). "NT" in experiments 1 and 2 represents 10 ng/ml BDNF and NT-4, respectively. Values represent axonal and dendritic branch points (mean + S.E.M. of I[~17 neurons in each experiment). Asterisk indicates significant difference from controls (P < 0.05, one-way ANOVA).
768
M.N. Patel and J. O. McNamara
(100 ng/ml). This finding is consistent with the ability of NT-3 to increase Fos-like immunostaining in the dentate gyrus of hippocampal explant cultures at the same concentration.9 While NT-3 is the preferred ligand for TrkC receptors, NT-3 also binds and activates TrkB receptors, although less potently and effectively than BDNF. 4° It seems plausible that axonal branching in our cultures is mediated by TrkB and not TrkC receptors, and the effect of NT-3 was mediated by its action on TrkB receptors. The effects of b F G F could be mediated by the direct interaction of b F G F with F G F receptor (e.g., FGFR1 or f i g ) expressed on dentate gyrus neurons.4s Alternatively, the morphoregulatory effects of b F G F observed here could be mediated indirectly through an action on astrocytes, since b F G F enhanced both size and numbers of astrocytes in these experiments. The absence of N G F responses in dentate gyrus neurons is consistent with previous reports of the lack of N G F effect on neurite outgrowth in cultured hippocampal neurons28 and the absence of TrkA receptors in dentate granule cells in vivo 1~.30 or in hippocampal neurons in v i t r o . 19
The absence of morphoregulatory effects of N G F on dentate dyrus neurons raises the question as to whether the seizure-induced expression of N G F in the dentate granule cells might exert morphoregulatory effects on other neuronal populations in t)il)o. 39 Importantly, seizures not only increase N G F mRNA but also increase N G F protein-like neurotrophic activity in hippocampal extracts isolated from rats following kainate-induced seizures,25 these extracts enhance survival of sympathetic neurons maintained in primary culture, an effect that can be inhibited by anti-NGF antibodies.25 Although it was not certain that the anti-NGF antibodies exerted their effects on N G F p e r se, the results of Lowenstein et al. 25 support the idea that N G F protein is expressed in the hippocampus following limbic seizures. One possibility is that N G F expressed in dentate granule cells exerts its effect on neuronal populations other than the granule cells themselves. The survival-promoting effects of N G F on cholinergic basal forebrain neurons39 have predicted the role of N G F as a target-derived neurotrophic factor for innervating cholinergic neurons of the basal forebrain. The trophic effects of hippocampal infusions of N G F on septal cholinergic neurons ~7 are consistent with this idea. The presence of sprouting of septal cholinergic afferents after kainate-evoked seizures may be one consequence of seizure-evoked expression of N G F mRNA. H'~2 Among the diversity of neurotrophic factors whose expression is enhanced by seizures, our findings suggest that b F G F and BDNF in particular may contribute to the axonal sprouting of dentate granule cells in the epileptic brain. This idea raises the interesting question as to the route of access of the growth factors to the receptors on the neurons. That is, the most prominent site of increased expression of b F G F immunoreactivity following seizures is in as-
trocytes in the molecular leyer of the dentate gyrus and to a lesser extent in the granule cells themselves. 14'45 Seizure-induction of b F G F immunoreactivity is paralleled in both distribution and time course by the induction of immunoreactivity of an F G F receptor, FGFR1:5 If b F G F contributes to the axonal sprouting of the granule cells in vivo, it will be interesting to determine whether b F G F expression in the granule cells exerts autocrine and/or paracrine effects or whether b F G F derived from astrocytes contributes to these effects. With respect to BDNF, kainate-induced seizures are associated with a redistribution of BDNF immunoreactivity from cell soma and dendrites of CA3 and dentate granule cells to the neuropil in the mossy fiber zone: 9 Together with the expression of full length TrkB receptor mRNA in the dentate granule cells, this led Wetmore et al. 49 to suggest that release of BDNF from soma and dendrites into axon terminal zones may promote axonal sprouting in a paracrine or autocrine fashion after kainate-induced seizures. The selective effects of the neurotrophic factors on branching of axons but not on dendrites in dentate gyrus neurons is curious. Although morphoregulatory effects of neurotrophic factors have been identified in diverse populations of neurons maintained in primary culture by numerous investigators, 16'3134'26'46'47 few studies28 have distinguished axons from dendrites. It seems likely that the morphoregulatory effects of neurotrophic factors in vivo will impact on the structure of both axons and dendrites, culminating in synapse formation. This raises the interesting possibility that the initial actions of these growth factors are exerted on the axon of dentate gyrus neurons; for example, the proteins synthesized in response to the neurotrophic factors may be preferentially targeted to the axons. Whether the axon specificity of these effects is specific to dentate gyrus neurons is currently under investigation. Although the absence of effects on dendritic branching is curious, the presence of morphoregulatory effects of b F G F and these neurotrophic factors on the axons of dentate hyrus neurons strengthens the likelihood that these factors contribute to the axonal sprouting of the granule cell axons in vivo. CONCLUSIONS
Two major findings emerge from this work. (1) Multiple neurotrophic factors selectively increased axonal but not dendritic branching of dentate gyrus neurons. (2) The rank order of effectiveness was: b F G F > BDNF > NT-4 > NT-3; N G R was ineffective. These in vitro findings parallel mossy fiber sprouting in t, ivo and are consistent with the idea that activity-driven gene expression contributes to axonal sprouting in diverse forms of epilepsy. Acknowledgements--This work was supported by a grant
from the National Institutes of Health, NS 32334.
Neurotrophic factors and axonal branching
769
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