Department of Physiology and Biophysics and Neuroscience Research Group, Faculty of Medicine, University of Calgary, Calgary, Alberta T2N 4N1, Canada
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ABSTRACT |
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Magoski, Neil S. and Andrew G. M. Bulloch. Trophic and contact conditions modulate synapse formation between identified neurons. J. Neurophysiol. 79: 3279-3283, 1998. We tested the ability of an identified interneuron from the mollusk, Lymnaea stagnalis, to reestablish appropriate synapses in vitro. In the CNS, the giant dopaminergic neuron, designated as right pedal dorsal one (RPeD1), makes an excitatory, chemical synapse with a pair of essentially identical postsynaptic cells known as visceral dorsal two and three (VD2/3). When the somata of the pre- and postsynaptic neurons were juxtaposed and cultured in vitro in defined medium, i.e., a soma-soma synapse, only an inappropriate electrical synapse was observed. The postsynaptic cell still responded to applied dopamine, the presynaptic transmitter, indicating that the lack of chemical synapse formation was not due to lack of dopamine receptors. When the somata were cultured apart in conditioned medium (medium previously incubated with Lymnaea CNS, thereby deriving trophic factors), the cells exhibited overlapping neurite outgrowth that resulted in an appropriate excitatory, chemical synapse from RPeD1 to VD2/3. On the other hand, when the cell pair was cultured in a soma-soma configuration, but in conditioned medium, a mixed chemical-electrical synapse was observed. Because conditioned medium could partially overcome the limitations of the soma-soma configuration and initiate chemical synapse formation, this data suggests that conditioned medium contains a factor(s) that supports synaptogenesis.
Neurons form connections with one another during both development and reinnervation after injury (Bulloch and Ridgway 1989 A laboratory raised stock of the mollusk, L. stagnalis was used (age ~1-2 mo). Dissections and electrophysiology were performed in normal Lymnaea saline [composition (in mM): 51.3 NaCl, 1.7 KCl, 4.1 CaCl2, 1.5 MgCl2, and 5.0 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES); pH 7.9].
The ability of RPeD1 to reestablish its synapse with VD2/3 first was tested by culturing the cells in defined medium (DM) such that the two somata were juxtaposed directly, i.e., in a "soma-soma" configuration (Haydon 1988
Studies using neurons from several invertebrate species have examined synapse formation between somata in vitro. In Helisoma, chemical soma-soma synapses were established between buccal neurons (Haydon 1988
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
; Haydon and Drapeau 1995
; Jacobson 1991
). Synapse formation is considered crucial in establishing specific types of neuronal connections (Haydon and Drapeau 1995
; Jessell and Kandel 1993
). Studies of invertebrate neurons have contributed significantly to our knowledge of the mechanisms that underlie synapse formation, these include work on the leech (Fernandez-de-Miguel and Drapeau 1995
; Haydon and Drapeau 1995
; Ready and Nicholls 1979
), and the gastropod mollusks, Aplysia californica (Camardo et al. 1983; Schacher et al. 1985
), Helisoma trivolvis (Haydon and Drapeau 1995
; Haydon and Kater 1988
; Zoran and Poyer 1997), and Lymnaea stagnalis (Bulloch and Syed 1992
).
; Schacher and Proshansky 1983
) or released from the CNS into conditioned medium (Bulloch and Syed 1992
; Ridgway et al. 1991
; Wong et al. 1981
), to induce neurite outgrowth. An issue yet to be thoroughly explored is whether or not contact between neurites is necessary for synapse formation or if the factors present in hemolymph or conditioned medium are more important requirements. For example, the cytokine, ciliary neurotrophic factor (CNTF) induces neurite outgrowth and contact but not synapse formation in Lymnaea neurons (Syed et al. 1996
). This suggests that neuritic contact alone is not necessarily sufficient for synapse formation. In fact, certain neurons from both Aplysia and Helisoma can form synapses between apposed cell somata (Haydon 1988
; Klein 1994
).
; Syed et al. 1990
), and the postsynaptic neurons are the giant neurons, visceral dorsal two and three (VD2/3) (Benjamin and Winlow 1981
). In the intact CNS, RPeD1 makes an excitatory, dopaminergic connection with VD2/3 (Magoski and Bulloch 1997
; Magoski et al. 1995
). The ability of this connection to reestablish itself in vitro under a variety of trophic and contact conditions, including defined medium versus brain-conditioned medium and soma-soma versus neurite-neurite configurations, is tested.
METHODS
Abstract
Introduction
Methods
Results
Discussion
References
. The CNS was placed in a mixture of 1.33 mg/ml collagenase/dispase and 0.67 mg/ml trypsin in defined medium (DM) for 20-30 min. The DM was serum-free 50% LiebowitzL-15 medium with added inorganic salts [containing (in mM) 40.0 NaCl, 1.7 KCl, 4.1 CaCl2, 1.5 MgCl2, and 5.0 HEPES; pH 7.9) and 20 µg/ml gentamicin. The CNS then was placed in a 0.67 mg/ml solution of trypsin inhibitor for 10 min. Identified neurons were isolated with a siliconized, wide-bore pipette attached to a micrometer syringe for vacuum or pressure, and plated on poly-L-lysine-coated 35-mm Petri dishes. The dishes contained 2 ml of DM or 1 ml of DM mixed with 1 ml of conditioned medium (CM)
thus a "CM" dish actually refers to a 1:1 mixture of CM and DM. The CM was made by incubating DM with 2 CNS/ml for 72 h. After plating, neurons were left in darkness and undisturbed for ~24 h.
, respectively. Electrophysiological data were collected with a dual-channel intracellular amplifier, the voltage being displayed on an oscilloscope and recorded on a chart recorder. Regardless of culture conditions, RPeD1 maintained its membrane potential between
50 and
60 mV, whereas VD2/3 had a range of
50 to
65 mV. Electrical coupling was measured by injecting a 0.5-3 s, 0.5-1 nA, hyperpolarizing, square current pulse into RPeD1. Thevoltage drop across RPeD1 (voltageRPeD1) and VD2/3 (voltageVD2/3)were determined and the coupling ratio (voltageVD2/3:voltageRPeD1), for the RPeD1 to VD2/3 electrical synapse was calculated. Dopamine was bath-applied and dissolved in saline containing 0.1% (wt/vol) sodium metabisulfite to prevent oxidation.
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
). With this particular form of contact and in DM, the neurons did not exhibit neurite outgrowth but remained essentially spherical. Note, it is typical of Lymnaea neurons not to extend neurites when cultured in DM alone. When the neurons were examined electrophysiologically, 1 day after plating, they always were found to be coupled electrically (n = 17; coupling coefficient = 0.26 ± 0.04, mean ± SE; Fig. 1, A and B). This type of connection was characterized by a lack of synaptic delay between the peak of the action potential and the inflection point of the postsynaptic potential (PSP) as well as an absence of a lengthy decay phase in the PSP. In no case did the cells display a chemical synapse. Regardless of whether or not the neurons were from the same or different CNS, there was no effect on the type of connection that formed. For five pairs of cells, the connection was examined not only at 1 day after plating, but also at 2, 3, and 4 days. At all time points, the connection was still electrical. Incidentally, attempts at examining the neurons immediately or even shortly after plating proved deleterious as the neurons were damaged easily
this was especially the case for VD2/3.
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FIG. 1.
Electrical connection between right pedal dorsal one (RPeD1) and visceral dorsal two and three (VD2/3) at a soma-soma synapse, in defined medium (DM), in vitro. A: injection of depolarizing current into RPeD1 produced a depolarization and electrotonic potentials in VD2/3. B: injection of hyperpolarizing current into RPeD1 produced a corresponding hyperpolarization in VD2/3. Membrane potentials: RPeD1 = 62 mV; VD2/3 =
56 mV. Bars denote duration of current injection into RPeD1. C: response of VD2/3 to bath-applied dopamine. Neuron VD2/3 was in vitro and at a soma-soma synapse cultured in DM. Action potentials were truncated. Membrane potential =
50 mV. Dopamine (DA, 100 µM) was applied at the bar.
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FIG. 2.
Chemical connection from RPeD1 to VD2/3 at a neurite-neurite synapse in CM in vitro. A: action potential evoked in RPeD1 produced an excitatory postsynaptic potential (EPSP) in VD2/3. B: when hyperpolarizing current was injected into RPeD1, it did not produce a hyperpolarizing voltage drop in VD2/3, indicating a lack of electrical coupling. Membrane potentials: RPeD1 = 64 mV; VD2/3 =
55 mV. Bars denote duration of current injection into RPeD1.
; Magoski and Bulloch 1997
; Magoski et al. 1995
; Werkman et al. 1991
). To determine if lack of chemical synapse formation was due to an absence of functional dopamine receptors on the postsynaptic cell, 100 µM dopamine was bath-applied to VD2/3 at the soma-soma synapse in DM. Dopamine reliably evoked a depolarization in VD2/3 (n = 8; Fig. 1C). This depolarization was similar to that induced by applying dopamine to VD2/3 in the intact CNS (refer to Magoski and Bulloch 1997
; Magoski et al. 1995
); furthermore, the depolarization mimicked the effects of RPeD1 stimulation on VD2/3 in the intact CNS (refer to Benjamin and Winlow 1981
; Magoski and Bulloch 1997
; Magoski et al. 1995
).
; Ridgway et al. 1991
; Wong et al. 1981
). If both neurons sprouted and extended neurites into the same area of the culture surface, the result was neurite-neurite contact. Again, neurons were examined electrophysiologically 1 day after plating. In all cases of neurite-neurite contact, a chemical synapse was detected, with an action potential in RPeD1 evoking an excitatory postsynaptic potential (EPSP) in VD2/3 (n = 5; Fig. 2A). The EPSP displayed the characteristic features of chemical transmission with a short but distinct latency between the peak of the action potential and the inflection point of the PSP as well as a slow decay phase of the PSP. Passage of hyperpolarizing current into RPeD1 did not produce a corresponding voltage drop in VD2/3, indicating that an electrical synapse had not developed (Fig. 2B). We also tested the chemical nature of the synapse by perfusing with a low Ca2+ saline (~1 µM free Ca2+); this resulted in complete elimination of the postsynaptic response (n = 2; data not shown).
RPeD1 in one case and VD2/3 in the other. In the other seven pairs, neither of the cells exhibited neurite outgrowth. Regardless of the presence of absence of outgrowth the resulting synapse was the same: when examined 1 day after plating, the synapse was observed to be a mixed chemical/electrical connection (n = 9; Fig. 3). Typically, the chemical component of the synapse dominated (Fig. 3A), but an electrical component could be seen at a more rapid time base (Fig. 3B) or on injection of hyperpolarizing current into RPeD1 (Fig. 3C). The electrical component of this synapse was weak, with a coupling coefficient of 0.10 ± 0.01. This coupling coefficient was significantly less than that observed at the soma-soma synapse in DM (P < 0.05, unpaired Student's t-test).
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FIG. 3.
Mixed chemical-electrical connection between RPeD1 and VD2/3 at a soma-soma synapse, in conditioned medium (CM), in vitro. A: action potential evoked in RPeD1 produces a large EPSP in VD2/3. B: when the synapse was observed on a more rapid time scale, an initial, short-latency electrical component could be observed, followed by a long-lasting chemical component. The electrical component is apparent just after the step depolarization of RPeD1 as the presynaptic cell depolarizes so does the postsynaptic cell. Spike in RPeD1 also produced a small electrotonic potential in VD2/3. Chemical component of the synapse had a very rapid latency (<5 ms). This is to be expected because the recording electrodes are close to the site of transmission and there is no attenuation of the voltage signal by the cable properties of pre- and postsynaptic axons. C: injection of hyperpolarizing current into RPeD1 produced a small, hyperpolarizing voltage drop in VD2/3, indicating the presence of weak electrical coupling. Membrane potentials: RPeD1 =
60 mV; VD2/3 =
60 mV. Bars denote the duration of current injection into RPeD1 or VD2/3.
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
), however, at ~40% of these synapses, electrical coupling also was detected. In the leech, soma-soma contact between Retzius neurons typically resulted in electrical coupling, although in 20% of the cases, weak chemical connections were established after 3 days in culture (Liu and Nicholls 1989
). Chemical synapses also were established between the somata of sensory and motor neurons from Aplysia (Coulson and Klein 1997
; Klein 1994
). It is important to note that all of those neurons were maintained in either hemolymph (for Aplysia and Helisoma) or in DM supplemented with serum (for leech) or on extracellular matrix (also for leech).
VD2/3 synapse in the intact CNS is dopamine (Magoski and Bulloch 1997
; Magoski et al. 1995
). It appears that the lack of chemical synapse formation at opposed somata in DM is not due to a lack of postsynaptic dopamine receptors because dopamine caused an appropriate depolarization in VD2/3 (Fig. 1C). Rather, this would suggest that in DM, RPeD1 does not express the machinery required for development or maintenance of synaptic transmission. Haydon and Drapeau (1995)
propose that some neurons presynthesize their secretory machinery and will readily form chemical synapses, whereas other cells require signals to either synthesize secretory machinery or render it sensitive to Ca2+. It would appear that for this pair of neurons, trophic signals are necessary for synapse formation.
; Camarado et al. 1983
; Eliot et al. 1994
; Lin and Glanzman 1994
; Rayport and Schacher 1986
; Schacher et al. 1985
), Helisoma (Haydon and Kater 1988
; Syed et al. 1992
; Zoran and Poyer 1996
), Lymnaea (Bulloch and Syed 1992
; Inoue et al. 1996
; Magoski et al. 1994
; Syed and Spencer 1994
; Syed et al. 1992
), and the leech (Ching et al. 1993
; Fuchs et al. 1981
). Again, in almost all of these cases, the neurons were plated in either hemolymph, CM, or DM supplemented with serum. Although these conditions are required for neurite outgrowth, it is impossible to determine whether contact per se or factors present in the medium are responsible for synapse formation. In rat hippocampal cultures, recent work suggests that dendritic (postsynaptic) filopodia actively initiate synaptic contact (Ziv and Smith 1996
)
supporting the idea that both outgrowth and proper neuronal architecture contribute to synaptogenesis.
; Haydon 1988
; Klein 1994
; Liu and Nicholls 1989
). Furthermore, the majority (7 of 9) of soma-soma pairs plated in CM did not exhibit neurite outgrowth, suggesting that for this pair of Lymnaea neurons there may be a neurite outgrowth "stop signal" associated with this type of contact. It should be noted that soma-soma pairing of Helisoma (Haydon 1988
) or Aplysia (Coulson and Klein 1997
; Klein 1994
) neurons could result in chemical synapse formation within minutes to hours. Also, some Aplysia neurons can form synapses when paired in the presence of a protein synthesis inhibitor (Coulson and Klein 1997
). This indicates that synapse formation for some neurons may not require trophic factor-induced protein synthesis. Interestingly, a recent report on synaptogenesis between the somata of RPeD1 and another identified postsynaptic cell, interneuron visceral dorsal four (VD4), revealed that chemical synapses could be established between the cell bodies of RPeD1 and VD4 in DM alone (Feng et al. 1997
). However, as we have demonstrated, when RPeD1 is paired with VD2/3, it requires exogenous signal(s) (from CM) to synthesize secretory machinery or render it sensitive to Ca2+. Thus the requirements for synapse formation appear to be cell specific, even for a single presynaptic neuron, where exogenous signals may be required depending on the target neuron.
; Klein 1994
), which superficially resemble neurites. Examination of RPeD1 (with Lucifer yellow injection) at its soma-soma synapses with VD2/3 (in either DM or CM) did not reveal such processes, although it is likely that they eluded detection with the epifluorescence microscope due to their small size.
). In this case, both CNTF and nerve growth factor (NGF) induce neurite outgrowth from RPeD1 and Lymnaea motor neurons; however, chemical synapse formation only occurs in the presence of NGF. Evidence suggests that CNTF does not promote the development of both dopamine secretion from RPeD1 nor dopamine receptors on the target motor neurons. Our data suggest that factor(s) in CM are necessary, but not wholly sufficient, for appropriate synaptogenesis. When CM is present along with neurite-neurite contact, appropriate chemical synaptic formation occurs without obvious inappropriate electrical coupling. This broadens the role for CM beyond that of inducing neurite outgrowth to include regulation of the complementary process of synapse formation. Our results can be generalized in so much that synapse formation would take place between neurites in vivo, and possibly some of the trophic factors present in CM also would be involved in both promoting neurite outgrowth and synapse formation.
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ACKNOWLEDGEMENTS |
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The authors thank G. C. Hauser for technical assistance, N. I. Syed for helpful discussion on aspects of this study, and N. M. Magoski for comments on earlier drafts of the manuscript.
This work was supported by grants from the Medical Research Council (MRC) of Canada to A.G.M. Bulloch. N. S. Magoski was a recipient of MRC, Alberta Heritage Foundation for Medical Research (AHFMR), and NeuroScience Network (Canadian Centers of Excellence) studentships. A.G.M. Bulloch is an AHFMR Senior Scientist.
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FOOTNOTES |
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Present address of N. S. Magoski: Yale University, Dept. of Pharmacology, School of Medicine, New Haven, CT 06520.
Address for reprint requests: A.G.M. Bulloch, Dept. of Physiology and Biophysics, University of Calgary, 3330 Hospital Drive N.W., Calgary, Alberta T2N 4N1, Canada.
Received 13 November 1997; accepted in final form 5 February 1998.
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