We explored whether the calcium imaging can be applicable to detection of spike activity of Aplysia wide-spread neurons. Well-used procedures, the membrane permeable acetoxymethyl (AM) types and the retrograde labeling could not be used for loading the calcium sensitive dye into many neurons, however, impalement of each cell body with a microelectrode containing high concentration of the dye solution in turn could load the dye iontophoretically into many large neurons in a relatively short time and into several small neurons in a very short time. Spike activity just induced a fluorescent increase in the cell body while depolarization without spikes induced it in the axon hillock. In the cell body the slope of the fluorescent increase was almost relative to the spike frequency while the period of it almost corresponded to the firing duration. Therefore, the phase relationship of the rhythmic firing responses in plural neurons could be precisely detected. Moreover stable responses could be repeatedly obtained for a long time from small neurons unsuitable for a long time recording with microelectrodes. Removal of the extracellular Ca2 completely suppressed the spike-induced increase in fluorescence in the cell body and application of nifedipine partly reduced it, suggesting contribution of L-type channels distributing in Aplysia wide-spread neurons.
INTRODUCTION
In vertebrate and invertebrate neurons the calcium imaging method has been widely used to study intracellular calcium regulation concerning synaptic activity such as transmitter release in presynaptic regions (Edmonds et al., 1990; Eliot et al., 1993; Dowdall et al., 1997) or receptor functions in postsynaptic regions (Regehr and Tank, 1992; Kyrozis et al. 1995; Spruston et al. 1995; Yuste and Denk 1995; Svoboda et al., 1997), and also concerning neurosecretion (Jonas et al., 1997) and release from intracellular calcium stores (Friel and Tsien, 1992; Jonas et al., 1997). In several species of animals this method has also been used to know neuronal spike activity (O'Donovan et al., 1993; McClellan et al., 1994; Fetcho and O'Malley, 1995; Lev-Tov and O'Donovan, 1995; O'Malley et al., 1996). Application of this method to studies for intracellular Ca2+ roles is easily acceptable because intra-cellular Ca2+ concentration ([Ca2+]i) is directly measured. In contrast, indirect detection of spike activity by this method requires assumption that the firing of interested neurons always cause an increase in [Ca2+]i quantitatively correlating with the firing frequency and the firing duration of neurons.
The marine gastropod Aplysia has been used to study general properties of neurons or neural networks for some specific behaviors because small number of neurons included in the nervous system enable us to identify each neuron easily and large cell bodies also enable us to use conventional electrophysiological techniques easily (Kandel, 1976). However, the conventional methods using microelectrodes have difficulty in recording of activity from many neurons simultaneously or long recording of activity from neurons whose cell bodies are small or hidden within neuropiles. The calcium imaging method may be a powerful technique to overcome these difficulties. In Aplysia californica several types of neurons including bursting neurons, peptidergic endocrine neurons and sensory neurons have demonstrated that repetitive firing of these neurons results in the increase in [Ca2+]i (Stinnakre and Tauc, 1973; Gorman and Thomas, 1978; Blumenfeld et al., 1990; Knox et al., 1996). Spike activity of these neurons will be exactly detected by the calcium imaging method. However, it is unknown whether the calcium imaging method is applicable to detection of spike activity of wide-spread neurons in Aplysia nervous system.
In the present experiments we initially tried the membrane permeable dyes (acetoxymethyl (AM) types) and the retrograde labeling to load the calcium sensitive dye into many cells but these were not appropriate for Aplysia neurons, similar to the other gastropods (Alkon et al. 1992; Ito et al., 1994). Alternatively, impalement of each cell body with a microelectrode in turn could load the dye iontophoretically into many large neurons in a relatively short time and into several small neurons in a very short time. Moreover we explored whether this methods could be quantitatively detect neuronal spike activity and how [Ca2+]i increased following spike activity by the use of already identified neurons in the buccal ganglia (Nagahama and Takata, 1988, 1989; Nagahama and Inoue, 1999). Some of the findings in this article have been presented in a preliminary communication (Yoshida et al., 1998).
MATERIALS AND METHODS
Animals
Aplysia kurodai, weighing 50–250 g, were collected from the coast of Yura in Awaji island or Mihama in Fukui. Animals were maintained in aquarium filled with aerated and filtered artificial seawater (ASW) at 14°C.
Preparation
For dissection, animals were anesthetized by injection of iso-tonic magnesium chloride at room temperature. The preparation comprised the buccal muscles and buccal ganglia. The buccal mass was cut into halves along the midline to separate paired symmetrical buccal musculatures. The cerebral-to-buccal connective (cbc) and the peripheral nerves were severed except the buccal nerves 2 and 3 (n2 and n3). The ganglia and muscle preparation was pinned to the Sylgard (Dow) surface of the recording chamber. All experiments were carried out at room temperature (22–24°C).
Electrophysiology and dye loading
For intracellular recordings and dye loading, glass microelectrodes filled with 4% Calcium Green-1 hexapotassium salt (Molecular Probes) in distilled water (10∼20 MΩ) were used. After impalement of a cell body with it, the dye was iontophoretically introduced with 10–20 nA of hyperpolarizing DC current for 0.5–10 min and spike activity of the neurons was induced by electrical nerve stimulation or direct current injection. The dye concentration was prepared to be considerably higher than that reported in the other studies because of two reasons. We lowered the electrode resistance as much as we can with pure dye aqueous solution because addition of other ion species reduced the electrode resistance but also prevented the dye from loading with electrophoresis. For nerve stimulation, polyethylene suction electrodes (50–150 μm in diameter) were used.
Calcium imaging with LSM
The recording chamber was mounted on a stage of a confocal laser scanning microscope (LSM: Zeiss, LSM-310). The neurons were visualized with a 10x objective (0.3 NA). When we used the confocal images, fluorescent signals from the cell bodies of neurons being out of the confocal plane were very small because the cell bodies of neurons within the ganglia were distributed in three-dimension. Therefore, we usually used this microscope without confocal observation. Calcium Green-1 was excited by 488 nm line of an argon laser. The emission fluorescence passed through a dichroic reflector (FT510) and a barrier filter (LP515) was detected. Brightness and contrast levels were adjusted to achieve minimal background fluorescence, visualization of the filled neuron, and sufficient dynamic range for increases in fluorescence. A change in fluorescence was measured from the ROI (region of interest) flame, where we addressed an interested region within 512×512 pixels of a full image, at 0.3–1.0 sec intervals for 30–90 sec. Fluorescent intensity was averaged over all pixels in ROI area. Changes in fluorescence were defined as ΔF/F (in percent), where F was average fluorescent intensity during 1 sec just before stimulation and ΔF was a time-dependent change in fluorescence. In the case of neurons showing spontaneous rhythmic change in fluorescence, only data showing the lowest level of the fluorescence just before stimulation were adopted for the analysis.
Calcium imaging with EFM
In some experiments, the recording chamber was mounted on a stage of an epifluorescence microscope (EFM; Olympus, BX50WI stage-fixed microscope), and a cell body was impaled with a glass microelectrode filled with ∼1% Calcium Green-1 diluted with 200 mM KAcetate. After the dye loading fluorescent change and membrane potential were simultaneously recorded. In this case we used lower concentration of dye solution with other ion species for lowering electrode resistance because we found that long impalement with a electrode containing high concentration of dye solution gradually increased the intracellular dye concentration with diffusion. SpectraMASTER (Olympus) used in the present experiments was a monochromatic light source originated from a Xenon light source and the output wavelength could be continuously varied between 300 and 700 nm with full control of the half width full height bandwidth between 5 and 30 nm. For the calcium imaging we used it as a light source of 488 nm with a bandwidth of 15 nm. The emission fluorescence passed through a dichroic reflector (DM510) and a barrier filter (BA515) was detected. The fluorescent image was acquired by a cooled CCD camera (Olympus, Olympix FE250) at a rate of 20∼25 flames/sec and the image sequences were stored and analyzed in the MERLIN imaging system (Olympus).
Solution
ASW used in the present experiments had the following composition (mM): NaCl 470; KCl 11; CaCl2 11; MgCl2 25; MgSO4 25; TrisHCl 10 (pH 7.8–7.9). In a Ca2+ free solution Ca2+ concentration was reduced to 0 mM by replacement of Mg2+ to maintain osmotic balance. In exploring the effect of ω-conotoxin GVIA, it was applied to the preparation in a Low Ca2+ (1.1 mM), Low Mg2+ (1 mM) ASW by replacement of Tris-HCl because of the reduced efficacy of the toxin in solutions containing high concentration of divalent cations. After 30 min application of the toxin, normal ASW was reintroduced and allowed to equilibrate for 15–20 min (Fossier et al., 1994). Nifedipine and amiloride were prepared as 10 mM and 500 mM stock solutions in dimethylsulfoxyde (DMSO) respectively. ω-conotoxin-GVIA or ω-agatoxin-TK was prepared as 100 μM stock solution in distilled water. These chemicals were purchased from Sigma. It has been reported that all chemicals and solutions used in this study had no effect on fluorescent properties of the dye (Dowdall et al., 1997; Jonas et al., 1997).
RESULTS
Application of the calcium imaging to the study of Aplysia nervous system
Initially, we tried well-used two procedures to load the calcium sensitive dye into many cells, one was the employment of the membrane permeable dyes (AM types) and the other was the retrograde labeling. In the use of the AM types we tried several kinds of dyes and widely changed the dye concentration, the loading time and the temperature (10–100 μM, 0.5–24 hr, 4–36°C) but any cell bodies were not loaded. In the retrograde labeling we used 3,000 MW dextran type of Calcium Green-1 reported as diffusing faster in the axon (Popov and Poo, 1992; Fritzsch, 1993), but a very few cells were loaded even after a transport of 24–48 hr although loading of much more cells was expected according to results of retrograde staining with the other chemicals such as cobalt chloride.
Alternatively we tried to inject the dye, Calcium Green-1, into neurons iontophoretically by impalement of each cell body with a microelectrode containing high concentration of the dye solution in turn and could easily load the dye into large or intermediate cell bodies (>50 μm) as many as we wished in a relatively short time (3–10 min/cell). In this procedure, photobleaching of the dye depending on the order of loading into cells did not affect the fluorescent signals in all cells. The buccal ganglia show symmetrical structures and include dozens of large or intermediate cell bodies of neurons (>50 μm) and hundreds of small cell bodies (10–50 μm, S-cluster neurons). Fig. 1A shows a typical result for the loaded large or intermediate cell bodies, in which 12 cell bodies were loaded. We previously showed that repetitive electrical stimuli of nerves such as the cbc produced the feeding-like response, in which rhythmic firing patterns similar to those during the ingestive response were induced in the identified neurons (Nagahama and Takata, 1988, 1990). Therefore, in order to know whether the calcium imaging method can be applicable to most of Aplysia neurons, the change in fluorescence (ΔF/F) of these neurons were explored during the feeding-like response. When repetitive stimuli were applied to the cbc nerve, the fluorescent intensity increased in most cells and rhythmic changes in fluorescence were induced in more than 6 cell bodies despite of low or high concentration of the loaded dye (Fig. 1A2). This result suggests that the intracellular dye concentration in all cells may be sufficient to detect fluorescent signals and the variation may not affect the detection. The rhythmic change in fluorescence usually lasted for 30 sec-1 min even after cessation of the stimuli and the increased fluorescence returned to the resting level 1–2 min after cessation of the rhythmic response. In these experiments, therefore, we repeated the stimuli in more than 5 min intervals. Next, in order to explore whether the calcium imaging method could detect spike activity of small neurons unsuitable for a long-time recording with microelectrodes, the dye was iontophoretically loaded to cell bodies of the S-cluster neurons with microelectrodes in a very short-time (0.5–2 min/cell). These neurons have been reported as sensory neurons sending afferent axons into the buccal nerves (Jahan-Parwar et al., 1983). We found that electrical stimulation of one of the buccal nerves increased the fluorescent intensity in the cell bodies and the stable response could be repeatedly obtained for a long time (Fig. 1B). These results suggest that the impalement procedure for the dye loading may be very useful in application of the calcium imaging to Aplysia wide-spread neurons.
Fig. 1
Application of the calcium imaging to Aplysia neurons. A: Pseudocolor images of 12 neurons loaded with a calcium sensitive dye, Calcium Green-1 in the buccal ganglion during resting level (1) and changes in fluorescence in these neurons induced by repetitive electrical stimuli of the ipsilateral cbc nerve (2). Color bar in A1 shows fluorescent intensity appeared in pseudocolor. The stimuli were applied 20 sec after onset of the measurement (2.5 Hz, 2V). B: Pseudocolor images of 2 small neurons in the S cluster shown in A1 and the fluorescent change in these neurons induced by electrical stimulation of the ipsilateral buccal nerve 2 (arrow head, 5 msec, 20 Hz, 40 pulses, 1.5 V). These experiments were performed with the EFM after the dye was loaded into all test neurons. Stimulus conditions in parenthesis show pulse duration, pulse frequency, pulse number, and stimulus intensity, respectively.
Relationship between spike activity and fluorescent change
In the next step, whether the change in fluorescence corresponded to the neuronal spike activity was explored. Then we compared the results obtained from the LSM and the EFM experiments. In the LSM experiments spike activity was initially recorded with a microelectrode and then the change in fluorescence in the cell body of the same neuron was explored later with the LSM. On the other hand, in the EFM experiments the spike activity and the change in fluorescence were simultaneously explored (see the Methods). Figs. 2A–B show typical results obtained from the MA1 neuron in two types of experiments. In the MA1, electrical stimulation of the ipsilateral n3 usually produced the high frequency of firing containing antidromic spikes and additional spikes induced by the excitatory postsynaptic potentials (EPSPs). When the MA1 was fired, a transient increase in fluorescence was synchronously induced in the cell body of the MA1. The relationship was also explored in the MA1, JO1 and JC2 when the ipsilateral esophageal nerve was electrically stimulated (Fig. 2C). Slow EPSPs were induced in the JO1 and JC2 and they fired several seconds after the stimulation while high frequency of firing was induced in the MA1 immediately after the stimulation. In the LSM recording of these neurons, fluorescence in cell bodies largely increased almost corresponding to the firing phase. Moreover, in the EFM experiments the spontaneous bursts of spikes in the MA1 well corresponded to the fluorescent increase in the cell body (Fig. 2D). These results support that an increase in fluorescence in the cell body region may accompany spike generation in test neurons and both types of microscopes can be sufficiently used for detection of the neuronal spike activity. It is noted that this method can not detect a single spike generation but can sufficiently detect the burst of spikes as shown in Fig. 2D.
Fig. 2
Changes in fluorescence following spike activity in test neurons. A: Spike activity in a MA1 (1) and a transient increase in fluorescence in the cell body of the same neuron (2) evoked by electrical stimulation of the ipsilateral buccal nerve 3 (bar, 5 msec, 10 Hz, 20 pluses, 1 V) separately obtained from the electrophysiological experiments and the LSM experiments. B: Simultaneous recordings of spike activity (1) and a change in fluorescence (2) in the cell body of a MA1 evoked by electrical stimulation of the ipsilateral buccal nerve 3 with the EFM (bar, 5 msec, 15 Hz, 30 pluses, 0.3V). C: Spike activity in a MA1, a JO1 and a JC2 (1) and transient increases in fluorescence in cell bodies of the same neurons (2) evoked by stimulation of the ipsilateral esophageal nerve (bars, 5 msec, 33 Hz, 20 pulses, 6 V). D: Simultaneous recordings of spontaneous rhythmic burst activity (1) and the change in fluorescence (2) in the cell body of a MA1 neuron with the EFM.
Dependence of the fluorescent change on measuring sites
A change in fluorescence was initially measured in a whole cell body region. We next explored an effect of measuring sites on the change in fluorescence following spike generation. In this study, the LSM with good space resolution was used. Fig. 3A shows an example of results obtained from the MA1 when the ipsilateral n3 was electrically stimulated. The comparison of the change in fluorescence in two locations of the JO1 when the esophageal nerve was stimulated is also shown in Fig. 3B. In both cases, the increase in fluorescence following spike activity was larger in the axon hillock than the middle of the cell body. However, it is noted that the fluorescence slightly increased just after stimulation in the axon hillock of the JO1 but not in the middle of the cell body. This period corresponded to small depolarization of the neuron with no spikes. These results suggest that in order to use the calcium imaging only for detection of neuronal spike activity, the change in fluorescence should be measured in the middle of the cell body despite the absolute small change because the similar small change in the axon hillock may correspond to only depolarization of the neuron.
Fig. 3
Dependence of a change in fluorescence on measuring sites. A: Changes in fluorescence in the cell body and the axon hillock of a MA1 when the ipsilateral buccal nerve 3 was stimulated (arrow head, 5 msec, 33 Hz, 10 pulses, 6 V). B1: Spike activity in a JO1 evoked by electrical stimulation of the ipsilateral esophageal nerve (arrow head, 5 msec, 33 Hz, 20 pulses, 6 V). B2: Changes in fluorescence in the cell body and the axon hillock of the same JO1 neuron evoked by the same stimulation as in B1. Measuring sites are surrounded by squares in top images.
We also found that the time course of the fluorescent change in the whole region of the cell body including the axon hillock was very similar to that in the middle of the cell body. It may come from the fact that the area of the axon hillock was much smaller than that of the cell body, and the change in fluorescence measured in the whole cell body region could not reflect the small change in the axon hillock.
Quantitative analysis of the spike-induced change in fluorescence
In order to ascertain whether the calcium imaging could quantitatively detect the neuronal spike activity, dependence of the change in fluorescence on firing frequency and duration were explored. We used two types of stimulation to produce spike activity, electrical nerve stimulation and depolarizing current injection. The LSM and the EFM were used for experiments of the nerve stimulation and the current injection, respectively. The ipsilateral n3 stimulation easily produced full size of spikes in the MA1 and the firing properties of this neuron were easily controlled. Therefore, the MA1 neuron was used for these experiments.
In the LSM experiments (n=5 preparations), the firing frequency of the MA1 was controlled by changing the frequency of train pulses of the nerve stimulation while the firing duration was kept constant by adjusting the pulse number. Each pulse evoked an antidromic spike and EPSP-induced several spikes. The measurement for each frequency of stimuli was repeated ten times. Fig. 4A shows time courses of the averaged spike number measured every 0.3 sec when the spikes were induced with different stimulus frequencies. The averaged spike number was almost steady and increased with increasing the stimulus frequency. Time courses of the averaged fluorescent change induced in the cell body of the same neuron by the same stimulus conditions are also shown in Fig. 4B. The averaged ΔF/F showed a transient increase and the peak value increased with increasing the stimulus frequency. When we plotted the averaged slope (%/s) of the ΔF/F increase against the averaged spike frequency (spikes/sec), a good linear relationship was obtained (Fig. 4C). We further explored effects of the spike frequency on the fluorescent increase following current-induced spikes (n=6 preparations). Figs. 4D shows simultaneous recordings of the membrane potential and the fluorescent change with the EFM in the cell body of a MA2 neuron having similar functions to the MA1 within the ganglia (Nagahama and Takata, 1989, 1990). The firing of the MA2 with current injection induced a transient increase in fluorescence. And increasing the injection current with constant duration caused increases in both the spike frequency and the change in fluorescence. Fig. 4E shows a good linear relationship between the averaged spike frequency and the averaged slope of the ΔF/F increase. In both types of experiments the ΔF/F increased until the neuron ceased the firing and the slope of the ΔF/F increase became steeper with increasing the spike frequency. In comparison between Figs. 4A and B, the gradual decrease in spike frequency corresponded to the gradual decrease in slope of the tangential line of the ΔF/F increase, i.e., to the upward curvature. In all test neurons the similar relationship was also obtained.
Fig. 4
Relationship between spike frequency and a change in fluorescence. A–C: Electrophysiological experiments and the LSM experiments performed separately. D–E: The EFM experiments. A: Time courses of the averaged number of spikes measured every 0.3 sec in a MA1. The neuron was stimulated 3 sec after onset of the measurement with 2.5, 5, 7.5, 10, 15 and 20 Hz stimulus pulses (5 msec, 1 V), and the firing duration was adjusted to 2 sec by changing pulse numbers. B: Time courses of the averaged change in fluorescence induced in the cell body of the same MA1 by the same stimulus conditions as in A. Measurement was repeated ten times for each condition in A and B. C: The averaged slope of the ΔF/F increase plotted against the averaged spike frequency. Vertical lines in A and B show the standard error of mean (SEM). D1: Two examples of spike activity in a MA2 evoked by injection of the depolarizing current (1 sec, 4–10 nA). D2: The change in fluorescence in the cell body simultaneously recorded with spike activity in D1. E: The averaged slope of the ΔF/F increase plotted against the spike frequency. The averaged slopes and the lines in C and E were obtained from the least-squares method.
Next, we explored a relationship between the firing duration and the fluorescent increase. In the LSM experiments (n=6 preparations), the firing duration was elongated by increasing the number of stimulus pulses with constant frequency (14.3 Hz). The measurement for each stimulus condition was repeated ten times and the time courses of averaged frequency of spikes are shown in Fig. 5A. In this preparation, the firing frequency was not constant during stimulation but initial time courses were almost the same in all stimulus conditions. Time courses of the averaged ΔF/F in the cell body of the same MA1 are also shown in Fig. 5B. The peak value of the averaged ΔF/F increased with increasing the stimulus number, in which the slope of the fluorescent change was almost the same but the period from the onset to the peak increased. The peak time almost corresponded to onset of the rapid fall in the firing frequency for each stimulus condition. When the averaged period of the ΔF/F increase was plotted against the averaged firing duration, a good linear relationship was obtained (Fig. 5C).
Fig. 5
Relationship between firing duration and a change in fluorescence. A–C: Electrophysiological experiments and the LSM experiments performed separately. D–E: The EFM experiments. A: Time courses of the averaged number of spikes measured every 0.3 sec in a MA1. The neuron was stimulated 3 sec after onset of the measurement. The numbers of stimulus pulses (5 msec, 14.3 Hz, 0.5 V) were 10, 20, 30, 40, 50, and 60, respectively. B: Time courses of the averaged change in fluorescence induced in the cell body of the same MA1 by the same stimulus conditions as in A. Measurement was repeated ten times for each condition in A and B. C: The averaged period of the ΔF/F increase as a function of the averaged firing duration. Vertical lines in A–C show the SEM. D1: Four examples of spike activity in a MA2 evoked by the depolarizing current injection (0.5–2 sec, 7 nA). D2: The change in fluorescence in the cell body simultaneously recorded with the spike activity in D1 with the EFM. E: The period of the ΔF/F increase as a function of the firing duration. The lines in C and E were calculated by the least-squares method.
In the EFM experiments (n=5 preparations) we further explored these relationship. The firing duration of a MA2 was elongated with increasing duration of the injection current but the firing frequency was kept constant with constant current strength (Fig. 5D1). In this case, the longer firing duration induced the longer ΔF/F increase and the larger peak (Fig. 5D2). Fig. 5E shows a good linear relationship between the firing duration and the period of the ΔF/F increase. In all test neurons the similar relationship was also obtained.
These results suggest that the slope of the ΔF/F increase may be almost relative to the spike frequency whereas the period of the ΔF/F increase may almost correspond to the firing duration.
Detection of phase relationship of the rhythmic firing patterns in plural neurons
We previously reported the firing patterns in the MA1 and JC2 neurons (Fig. 6A) during the feeding-like response which was induced by repetitive electrical stimuli of the cbc (Nagahama and Takata, 1988, 1990). Fig. 6D shows a typical result of simultaneous intracellular recordings of rhythmic spike activity in these neurons, in which the MA1 firing always preceded the JC2 firing. In the present experiments, therefore, we simultaneously measured the fluorescent changes in these neurons during the feeding-like response in order to know whether the calcium imaging can detect phase relationship of rhythmic bursts of spikes in plural neurons. During the response rhythmic fluorescent changes were induced in both neurons (B), and the faster speed of representation of these recordings (C; 30–60 s in B) shows that the peak fluorescent time in the MA1 always preceded to that in the JC2 at each cycle. Considering the quantitative relationship between the spike generation and the fluorescent change, this result suggests that the JC2 firing may be behind the MA1 firing. Therefore, the deduction from the calcium imaging was well consistent with the real firing patterns and this method may be very useful to explore the phase relationship of firing patterns in plural neurons. In addition, the basal fluorescent intensity of the MA1 largely increased but then gradually decreased during the rhythmic response induced by the electrical stimulation. This change may correspond to the high frequency of sustaining firing of the MA1 just after onset of the repetitive electrical stimulation as reported previously (Nagahama and Takata, 1990). And the gradual decrease in the size of the rhythmic fluorescent change in the JC2 may also correspond to the gradual decrease in the spike activity of this neuron during the rhythmic response induced by the electrical stimulation as reported in the same study. These results also support the usefulness of the calcium imaging to detect neuronal spike activity.
Fig. 6
Simultaneous measurement of changes in fluorescence in cell bodies of the MA1 and JC2 during the feeding-like response. A: Drawing of the buccal ganglion showing location of cell bodies of these neurons. B: Simultaneous recordings of changes in fluorescence in the cell bodies of these neurons during the rhythmic response induced by repetitive electrical stimuli of the ipsilateral cbc nerve (5 msec, 2 Hz, 1 V). The stimulation was applied 10 sec after onset of the measurement. Faster speed of representation of a part of B (30–60 sec) is shown in C. The peak time of the change in fluorescence in the MA1 always preceded that in the JC2. Vertical dotted lines show the points in time when the ΔF/F in the JC2 attained its peak value. D: Simultaneous intracellular recordings of the rhythmic burst of spikes in the MA1 and the JC2 during the feeding-like response induced by stimulation of the same nerve. Recordings B and D were obtained from different preparations.
Origin of [Ca2+]i increase following spike generation
Our present results suggest that the [Ca2+]i in the cell body may increase following spike generation. Therefore, we further explored origin of the [Ca2+]i increase. Firstly, Ca2+ was removed from the bath solution by replacement of Mg2+ to maintain osmotic balance. Fig. 7A shows intracellular recordings and fluorescent changes in the cell body of the same MA1 separately recorded with the LSM when the buccal ganglia were bathed in ASW or Ca2+ free solution. The n3 nerve stimulation still induced spike activity even in the absence of extracellular Ca2+ but the fluorescent change completely disappeared in this solution. Fig. 7B also shows simultaneous recordings of the membrane potential and the fluorescent change in the cell body of the JOm neuron (Nagahama & Inoue 1999) when the ipsilateral n2 including its axon was electrically stimulated in ASW or Ca2+ free solution. Even in this neuron, the fluorescent change completely disappeared in Ca2+ free solution without reducing spike activity. Moreover, similar results were obtained from all test neurons. These results suggest that the extracellular Ca2+ may flow in accompanying spike generation through some voltage-gated Ca2+ channels (VGCC).
Fig. 7
Effects of removal of the extracellular Ca2+ on the spike-induced increase in fluorescence. A1: Intracellular recordings of spike activity in a MA1 induced by electrical stimulation of the ipsilateral buccal nerve 3 (5 msec, 14.3 Hz, 30 pulses, 0.3 V) when the buccal ganglia were bathed in ASW and Ca2+ free solution. A2: Changes in fluorescence in the cell body of the same MA1 induced by the same stimulus condition in these solutions with the LSM. B1: Intracellular recordings of spike activity in a JOm induced by stimulation of the ipsilateral buccal nerve 2 (5 msec, 20 Hz, 40 pulses, 0.4 V) in ASW and Ca2+ free solution. B2: The changes in fluorescence in the cell body simultaneously recorded with B1 by the use of the EFM.
Types of VGCCs contributing to Ca2+ influx
Electrophysiological and pharmaceutical studies in vertebrate neurons have shown that the VGCCs can be classified into several types (Nowycky et al., 1985; Regan, 1991; Teramoto et al., 1995). Moreover, the same pharmaceutical agents have been demonstrated to be applicable to classify the VGCCs in invertebrate neurons (Edmonds et al., 1990; Fossier et al., 1994; Ma and Koester, 1995). In the next step, therefore, we explored types of the VGCCs contributing to the Ca2+ influx following spike generation by the use of several blockers.
Application of Ni2+ (100 μM) or amiloride (500 μM) to the external solution can block T-type Ca2+ channels selectively (Tang et al., 1988; Viana et al., 1993; Kits et al., 1997; Todorovic and Lingle, 1998), but any effects of these blockers on the spike-induced increase in fluorescence in the MA1 were not observed even in the sufficient concentration. Moreover, we also explored the effect of selective blockers for N-type Ca2+ channels or P-, Q-type Ca2+ channels, ω-conotoxin-GVIA (5 μM) and ω-agatoxin-TK (300 nM, Teramoto et al., 1995), respectively. However, no effect on the fluorescent increase was obtained even in the sufficient concentration. In contrast, a typical blocker for L-type Ca2+ channels, nifedipine (10 μM) considerably reduced the fluorescent increase following spike generation in the cell body of MA1 although it did not change spike activity in the neuron (Figs. 8A–B). Nifedipine irreversibly reduced the fluorescent increase following spike generation. The effects of nifedipine averaged over all MA1 neurons used in the present experiments are shown in Fig. 8C together with the averaged effects of reducing extracellular Ca2+. The averaged percentage for nifedipine relative to the control was 56.0± 4.7% (n=8) and the difference from the control was significant (P<0.001, two sample t-test). Moreover, the similar results were obtained from all test neurons. These results suggest that L-type Ca2+ channels may partly contribute to the Ca2+ influx following spike generation in most neurons.
Fig. 8
Effects of an L-type channel blocker, nifedipine on the spike-induced increase in fluorescence in the cell body of the MA1. A1: Spike activity in a MA1 evoked by electrical stimulation of the ipsilateral buccal nerve 3 (5 msec, 14.3 Hz, 30 pulses, 0.3 V) when the buccal ganglia were bathed in ASW and ASW containing 10 μM nifedipine. A2: The LSM recording of an effect of nifedipine on the spike-induced change in fluorescence in the cell body of a MA1 evoked by the same stimulation as in A1. In A1 and A2 the different neurons were used because nifedipine irreversibly reduced the increase in fluorescence following the spike generation. B: Simultaneous recordings of spike activity (1) and the change in fluorescence (2) in the cell body of the same MA1 evoked by electrical stimulation of the ipsilateral buccal nerve 3 (5 msec, 15 Hz, 30 pulses, 0.3V) when the ganglion was bathed in ASW and ASW containing 10 μM nifedipine. C: Summary of averaged effects of extracellular Ca2+ removal and nifedipine on the spike-induced increase in fluorescence in the cell body of the MA1. The peak values of the transient increases in Ca2+ free solution or ASW containing 10 μM nifedipine were compared with those in ASW, and the percentage was averaged over all preparations. Vertical lines show the SEM (n=5 for Ca2+ free, n=8 for nifedipine).
DISCUSSION
In the present experiments we explored whether the calcium imaging method could be used for detection of spike activity in Aplysia wide-spread neurons. Initially, we tried well-used two procedures to load the calcium sensitive dye into many cells, the employment of the membrane permeable dyes (AM types) and the retrograde labeling. In the use of the AM types of several dyes no neurons were loaded although conditions of the dye concentration, the loading time and the temperature were widely changed. This may come from the reason that the dye can not pass through the membrane of Aplysia neurons or the AM type can not be cleaved to cell-impermeant products owing to lack of intracellular esterase.
In the retrograde labeling we used 3,000 MW dextran type diffusing faster (Popov and Poo, 1992; Fritzsch, 1993), but the dye could not be loaded into the expected many neurons. This may come from extreme dispersion of axonal diameters of the neurons. Alternatively, we could load the dye iontophoretically into large or intermediate neurons as many as we wished by impalement of each cell body with a microelectrode containing high concentration of the dye solution in turn in a relatively short time (1–10 min/cell) or into several small neurons unsuitable for a long-time electrophysiological recording by the same procedure in a very short time (0.5–2 min/cell). And we ascertained that the fluorescent intensity in most neurons increased following spike activity and we could repeatedly obtained the stable response for a long time. Therefore, this impalement procedure may be very useful for application of the calcium imaging to Aplysia neurons.
In the present experiments we used two types of microscope systems, the LSM and the EFM for the calcium imaging. Although the LSM is superior to the EFM in the space resolution but inferior in the time resolution, both systems could be used for detection of spike activity. The EFM is also superior to the LSM in the point that the membrane potential and the fluorescent change can be simultaneously recorded. This method enabled us to stimulate the neuron directly with the current injection. In the LSM we alternatively stimulated the nerves to produce neuronal spikes. In the use of the LSM and FEM we also found that these systems could not detect a single spike generation but could detect a burst of spikes. In the study of neuronal mechanisms generating some Aplysia behaviors, the neurons usually generate bursts of spikes. Therefore, these systems may be sufficient to detect the neuronal spike activity concerning behaviors.
In invertebrate nervous systems the nerves originated from the ganglia usually include afferent sensory axons and efferent motor axons. Therefore, the electrical nerve stimulation often produced both antidromic spikes and EPSP-induced spikes, and the change in [Ca2+]i caused by postsynaptic activity may be additionally detected. In the present experiments, we found that the fluorescent change was always larger in the axon hillock than the cell body. This may come from the difference in the surface area-to-volume ratio for the cell body and the axon hillock. However, in the experiments of the JO1 the increase in the axon hillock already started at the depolarizing phase without spike activity while the increase in the cell body just started at the onset of spike activity. Our results may be consistent with the results obtained from vertebrate neurons in which the [Ca2+]i increase following spike activity in the cell body is smaller than that in the dendritic regions (Jaffe et al., 1992; Lev-Ram et al., 1992; Regehr and Tank, 1992; Midtgaard et al., 1993; Svoboda et al., 1997). Invertebrate neurons generally show different structures for dendritic branches from vertebrate neurons, and the branches do not leave from the cell body. Therefore, all synaptic informations tend to concentrate on the vicinity of the axon hillock probably contributing to spike triggering and such synaptic activity may contribute to an additional [Ca2+]i increase in the axon hillock.
There may be the other possibility that low threshold voltage-gated calcium channels may exist in high density in this region. In contrast, the fluorescent change in the cell body may mainly correspond to the spike generation alone. These results suggest that for our purpose the fluorescent change should be measured in the cell body region despite a small change because the similar small change in the axon hillock may correspond to only depolarization of the neuron.
In detection of spike activity it has been demonstrated that the calcium imaging method gives a good relationship between the fluorescent change and the firing feature in vertebrate neurons (Lev-Tov and O'Donovan, 1995; O'Donovan et al., 1993; O'Malley et al., 1996). The present results also supported a good relationship between them and demonstrated that the period of the ΔF/F increase may nearly correspond to the firing duration while the slope of the ΔF/F increase may be almost relative to the spike frequency. These relationship may be very useful to know firing features of neurons by the calcium imaging instead of intracellular recording with microelectrodes. Actually, we demonstrated that the simultaneous measurement of the fluorescent change in cell bodies of the MA1 and JC2 during the feeding-like response made it possible to detect the firing patterns in these neurons. In the present experiments we also found the slow decay of the fluorescence after cessation of the spike activity. It may make trouble for us to detect the change in fluorescence when the period of the rhythmic response is very short or the burst duration of the neuron is as long as the period. In the Aplysia feeding rhythm, however, the rhythmic period is usually longer than 4 sec and the burst duration is less than 70% of the period. Therefore, this method can sufficiently detect the firing patterns in most of neurons during the rhythmic response concerning feeding behavior.
The slow decay of the fluorescence may result from the low dissociation constant (Kd=190 nM) of Calcium Green-1 (Regehr and Atluri, 1995) in addition to the real slow decay of the [Ca2+]i accompanying the intracellular Ca2+ regulation. In the present experiments this dye was used for the large change in fluorescence following spike generation and good relationship between the spike frequency and the change in fluorescence. It may come from the property of the detectable concentration range appropriate to Aplysia neurons. The calcium imaging may suggest the calcium accumulation during rhythmic firing in addition to detection of the firing pattern. The cumulative Ca2+ in the cell body may play an important role as a second messenger or in the gene expression. It may also contribute to generate rhythmic bursts itself. Therefore, in future the relationship between the decrease in [Ca2+]i and the decay time of the fluorescence for Calcium Green-1 should be explored in detail.
In the present experiments how the spike generation increased the [Ca2+]i in the cell body was further explored. The removal of extracellular Ca2+ induced predominant reduction of the spike-induced increase in fluorescence in all test neurons, suggesting that the Ca2+ influx may be mainly responsible for it. The most possible candidates will be the VGCCs classified into several types in vertebrate and invertebrate neurons (Nowycky et al., 1985; Edmonds et al., 1990; Regan, 1991; Fossier et al., 1994; Ma and Koester, 1995; Teramoto et al., 1995). Actually, in several species of animals specific types of VGCCs have been reported to contribute to the [Ca2+]i increase in whole regions of neurons following spike activity (Jaffe et al., 1992; Lev-Ram et al., 1992; Regehr and Tank, 1992; Midtgaard et al., 1993; O'Donovan et al., 1993; Viana et al., 1993; McClellan et al., 1994; Yuste et al., 1994). In the present experiments using typical blockers for the VGCCs, application of nifedipine partly reduced the spike-induced increase in fluorescence in cell bodies of all test neurons. These results strongly suggest that L-type Ca2+ channels may contribute to the Ca2+ influx following spike generation in cell bodies of Aplysia wide-spread neurons. This may be consistent with the results obtained from the cell body of CA1 pyramidal cells in vertebrates (Regehr and Tank, 1992). In the buccal ganglia of Aplysia californica the N and P-type Ca2+ channels have been reported to contribute to transmitter release from presynaptic terminals of the B4/B5 neurons, probably equivalent to the MA1 neurons in Japanese species (Fossier et al., 1994). In contrast, we could not observe the effects of the same blockers for these channels on the responses in the MA1 cell body. Therefore, the N and P-type Ca2+ channels may be localized in the synaptic terminals of the MA1. In the present experiments it is also noted that nifedipine could not sufficiently block the spike-induced increase in fluorescence in comparison with the effects of the removal of the extracellular Ca2+. The other types of Ca2+ channels on which the examined blockers could not act may also contribute to the Ca2+ influx following spike generation. In invertebrate Lymnaea neurons, the VGCCs unknown in vertebrate have been reported (Kits et al., 1997). Such types of channels may additionally contribute to the Ca2+ influx in our system. In the other case, there may be a possibility of Ca2+ entry through Na+ channels (Baker et al., 1971; Brown et al., 1975). Further detailed study will be required.
L-type Ca2+ channels have been reported to be widely distributed throughout animal neurons and our present study will strongly support that the calcium imaging method may be applicable to detect and estimate the spike activity of Aplysia wide-spread neurons.
Acknowledgments
This work was supported by Grant-in-Aid for Scientific Research on Priority Areas (Microbrain) from Japan Ministry of Education, Culture, Sports, Science and Technology to T. N.
