Ciliary Neurotrophic Factor, Unlike Nerve Growth Factor, Supports Neurite Outgrowth but not Synapse Formation by Adult Lymnaea Neurons

Naweed Syed, ’ Peter Richardson,* and Andrew Bulloch’,*

T2N 4N1 and Division of Neurosurgery, Montreal General Hospital, Montreal, Quebec, Canada, H3G 1A4

Neuroscience Research Group, University of Calgary, Faculty of Medicine, Calgary, Alberta, Canada, 1

SUMMARY

The nerve growth factor ( N G F ) family and ciliary neuro- trophic factor (CNTF) support survival and /or neurite outgrowth of many cell types. However, it is not known whether the neurite outgrowth induced by neurotrophic factors results in the formation of synapses. We tested NGF and CNTF for their ability to induce neurite out- growth and synapse formation in vitro by interneurons from the mollusc Lymnaea. Dopaminergic and peptider- gic interneurons survived in the absence of neurotrophic

factors but exhibited robust outgrowth in response to both NGF and CNTF. Chemical synapses formed be- tween these interneurons and their target neurons cul- tured in NGF, but synapses were absent in CNTF. Sur- vival, neurite outgrowth, and synaptogenesis are there- fore differentially regulated in these neurons. o 1996 John Wiley & Sons, Inc. Keywords: neurotrophic factor, outgrowth, synaptogene- sis, culture, mollusc.

INTRODUCTION

Molecules from the nerve growth factor (NGF) family (the neurotrophins) and ciliary neuro- trophic factor (CNTF) support survival and neu- rite outgrowth in vitro from many cell types (Korsching, 1993). It is usually impossible to sep- arate survival from neurite outgrowth as distinct functions of neurotrophic factors. One exception is provided by adult sensory neurons that depend on NGF/ brain-derived neurotrophic factor ( BDNF) for neurite outgrowth, but not survival (Lindsay, 1988). Here we show that CNTF can also support neurite outgrowth independently of survival.

Recent observations have indicated a role for neurotrophic factors in synapse formation and syn- aptic plasticity. In vitro the neurotrophins or

Received May 24, 1995; accepted October 17, 1995 Journal ofNeurobiology, Vol. 29, No. 3, pp. 293-303 ( 1996) 0 1996 John Wiley & Sons, Inc. CCC 0022-3034/96/030293-11

* To whom correspondence should be addressed.

CNTF, or both, modulate the spontaneous release of acetylcholine at developing neuromuscular junctions (Lohof et al., 1993; Stoop and Poo, 1995 ) , acetylcholine release from the central ner- vous system (CNS) synaptosomes (Knipper et al., 1994b), synaptic transmission between cultured hippocampal neurons (Girod et al., 1994; Lessman et al., 1994), and synaptic transmission in the hip- pocampal slice (Knipper et al., 1994a; Kang and Schumann, 1995). In v i v a the period of synaptic plasticity in the cat visual cortex is extended by NGF (Maffei et al., 1993). These observations led us to pose the question: “Does the neurite out- growth induced by two neurotrophic factors (NGF and CNTF) result in the formation of functional synapses?”

The characterization and function of neuro- trophic molecules in the invertebrates has received relatively little attention. Evidence supports the ex- istence of neurotrophins in both Drosophila (Hayashi et al., 1992) and Lymnaea (Ridgway et al., 199 1 ). For example, brain-conditioned me- dium is required for sprouting by Lymnaea neu-

293

294 Syed et al.

rons. Preabsorption of conditioned medium, how- ever, with an affinity-purified murine NGF antise- rum removes the factor that induces sprouting by motoneurons and interneurons (but not neurose- cretory cells) (Ridgway et al., 1991). Moreover, murine 2.5s NGF (at 50 to 400 ng/ml) induces sprouting by Lymnaea motoneurons and interneu- rons. The relatively high doses of NGF required for Lymnaea presumably reflect a ligand/receptor mismatch. However, it is reasonable to predict that this molecular family exists in the invertebrates, since they are found in primitive vertebrates (Hallbook et al., 199 1 ); also, many other develop- mentally important molecules are conserved phy- logenetically, such as laminin ( Masuda-Nakagawa et al., 1988; Miller and Hadley, 1991 ), fibronectin (Mattson and Kater, 1988), cell adhesion mole- cules (Schacher et al., 1990), and tyrosine kinase receptors (Pulido et al., 1992) (reviewed by Bul- loch et al., 1994). Invertebrates such as the leech and the molluscs, Aplysia and Lyrnnaea and Heli- soma, provide opportunities to study synapse for- mation (for example, between interneurons and target neurons) not yet feasible in higher animals (Bulloch and Syed, 1992). For example, we have shown that Lymnaea interneurons reform specific synapses in vitro on target neurons from both Lym- naea and Helisoma (Syed et al., 1990, 1992a). In this study we extend our examination ofthe actions of neurotrophic molecules in molluscs with assays of neurite outgrowth and synapse formation by in- terneurons cultured in CNTF and NGF. Our data provide evidence that outgrowth and synapse for- mation can be differentially regulated. Some of these data were presented in abstract form (Bulloch et al., 1992).

METHODS

Animals

Animals were from our laboratory stock of Lymnaea stugnulis (originally derived from the Free University of Amsterdam) that was maintained as described by Ridg- way et al. (1991). Snails had a shell length of 15 to 20 mm (approximate age 2 to 4 months).

Cell Culture

The cell culture procedure was as described by Ridgway et al. ( I99 1 ) and Magoski et al. ( 1994). In brief, ganglia were sterilely isolated, washed in antibiotic saline, and treated with collagenase and trypsin ( 1.33 and 0.67 mg/ ml, respectively) for 30 to 40 min, and then with 0.67

mg/ml trypsin inhibitor for 10 min, all enzymes being in defined medium. Defined medium consisted of serum- free 50% Liebowitz- 15 medium (GIBCO, special order) with added inorganic salts (sodium chloride, 40 mM; potassium chloride, I .7 mM; calcium chloride, 4.1 mM; magnesium chloride, 1.5 mM; N-2-hydroxyethyl-piper- azine-N’-2-ethanesulfonic acid, 10 mM; final pH 7.9) and 20 phfofgentamicin. Ganglia were then pinned out in defined medium with 20 m M glucose. After removal of the inner sheath, identified neurons were removed one at a time with suction via a fire-polished pipette. Neurons were then transferred to poly-L-lysine-coated glass cov- erslips that had been mounted over holes drilled in Fal- con 300 l culture plates. Schematic diagrams were made of each plate to depict the position of each neuron with respect to an ink line on the side of the plate. Neurons were plated into defined medium (DM ) or DM supple- mented with either murine 2.5s NGF or rat CNTF. The purified mouse NGF was a gift from Dr. R. Murphy, whereas we tested both purified and recombinant CNTF (Gupta et al., 1992) (the majority of experiments used the latter). Neurons were considered to have sprouted if they exhibited one or more neurites of more than two soma diameters in length (our usual criterion; compare Ridgwayet al., 1991).

Special culture plates were used for the dopamine re- lease experiments (see later). To create a nonadhesive surface for neurons, the area of the plastic surface sur- rounding the coverslip was coated for 20 min with fresh, filtered (0.2 pM Millepore filter) Lymnaea hemolymph. The somata of assay neurons were then temporarily placed in this area before being impaled with an electrode and moved close to the growth cone to be tested.

Time lapse microphotography of growth cones was with a Panasonic VCR (AG 6720A) attached to a Zeiss Axiovert inverted microscope (using Nomarski optics) via a Hitachi CCD camera. The rate of advance of growth cones from several neurons in close proximity was monitored via a X I 0 objective, 14 to 18 h postplat- ing, for a period of 3 to 6 h.

Electrophysiology

Conventional electrophysiological techniques were used. Electrodes were filled with saturated potassium sulfate and permanent records obtained with a Gould 2200s pen recorder. A chemical synapse was considered to have developed if action potentials in an interneuron could evoke one-for-one potentials in a target neuron with the long, constant delay (50 to 100 ms) and amplitude ( 1 to 10 mV) characteristic of postsynaptic potentials ( PSPs) in L,yrnnaea both in vivo and in vitro (Syed et al., 1990, 1992a, 199213; Magoski et al., 1995). Dopamine was pressure applied to neurites via wide-bore ( 1 to 2 p M ) pipettes filled with 10 pMdopamine connected to an Ep- pendorf microinjector system (model 5242 ). In order to restrict the flow of dopamine to neurites, these pipettes were placed in a fast stream of sterile saline that was de-

CNTF, NGF, and Synapse Formation by Lymnaea Neurons 295

livered and recovered at I0 to 15 ml /min by two chan- nels of a peristaltic pump (LKB Microperspex 2 132). To assay dopamine release a freshly isolated soma of a vis- ceral I motoneuron was placed in the nonadhesive area (see before) of a culture plate containing the sprouted interneuron. This soma was then impaled with a micro- electrode (whose tip had been previously dipped into a 0.2% solution of poly-L-lysine) and positioned within 10 to 50 pMof large motile growth cones of the interneuron. The interneuron was then stimulated with a long depo- larizing current pulse to evoke a train of action potentials (this method was adapted from the previously developed assay for transmitter release described by Haydon and Man-Son-Hing, 1988). In each experiment the test soma was held in place for at least 30 min during multiple stim- ulation trials. For experiments involving both dopamine release by interneurons and dopamine responses by post- synaptic neurons, the relevant neurons were plated in isolation. In our hands freshly isolated somata exhibit a similar sensitivity to dopamine as neurons in isolated ganglia (that is, bath application of 100 pM dopamine elicits a 5 to 10 mV depolarization in both cases). The criterion for a positive response in both dopamine release and dopamine response assays was a clear and reproduc- ible signal above the background noise level.

RESULTS

This study progressed in three stages. First, since CNTF (unlike NGF) was untested on Lymnaea, we examined whether CNTF can induce out- growth from specific Lymnaea neurons in vitro. Second, we tested interneurons cultured in either NGF or CNTF for their ability to form specific syn- apses in vitro. Third, since interneurons failed to form synapses in CNTF, we examined some of the possible underlying mechanisms.

Specific Lymnaea Neurons Respond to CNTF

Identified Lymnaea neurons were isolated and cul- tured in defined medium supplemented with CNTF at concentrations from 250 to 4000 pM. In preliminary trials CNTF was added prior to plating of neurons, but it appeared to impair adhesion to the poly-L-lysine-coated surface of plates. In all ex- periments reported here, CNTF was added to plates about 5 h after plating the neurons, at which point most neurons were firmly attached. In paral- lel control experiments, neurons were plated in DM, or in DM supplemented with murine 2.5s NGF (400 ng/ml ) .

Whereas little neurite outgrowth occurred in DM, many Lymnaea neurons exhibited rapid neu-

Table 1 to CNTF and NGF

Sprouting by Lymnaea Neurons in Response

Cells Sprouted (%)

CNTF NGF DM Cell Type (n) (n ) (a)

~~ ~

Motoneurons Visceral HIJK Pedal A Cerebral A Pedal F/G

Interneurons RPeD 1 VD4

RPB VF

Neurosecretory cells

81 (54) 66(160)

5 (47) 7 (27)

78 (18) 83 (12)

18 (33) 14 (43)

79(29) 6(17) 82(45) 4(56) 89(38) - 66(35) -

85 (20) 0(6) 82(17) 017)

6(34) O(28) 7(28) O(22)

rite outgrowth in response to CNTF. This response was specific in that it was limited to members of two classes of neurons, motoneurons and interneu- rons, the neurosecretory cells being unresponsive [Fig. 1 (A); Table I] . Among the motoneurons, neurite outgrowth was limited to specific groups of cells (pedal A, visceral HIJK), other groups (cerebral A and pedal F/G cells) showing little re- sponse. The two types of interneurons tested, how- ever, both showed outgrowth in virtually all cases, making these ideal for subsequent tests of synapse formation.

In parallel control experiments with NGF, re- sults were similar to our previous studies (Table 1 ) (Ridgway et al., 199 1 ) . As expected, all motoneu- rons and interneurons, but not neurosecretory cells, responded to NGF. Thus, the spectrum of neurons that respond to CNTF overlaps with, but differs from, that which respond to NGF. In these studies all neurons that responded to CNTF also responded to NGF, but not vice versa.

To obtain a dose response to CNTF, we ana- lyzed the pedal A cluster motoneurons, which can be readily cultured in reasonable numbers (Ridgway et al., 199 1 ). These neurons respond to CNTF over a concentration range of 250 to 1000 p M (Fig. 2) . Whereas more than 90% of pedal A neurons respond to CNTF at 1000 pM, the half- maximal response was approximately 500 p M . The steepness of this dose-response curve is remi- niscent of that to NGF (Ridgway et al., 199 1 ).

A number of striking features were observed in neurites induced by CNTF. The growth cones of neurites in CNTF were small and neurites had lat- eral filopodial-like extensions. All neurons cul-

296 Syed et al.

Figure 1 Response of Lymnaea neurons to CNTF and NGF. ( A ) Morphology of motoneu- rons, an interneuron, and neurosecretory cells after 1 day of culture in 1 n M CNTF. Each neuron was plated individually and its position mapped in the culture plate (see Methods). Extensive sprouting is evident from both the motoneurons (pedal A cluster, left hand three cells; visceral I cluster, top right hand cell) and the interneuron (RPeD1, middle upper cell) (note the extensive overlap of processes from neuron RPeDl and the VI neuron). The two neurosecretory cells (from right parietal B and visceral F cluster, lower two right-hand cells, respectively) were scored as not sprouted (the lower neuron has two processes of less than two soma diameters in length; the upper has not extended processes, rather a neurite from another neuron has extended beneath it’s soma. ( B and C). Examples of growth cones in NGF (400

CNTF, NGF, and Synapse Formation by Lymnaea Neurons 297

CNTF Dose Response

Figure 2 Dose response to CNTF. The graph shows the response of pedal A neurons to various concentrations of CNTF. Each data point is from several plates; total number of neurons = 31 (250 pM), 63 (500 pM), 56 (750pM),and36( lOOOpM).

tured in CNTF exhibited growth cones that were small compared with those observed in NGF. These morphological properties are exemplified by high magnification photomicrographs of primary neurites extending in response to CNTF and NGF [Fig. 1 (B, C)] . The growth cones in NGF are more typical from Lymnaea neurons sprouting in brain- conditioned medium (not shown). It was also no- table that growth cones appeared to advance more rapidly in CNTF than in NGF. Time lapse video recordings of growth cones on primary neurites from pedal A neurons extending 14 to 18 h after plating confirmed this impression. The estimated

advance rates of these growth cones were 49 _t 9 and 27 f 6 pm/h in CNTF and NGF, respectively (n = 12and ll;p<O.OOOl,Student’sttest). The rate of advance in NGF is comparable to that in conditioned medium (Syed, unpublished ob- servation).

Synapse Formation: CNTF versus NGF

We examined the formation of synapses between two identified Lymnaea interneurons and target neurons in cell culture. These neurons form appro- priate chemical synapses on target neurons when cultured in brain-conditioned medium ( Syed et al., 1992a), but nothing was known regarding the role of specific neurotrophic factors in synaptogenesis.

In the first set of experiments we cultured Lym- naea interneurons and their target neurons in DM supplemented with either CNTF or 2.5s NGF. We tested both a dopaminergic interneuron [right pedal dorsal 1 (RPeDl )] cultured together with normal target neurons from the visceral I and J ( VI and VJ) cluster (Winlow et al., 198 1 ), as well as a peptidergic interneuron [visceral dorsal 4, (VD4)] cultured together with its normal target neurons from the pedal A ( PeA) group (Syed and Winlow, 1989). All of these cell types exhibited neurite out- growth in response to CNTF [ Fig. 1 (A)] and also NGF as expected from our previous results. These neurons responded to NGF by forming chemical synapses after 1 day of culture in all cases tested (Figs. 3 and 4), but no synapses were observed in cultures in which outgrowth was induced by CNTF (Fig. 3 ) even after periods as long as 5 days. Both excitatory (Figs. 3 and 4) and inhibitory (Fig. 4) synaptic connections were observed in NGF with no evidence of electrical coupling. These interac- tions had the one-for-one action potential to PSP relationship, long, constant delay (50 to 100 ms) and amplitudes (1 to 10 mV) characteristic of chemical synapses between Lymnaea neurons both in vivo and in vitro (Syed et al., 1990; 1992a, 1992b; Magoski et al., 1995 ) . The failure of synap- togenesis in CNTF occurred despite the presence of overlapping neuritic processes between inter-

ng/ml) and CNTF ( 1 nM), respectively. in both cases the growth cones are on primary neu- rites of a single pedal A motoneuron cultured for 1 day. Note the unusually compact nature of the growth cones in CNTF as well as lateral filopodial-like extensions from neurites. Scale bar: ( A ) = 200 pm; (B, C) = 50 pm.

298 S-ved et a/.

NGF

I \

1 s RPeD 1

CNTF

RPeA VI

10 I r n

Figure 3 Dual intracellular recordings from interneurons and target neurons after 1 day of culture. Stimulation of interneuron VD4 evoked excitatory postsynaptic potentials ( EPSPs) in the target neuron ( RPeA) after culture in NGF but not CNTF (left panels). Similarly, an excitatory connection developed from interneuron RPeDl to its target neuron (VI) in NGF but not CNTF (right panels) (for RPeD1, 14 cell pairs were tested in both NGF and CNTF for VD4, seven cell pairs were tested in NGF and four in CNTF). Bursts of action potentials were evoked in neuron VD4 and RPeDl with long depolarizing current injections (at bars).

neurons and their target neurons in CNTF cultures [Fig. 1 (A) ] .

Failure of Synapse Formation in CNTF: Mechanisms

To investigate the basis of this differential synapto- genic response to NGF and CNTF, we examined both presynaptic and postsynaptic functions of one of the interneurons, the giant dopamine neuron (RPeD 1 ), and its target neurons in culture. Dopa- mine is the only known transmitter of this in- terneuron (Magoski et al., 1995). To test whether dopamine is released by neurons cultured in NGF or CNTF, freshly isolated somata of target neurons were impaled with a microelectrode and juxta- posed close to growth cones of interneurons in cul- ture (Haydon and Man-Son-Hing, 1988). Action

potentials were then elicited in the interneuron by intracellular stimulation to attempt to evoke the re- lease of transmitter, which would be expected to cause a nonsynaptic depolarization of the newly positioned target soma. Depolarizing responses were observed in all recordings from neurons cul- tured in NGF, but were absent in all cases when neurite outgrowth had been evoked by CNTF (Fig. 5 ) . As expected in this type of nonsynaptic interac- tion, no delay was evident (compare the synaptic delays in Figs. 3 and 4) . In this assay, the growth cones of neurites extending in response to CNTF do not appear to release dopamine.

We next examined the ability of neurites from target neurons cultured in either NGF or CNTF to respond to transmitter. Whereas dopamine appli- cation caused the expected depolarization of neu- rons cultured in NGF, neurons cultured in CNTF

CNTF, NGF, and Synapse Formation by Lymnaea Neurons 299

/ RPeD1 /

\

L

250 ms (a) 500 ms (b)

Figure 4 Further examples of chemical synapses between neurons after 1 day of culture in NGF (400 ng/ml) at high chart recorder speeds. ( A ) The one-for-one action potential to EPSP relationship and constant delay is evident in this paired recording from a RPeDl interneuron and a VI target neuron. ( B ) An example of an inhibitory chemical synapse from RPeDl to another target neuron ( a visceral J neuron). Action potentials were driven in RPeDl with depolarizing current pulses (between the dots; bridge out of balance in A). These records also emphasize the lack of electrical coupling that would be evident as both slow depolarizations of the postsynaptic neuron coincident with the current applied to the interneuron and fast depolarizations in the target neurons that would coincide with the presynaptic action poten- tials.

failed to respond to dopamine (Fig. 6). Thus, neu- rites extending in response to CNTF lack func- tional dopamine receptors or coupled pathways.

We then asked if NGF can reverse or overcome the failure of CNTF to induce synapse formation. Neurite outgrowth was induced by CNTF for 1 day, after which the culture medium was ex- changed to one containing NGF. Whereas synapse formation did not occur after 1 day of culture in CNTF, connections were subsequently detected af- ter exposure of the same neurons for an additional

day to NGF (five cell pairs tested in four cultures; data not shown).

DISCUSSION

Several signaling pathways activated by the neuro- trophins and CNTF have been discovered. The neurotrophins initiate signaling via trk receptor ty- rosine kinases, whereas CNTF acts via soluble Jak/ Tyk tyrosine kinases that are preassociated with its

300 Syed et al.

Detection of doparnine release

RPeDl h CNTF

VI

RPeDl I 1 mV

40

1 0 s

Figure 5 Assay of transmitter release from RPeD1 in- terneurons cultured for 1 day in NGF or CNTF. Stimu- lation of RPeD 1 evokes a nonsynaptic depolarization of the assay target neuron (VI ) when the interneuron was cultured in NGF ( n = 5 ) but not CNTF ( n = 13) .

receptor complex (reviewed by Kaplan and Ste- vens, 1994; Stahl and Yancopoulos, 1994). Once activated, these kinases in turn initiate multiple in- tracellular signaling pathways, some of which lead to transcriptional regulation. It is notable that many of the signal molecules activated by CNTF are also activated by the neurotrophins. This com- bination of convergent and separate signal path- ways is presumed to underlie the ability of the neu- rotrophins and CNTF to exert common, separate, or synergistic actions (Korsching, 1993; Stahl and Yancopoulos, 1994). We have now shown that NGF can support both neurite outgrowth and syn- apse formation by specific Lywlnaea neurons. On the other hand, CNTF evokes outgrowth that does not result in synaptogenesis. Both presynaptic and

postsynaptic mechanisms appear to be impaired in neurons cultured in CNTF. These deficiencies must reflect the failure of CNTF to activate signal- ing molecules in Lymnaea neurons that are acti- vated by NGF. As a caveat, it should be empha- sized that these results could, in principle, reflect the inability of rat CNTF to mimic the endogenous Lymnaea ligand. Alternatively, or additionally, our culture conditions or the adult nature of the neurons may be limiting in some respect. Whereas the demonstration of differential synapse forma- tion is the most important aspect of this study, this also appears to be the first demonstration that CNTF, like NGF, can support outgrowth indepen- dently of survival (see Introduction).

We had shown previously that Lymnaea and HeEisoma neurons reform appropriate chemical synapses when plated in brain-conditioned me- dium (Syed et al., 1990, 1992a). Since we had also shown that outgrowth in Lymnaea conditioned medium is due to an NGF-like molecule (and can be mimicked by murine NGF; see Introduction), it was not surprising that murine NGF itself sup- ported synapse formation in this study (it should be pointed out that we have not as yet looked for a CNTF-like molecule in Lymnaea, a ligand/ receptor mismatch may account for the relatively high doses needed in this study). Recently, differ- ential regulation of voltage-gated ion channels by NGF and CNTF was observed in a neuroblastoma cell line: NGF increased sodium, calcium, and po- tassium currents, whereas CNTF increased only the latter (Lesser and Lo, 1995). A low level of calcium channel expression could explain the a p parent inability of CNTF-induced growth cones of Lymnaea to release transmitter (Fig. 5 ) . Conceiv- ably, during outgrowth in vivo, CNTF-induced neurites might extend through the nervous system and avoid inappropriate targets. Under the influ- ence of target-derived factors (such as neuro- trophins), these neurites may gain the ability to form synapses on appropriate targets.

Our results show that target neurons extending neurites in response to CNTF fail to exhibit the ex- pected response to dopamine (Fig. 6) . Whether this reflects a lack of newly synthesized receptors or coupling mechanisms remains to be determined. Another consideration is whether dopamine re- sponsiveness still exists in the somata of the target neurons or whether down-regulation of receptors or other molecutes occurs. Although our fast per- fusion system was designed to restrict flow of transmitter to neurites, it is difficult to rule out the possibility that somata were exposed to transmit-

CNTF, NGF, and Synapse Formation by Lymnaea Neurons 301

- p p min t n ti n rit

NGF

h

CNTF

I 10s

Figure 6 Assay for transmitter response from target neurons cultured for 1 day in either NGF or CNTF. Depolarizing responses to dopamine occurred in neurites that had extended in re- sponsetoNGF(n = 5)but not CNTF(n = 7) .

ter. Resolution of this issue will require further ex- periments. For example, cultured neurons could be exposed to CNTF under culture conditions that prevent adhesion and therefore sprouting. The re- sponsiveness of these somata could then be directly examined.

Our observations provide new insight into pos- sible functions of the neurotrophins in synapse for- mation. Ironically, although NGF was first associ- ated with neurite outgrowth in vitvo, whether or not it functions in outgrowth (in addition to prevent- ing apoptosis) during development in vivo is un- known (Barde, 1994). Taken together with the ob- servations that both activity and hormones can reg- ulate neurotrophin mRNA levels in central nervous system (Lindholm et a]., 1994), our work supports a role for neurotrophins in synaptic plas- ticity (see Introduction).

In addition to its effects on neuronal gene regu- lation, diverse acute effects of NGF have been re- ported. For example, NGF prolongs action poten- tials of cultured neurons from dorsal root ganglia, apparently via release of endogenous opiates (Shen and Crain, 1994). In Lyrnnueu we have recently observed that acute application of NGF can en-

hance voltage-activated calcium currents in some motoneurons (Wildering et al., 1995). The latter data complement work showing that NGF can modulate calcium currents (Johnson et a]., 1992) and intracellular calcium (Berninger et al., 1993, 1994) in vertebrate cells. When considered to- gether with our observations, it is apparent that neurotrophic factors may play diverse roles in de- velopment and plasticity of the nervous system.

We thank Garry Hauser for technical assistance, Neil Magoski and Wic Wildering for criticism of the text, and Sylvia Baldwin for manuscript preparation. Supported by grants from the Neuroscience Network (Canadian National Centres of Excellence) (to A.B.), the Medical Research Council (Canada) (to A.B.), and the Natural Sciences and Engineering Research Council (to N.S.).

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