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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.
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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
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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.
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Monoamine release in sea pansy neurons
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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.
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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.
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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 ˆ 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.
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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).
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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.
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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
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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.
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