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Journal of Neurochemistry, 2001, 76, 1774±1784 Monoamine release by neurons of a primitive nervous system: an amperometric study Marc-Antoine Gillis and Michel Anctil DeÂpartement de sciences biologiques and Centre de recherches en sciences neurologiques, Universite de MontreÂal, MontreÂal, Canada Abstract We measured monoamine release from dissociated neurons of the sea pansy Renilla koellikeri, a representative of the most evolutionarily ancient animals with nervous systems, by real-time monitoring of exocytosis using the amperometric method with carbon-®ber microelectrodes. Depolarizationinduced, as well as spontaneously active, neurons exhibited calcium-dependent exocytotic events at both the soma and the terminal bulb of neuritic processes. All spontaneously active neurons exhibited a bursting activity pattern in which amplitudes of exocytotic events appeared to be distributed in a quantal-like fashion. Fast Fourier transform analysis of bursting activity in 20 such neurons revealed burst harmonics with a major frequency of 8 Hz and a dominant rate of 95 Hz for individual exocytotic events within bursts. The results suggest that exocytotic transmitter release is as ancient as neurons and that endogenously bursting neurons in the sea pansy are as complex as those of higher animals. In addition, the observation that both soma and neuritic terminals of the same neuron can release transmitter suggests that local release sites in these cnidarian neurons are not critical for nerve net function. Keywords: amperometry, bursting neurons, cnidarian neurons, exocytosis, monoamines, sea pansy. J. Neurochem. (2001) 76, 1774±1784. Cnidarians display a range of behaviors from simple re¯exes to relatively complex rhythmic activities that are controlled by the ®rst nervous systems believed to have emerged in the course of metazoan evolution (Mackie 1990). The basic organization of these nervous systems is usually represented as two-dimensional plexuses of neurons with nonpolarized processes forming chemical synapses at their crossing points (Anderson 1985; Anderson and GruÈnert 1988). In hydrozoan cnidarians there are also plexuses in which neurons are interconnected by gap junctions (Spencer and Satterlie 1980; Spencer 1981). Our current understanding of chemical synaptic transmission in cnidarian nerve nets is based primarily on electrophysiological studies in which postsynaptic potentials were recorded intracellularly from a few favorable jelly®sh preparations. These include the neuromuscular junction of Polyorchis (Spencer 1982), the interneuronal and bidirectional synapse of the Cyanea motor nerve net (Anderson 1985) and the locomotor synaptic circuitry of Aglantha (Mackie and Meech 1995a, b). All these preparations exhibit physiological properties similar to those of conventional synapses in higher animals (see Anderson and Spencer 1989 and Mackie 1990 for review). None of these contributions directly examined the presynaptic steps of synaptic transmission in cnidarians. What are the capabilities of transmitter release of cnidarian neurons, and how do they compare with those of neurons from more advanced phyla? Measuring transmitter secretion directly and in real time has advantages over postsynaptic potentials as the latter can be poor mirrors of exocytotic events. For example, successive postsynaptic potentials that decrease in amplitude cannot be interpreted unambiguously as re¯ecting reduced transmitter release because desensitization of postsynaptic receptors may account for the decrease. Receptor desensitization in turn may mask the presence of spontaneously secreting tonic neurons. Assessing the quantal nature of secretion through postsynaptic responses 1774 Received August 28, 2000; revised manuscript received November 10, 2000; accepted November 13, 2000. Address correspondence and reprints requests to Dr Michel Anctil at DeÂpartement de sciences biologiques, Universite de MontreÂal, Case postale 6128, Succ. Center-Ville, MontreÂal, QueÂbec, Canada H3C 3J7. E-mail: [email protected] Abbreviations used: ASW, arti®cial sea water; DMEM, Dulbecco's modi®ed Eagle's medium. q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 76, 1774±1784 Monoamine release in sea pansy neurons may also raise problems, not least of which is the loose organization of cnidarian synapses (Anderson and GruÈnert 1988; Westfall 1996) which may obscure spatial and temporal parameters of quantal release. For these reasons we used amperometry with carbon ®ber microelectrodes to study monoamine exocytosis and the pattern of secretion of cnidarian neurons in culture using the sea pansy Renilla koellikeri as experimental preparation. This preparation is particularly suitable because monoamines were detected using HPLC and other techniques (De Waele et al. 1987; Pani and Anctil 1994a, b) and monoamines have been visualized immunohistochemically in neurons in the sea pansy (Umbriaco et al. 1990; Pani et al. 1995). Our results show that monoamine release from sea pansy neurons occurs as exocytotic events in a quantal-like fashion, and that endogenously generated exocytotic bursts occur in some of these neurons. In addition, we found that cell bodies were as capable as neurites of generating exocytotic events, thus suggesting that some cnidarian neurons use multiple sites to release transmitter. Materials and methods Sea pansies, Renilla koellikeri Pfeffer, were collected by Marinus, Inc. (Long Beach, CA, USA) and shipped to Montreal where they were placed in an aquarium in which the sea water temperature was maintained between 14 and 228C and the photoperiod controlled according to the seasonal settings of the animal's natural environment. Sea water (Instant Ocean) was prepared according to the supplier's instructions to reach an osmolarity of 1024 mOsm and pH 8. Cell culture For each cell culture preparation, the column of 30 autozooid polyps was excised from sea pansies anesthesized in an equal mixture of sea water and 0.37 mol MgCl2. Tissues were washed in arti®cial sea water (ASW: 395 mmol NaCl, 8.5 mmol KCl, 50 mmol MgCl2, 10 mmol CaCl2, 26 mmol Na2SO4 and 10 mmol Hepes), minced and incubated in 2 mL ASW containing 7.4 mg papain and 1 mg dithiothreitol (all from Sigma) for 90 min at room temperature with shaking. Cell suspensions were washed three times after centrifugation at 15 000 g in ASW and 66 mg/L gentamycin was added to the suspension after the last wash. Cells were transferred on the bottom of plastic Petri dishes (Sarstedt) previously coated with 1 mg/mL poly-l-lysine and were allowed to settle at 158C for up to 7 days. No experiment was performed within the ®rst 24 h of culture. Because of the small size of sea pansy cells and of the small differences in size between cell types, it was not possible to obtain neuron-enriched cell suspensions. Electrode preparation and experimental set-up For amperometry electrodes were built according to Chow and von Ruden (1995) with modi®cations. Carbon graphite ®bers (P55S, Amoco), 2 cm in length and 7 mm in diameter, were inserted in polypropylene pipette tips which were then heated and pulled to seal the carbon, except for a protruding length of 50 mm, and to produce a pointed, rigid tip. The pipette tip was ®lled with silver epoxy (EPO-TEK, Epoxy Technology, Billerica, MA, USA) 1775 and a copper wire 5 cm long and 0.5 mm in diameter was inserted into the tip so as to dip in the epoxy. The resin was baked for 12 h at 808C, followed by the soldiering of a male BNC connector to the copper wire to connect the electrode to the ampli®er. Transient capacitance was tested with a 1-ms square stimulation of 50 mV. Electrode responses were also tested with 10 mmol of epinephrine prior to experiments. Electrode signals were linearly related with epinephrine concentrations between 10 mmol and 0.1 mmol. To deliver solutions micropipettes were prepared from glass hemo capillaries (Sarstedt) pulled on a Narishige PP83 puller to obtain tips of 3 mm diameter. Solutions were ejected by pressure using a picospritzer II (General Valve). The propulsion gas was nitrogen and the ejection pressure was 6 psi. Petri dishes containing cell suspensions (usually 2 days old) were mounted on a Nikon Eclipse TE 300 inverted microscope. The electrode and the ejection pipette were mounted on Narishige WR88 micromanipulators and moved near selected cells. Throughout the experiments the cell cultures were perfused with ASW at 188C. Biological validation of the carbon electrodes was with PC12 cells (courtesy of Dr P. Barker, Montreal Neurological Institute). Cells were maintained in Dulbecco's modi®ed Eagle's medium (DMEM) supplemented with 10% heat-inactivated horse serum, 5% fetal bovine serum and 0.2% of an antibiotics/antimycotics solution (all from Sigma) at 378C in an atmosphere of 7.5% CO2 and 100% humidity. Before use, the cells were preincubated in the culture medium supplemented with 0.1 mmol dopamine and 0.1 mmol norepinephrine for 30 min, followed by washes in the regular culture medium. Data acquisition and analysis Currents generated at the carbon electrode were processed through a EPC7 patch-clamp ampli®er (List Medical) modi®ed to hold potentials at up to 1 V. To maximize electro-oxidation signals of monoamines, the holding potential was routinely set at 750 mV vs. the Ag/AgCl reference electrode in the bath. Signals were recorded and saved with a BIOPAC MP100 data acquisition system set at a frequency of 5 kHz. Data were analyzed using acknowledge 3.5 software. Noise suppression was effected by a non recursive numerical ®lter (coef®cient 31) equivalent to a low-pass ®lter at 200 Hz. Changing the low-pass from 400 to 200 Hz produced only negligible alterations in the shape and amplitude of the signals. Chemiluminescent monitoring of catecholamine release To test the competence of cells to release monoamines in culture, and to provide independent evidence of monoamine release, catecholamines released in the culture medium were measured using the chemiluminescence method of IsraeÈl and Tomasi (1999). A stock solution of lactoperoxidase (Sigma) was prepared by dissolving 1 mg of the enzyme in 1 mL distilled water. From this stock 100-mL aliquots were stored. Luminol was prepared in low light by dissolving 18 mg in three drops of 1 mol NaOH, followed by dilution in 100 mL of 0.2 mol Tris buffer (pH 8.6). Just prior to the assays, 30 mL of the luminol solution was mixed with 50 mL of a lactoperoxidase aliquot. The assay was performed by adding 10 mL of this mixture to 2 mL of cell suspension in assay vials. The reaction induced a chemiluminescence that was allowed to decay to a stable level. Stimulants were then added and the luminescent responses recorded with a LKB 1250 luminometer. Data were stored by linking the luminometer with the BIOPAC MP100 data q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 76, 1774±1784 1776 M.-A. Gillis and M. Anctil Fig. 1 Photomicrograph of a 2-day culture of sea pansy cells showing a variety of neurons representative of those from which amperometric recordings were obtained. acquisition system and analyzed with the acknowledge 3.5 software. The chemiluminescent assay was validated by adding 2 mL of ASW containing 0.1±100 mmol of epinephrine in the assay vials and by plotting dose±response curves from the data thus generated. Results Cultured neurons Sea pansy neurons were recognizable from other cells in culture because they readily grew ®ne processes (neurites) Fig. 2 Chemiluminescent signals representing catecholamine release from a suspension of dissociated sea pansy cells. The luminol±lactoperoxidase mix was ®rst added to the suspension (Mix), bearing varicosities (Fig. 1). They were capable of readily adhering to the bottom of culture dishes and initiating neurite growth within 24±48 h of cell dispersion. Neurite growth was also shown to be directed through growth cones toward other cells, many of which were neurons. The majority of observed neurons exhibited a soma containing polymorphic granules and from which a ciliary cone as well as a variable number of neurites extended. The diversity in neuronal morphology was similar to that described previously in another anthozoan species, the sea anemone Calliactis parasitica (Saripalli & Westfall 1996). Of a total of 346 neurons sampled in culture, 53% were bipolar or pseudounipolar, 31% were unipolar and the remaining 16% were multipolar. After 48 h in culture neuronal survival appeared to decrease as some of the cells changed shape and lost their adhesion to the dish bottom. Therefore, 48-h-old cultured neurons with morphological evidence of maturity (presence of varicose neurites but no growth cone) were routinely used for recording. Chemiluminescent monitoring of release in culture The capacity of sea pansy cell cultures to release monoamines was tested using the chemiluminescence method of IsraeÈl and Tomasi (1999) which allowed recording in real time. Mass depolarization of cultured cells by 80 mm KCl consistently induced catecholamine release in culture media using this method (Fig. 2). The luminescence signals appeared 0.3±0.5 s after stimulation and rose to peak within causing a transient light signal, 80 mmol KCl and arti®cial sea water (ASW) were then added at the times indicated by arrowheads. q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 76, 1774±1784 Monoamine release in sea pansy neurons 1777 Fig. 3 Representative amperometric recording of spike activity from a bipolar neuron of the sea pansy. This neuron was silent until after a 1-s pulse of 50 mmol KCl was ejected. The time duration of ejection pulses is indicated by a bar in this and subsequent ®gures. (Inset) Enlarged time scale of a single spike with the fast (6.4 ms) rise and slower decay typical of exocytotic events. 1.9±3.0 s. The exponential fall of the response, which was completed in 22±49 s, probably re¯ected a decay of the chemiluminescent reaction as it was also observed in the small response artifacts elicited by injecting ASW instead of KCl in the culture medium (Fig. 2). The mass release responses were not readily subject to fatigue, as indicated by the similar amplitudes of consecutive responses in the two pairs of responses shown in Fig. 2. Fig. 4 Amperometric recording of an active neuron subjected to an ejection pulse of Ca21-free sea water. Note the reduced rate of spikes after 5 s exposure, followed by a complete elimination of spikes. Some spiking resumed 15 s after the end of the ejection pulse, presumably due to diffusion of the Ca21-free medium away from the cell. q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 76, 1774±1784 1778 M.-A. Gillis and M. Anctil Fig. 5 Effects of various stimulants on the spike activity of three different neurons of the sea pansy. Note that the spiking response to the depolarizing agent veratridine (10 mmol) is almost instantaneous (a), whereas responses to the calcium ionophores A23187 and ionomycin (both at 1 mmol) occur after delays of seconds (b and c). The tonic rise of the trace in (b) is an artifact due to a direct interaction of the ionophore with the carbon probe. q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 76, 1774±1784 Monoamine release in sea pansy neurons Amperometric monitoring of release Only 2.3% (106) of all cultured sea pansy neurons probed by amperometry exhibited spontaneous (19%) or induced (81%) secretory events. Of these 106 cells, 48% were unipolar, 43% bipolar and 9% multipolar. No signal was ever recorded from probed non-neuronal cells. Almost all responsive bipolar neurons had a round or oblong soma 11 mm in diameter and short neurites (13 mm long). The amperometric events recorded from such a cell are shown in Fig. 3. A 1-s ejection pulse of 50 mm KCl within 70 mm of that neuron induced a series of spikes of variable amplitude and frequency. All monitored neurons that were not spontaneously active were completely quiescent before stimulation. The ®rst spikes appeared 0.5 s after the beginning of the ejection pulse, and the whole response lasted < 3 s. An individual transient of relatively large amplitude from this response is shown in Fig. 3 (inset). The rise time of individual spikes ranged from 1.8 to 10 ms, and that shown in the inset was in the mid-range (6.4 ms). The total duration of the spikes was highly variable (32 ^ 21 ms, N 20), probably due to variable geometries of microelectrode placement affecting the time course of dispersion of released transmitter (trace decay). Although this and most of the other recordings shown below are from the soma, similar spikes were detected when the electrode was abutted against the terminal bulb of neurites from the same neuron. As a biological control, we recorded spikes from 10 PC12 cells identically exposed to KCl (not shown). The 1779 KCl-induced spikes appeared in a manner similar to those associated with sea pansy neurons, but their occurrence spread over a longer duration. The individual spikes also lasted longer, their rise time ranging from 37 to 73 ms. The recorded spikes were calcium dependent. Ejecting calcium-free, EDTA-enriched ASW onto KCl responsive or spontaneously active neurons momentarily eliminated the production of these spikes, the latter apparently resuming after the ejected calcium-free ASW has suf®ciently dissipated away from the recorded cell (Fig. 4). Another depolarizing agent, veratridine, induced spikes within 17±30 ms of ejection (Fig. 5a). Contrary to the KCl responses, only one veratridine-induced spike was generated for each ejection pulse, lasting up to 200 ms each. In addition, the ionophores A23187 (Fig. 5b) and ionomycin (Fig. 5c) generated a series of spikes, ionomycin being the most potent. Response delays were relatively long (1±9 s) and the response duration ranged from 3 to 20 s. Antho-RFamide, a peptide native to numerous neurons in the sea pansy (Grimmelikhuijzen and Groeger 1987; Anctil and Grimmelikhuijzen 1989), induced from 15 neurons only a few spikes over a period up to 30 s, after a response delay of 4±18 s (Fig. 6). Neurons secreting in rhythmic bursts In 20 of the 106 neurons in which events were recorded, activity was ongoing from the moment the carbon electrode tip became contiguous with the target cells (Fig. 7a). This Fig. 6 Effect of the native neuropeptide Antho-RFamide (10 mmol) on spike activity of a sea pansy neuron. Note the sparseness of spiking activity. q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 76, 1774±1784 1780 M.-A. Gillis and M. Anctil spontaneous activity, although variable in spike amplitude, never wavered throughout the recording period and appeared to be refractory to KCl depolarization (not shown). Closer scrutiny of these events on a spread time scale shows that their amplitudes appear to vary from one another as multiples of a unit spike of < 1.5 pA (Fig. 7b). This amplitude distribution was also observed in ®ve other neurons and is suggestive of a quantal mode of secretion. However, it is apparent that the distribution of events is heavily skewed toward the lowest amplitude range (Fig. 7b) and frequency analyses of amplitude ranges in several thousand such events compiled from recordings of six spontaneously active neurons failed to conclusively demonstrate a quantal distribution as in the classical model of miniature endplate potentials (Boyd and Martin 1956). As shown in Fig. 7(a), monitoring events on a compressed time scale gives the impression of randomly generated events in spontaneously active neurons. Spreading the time scale unmasked the bursting nature of these neurons (Fig. 8a). Bursts lasted 20±125 ms and were comprised of between two and eight single spikes. The rise time of the single spikes ranged from 3.4 to 8.0 ms but their total duration was invariably 15 ms (Fig. 8b). A fast Fourier transform analysis of the rate of occurrence of bursts and single spikes from spontaneously active neurons reveals two sharply distinct frequency classes, one associated with bursts in the low frequency range and the other with single spikes in the high frequency range (Fig. 8c). In the transform for the neuron shown in Fig. 8(c), the three frequency peaks for bursting are multiples of each other (at 8.7, 18.0 and 27.3 Hz) and therefore represent harmonics. When this analysis was applied to the combined data of the 20 bursting neurons it became apparent that the major frequency peak for bursting is 8 Hz, whereas that for spiking within bursts is Fig. 7 Amperometric traces of a spontaneously active neuron (a) show evidence in an expanded time scale (area delimited by dashed lines) of spike amplitude jumps representing multiples of an unitary spike amplitude (b, horizontal lines). q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 76, 1774±1784 Monoamine release in sea pansy neurons Fig. 8 Analysis of the rate of events in spontaneously occurring burst exocytosis from sea pansy neurons. Interburst (a) and interspike intervals within burst framed by dashed lines (b) are segregated when a fast Fourier transform is applied to rate of events compiled from a single neuron (c) in which . 200 bursts were analyzed. Note that 1781 spiking rates within bursts are more variable than bursting rates and that the three peaks in the low frequency range represent three burst harmonics. (Inset) The frequency peaks based on event rate data cumulated from 20 neurons. q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 76, 1774±1784 1782 M.-A. Gillis and M. Anctil 96 Hz (Fig. 8c, inset). There was little variance between neurons within each frequency class as evidenced by the small standard deviations (Fig. 8c, inset). Discussion Cnidarian neurons examined thus far share several basic electrophysiological properties with those of higher animals, including those associated with synaptic transmission (Anderson and Spencer 1989). In this study we show that a key presynaptic event, transmitter release, appears to unfold in neurons of a cnidarian species according to the conventional exocytotic model of transmission, with suggestive evidence of a quantal distribution of release events. We also reveal the existence in this cnidarian of intrinsically active bursting neurons. Monoamine releasing neurons in culture The sea pansy neurons from which amperometric recordings were attempted proved to be amenable to primary culture conditions compatible with physiological viability. Because it was not possible to isolate nerve cells from other cell types and because there are no uniquely identi®able monoaminergic neurons in the sea pansy, we had to randomly screen cultured neurons for their ability to generate amperometric signals. The low yield of positive neurons (2.3%) suggests that monoamine-containing neurons constitute just a small fraction of the neuronal population of the sea pansy. This view is supported by biochemical and immunohistochemical evidence indicating that the nervous systems of cnidarians are predominantly peptidergic (Grimmelikhuijzen et al. 1992) and that neurons containing the neuropeptide Antho-RFamide are considerably more numerous and widespread in the nervous system of the sea pansy than monoaminergic neurons (Grimmelikhuijzen and Groeger 1987; Umbriaco et al. 1990; Pani et al. 1995). It is surprising therefore that Antho-RFamide had only a small excitatory effect on monoamine release in this study. The responsive neurons were morphologically diverse, the majority being equally divided between unipolar and bipolar shapes. This is mirrored by the morphological diversity of monoaminergic neurons in the sea pansy: serotonin-immunoreactive neurons exhibit unipolar as well as bipolar shapes similar to those seen here (Umbriaco et al. 1990), and norepinephrine-immunoreactive neurons exhibit bipolar shapes (Pani et al. 1995). We were unable to directly identify the released monoamines using cyclic voltammetry. Although the exact identity of the chemical species generating the amperometric signals is unknown, it is highly likely that catecholamines were at least partly involved because catecholamines were detected by the chemiluminescence method in culture media in which sea pansy neurons were depolarized by KCl, and because release of preloaded tritiated norepinephrine from sea pansy tissues has been reported previously (Pani et al. 1995). Exocytotic properties of stimulable release Depolarization of otherwise silent sea pansy neurons by KCl or veratridine induced several transient amperometric events (spikes) that bear similarities to exocytotic release in other preparations, including a relatively fast rise time (# 7 ms) re¯ecting the ejection of vesicular contents and transmitter diffusion to the electrode tip, and a decay of longer duration. That our recording system was able to detect spikes re¯ecting exocytotic release was validated by recording KCl induced spikes in PC12 cells with kinetics similar to those recorded by Zerby and Ewing (1996). This was further validated by the suppression of spontaneously generated spikes from sea pansy neurons within a few seconds of exposure to calcium-free ASW. Conversely, facilitating calcium entry in sea pansy neurons with ionophores by itself elicited many spikes. Thus, sea pansy neurons seem to display conventional properties of transmitter release by exocytosis. The exocytotic events measured from depolarized sea pansy neurons share dynamic properties with those of other invertebrate neurons such as the leech Retzius cells (Bruns and Jahn 1995) and the giant dopaminergic neuron of the snail Planorbis (Chen and Ewing 1995; Chen et al. 1995, 1996). There are differences in spike amplitude, latency and rise time that can be attributed to the varying recording geometries among preparations, as well as to substantial differences in cell sizes and carbon-®ber electrode performance. One discrepancy with these preparations is the shorter duration of the responses of sea pansy neurons which never outlast the duration of KCl ejection. This suggests a high threshold for KCl depolarization such that even a small dispersion of the local KCl build up at the end of the ejection period may be suf®cient to lower the depolarization level below threshold. Some cnidarian neurons are known to necessitate overshooting depolarizations to induce postsynaptic currents (Anderson 1985), and these stimulable sea pansy neurons may belong to that category. Neurons displaying bursts of exocytosis and quantal-like release We found a distinct population of sea pansy neurons that, contrary to the stimulable neurons just mentioned, were spontaneously active and refractory to KCl stimulation. Their activity was characterized by a continuous barrage of spikes of varying amplitude. It became apparent on closer scrutiny that these spikes exhibited two striking properties: their amplitudes fell into size classes that appeared to be multiples of each other and they were ®red in rhythmic bursts. The ®rst property is expected of neurons releasing transmitter in a quantal fashion (Fatt and Katz 1952). We were unable to produce frequency histograms of spike amplitudes from stimulable neurons, as in the conventional q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 76, 1774±1784 Monoamine release in sea pansy neurons method of Boyd and Martin (1956), because they generated too few spikes for that purpose. In contrast, the number of events in spontaneously active neurons allowed such a quantitative analysis. Although the analysis was inconclusive, the recorded events suggest a quantal-like mode of transmitter release. An earlier attempt to demonstrate quantal release by recording epps from synapses of a scyphozoan motor nerve net was also suggestive of quantal release, but the analysis was equally inconclusive (Anderson 1988; Anderson and Spencer 1989). The second property of spontaneously active neurons, secretion in rhythmic bursts, is interesting because it is reasonable to assume that this release pattern is driven by oscillations in bursts of the membrane potential. This appears to be the ®rst direct report of endogenously active bursting neurons in an anthozoan. The small size of anthozoan neurons has always precluded in situ intracellular recordings and the numerous studies using extracellular recordings (Satterlie and Spencer 1987 for review) may have overlooked bursting activities if they are driven by membrane potential oscillations not leading to action potentials. Bursting in sea pansy neurons is regular although burst duration and the number of spikes per burst are variable. However, the spiking rates within a burst and the harmonics of bursting showed little variance among neurons sampled (Fig. 8c, inset), thus demonstrating the robustness of the rhythms. The physiological signi®cance of the sea pansy bursting neurons is dif®cult to infer because their activity was recorded in culture where they were devoid of synaptic input. Neurones ®ring action potentials in bursts were observed in situ in the hydrozoan jelly®sh Polyorchis only when subjected to photic stimulation (Spencer and Arkett 1984) or calcium-free sea water (Anderson 1979). Thus the possibility remains that bursting is only a property of sea pansy neurons in culture. If bursting also occurs in these neurons in situ, they could serve to generate rhythmic behaviors or secrete neurohormones (Levitan and Kaczmarek 1991). The sea pansy displays a rhythmic behavior, peristalsis, which is involved in feeding and is known to be modulated by monoamines (Anctil 1989, 1994). Another potential role for these neurons is the priming of the colonial nerve net of the sea pansy which coordinates defensive behaviors such as bioluminescence, individual polyp retraction and colonial mass contraction (Parker 1920). Anderson and Case (1975) observed that sea pansy nerve net impulses are through-conducted, that is, they are conducted without decrement throughout the colony, thus apparently negating the need for repetitive stimulation to facilitate interneuronal transmission. They proposed the existence of spontaneously active neurons in the nerve net that serve to keep the colony in a facilitated state, such that any ®rst stimulus may trigger through-conducted impulses in a naõÈve animal. The bursting neurons of the sea pansy are obvious candidates for this role because of their continuous, endogenous activity and 1783 because many norepinephrine-immunoreactive neurons were observed in the colonial nerve net of the sea pansy (Pani et al. 1995). Monoamine release from neuronal cell bodies The observation that spikes were detected from the soma as well as from neuritic terminals from the same neuron suggests that these neurons may release transmitter from multiple sites. Although this property may be limited to cultured neurons, the latter appeared to be developmentally mature, displaying varicose swellings that are typical of functional release sites in monoamine neurons within intact nervous systems. Chen et al. (1995) made similar observations on dopamine neurons from the snail Planorbis and suggested that primitive invertebrate neurons may not be able to suppress soma exocytosis after maturation or be able to use presynaptic autoreceptors to limit the number of exocytotic events. The lack of inhibitory autoreceptors in the sea pansy is supported by earlier observations that norepinephrine increased rather than decreased the release of tritiated norepinephrine, an effect reversed by adrenergic antagonists (Pani et al. 1995). Cnidarian synapses within nerve nets are usually formed where neurites from two neurons cross over each other (Anderson and Schwab 1981, 1983), and those synapses are known to contain few vesicles (Anderson and GruÈnert 1988; Westfall 1996), apparently too few to account for the levels of postsynaptic activity recorded (Anderson 1985; Anderson and Spencer 1989). Although synaptic contacts have been reported between neurites and neuronal somata in the jelly®sh Cyanea (Anderdon 1985), it is unclear whether transmission was uni- or bidirectional at these contacts. The routine recording of exocytotic events from somata in this study raises questions about the potential role of transmitter release from the soma in overcoming shortfalls of synaptic release at varicose terminals on neurites. Alternatively, transmitter release from the soma may serve a neuroendocrine function, thus allowing transmitter diffusion over a large area of the nerve net and modulation of nerve net activity, such as prefacilitation of through-conduction. Acknowledgements We thank Dr Philip Barker of the Montreal Neurological Institute for the gift of the PC12 cell line cultures used in this study, and Dr BenoõÃt Lussier for advice. This study was supported by a research grant from the Natural Sciences and Engineering Research Council of Canada to MA. References Anctil M. 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