1Department of Biological Sciences and 2Department of Cell Biology and Anatomy, University of Calgary, Calgary, Alberta T2N 1N4, Canada
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ABSTRACT |
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Saver, Michelle A., Jerrel L. Wilkens, and Naweed I. Syed. In situ and in vitro identification and characterization of cardiac ganglion neurons in the crab, Carcinus maenas. The aim of this study was to investigate the intrinsic membrane properties and hormonal responses of individual central pattern generating neurons in the cardiac ganglion of the shore crab Carcinus maenas. Because the cardiac ganglion in this crustacean species is buried within the heart musculature and is therefore inaccessible for direct morphological and electrophysiological analysis, we developed two novel in vitro preparations. First, to make the ganglion accessible, we established a brief enzymatic treatment procedure that enabled us to isolate the entire cardiac ganglion, in the absence of muscle tissue. Second, a cell culture procedure was developed to isolate individual neurons in vitro. With the use of both isolated ganglionic and neuronal cell culture techniques, this study provides the first direct account of the neuroanatomy of the cardiac ganglion in shore crabs. We demonstrate that cultured neurons not only survived the isolation procedures, but that they also maintained their intrinsic membrane and transmitter response properties, similar to those seen in the intact ganglion. Specifically, we tested the peptides proctolin, crustacean cardioactive peptide, the FLRFamide-related peptide F2, and an amine (serotonin) on both isolated ganglion and in vitro culture neurons. We measured changes in neuronal burst rate, burst amplitude, pacemaker slope, and membrane potential oscillation amplitude in response to the above four hormones. Each hormone either increased neuronal activity in spontaneously bursting neurons, or induced a bursting pattern in quiescent cells. The in vitro cell culture system developed here now provides us with an excellent opportunity to elucidate cellular, synaptic and hormonal mechanisms by which cardiac activity is generated in shore crabs.
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INTRODUCTION |
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Rhythmic behaviors such as respiration
(Bulloch and Syed 1992; DiCaprio and Fourtner
1984
, 1988
; Funk and Feldman
1995
), locomotion (Getting 1989
; Grillner
et al. 1995
; Kiehn 1991
), feeding
(Willows 1980
), and heartbeat (Arbas and
Calabrese 1987
; Hartline 1979
for review) are
controlled by networks of neurons, called central pattern generators
(CPGs) (see Delcomyn 1980
; Getting 1989
;
Kristan 1980
; Pearson 1985
,
1993
). Identification of various CPG neurons and
characterization of their intrinsic membrane and synaptic properties is
critical for our understanding of the cellular basis of most rhythmic
behaviors. A variety of vertebrate (Funk and Feldman
1995
; Grillner et al. 1995
; Ramirez and
Richter 1996
) and invertebrate (Calabrese et al.
1989
; Harris-Warrick and Flamm 1987
;
Kristan 1980
; Marder 1987
;
Selverston 1987
; Syed et al. 1990
,
1992
; Turrigiano and Marder 1993
) species
have been used in the past to understand how CPG neurons initiate,
maintain, terminate, and modulate various rhythmic behaviors. In the
vast majority of preparations studied to date, however, the precise identity, number, and nature of synaptic connections between CPG neurons have not been fully deduced. This is due to the fact that in
most cases a large number of CPG neurons are involved in any given
behavior, and their synaptic connections are often complex and
difficult to resolve in the intact brain.
The cardiac ganglion (CG) located in the decapod crustacean heart, on
the other hand, offers a simpler preparation where a nine-celled CPG is
located at some distance from the CNS. Moreover, the neuronal somata
are large and physically distant from one another. The decapod CG
usually consists of four pacemaker cells (small cells, SCs) and five
motor neurons (large cells, LCs) (reviewed in Hartline
1979). The isolated CG reliably produces bursts of impulses in
the absence of synaptic activity from the CNS (Welsh and Maynard
1951
), and therefore offers an excellent opportunity to
investigate the intrinsic membrane and synaptic properties of this
rather simple CPG network both in crabs (Portunus
sanguinolentus) (Tazaki and Cooke 1979a
-c
) and
lobsters (Homarus americanus) (Berlind 1985
,
1989
; Tazaki and Cooke 1986
). In these
larger crustaceans, the CG is easily discernible under a dissection
microscope and can be manually exposed by teasing away the heart muscle
tissue surrounding the ganglion (Tazaki and Cooke
1979a
). To examine the intrinsic properties of individual
ganglionic cells, previous investigators either ligatured
(Tazaki and Cooke 1983b
), or transected the ganglion to
separate neurons from one another within the ganglion (Sullivan
and Miller 1984
). Alternatively, groups of neurons were pharmacologically isolated by creating a two-pool system in which neurons in each pool were bathed independently (Berlind
1985
, 1989
; Sullivan and Miller
1984
; Tazaki and Cooke 1986
). In these studies,
both SCs and LCs were found to generate driver potentials, which
underlie spontaneous bursting activity in the network
(Berlind 1985
, 1989
; Tazaki and
Cooke 1979b
,c
).
Although the above studies on crustacean heart preparations contributed
significantly toward our understanding of various neuronal properties
underlying CG burst generation, most issues concerning the role(s) of
intrinsic versus network properties of neurons still remain unresolved.
For instance, neither ligature nor two-pool preparations could create a
completely "isolated" environment, and hence the synaptic
connections between the neurons may have still persisted in these
studies (Panchin et al. 1993; Turrigiano and
Marder 1993
). In addition, tight ligatures around the CG may
have also altered the physiological and/or pharmacological properties
of these so-called isolated cells (Sullivan and Miller 1984
). This potential problem could have been resolved by
dissociating neurons in primary cell culture. However, as compared with
molluscan species such as Helisoma (Wong et al.
1981
), Aplysia (Dagan and Levitan
1981
), and Lymnaea (Syed et al.
1990
), the cell culture techniques for most crustaceans have
enjoyed little success, with a few notable exceptions (Cooke et
al. 1989
; Grau and Cooke 1992
; Panchin et
al. 1993
; Turrigiano and Marder 1993
).
This study was designed to understand the intrinsic properties of
various CG neurons in the crab Carcinus maenas and to
determine how these are modulated by various cardioactive substances.
However, as compared with their larger counterparts, the CG in smaller crabs (such as C. maenas) is buried within the heart
musculature and is therefore not discernible even under a high-powered
dissection microscope. To overcome this potential problem, we first
developed an enzymatic procedure that allowed us to isolate the entire
CG from the rest of the heart. Subsequently, intracellular recordings were made from the isolated CG neurons and various hormones (serotonin, 5-HT; proctolin, PR; crustacean cardioactive peptide, CCAP; and SDRNFLRFamide, peptide F2) were tested for their modulatory roles. Further techniques were developed to isolate individual neurons from
the intact ganglion in primary cell culture. The isolated neurons not
only survived enzymatic and dissociation procedures, but also exhibited
electrophysiological and pharmacological properties that were similar
to those observed in the intact ganglia. Moreover, the aforementioned
hormones that are known to influence heart rate and contractility in
higher order crustaceans (F2: Trimmer et al. 1987;
Mercier and Russenes 1992
; all others reviewed by Cooke 1988
) were tested for their modulatory roles.
These responses were also compared with those obtained from the intact
ganglia. The ganglionic and primary cell culture techniques developed
in this study provide a useful model system in which to explore
cellular and synaptic mechanisms by which rhythmic heart activity is
regulated in the crab C. maenas.
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METHODS |
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Heart isolation procedure
Adult male and female shore crabs (n = 139) were used in this study. All legs were autotomized before exsanguination of the animal. The heart was accessed by removing the overlying carapace and connective tissues. The alary ligaments, which suspend the heart in the pericardial sinus, were cut and the heart was removed. Finally, the ventral heart wall was cut open and the heart was pinned in a silicone elastomer (Sylgard) dish. The isolated heart was bathed with C. maenas saline (in mM: 433 NaCl, 12 KCl, 12 CaCl2 · 2H2O, 20 MgCl2 · 6H2O, 10 HEPES, adjusted to pH 7.60 with 2 N NaOH).
Ganglionic isolation
To isolate the CG, the heart was rinsed in 3 ml antibiotic
saline (ABS, 150 µg/ml gentamycin sulfate added to regular C. maenas saline) for two, 10-min washes, each in a fresh Falcon 3001 plastic dish. All experiments were performed under sterile cell culture conditions (Ridgway et al. 1991). The heart was
then placed in a Falcon Blue-Max 15-ml tube containing collagenase
(type II, 1 mg/ml) in 3 ml defined medium (DM). DM was made by adding
2× Leibowitz L-15 medium to 2× C. maenas saline; pH was
then adjusted to 7.60 with 2 N NaOH. DM was tested with an osmometer to
confirm that the osmolarity was around 1,000 mOsm. During enzyme
treatment, the tubes were refrigerated (4°C) for 10 min, then removed
and shaken at room temperature for a further 10 min. Refrigeration was
considered necessary to slow the enzymatic degradation of heart muscle
cells. After collagenase treatment, the tube's contents were added to
a fresh Falcon dish containing 3 ml of DM. At this stage, the CG could
easily be recognized among completely fragmented heart muscle cells.
After an additional DM wash, the CG was pinned at its distal branches
on a Sylgard dish containing 3 ml DM.
Unless otherwise stated, the following procedures were performed at room temperature (~20-23°C). To soften the connective tissue sheath, the CG was bathed in DM containing both protease (type IX, 3.33 mg/ml) and trypsin (type III, 2 mg/ml) for 10 min. Enzyme treatment was followed by three consecutive washes in DM (3 ml each wash). The ganglion was desheathed, first using a pair of fine forceps and then by two sharp microelectrodes. Intracellular microelectrode recordings were made from the desheathed ganglion.
To aid neuronal visualization, some preparations (n = 15) were stained with methylene blue dye. Ganglia were isolated and desheathed as outlined above, and a few drops of methylene blue were added to the preparation dish. Ganglia were then refrigerated overnight, and, subsequently, the positions of labeled somata visible under a dissection microscope were traced on a drawing sheet.
Single cell isolation
As for the above protocols, all procedures were carried out under sterile conditions. No further enzyme treatment was required beyond the aforementioned procedures to isolate neurons from the ganglion. Neurons were first identified visually by their position in the ganglion and were subsequently removed by applying gentle suction through a fire-polished and Sigmacote-treated (Sigma cat. no. SL-2) pipette that was held in a micromanipulator (MM-33). The isolated soma with its accompanying axon stump was plated in a poly-L-lysine (MW = 50,200)-coated Falcon 3001 dish containing 2-3 ml of DM. A maximum of three identified neurons were plated in any given dish, and their positions were marked on the culture dish. The isolated cells were allowed to adhere to the poly-L-lysine-coated dish overnight and were subsequently penetrated with microelectrodes. Because SCs were often difficult to find, fewer were isolated.
Photographs were taken on a Zeiss Axiovert 135 inverted microscope with visible light at ×10-40 magnification. Tech pan film (50 ASA) was used in a Contax camera.
Electrophysiology and hormone applications
For both the isolated ganglia and cultured CG neuron
preparations, sharp microelectrodes filled with a saturated solution of
K2SO4 (resistance 10-40 M) were used. DM
was replaced with regular C. maenas saline before
intracellular impalements, and this was continuously perfused
throughout the experiment. Signals were amplified on a NeuroData (Model
IR-283) amplifier, displayed on a Gould 2-channel chart recorder (Model
2200S), and simultaneously stored on a VCR (Sony Model 420 K, A. R. Vetter) for playback and data analysis.
After microelectrode penetration, the cells were immediately hyperpolarized (range of injected current: 0.15-0.5 nA) to reduce spontaneous activity of the intact CG neurons, and this current was maintained throughout the experiment. The above range of hyperpolarizing current was sufficient to completely silence the isolated cells.
The neurohormones 5-HT, proctolin (Sigma Chemical, St. Louis, MO), CCAP
(Peninsula Laboratories, Belmont, CA), and F2 (a gift from Dr. Ian
Orchard, University of Toronto) were tested. Each neurohormone solution
was diluted in C. maenas saline to a final (pipette)
concentration of 104 M. Pipettes were fire-polished and
back-filled with hormone. Each hormone was applied directly onto an
individual somata using pressure application (WPI PV800 pneumatic
PicoPump), and the aforementioned hormones were tested at least 2-3
times on each cell, with sufficient wash out time in between each
application to reestablish baseline activity (~10-40 min).
From raw data recordings of LC neurons, either in situ or in vitro, the burst rate, amplitude, pacemaker slope, and oscillation amplitude were measured during control and after hormone treatments. Amplitudes and rates were simply measured from chart records, whereas the pacemaker slope required calculation. The pacemaker potential was arbitrarily defined as the depolarizing phase that began at the end of a postburst afterhyperpolarization, but before the driver potential depolarization of the next burst. Slope was then calculated as the rise/run for this depolarizing pacemaker phase. A model of the events underlying the burst in a LC ganglionic neuron is shown in Fig. 1A.
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RESULTS |
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Neuronal morphology
Because the cardiac ganglion in C. maenas is not discernible in the heart musculature under a dissection microscope, the precise location, position, and number of cells have not been examined to date. In this study, using an enzyme treatment procedure, we were able to isolate the CG from its surrounding heart musculature. Methylene blue staining coupled with visual inspection allowed us to produce a map depicting the precise number, location, and position of CG neurons (Fig. 1). We used methylene blue in the early stages of this study primarily to locate the SCs. After staining several ganglia with methylene blue, we were able to pinpoint the area in which they resided. Once we were more experienced at finding the small cells, we no longer needed methylene blue to pinpoint them but could do it using unstained ganglia. The entire ganglion was surrounded by a thick connective tissue sheath, which was removed via fine forceps to aid neuronal visualization. Rather than a typical nine-celled CG, as observed in other crabs and lobsters, no more than eight cells were ever observed in any given C. maenas ganglion. LCs 1 and 2 were located in the left and right "Y" branches, respectively. LC3 was present at the junction of the Y, and was physically distant from LCs 4 and 5, which were usually located side by side at the bottom of the ganglionic trunk. SCs were only observed in a handful of preparations (21 of 139) and were always clustered below LCs 4 and 5. The SCs were encased in a "pocket" of connective tissue that was buried deep within the main trunk of the CG at its distal end. Each LC was covered by a connective tissue sheath surrounding its somata, whereas the SCs were devoid of such a sheath.
About one in every four ganglia showed anatomic anomalies. Sometimes two large cells were located in one Y branch (3/139), whereas in some other preparations LCs 4 and 5 (the "twin cells") were located anterior and posterior to one another. Although this appearance is typical in lobster CGs, it was not commonly observed in C. maenas ganglia (only 20/139 ganglia). In two cases, the ganglia were unbranched and the cells were aligned down the main trunk. In four ganglia, there were two LCs in place of one at the position of LC3. The SCs, however, were always located below the most posterior LCs, and were clustered in a group.
Electrophysiological properties of the isolated CG
To determine the electrophysiological properties of CG neurons in
the intact ganglion, direct intracellular recordings were made from
freshly isolated ganglia (a total of 23 preparations). Most LCs showed
spontaneous spike activity (27 of 30 cells, Fig. 2A), and their membrane
potential (Vm) ranged from 12.3 to
65 mV,
the average being
37.4 ± 4.8 (SE) mV. Cells with low
membrane potentials had a similar morphology, similar physiological
activity, and similar responses to hormones as those cells that were
more polarized. These low membrane potentials may have resulted from incomplete electrode penetration, but this seems unlikely.
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Figure 2A shows the most common LC waveform: small amplitude
and short-duration bursts superimposed on a large amplitude driver potential, large afterhyperpotentials, and slow pacemaker potentials (see Table 1 for average burst amplitudes
and pacemaker slopes). This bursting activity, however, varied
from preparation to preparation. For instance, some ganglia fired
slowly with diffuse bursts that occasionally produced larger spikes
(Fig. 2B). Some LCs exhibited larger spikes arising from the
driver potential (Fig. 2C), whereas still others had
postsynaptic potential (PSP)-like events during the interburst
period (Fig. 2D). Because of this variability in burst
pattern from cell to cell, some data were presented here as individual
raw data traces. A few (5 of 30) LC recordings showed spontaneous
membrane potential oscillations in addition to bursting activity. As
opposed to driver potentials, which were sustained depolarizations of
~20 mV amplitude and 200-250 ms in duration, spontaneous membrane
potential oscillations were smaller (~2-5 mV) and slower (500 ms)
membrane potential fluctuations that occurred during the interburst
interval. The values for resting burst rate, burst amplitude, pacemaker
slope, and amplitude of membrane potential oscillations as recorded
from the LCs are given in Table 1.
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Hormonal modulation of CG neurons
To determine whether various cardioactive substances found in crabs affect CG neuronal properties, 5-HT, CCAP, proctolin, and F2 were pressure ejected directly onto the LC somata during the intracellular recordings. In general, all hormones significantly increased burst rate and pacemaker slope (P = 0.0001-0.010 for burst rate, P = 0.005-0.029 for pacemaker slope, Figs. 3 and 4). The hormone-induced changes in burst amplitude were, however, not significant (Fig. 3). Similarly, in preparations that exhibited spontaneous membrane potential oscillations, hormone treatment did not significantly alter the amplitude of these oscillations. Control pressure pulses of saline did not alter any of the above aspects of neuronal excitability (Fig. 5A).
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The time course of hormone effects on these burst parameters was similar for all peptides (i.e., the responses were immediate in their onset, and reached peak amplitude within 30 s to 1 min after pressure application). All hormones regularized the rate of LC bursting and augmented the responses of the LCs, especially in slowly bursting preparations. An example of this augmentation effect is shown in Fig. 6, where CCAP treatment induces a change in the burst pattern from PSP-like activity to true bursts with more spikes. Hormonal effects were distinguished from one another, based not only on the time course, but also on changes in burst rate, burst amplitude, pacemaker slope, and amplitude of membrane potential oscillations.
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F2 was unique in that it was the only hormone tested that caused desensitization when applied repeatedly to the same cell. As shown in Fig. 7, the first application of F2 caused a 47% increase in burst rate, whereas the second application after a prolonged wash period (17 min) produced a smaller response (24% increase). Desensitization was observed in 5 of 16 cells that received 2 successive F2 treatments. Interestingly, when proctolin was applied to a neuron following an F2 trial, proctolin responses were prolonged; lasting up to 10 min. This was observed in 4 of the 5 ganglionic preparations in which hormones were applied in this order (data not shown).
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Serotonin (5-HT) is found in the pericardial organs of the crab and
modulates heart rate (Cooke 1988; Cooke and
Hartline 1975
; Wilkens and McMahon 1992
). To
test whether 5-HT also modulated CG neuronal activity, this biogenic
amine was tested directly on the CG neurons. Typically, there was a 10- to 30-s delay in the onset of the response to 5-HT, which lasted for
3-4 min (Fig. 4C). Responses to peptide applications were,
however, immediate in their onset and lasted for a maximum of 2-3 min
(Fig. 4).
The above data were obtained from LCs that were isolated from the
surrounding cardiac muscles, regulatory nerves (cardioinhibitor and
cardioaccelerators), and endogenous hormones (pericardial organ
secretions). Because strong electrotonic and chemical synaptic connections exist between LCs and SCs (reviewed in Hartline
1979) in the intact ganglion, it was therefore difficult to
demonstrate unequivocally that the responses to the various
cardioactive substances were indeed direct. To study direct hormonal
effects on CG neurons, individual somata were carefully microdissected
from the intact ganglion and maintained in primary cell culture.
In vitro isolation and characterization of CG neurons
Neuronal somata of individually identifiable LCs were
isolated and plated in culture (Fig. 8).
Electrophysiological recordings were made from cultured neurons
(n = 15) at 18-24 h. There was no evidence of growth
or sprouting in culture for either the LCs or SCs during this time
period. Only those isolated neurons that appeared morphologically
healthy (spherical and had longer axon stumps) were used for
electrophysiological analysis. The resting membrane potentials of
cultured LC neurons (range: 10 to
56 mV, average
34.2 ± 5.6 mV) were similar to those in the isolated ganglion. An example of
spontaneous activity in a cultured LC, including pacemaker
depolarizations and driver potentials, is illustrated in Fig.
9A. The small deflections
riding on top of the driver potential appear to be aborted spikes. The
main difference between cultured cells and isolated ganglionic neurons
was that over half (8 of 15) of the cultured LCs showed membrane
potential oscillations (Fig. 9B); these were observed in
only 5 of 30 isolated CG preparations. Table 1 shows the average
resting burst rate, burst amplitude, pacemaker slope, and membrane
oscillation amplitude recorded from cultured cells.
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Because the SCs were difficult to see in the intact ganglion, fewer were isolated. None of the SCs plated exhibited membrane potential oscillations. Moreover, the cellular appearance and spike pattern of SCs were very different from those of LCs (Fig. 9C). For instance, all SCs (n = 3) fired tonically at a faster rate (3.0 ± 0.3 Hz), and their spikes were of larger amplitude than those recorded from LCs. Pacemaker slope was 14.92 ± 3.26 mV/s (n = 2). These values for SC pacemaker slope were slightly greater than the slopes calculated for LCs.
Hormonal modulation of isolated CG neuronal activity in primary cell culture
To determine whether hormonal effects seen in the CG neurons were direct, we tested their effects on the individually isolated cells. As with the isolated ganglion preparation, all data for hormonal effects on cultured neurons were obtained from LCs (n = 15). The time course of the onset and recovery of each hormone's effects were similar to those observed in the isolated ganglia. Before hormone treatment, most of the cultured neurons were quiescent but later exhibited bursting activity in response to hormone application. These changes in burst rate were significant (P = 0.001-0.008, Fig. 10). Control saline applications had no effect on the burst characteristics of cultured LCs (Fig. 5B).
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On three different occasions, all four hormones were tested on a single neuron (Fig. 11). For oscillating cells, CCAP was the only hormone that significantly increased the amplitude of oscillation (67%, P = 0.0072). In fact, CCAP significantly increased all measured variables (rate, P = 0.001; amplitude, P = 0.017; slope, P = 0.004; Figs. 10 and 11). Proctolin, CCAP, and 5-HT significantly increased pacemaker slope (P = 0.002-0.004, Fig. 10). Desensitization was observed in two of four experiments in which F2 was applied twice in succession; no other hormone produced desensitizing responses. All hormones increased burst amplitude; this change was significant for F2 and CCAP (P = 0.007 and P = 0.017, respectively; Fig. 10).
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In comparing Figs. 3 and 10, which summarize the effects of hormones on the isolated ganglion and isolated LCs, respectively, some differences were noted. The results were qualitatively similar in that hormones were excitatory in both preparations, and increased burst rate, burst amplitude, pacemaker slope, and amplitude of membrane potential oscillations. However, greater increases in burst rate, amplitude, and pacemaker slope were recorded in response to these same hormones in cultured cells, which appeared more responsive to hormonal actions.
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DISCUSSION |
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In vitro cell culture system
In the past, attempts to examine basic electrophysiological characteristics of the CG neurons from smaller crabs (such as C. maenas), have been hampered by the fact that their ganglia are almost impossible to visualize under a dissection microscope. This, however, can now be easily achieved by using a simple enzymatic technique that was developed during this study. To further characterize the intrinsic membrane and hormonal response properties of the CG neurons, we developed an in vitro isolation technique, where individually identifiable CG neurons were extracted from the intact ganglia and maintained in primary cell culture. This, to the best of our knowledge, is the first study to accomplish these goals in a crustacean preparation.
Techniques to isolate individual neurons from invertebrate species were
originally developed in mollusks (Dagan and Levitan 1981; Wong et al. 1981
) and leeches
(Fuchs et al. 1981
). Specifically, some of these culture
techniques were described for molluscan neurons as early as the 1970s
and 1980s (Haydon et al. 1984
; Kaczmarek et al.
1979
; see review by Bulloch and Syed 1992
). Not
only have molluscan, insect, and annelid neurons been shown to survive
in cell culture, they also exhibit neurite outgrowth and reestablish specific synapses (Bulloch and Syed 1992
; Fuchs
et al. 1992
; Syed et al. 1990
; Thomas et
al. 1987
; Wong et al. 1981
). Generally, the
above cell culture models have proved highly beneficial for understanding mechanisms underlying learning, memory, growth cone behavior, synaptic plasticity, regeneration, and the formation and
function of synapses (Bulloch and Syed 1992
;
Moffett 1995
, 1996
).
Cell culture techniques for crustacean neurons are, however, still in
their infancy. Only crab (Cardisoma carnifex) and lobster (Panulirus marginatus) eyestalk X-organ peptidergic neurons
(Cooke et al. 1989; Grau and Cooke 1992
),
and lobster stomatogastric (STG) neurons (Panulirus
interruptus) (Panchin et al. 1993
;
Turrigiano and Marder 1993
) have been examined
morphologically and electrophysiologically in primary cell culture.
Grau and Cooke (1992)
alluded to problems with adhesion
of STG neurons (but not X-organ neurons), due to the thick glial sheath
surrounding each neuron. Some difficulties with cellular adhesion and
microelectrode penetration were also encountered during this study.
However, once attached to the poly-L-lysine substrate, the
cells usually adhered firmly and exhibited electrophysiological features that were characteristic of neurons recorded from the isolated
ganglia. Attempts were not made to identify trophic factors that
promote neurite outgrowth from these cells.
In the present study, the electrophysiological recordings were made
from neurons that were maintained in culture for one day. In contrast
to molluscan neurons (Syed et al. 1990), there was no
evidence of growth or sprouting in culture for either small or large
C. maenas cardiac ganglion neurons during this time period. After one day in culture, cells generally exhibited bursting behavior that was similar to that recorded from the isolated ganglia. This contrasts with previous studies on the lobster STG, where neurons were
initially silent in culture for a few days (inexcitable), but
subsequently regained tonic firing and bursting activity
(Panchin et al. 1993
; Turrigiano and Marder
1993
; Turrigiano et al. 1994
, 1995
). Another finding from the present study that
contrasts with previously published data relates to the spontaneous
bursting activity of the LCs. In the earlier studies, either chemically or electrotonically mediated inputs from SCs or LCs were considered necessary for LC burst generation (Tazaki and Cooke
1983c
; reviewed in Cooke 1988
). In the present
study, however, both SCs and LCs isolated from all synaptic inputs
showed spontaneous bursting patterns, suggesting that they exhibit
intrinsic membrane conductances necessary for burst generation. This
does not, however, underscore the importance of synaptic interactions
in the intact ganglion, which may serve to coordinate the activities of
LCs and SCs during rhythm generation.
Although enzyme treatments are standard protocols in cell culture
studies, they are also known to affect ionic fluxes across the membrane
and therefore, may perturb neuronal excitability and synaptic
transmission (Hermann et al. 1997). In the present study, neurons appeared both morphologically and electrophysiologically healthy despite the enzymatic treatments. For instance, the membrane potentials recorded from the enzymatically treated LCs (range:
12 to
65 mV in isolated ganglia, vs.
10 to
56 mV for cultured cells)
were similar to those recorded from lobster and crab CG neurons that
were isolated without the enzymatic treatments. Values were reported to
range from
42 to
60 mV in Homarus americanus lobsters
(Miller and Sullivan 1981
) and were documented as
54 mV in portunid crabs (Tazaki and Cooke 1979a
).
Furthermore, the bursting activity of the LCs, which is another measure
of excitability, was similar in the present study to that of previously
published data (Benson 1980
; Berlind
1985
; Miller and Sullivan 1981
; Tazaki and Cooke 1983b
). Finally, as detailed below, cell responses to hormone applications also matched previous studies (Cooke and Hartline 1975
; Freschi 1989
; Lemos and
Berlind 1981
; Miller and Sullivan 1981
). Because
we could not isolate the CG from C. maenas hearts without
enzymatic treatment, the effects of enzymes on hormonal responses could
not be compared. Taken together, the above data clearly validate the
utility of both isolated ganglia and cultured cells for further studies
on intrinsic membrane and network properties of this simple CPG network.
Burst characteristics of both in situ and in vitro isolated
neurons recorded in this study were similar. In the past, it has been
suggested that there may be functional diversity among the large
ganglionic neurons, including the speculation that LC5 may have some
impulse-generating ability in the Japanese spiny lobster (Kuramoto and Kuwasawa 1980). However, in the data
collected in the present study, no individual differences were noted in
the physiological activity or responses to hormones among LCs 1-5. There were, however, two main differences between in vitro and in situ
preparations. First, at rest, most in vitro neurons were quiescent
(i.e., not firing spikes), whereas in situ cells fired spontaneously.
Second, in vitro neurons were more likely to show spontaneous membrane
oscillations than in situ neurons. There were some qualitative
differences in hormonal responses (Figs. 3 and 10). For instance,
hormonal applications to neurons in the isolated ganglia coordinated
the bursting patterns and resulted in augmented cellular responses.
These responses might be attributed to hormonal effects on synaptic
interactions between the neurons in the intact ganglia. Because
individually cultured cells were devoid of axonal and dendritic
branches, gap junctions, and chemical synaptic connections to other
neurons, these features may serve to decrease
Rinput and hence enhance cell excitability. It
was, however, interesting to note that neurons in vitro exhibited more dramatic changes in their bursting behavior than the in situ
preparation in response to direct hormone application. These data
suggest that hormones may directly alter specific ion conductances that underly neuronal excitability. Thus not only did hormones appear to
alter synaptic properties, as indicated by more coordinated and regular
bursts in the isolated ganglion, they also appear to have modulated the
neuronal excitability of individual cells.
Morphology and electrophysiology of small cells
This study provides the first anatomic description of the SCs in
C. maenas. All SCs observed here were clustered below LCs 4 and 5, were encased in a pocket comprised of connective tissue, and
were buried deep within the CG. However, in vitro isolated SCs showed
no evidence of the thick glial sheath that covered individual LCs;
rather, these neurons were devoid of any covering (see Fig. 8).
Although only four SC recordings were made from both in situ and in
vitro preparations, some generalizations can be drawn about their
firing characteristics. In both preparations, the SC firing pattern was
very different from that of LCs and of previously reported SC activity.
In short, SCs fired tonically, in single spikes occurring at regular
intervals, whereas rhythmic bursting was recorded from LCs in the
present study and in SCs reported in other studies (Tameyasu
1976; Tazaki and Cooke 1979a
). The SC pacemaker
potentials recorded in the present study were faster and steeper (Fig.
9C) than those observed in SCs from other species
(Tameyasu 1976
; Tazaki and Cooke 1979a
).
Moreover, we showed that SCs fired tonically and did not generate slow
driver potential depolarizations; this contrasts with other studies
that have recorded prolonged SC driver potentials with superimposed spikes (Tameyasu 1976
; Tazaki and Cooke
1979a
). Our data are consistent with previous studies on the
rate of spike trains in SCs, which was twice (or more) that of the LC
firing (Tazaki and Cooke 1979a
). Because cultured SCs
fired tonically, in the absence of any synaptic inputs, our data
clearly show that contrary to previous thinking, these cells have the
intrinsic ability to fire. In cell culture, this spontaneous activity
is tonic, whereas, in the intact ganglion, the SCs fire in bursts
(Tameyasu 1976
; Tazaki and Cooke 1979a
), implying that in the intact ganglion network interactions must coordinate SC activity.
Hormonal modulation of large cell activity
PROCTOLIN (PR).
The peptide proctolin is present in shore crab pericardial organs (POs)
(Stangier et al. 1986), where it exerts myotropic effects on the heart contractility (Saver 1997
).
Specifically, proctolin was shown to increase heart rate,
electromyogram amplitude, and intracellularly recorded excitatory
junction potentials in intact, open, and isolated C. maenas
heart preparations (Saver 1997
). Our previous findings
(Saver 1997
; Saver and Wilkens 1998
; Saver et al. 1998
)
suggested that one site of action for proctolin is at the CG. In
isolated lobster CGs, proctolin depolarizes LCs and increases their
burst frequency and pacemaker potentials (Freschi 1989
;
Miller and Sullivan 1981
). In tetrodotoxin-silenced
lobster cardiac ganglia, proctolin application evoked depolarization
and repetitive driver potentials (Miller and Sullivan
1981
). Doublet bursts were often observed with proctolin
treatment. Proctolin-induced effects on isolated ganglia and cultured
cells observed in the present study were consistent with earlier
literature. For instance, the present study showed that proctolin
increased all burst parameters and induced immediate bursting activity
in previously silent cells. The main difference between the present
findings and those of previous studies was the time course of proctolin
action. We recorded rapid responses to proctolin (within 0-10 s),
whereas slower onset (60-90 s) and longer-lasting effects (10-20 min)
were reported elsewhere (Freschi 1989
; Sullivan
and Miller 1984
). This differential time course for peptidergic
responses can be attributed to different methods and amounts of peptide
application. In the current study, small amounts of proctolin
(estimated to be 0.5-1 µl) were pressure applied ("puffed")
directly onto the LC soma under investigation. In other studies,
proctolin was either bath applied for 2-3 min, or small pulses
(aliquots of 50-100 µl) were delivered "upstream" of the
preparation (Miller and Sullivan 1981
; Sullivan
and Miller 1984
). Another likely explanation for the faster
onset and recovery could be the fact that in our present study the
ganglion was stripped clear of its connective tissue sheath (although
each neuron soma was still surrounded by its own sheath). This in turn
may have facilitated quicker access for all solutions to the preparation.
CCAP.
Previously, CCAP was tested on intact or isolated C. maenas
hearts, where it produced chronotropic responses (Saver
1997; Stangier 1991
; Wilkens and Mercier
1993
). The chronotropic nature of CCAP's effects suggests that
its site of action is exclusively at the CG. Interestingly, other
crustaceans such as the lobster H. americanus and the
crayfish Procambarus clarkii, show negligible heart rate
responses to this nonapeptide (Wilkens, unpublished observations).
These species-specific effects of CCAP are attributed to different
concentrations of CCAP in the POs of each species. In C. maenas, the POs contain greater amounts of CCAP than other crustaceans (Stangier 1991
; Stangier et al.
1987
), which confirms the notion that these animals
utilize CCAP and have receptors for it.
5-HYDROXYTRYPTAMINE (5-HT).
The amine 5-HT was tested previously on isolated CGs from the lobsters
H. americanus (Cooke and Hartline 1975;
Lemos and Berlind 1981
) and Panulirus
japonicus (Kuramoto and Yamagishi 1990
), where it
increased neuronal burst frequency. In this study, 5-HT application to
LCs (both in situ and in vitro) significantly increased burst rate and
pacemaker slope. Most 5-HT applications produced large increases in
burst rate that lasted for 3-4 min. Burst duration and number of
spikes per burst were not calculated in this investigation.
PEPTIDE F2.
The actions of FMRFamide-related peptides (FaRPs) have been examined on
various heart preparations in several different crustaceans species
(blue crabs, Krajniak 1991; crayfish, Mercier and
Russenes 1992
; lobster, Trimmer et al. 1987
). In
particular, the peptide F2 (SDRNFLRFamide) was first isolated and
sequenced from lobster, where it was found in high concentrations in
the POs, and was shown to function as a cardioexcitor (H. americanus) (Kobierski et al. 1987
; Trimmer
et al. 1987
). F2 increases spontaneous contraction rate and
amplitude in isolated crayfish hearts (P. clarkii)
(Mercier and Russenes 1992
). In the present study, F2
increased LC burst rate and pacemaker slope in both intact ganglia and
cell culture preparations. These data are therefore consistent with
other functional observations and suggest a role for this peptide at
the neuronal level.
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ACKNOWLEDGMENTS |
---|
We thank Dr. Gaynor Spencer for reading earlier drafts of this manuscript and W. Zaidi for techincal support. N. I. Syed is an Alberta Heritage Foundation for Medical Research Senior Scholar.
Support for this research was obtained from the Natural Sciences and Engineering Research Council of Canada for J. L. Wilkens and from the Medical Research Council of Canada for N. I. Syed. M. A. Saver received generous scholarship funding from the Burns Memorial Fund and from Royal Canadian Legion Branch #255.
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FOOTNOTES |
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Address for reprint requests: N. I. Syed, Dept. of Cell Biology and Anatomy, University of Calgary, Health Sciences Centre, 3330 Hospital Dr. N.W., Calgary, Alberta T2N 4N1, Canada.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 10 June 1998; accepted in final form 8 March 1999.
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REFERENCES |
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