| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Department of Biology, Emory University, Atlanta, Georgia
Submitted 17 October 2005; accepted in final form 29 March 2006
 |
ABSTRACT |
|---|
|
|
|
INTRODUCTION |
|---|
|
), their properties, distribution, molecular structure, and functions of the channels have been exhaustively investigated. A wide variety of kinetically distinct LVA Ca channels has been reported in different neurons, and the cellular localization of the three identified members of the LVA Ca channel family varies, with different neurons possessing from one to all three identified LVA Ca channels (Huguenard 1996; Huguenard and Prince 1992; Lee et al. 1999b; Pan 2000, 2001; Perez-Reyes 2003; Talley et al. 1999; Tarasenko et al. 1997). Typically, LVA Ca channels are localized mainly to the dendritic tree and serve to support spiking or the pacemaker activity underlying bursting (Erickson et al. 1993; Fisher and Bourque 2001; Huguenard 1996; McCormick and Bal 1997; Yunker 2003a). In some systems, LVA channels are involved in synaptic transmission (Calabrese 1998; Pan et al. 2001; Uhrenholt and Nedergaard 2005).
In leeches, two bilateral pairs of reciprocally inhibitory heart interneurons in segmental ganglia 3 (G3) and 4 (G4) form the core oscillators of the motor pattern-generating network for heartbeat. These neurons possess, in addition to HVA Ca currents, two types of LVA Ca currents, one rapidly activating/inactivating, ICaF, and another slowly activating/inactivating, ICaS. The LVA channels corresponding to these LVA currents appear to be widely distributed throughout the neuritic tree (Angstadt and Calabrese 1991; Ivanov and Calabrese 2000; Lu et al. 1997; Olsen and Calabrese 1996). Oscillator heart interneurons are active in alternating bursts and inhibit one another by both graded and spike-mediated transmission. Although HVA Ca currents underlie spike-mediated transmission (Lu et al. 1997), LVA Ca currents serve to: 1) generate the plateau potential that drives bursts of action potential (Arbas and Calabrese 1987; Olsen and Calabrese 1996); 2) mediate graded transmission (Angstadt and Calabrese 1991); and 3) modulate spike-mediated transmission through homosynaptic enhancement (Ivanov and Calabrese 2003). Although the properties of ICaF and ICaS in heart interneurons and their involvement in synaptic transmission have been extensively characterized in detailed voltage-clamp and Ca imaging studies (Angstadt and Calabrese 1991; Ivanov and Calabrese 2000; Lu et al. 1997; Olsen and Calabrese 1996), the structural/functional organization of synaptic release sites and a precise definition of the role of the individual LVA Ca currents in graded transmission and the modulation of spike-mediated synaptic strength have not been thoroughly investigated.
Such an investigation requires developing usable agents and tools to separate the different LVA Ca channels and the HVA Ca channels, and their effects in heart interneurons. Even in vertebrates, despite the availability of a wide spectrum of selective activators and blockers of Ca channels, study of LVA channels has been challenging (Huguenard 1996; Yunker 2003b) and there is an almost complete lack of such agents for invertebrates (Jeziorski et al. 2000; Kleinhaus and Angstadt 1995; Lu et al. 1997; Staras et al. 2002; Wicher and Penzlin 1997).
To address these issues, in this study, we monitored simultaneously pre- and postsynaptic Ca currents and, in some cases, changes of intracellular Ca fluorescence in leech heart interneurons of isolated G3 or G4. Using different stimulating paradigms and inorganic Ca channels blockers, we were able to evaluate the effects of ICaF and ICaS on the amplitude and time course of inhibitory synaptic transmission. We found that 1 mM Ni2+ selectively blocks LVA Ca currents and associated graded synaptic release, but not HVA Ca current and associated high-threshold/spike-mediated release. However, 1 mM Ni2+ does affect homosynaptic enhancement of spike-mediated transmission similar to intracellular Ca2+ chelators, corroborating that this plasticity is mediated by Ca2+ entering by LVA Ca channels. We show that fast and slow low-threshold (LVA) Ca channels evoke transmitter release from the same release sites but have different degrees of efficacy in promoting release. We argue that Ca2+ from multichannel domains arising from ICaF channels that are clustered near but not directly associated with the release trigger, and Ca2+ radially diffusing from generally distributed ICaS channels interact at common release sites to mediate graded transmission.
|
|
METHODS |
|---|
|
Adult leeches (H. medicinalis) were obtained from Leeches USA and Biopharm and maintained in artificial pond water (Leeches USA) at about 15°C.
Preparation
Leeches were anesthetized in cold saline, after which individual ganglia (midbody ganglion 3 or 4) were dissected and pinned in clear, Sylgard-coated open bath recording/imaging chamber (RC-26, Warner Instrument) with a working volume of 150 µl. The sheath on the ventral surface of the ganglion was removed with microscalpels. Ganglia were superfused continually with normal leech saline (Nicholls and Baylor 1968) containing (in mM) 115 NaCl, 4 KCl, 1.8 CaCl2, 10 glucose, and 10 N'-2-hydroxyethylpiperazine-N'-2-ethanesulfonic (HEPES) acid buffer, adjusted to pH 7.4 with NaOH or HCl. The preparation was mounted ventral side up on the stage of an Olympus BX50WI fluorescent microscope with an Olympus 40x/0.80W water immersion objective.
Electrophysiology
Heart interneurons were penetrated with thin-walled (1 mm OD, 0.75 mm ID) borosilicate microelectrodes (A-M Systems). and identified by the posterolateral position of their somata on the ventral surface of the ganglion and by their characteristic pattern of rhythmic bursting. In all experiments, the recording microelectrode, inserted into a postsynaptic cell, was filled with 4 M K-acetate, 20 mM KCl (unbuffered, pH 8.4). For presynaptic current-clamp experiments, the recording microelectrode inserted into a presynaptic cell was filled with the same solution as the postsynaptic cell, and for presynaptic voltage-clamp experiments, the "presynaptic" microelectrode was filled with 1 M K-acetate, 1.5 M tetraethyl ammonium acetate (TEA-acetate), and 1.5 Cs-acetate (unbuffered, pH 7.9) to block outward currents. Microelectrodes were coated along their shanks with Sylgard 186 (Dow-Corning) and had resistances of 20–45 M
and time constants of 0.5–1.5 ms when capacity compensated.
Once the cells were penetrated with recording microelectrodes, for all experiments except those in Fig. 11 (see following text), the superfusate was immediately switched to Na+-free/5 mM Ca2+ saline containing (in mM): 110.0 N-methyl-D-glucamine (NMDG), 4.0 KCl, 5.0 CaCl2, 10.0 glucose, 10.0 HEPES acid buffer, adjusted to pH 7.4 with KOH or HCl. In a few of these experiments, Ca2+ in the saline was reduced to 2 mM with suitable osmotic adjustment of NMDG to 115.0 mM. In some cases, 150 µM Cd2+, 1 mM Ni2+, or both were added to the saline.
|
|
|
|
|
|
|
|
|
|
|
All stimulus protocols were generated using the pCLAMP program CLAMPEX. The usual voltage-clamp protocol consisted of voltage pulses from a holding potential of –70 mV to various depolarizing voltages, or from different holding potential to a fixed depolarizing potential. Various approaches were used, from single voltage pulses/steps to combined pulses/steps. Software-controlled leak subtraction was implemented as previously described (Ivanov and Calabrese 2003). For the experiments of Fig. 11, the presynaptic current-clamp protocol used to study spike-mediated transmission was built to simulate normal burst activity as described previously (Ivanov and Calabrese 2003). These experiments were carried out in 5 mM Ca2+, 20 mM Mg2+ saline that contained (in mM) 80.5 NaCl, 4.0 KCl, 5.0 CaCl2, 20 MgCl2, 10.0 glucose, 10.0 HEPES acid buffer, adjusted to pH 7.4 with KOH or HCl. This elevated divalent ion solution effectively suppresses spontaneous spike activity in heart interneurons but does not appreciably affect their synaptic transmission (Nichols and Wallace 1978a). More details on all stimulus protocols used are provided in the RESULTS section.
In all experiments, the postsynaptic cell was in voltage clamp, typically held at –40 mV.
Ca imaging
In some experiments, we monitored changes in intracellular Ca2+ with the fluorescent indicator Calcium Orange (Molecular Probes). In these experiments, one cell (presynaptic) was iontophoretically filled with Calcium Orange [see Ivanov and Calabrese (2000, 2003) for details of methods and indicator properties] and then repenetrated after 5–15 min with a recording microelectrode. Changes of Calcium Orange fluorescence were continuously monitored and recorded with an ICCD-350f CCD camera (Video Scope International), connected to the fluorescent microscope mentioned above, equipped with an Olympus U-MNG (exciter filter BP 530–550 nm, dichroic mirror DM 570 nm, barrier filter BA 590 nm) filter set, 10% neutral density filter, and Olympus 40x/0.80W water immersion objective and Axon Imaging Workbench 4.0 (AIW 4.0) software with a Digidata 2000 interface (Axon Instruments) on a Pentium III (Intel) computer. Intensifier gain and black (baseline) levels were adjusted to achieve minimal background fluorescence, convenient visualization of the filled neuron, and sufficient dynamic range for monitoring fluorescence changes.
Our setup permits the acquisition of full-frame images of 640 x 480 pixels size at a resolution of 0.379 µm2 for 1 pixel (395 x 295 µm for full frame) with the Olympus 40x/0.80W water immersion objective. Changes of fluorescence were recorded from the approximate synaptic contact region of a heart interneuron (600–1,200 pixels, 235–470 µm2), as described by Ivanov and Calabrese (2003). In all experiments, the maximal available acquisition rate (video rate, 30 Hz) was used, yielding a time resolution of 33 ms. Video signals were accumulated for 33 ms per image, without any kind of gating, using the DC mode of the camera.
To synchronize the acquisition of electrophysiological data and Ca fluorescence recording, the Digidata 2000 and Digidata 1200/1320A were connected using a DIO-3 cable interface (Axon Instruments) that permits one program to trigger the other. In our experiments, we used pCLAMP 7.0/8.0 protocols to trigger data acquisition by Axon Imaging Workbench, which, in turn, controlled the shutter for the imaging lamp.
Data analysis
All stored data were analyzed on the same computer using pCLAMP program CLAMPFIT and Origin 7.5 (OriginLab) software. Calcium fluorescence data are presented mainly as changes in fluorescence (
F/F), but in some cases as fluorescence (F); in this latter case, the data are presented in units of absolute fluorescence on a scale from 0 to 255 fu (fluorescence units). Statistical analyses used one-way ANOVA, factorial ANOVA, repeated-measures ANOVA, and ANCOVA with post hoc comparisons made by Student's t-test with Bonferroni correction for multiple comparisons (Bonferroni test), linear regression with 95% confidence interval, and linear correlation analysis, all performed with Statistica 6 and Origin 7.5 software. Results are presented/plotted as means + SD.
|
|
RESULTS |
|---|
|
To parse the relative contributions of fast and slow LVA Ca currents in leech heart interneurons to graded synaptic transmission and to determine whether they share common release sites, it was first necessary to develop tools to separate fast (ICaF) and slow (ICaS) LVA Ca currents. Subtraction techniques illustrated in Fig. 1 proved useful to effect this separation. Presynaptic heart interneurons were voltage clamped at –70 mV and postsynaptic heart interneurons at –40 mV, whereas depolarizing steps to –40 mV, progressively increasing in duration from 50 to 450 ms with 50-ms increment were applied presynaptically and pre- and postsynaptic currents were recorded (Fig. 1A). This type of experiment was performed in some preparations with maximal duration of depolarization 
850 ms and 100-ms time increment. Although the 50-ms depolarization to –40 mV was insufficient to evoke the full time course of ICaF (rapidly inactivating component of LVA Ca current) and thus evoke a fully developed gIPSCF (fast component of graded inhibitory postsynaptic current), the 100-ms depolarization evoked a fully developed ICaF with mean time-to-peak of 53.3 (SD 4.47) ms (n = 12), and gIPSCF with mean time-to-peak of 98.2 (SD 9.63) ms (n = 12); these characteristics did not change with increase in the duration of depolarization. Depolarizing steps with duration over 100 ms led to the appearance of distinguishable ICaS (slowly inactivating component of LVA Ca current) that manifested as a secondary peak in the Ca current recording and a corresponding gIPSCS (slow component of graded inhibitory postsynaptic current) that manifested as a shoulder or plateau in the gIPSC (Fig. 1A). To minimize the effects of variability among individual traces that occurred during these experiments, we normalized pre- and postsynaptic currents (ICa and gIPSC) obtained in each individual trace by their own individual maximal (peak) values [ICaF (P) and gIPSCF (P)] that corresponded to peak ICaF and IPSCF, elicited by 100-ms depolarization. To evaluate ICaS and corresponding gIPSCS, we subtracted normalized recordings of ICaF and corresponding gIPSCF obtained with a 100-ms presynaptic depolarization from normalized recordings obtained with depolarizing steps of longer duration. These subtracted results are presented in Fig. 1C, whereas in Fig. 1B we show ICaF and gIPSCF, recorded during 100-ms depolarizing steps for comparison. The ICaS obtained by this subtraction became maximal (peaked) at 277 (SD 11.21) ms (n = 6) and the corresponding maximal gIPSCS at 405.2 (SD 14.89) ms (n = 6) during the 300-ms depolarizing step; these maximal values did not change with longer depolarizing steps. To verify the repeatability of this method for estimating fast and slow ICa and the corresponding gIPSCs, we averaged pre- and postsynaptic currents recorded in seven different preparations during 100- and 350-ms depolarizing steps and obtained ICaS and gIPSCS, as described above (Fig. 1D).
To evaluate further the postsynaptic effects of ICaF and ICaS, we integrated over time normalized ICaF and ICaS, and their corresponding gIPSCs evoked by depolarizing steps of different durations (Fig. 1E). The integrated postsynaptic response corresponding to ICaF (gIPSCF) evoked by 100-ms depolarizing steps was significantly greater than the integrated postsynaptic response corresponding to ICaS (gIPSCS), when evoked by depolarizing steps at 350 ms [25,116 (SD 3.576) vs. 15,908 (SD 6.510), P = 0.004231, Bonferroni test], whereas the corresponding integrated ICaF and ICaS were not different [6.535 (SD 1.286) vs. 6.763 (SD 1.868), P = 0.785442, Bonferroni test]. After this time, an increase in depolarization duration (850 ms) led to only a very small increase in integrated gIPSC, whereas the integrated ICaS increased monotonically. Moreover, the time course of changes in the integrated ICaS and the corresponding gIPSCS in response to increasing depolarization were significantly different (P = 0.000001, repeated-measures ANOVA).
Thus the rapid initial transfer of smaller amount of charge by ICaF (i.e., smaller Ca2+ entry) evoked a larger postsynaptic response than the delayed transfer of a larger amount of charge by ICaS (i.e., larger Ca2+ entry). Such differences in effects of ICaF and ICaS on synaptic transmission could result from: 1) the initial ICaF-evoked neurotransmitter release may severely deplete the readily releasable pool of synaptic vesicles that is common for Ca2+ entering by both LVA Ca currents, without sufficient replenishment by vesicles from reserve pools; 2) ICaF and ICaS release neurotransmitter at spatially segregated release sites with different release probabilities; 3) ICaF channels are localized in closer effective proximity to the secretory trigger than ICaS channels at common or segregated release sites; and/or 4) because of the cooperative binding of Ca2+ to the release trigger (Dodge and Rahamimoff 1967) (power-law dependency of release on intracellular Ca), postsynaptic responses evoked by ICaF (rapid brief charge transfer) are much larger and faster than those evoked by ICaS (slow prolonged charge transfer). Thus the amount and the rate of increase of intracellular Ca2+ are critical for neurotransmitter release.
What determines the amplitude and time course of graded transmission under presynaptic voltage clamp: Ca2+ influx by LVA Ca channels or the availability of readily releasable synaptic vesicles?
To begin to sort out the possibilities outlined above, we used presynaptic holding potential to set the inactivation levels of ICaS and ICaF and thus vary the amounts of Ca2+ they provide at a given test step potential. This procedure allowed us to begin to dissect how the availability of Ca2+ and/or of readily releasable synaptic vesicles limits the amplitude and time course both the fast and slow components of graded transmission (gIPSCF and gIPSCS). We held the postsynaptic cell at –35 mV and applied depolarizing steps to –35 mV (2 s) to the presynaptic cell from progressively more depolarized holding potentials (from –70 to –45 mV at 5-mV increments) (Fig. 2). With increasing depolarization of the presynaptic holding potential from –70 to –45 mV, the peak amplitude of ICaF [ICaF (P)] and the magnitude of ICaS, measured at 1 s [ICaS (1 s)] and at 2 s [ICaS (2 s)] as well as the corresponding gIPSCs that were evoked by the presynaptic depolarizing steps all progressively declined (Fig. 2A). Averaged data normalized by the values obtained from presynaptic holding potential of –70 mV (ICas and corresponding gIPSCs; n = 8 preparations) are shown in Fig. 2B.
Normalized ICaS (1 s) and ICaS (2 s) decreased steadily and to a similar extent with increasing presynaptic holding potential (no significant difference P = 0.368178; repeated-measures ANOVA). ICaF (P) also decreased with increasing presynaptic holding potential but this was more gradual at the more negative holding potentials and was significantly different from the course of decrease in both ICaS (1 s) and ICaS (2 s) [ICaF (P) vs. ICaS (1 s): P = 0.000001; ICaF (P) vs. ICaS (2 s): P = 0.00077; repeated-measures ANOVA], being less steep (Fig. 2B1). The decrease in peak gIPSCF values [gIPSCF (P)] and the decrease of gIPSCS values, measured at 1 s of depolarization [gIPSCS (1 s)] with increasing presynaptic holding potential were similar (no significant difference P = 0.113507, repeated-measures ANOVA); their amplitudes remaining relatively constant at a holding potential of –60 mV and below. The decrease in gIPSCS values measured at 2 s of depolarization [gIPSCS (2 s))] with holding potential was more steady and significantly differed from that of both the gIPSCF (P) and the gIPSCS (1 s) (P = 0.0.00076 and P = 0.0000001, respectively; repeated-measures ANOVA) (Fig. 2B2).
The decrease in graded transmission with increasing depolarization of presynaptic holding potential is not surprising: depolarization of the presynaptic holding potential leads to inactivation of LVA Ca currents (Angstadt and Calabrese 1991) and thus to a decrease in the corresponding postsynaptic responses. Figure 3, A–C illustrates how the changes in the components of LVA Ca current [ICaF, ICaS (1 s), and ICaS (2 s)] and their corresponding postsynaptic responses [gIPSCF (P), gIPSCS (1 s), and gIPSCS (2 s)] co-vary. Data were binned according to holding potential, which increases right to left. The gIPSCF (P) and gIPSCS (1 s) show very little change at the most negative presynaptic holding potentials (–70, –65, –60 mV: three right-handmost points), whereas LVA Ca currents progressively decrease (albeit ICaF, less) but are nevertheless at high values. Such a saturation in the postsynaptic responses mediated by ICaF (P) and ICaS (1 s) (Fig. 3, A and B) suggests that it is not Ca2+ influx, but rather the availability of readily releasable synaptic vesicles that limit amplitude of postsynaptic responses at the early stage of the test depolarization (to 
1 s of depolarization) at the lowest presynaptic holding potentials used. Saturation is not evident in the relation between ICaS (2 s) and gIPSCS (2 s) (Fig. 3C), indicating that by 2 s graded transmission is no longer limited by the availability of readily releasable synaptic vesicles but by Ca2+ influx as ICaS inactivated during the test step. At more depolarized holding potentials, resulting from inactivation of LVA Ca currents, Ca influx becomes limiting and gIPSCs and ICas decline in step (see Olsen and Calabrese 1996).
The time-to-peak of ICaF and gIPSCF increased progressively with increasing presynaptic holding potential (Fig. 2C), but the increase in time-to-peak of gIPSCF was larger and steeper than the increase in time-to-peak of ICaF (P = 0.0000001, repeated-measures ANOVA). The binned (according to holding potential) nonnormalized gIPSCF (P) correlated with binned ICaF (P) time-to-peak (Fig. 3D) (R = –0.993775, adjusted r2 = 0.984487, P < 0.000058). Such a strong correlation of the amplitudes of the postsynaptic responses (gIPSCF) with ICaF (P) time-to-peak is consistent with the observations of Felmy et al. (2003) and Bollmann and Sakmann (2005), who pointed out that the amount and timing of neurotransmitter release depend on both amplitude and time course of the intracellular "Ca signal" at release sites (see also Lin and Faber 2002; Simmons 2002).
Do ICaS and ICaF evoke release from the same readily releasable pool of synaptic vesicles?
The analysis of the previous section suggests that ICaS and ICaF evoke release from the same readily releasable pool of synaptic vesicles. To test this hypothesis we used conditioning low-threshold presynaptic depolarizations of various durations to both deplete the readily releasable pool and inactivate LVA Ca currents and followed them by brief test pulses at various intervals to evoke ICaF and the associated gIPSCF. We then monitored the recovery of ICaF and of the associated gIPSCF after the various conditioning depolarizations to determine the extent to which the recovery of ICaF from inactivation and the readily releasable pool from depletion could be dissociated. If ICaF recovers more rapidly than the gIPSCF, it would indicate that a common readily releasable pool was depleted by the conditioning and the test depolarization. We held the presynaptic cell at –70 mV and applied conditioning and test depolarizing pulses to –40 mV. The postsynaptic cell was held at –40 mV.
In the first experiments, depolarizing test pulses, applied 200 ms after conditioning depolarization of either 4-s duration (Fig. 4A1) or 200-ms duration (Fig. 4A2), evoked strongly reduced ICaF and corresponding gIPSCF. In both cases, increase in time interval between conditioning depolarization and the test depolarization led to progressive recovery of ICaF and corresponding gIPSCF. ICaF (P) and gIPSCF (P) were back to their initial amplitudes in 8–12 s [Fig. 4, B1 and B2; ICaF (P) and gIPSCF (P) were normalized by values obtained during conditioning depolarization in each run; data were averaged over four experiments in both cases]. In experiments with a conditioning depolarization of 4-s duration, normalized ICaF (P) and gIPSCF (P) in response to the subsequent test depolarization changed in step with each other. The amplitudes of both the ICaF (P) and gIPSCF (P) in response to the test depolarizations were strongly dependent on the time interval between conditioning depolarization and the test depolarization (P = 0.000005, repeated-measures ANOVA), without any significant differences in time courses of changes in ICaF (P) and gIPSCF (P) amplitudes (P = 0.906677, repeated-measures ANOVA) (Fig. 4B1). The amplitude of ICaF (P), evoked by first test depolarization (200 ms after conditioning depolarization), was significantly different from the amplitude of ICaF (P), evoked by the conditioning depolarization and the two other test depolarizations (P = 0.00096, P = 0.00883, P = 0.002218, respectively; Bonferroni test). A similar result was obtained for the corresponding gIPSCF (P) (P = 0.001715, P = 0.01535, P = 0.005043, respectively; Bonferroni test). In the experiments with a conditioning depolarization of 200-ms duration, the decrease in normalized ICaF (P) in response to the first test depolarization was smaller than the decrease in the corresponding normalized gIPSCF (P). The amplitudes of both ICaF (P) and gIPSCF (P) in response to the test depolarizations were strongly dependent on the time interval between conditioning depolarization and the test depolarization (P = 0.0000001, repeated-measures ANOVA), but the time courses of changes in ICaF (P) and gIPSCF (P) amplitudes were significantly different (P = 0.015628, repeated-measures ANOVA). The amplitude of ICaF (P), evoked by the first test depolarization (200 ms after conditioning depolarization), was significantly smaller than the amplitudes of ICaF (P), evoked by conditioning depolarization and the last test depolarizations (P = 0.0085, and P = 0.010393, respectively; Bonferroni test). The corresponding gIPSCF (P) was significantly smaller than the gIPSCF (P) evoked by conditioning depolarization and from the three other test depolarizations (P = 0.000007, P = 0.000694, P = 0.000048, and P = 0.000019, respectively; Bonferroni test). The amplitude of this normalized gIPSCF (P) was significantly smaller than the corresponding normalized ICaF (P) (P = 0.009843; Bonferroni test).
The results show that recovery in the strength of graded synaptic transmission between interneurons after a prolonged conditioning depolarization (seconds) followed the recovery of ICaF (with its complete deinactivation time not <8–12 s). After a brief conditioning depolarization (hundreds of milliseconds), however, because the recovery of the gIPSCF lagged the recovery of ICaF, the replenishment of a readily releasable vesicle pool appears to limit the recovery of synaptic strength. This finding indicates that ICaF can severely deplete the pool of readily releasable synaptic vesicles available for release by subsequent ICaF (Fig. 1).
We next sought to determine whether ICaS could deplete the readily releasable pool of vesicles available to ICaF. We thus modified the voltage-clamp protocols of Fig. 4 so that the depleting effects of these two components of LVA current could be compared. First we performed the experiments (n = 5) illustrated in Fig. 5A to more carefully determine the time course of ICaF and gIPSCF recovery after a brief conditioning depolarization that activated only ICaF. In independent traces, we held the presynaptic cell at –70 mV and applied a conditioning depolarizing step of 100 ms to –40 mV. Test depolarizing pulses of 100 ms to –40 mV were applied after a progressively increasing (500-ms increments) time interval of 200 to 3,200 ms. Simultaneous recordings of ICaF and gIPSCF, normalized by ICaF (P) and gIPSCF (P) recorded during the conditioning step and averaged over the five experiments, are presented in Fig. 5A1. Figure 5A2 presents plots of the test ICaF (P) and gIPSCF (P), extracted from the individual traces constituting the recordings presented in Fig. 5A1 versus time. The time courses of changes in amplitudes of both the test ICaF (P) and the corresponding gIPSCF (P) [Fig. 5A1 (middle column, Pre(ICa), right column, Post(gIPSC)), and Fig. 5A2] were not significantly different (P = 0.054333, repeated-measures ANOVA) but there was a significant difference in the normalized amplitudes of ICaF (P) and gIPSCF (P) seen in response to the first test depolarization (P = 0.014218, Bonferroni test). The gIPSCF (P) evoked by the sixth test depolarization returns to the level of the gIPSCF (P) evoked by conditioning depolarization (P = 1.0, Bonferroni test). Until this time, the amplitudes of "test" gIPSCF (P) were significantly smaller than amplitudes of the "conditioning" gIPSCF (P) (P varied from 0.0000001 to 0.00016, Bonferroni test). At the same time, only ICaF (P) evoked by the first test depolarization was significantly decreased (P = 0.0000001, Bonferroni test) compared with the "conditioning" ICaF (P), and after that there were no significant differences (P varied from 0.100913 to 1.0, Bonferroni test). These results corroborate the findings of Fig. 4, indicating that there is significant depletion of the readily releasable pool available for released by ICaF shortly after strong release evoked by ICaF.
Next we performed the experiments (n = 7) illustrated in Fig. 5B in which we held the presynaptic cell at –70 mV and applied a conditioning depolarizing step to –40 mV of progressively increasing duration, from 100 to 3,100 ms (500-ms increments) and then 200 ms later applied a 100-ms test depolarizing pulse to –40 mV. In this protocol the conditioning step first evokes only ICaF but progressively evokes ICaS for longer intervals, whereas the test pulse evokes only ICaF. Simultaneous recordings of LVA ICa and gIPSCs, normalized in independent traces by ICaF (P) and gIPSCF (P) recorded during conditioning depolarizing step, and averaged over seven experiments, are presented in Fig. 5B1. Figure 5B2 presents plots of the test ICaF (P) and gIPSCF (P), extracted from the individual traces constituting the recordings presented in Fig. 5B1, versus time. Although the test ICaF (P) decreases steadily with the increase in duration of conditioning depolarizing step [Fig. 5B1, middle column, Pre(ICa), and Fig. 5B2], the gIPSCF (P) after an initial decrease that bottoms out with the 1,100-ms conditioning pulse [Fig. 5B1, right column, Post(gIPSC), and Fig. 5B2]. The normalized amplitudes of ICaF (P), evoked by test depolarizations, and the corresponding gIPSCF (P) were significantly different (P = 0.0000001; repeated-measures ANOVA) with different time courses (P = 0.0000001; repeated-measures ANOVA). Most important, the decrease in the normalized amplitudes of each (except the very last one) ICaF (P) elicited by test depolarizations were significantly smaller than the decrease in the normalized amplitudes of the corresponding gIPSCF (P) values (P ranged from 0.0000001 to 0.0025671; Bonferroni test). We interpret these differences in normalized amplitude and time course between the test ICaF and gIPSCF as indicating that release evoked by ICaS depletes the readily releasable pool of vesicles available to be released by ICaF, especially for conditioning steps 1,100. Nevertheless, as the conditioning pulse extends beyond 1,000 ms and both ICaS (inactivation) and gIPSCS (inactivation of ICaS and pool vesicle depletion) wane, ICaF-evoked release begins to recover as a result of recovery of the readily releasable vesicle pool, presumably through increased vesicle recycling or mobilization (Schneggenburger et al. 2002; Thomson 2000, 2003).
This interpretation is supported by a comparison of the time course of normalized amplitudes of the test gIPSCF (P) for the two voltage-clamp protocols in Fig. 5, A and B. As illustrated in Fig. 5C, the time course of recovery of normalized amplitudes of the gIPSCF (P) from Fig. 5, A1 and B1 are significantly different (P = 0.0000001, repeated-measures ANOVA), with significant differences in amplitudes of gIPSCF (P) evoked by second and all subsequent test depolarizations (P varied from 0.0000001 to 0.001104, Bonferroni test) (Fig. 5C). Sustained ICaS-related synaptic transmission (Fig. 5B) led to much deeper depletion of the readily releasable pool of synaptic vesicles available for release by ICaF than intensive but brief ICaF-related synaptic transmission (Fig. 5A), thus indicating that ICaF and ICaS do release vesicles from common release sites.
Do multichannel Ca domains cooperate in fast graded transmission between heart interneurons?
In the experiments illustrated in Fig. 6 (n = 3), we tried to determine the type of Ca domain (Augustine 2001) that controls the synaptic transmission evoked by ICaF and to evaluate potential cooperativity between ICaF channels. Brief depolarized steps from a holding potential of –70 to –40 mV (5 to 100 ms) applied presynaptically evoked a progressively increasing ICaF and a corresponding gIPSCF (Fig. 6, A and B). Both amplitudes of the gIPSCF (P) and ICaF (P) (Fig. 6A) significantly increased with increasing pulse duration (P = 0.002881, factorial ANOVA; averaged data not shown) as did both integrated gIPSCF and ICaF (Fig. 6B) (P = 0.0021618; factorial ANOVA). The log10 nonaveraged integrated gIPSCF plotted versus the log10 nonaveraged integrated ICaF (Hill coordinates) (Fig. 6C), yields an exponent of dependency equal to 1.7 (R2 = 0.76, P = 0.0001). The log10 averaged integrated gIPSCF plotted versus the log10 averaged integrated ICaF (Hill coordinates) yields an exponent of dependency equal to 1.95 (R2 = 0.99, P = 0.0048). Similar results were obtained for log/log plots of gIPSCF (P) versus ICaF (P) (nonaveraged data: exponent = 1.81, R2 = 0.54, P = 0.00099; averaged data: exponent = 2.58, R2 = 0.796, P = 0.0267; data not shown). These data suggest the involvement of more than one (presumably, at least two) ICaF channels in gating transmitter release at the early stages of graded synaptic transmission (Augustine 2001; Augustine et al. 1991; Bertram et al. 1999; Borst and Sakmann 1999; Fedchyshin and Wang 2005; Gentile and Stanley 2005). Thus multichannel calcium domains appear to mediate ICaF-related graded synaptic transmission between heart interneurons.
How do the rate and amount of Ca2+ influx by LVA Ca channels affect graded synaptic transmission between heart interneurons?
To evaluate how the rate and amount of Ca entering by LVA Ca channels affects graded transmission, we compared graded release at 2 and 5 mM external Ca2+ (Angstadt and Calabrese 1991) with simultaneous recordings of presynaptic intracellular Ca signal, presynaptic LVA ICa, and gIPSCs. The presynaptic cell in these experiments was filled with Calcium Orange and held at –70 mV and the postsynaptic cell was held at –40 mV. As illustrated in Fig. 7A (n = 5), superfusion (3 min) with 2 mM Ca2+ saline had a significant effect on LVA Ca currents, ICaF and ICaS, evoked by presynaptic depolarization to –40 mV (P = 0.002345, factorial ANOVA), and their corresponding gIPSCs (P = 0.00512, factorial ANOVA). ICaF (P) was reduced in 2 mM Ca2+ saline by roughly 50% [P = 0.010533; Bonferroni test (one-way ANOVA)], but ICaS (1 s and 2 s) were both virtually unchanged [P = 1.000 and P = 1.000, respectively; Bonferroni test (factorial ANOVA and one-way ANOVA)] (Fig. 7B, 2a–2c). The corresponding gIPSCF (P) was reduced by nearly 50% [P = 0.04976; Bonferroni test (one-way ANOVA)] and gIPSCS (1 s and 2 s) were not significantly affected [P = 1.000 and P = 1.000, respectively; Bonferroni test (factorial ANOVA and one-way ANOVA)]. The presynaptic fluorescent Ca signal (F/F) was not significantly affected (P, 1 s and 2s) in 2 versus 5 mM Ca2+ saline (Fig. 7B, 1a–1c) [P = 0.110322; Bonferroni test (factorial ANOVA and one-way ANOVA)]. The time-to-peak, however, of ICaF, gIPSCF, and F/F were all significantly increased [P = 0.04905, P = 0.01561, and P = 0.006769, respectively; Bonferroni test (one-way ANOVA)] (Fig. 7B, 1d–3d). These observations suggest that the rate of increase in internal Ca2+ concentration may be as important as the absolute amount of the increase in mediating release, corroborating data presented above in Figs. 2 and 3. In the present experiments, the delay of the intracellular Ca fluorescent signal time-to-peak was associated with a prominent decrease in the postsynaptic responses despite only a moderate reduction of F/F. ANCOVA (P = 0.039602) confirms a strong effect of changes in F/F (P) time-to-peak on amplitude of gIPSCF (P).
Ni2+ (1 mM) is an effective blocker of LVA Ca currents and associated graded synaptic transmission in leech heart interneurons
Whereas HVA Ca channels in heart interneurons can be selectively blocked by 100 µM Cd2+ (Lu et al. 1997), no such tool was available for LVA Ca channels. On other hand, Ni2+ has been widely used as a relatively selective blocker of LVA Ca channels (Huguenard 1996; Lee et al. 1999a; Perez-Reyes 2003; Yunker 2003b). To determine whether it is possible to use Ni2+ to manipulate LVA Ca currents in leech heart interneurons, we performed the experiments illustrated in Fig. 8. Individual heart interneurons were voltage clamped at –70 mV and 2-s depolarizing steps to –30 mV were applied to evoke LVA Ca currents. Recordings were performed during superfusion with control saline and after 5-min superfusion with saline containing Ni2+ at different concentrations.
Preliminary experiments showed that Ni2+ at concentrations of 2 mM quickly and effectively blocked both HVA and LVA Ca currents, and in concentrations of 0.5 mM had little or no effect (n > 15; data not shown). At the same time, 1 mM Ni2+ had a very reproducible effect on LVA Ca current, and at least in 75% of experiments strongly blocked ICaF with a somewhat lesser effect on ICaS (n > 15). Figure 8A1 presents typical recordings from experiments demonstrating the effect of 1 mM Ni2+ on LVA ICas (n = 5). Depolarization to –30 mV under control conditions evoked typical ICaF and ICaS (Angstadt and Calabrese 1991; Lu et al. 1997; Olsen and Calabrese 1996). The LVA ICas recorded were very stable and reproducible: responses to two depolarizing steps applied with a 3-min interval were virtually indistinguishable. Ni2+ (1 mM) progressively blocked all LVA Ca current. Superfusion with 1 mM Ni2+ progressively blocked ICaF (Fig. 8A2), by almost 50% at 5 min [P = 0.000054; Bonferroni test (one-way ANOVA)] (Fig. 8B1). ICaS was more resistant to the effect of 1 mM Ni2+, but nevertheless 1 mM Ni2+ had a significant blocking effect (P = 0.008534; factorial ANOVA). ICaS, recorded at 277 ms from the beginning of depolarization [ICaS (277 ms), estimated maximum of ICaS; see above], was reduced by 40% (P = 0.048294; Bonferroni test) and ICaS (1 s) was reduced insignificantly by about 20% (P = 1.00000; Bonferroni test) (Fig. 8, B2 and B3). A differential effect of 1 mM Ni2+ on ICaF versus ICaS was confirmed by factorial ANOVA (P = 0.000638). These observations show that 1 mM Ni2+ is an effective blocker of LVA Ca currents with a predominant effect on ICaF.
To evaluate the effect of block of LVA Ca channels by 1 mM Ni2+ on graded synaptic transmission, we held the presynaptic cell at –70 mV (the postsynaptic cell was held at –40 mV) and applied presynaptic depolarizing steps (2 s) to –55 mV and up to –30 mV in 5-mV increments (Fig. 9). Similar to our previous results (Angstadt and Calabrese 1991; Ivanov and Calabrese 2000), under control conditions (superfusion with 5 mM Ca2+/0 mM Na+ saline), incremental depolarization evoked a progressive concomitant increase in presynaptic ICaF and ICaS [for ICaF (P), ICaS (277 ms), and ICaS (1 s) P = 0.0000001 for all three; repeated-measures ANOVA], and in the gIPSC [for gIPSCF (P) and gIPSCS (1 s) P = 0.0000001, and for gIPSCS (405 ms) P = 0.000001; repeated-measures ANOVA] (see Fig. 9A for examples of recordings and Fig. 9B for averages over seven experiments). Some decrease in LVA Ca current at depolarizing potentials > –40 mV is probably a result of contamination with outward currents (Angstadt and Calabrese 1991; Lu et al. 1997). Superfusion (3 min) with 1 mM Ni2+ (Fig. 9, A and B, 1 mM Ni2+) strongly blocked both ICaF and ICaS (277 ms) [for both ICaF (P) and ICaS (277 ms) P = 0000001; repeated-measures ANOVA], with a smaller effect on ICaS (1 s) (P = 0.051321; repeated-measures ANOVA), significantly reduced synaptic transmission [for gIPSCF (P), gIPSCS (405 ms), and gIPSCS (1 s), P = 0.000001, P = 0.000419, and P = 0.001302, respectively; repeated-measures ANOVA], and up-shifted the dependency of the postsynaptic response on presynaptic depolarization: a much more depolarizing potential had to be applied presynaptically to evoke release than in the absence of Ni2+. Whereas in control presynaptic depolarization to –55 mV evoked a detectable gIPSCF, and depolarization to –50 mV evoked detectable gIPSCs of both kinds, in 1 mM Ni2+-containing solutions, the first gIPSCs were detectable only at depolarization to –45 mV (Fig. 9B); at any given presynaptic depolarizing potential the evoked gIPSCF (P) values were reduced, compared with control (P = 0.035947; repeated-measures ANOVA). This effect on gIPSCS (405 ms) and gIPSCS (1 s) was not significant (P = 0.099433 and P = 0.204156; repeated-measures ANOVA). Moreover, the time-to-peak for ICaF and the corresponding gIPSC were greatly increased (P = 0.009406 and P = 0.000015, respectively; repeated-measures ANOVA) (Fig. 9B). Thus the effect of 1 mM Ni2+ on graded synaptic transmission follows from its ability to block presynaptic LVA Ca currents.
Can 1 mM Ni2+ be used to separate synaptic transmission mediated by LVA and HVA Ca currents in leech heart interneurons?
LVA Ca currents are completely inactivated at holding potentials above –50 mV (Angstadt and Calabrese 1991), but additional depolarization to much higher potentials evokes a postsynaptic response that has been attributed to presynaptic HVA Ca current (ICaHT) (Simon et al. 1994) that is selectively blocked by 150 µM Cd2+ (Ivanov and Calabrese 2000; Lu et al. 1997). We took advantage of these findings to confirm the selective block of LVA Ca currents by 1 mM Ni2+and to corroborate that the postsynaptic response to HVA depolarization is mediated by activation of an ICaHT. In these experiments (n = 4), we also monitored the intracellular Ca fluorescent signal by filling the presynaptic cell with Calcium Orange. The presynaptic LVA Ca currents were inactivated by clamping the presynaptic cell at a potential of –40 mV for 20+ s, after which 2-s depolarizations to –10 mV were applied. The postsynaptic cell was held at –35 mV. Superfusion with 1 mM Ni2+ did not noticeably affect IPSCs evoked by high-threshold presynaptic depolarization (Fig. 10, A and B). Averaged across these experiments (n = 4), the htIPSC integrated over time in 1 mM Ni2+-containing saline [0.49 nA · s (SD 0.33)] versus in Control saline [0.44 nA · s (SD 0.31)] were not significantly different [P = 0.821372; Bonferroni test (one-way ANOVA)]. Subsequent superfusion with 1 mM Ni2+ and 150 µM Cd2+, however, nearly completely blocked the postsynaptic response to high-threshold depolarization (Fig. 10, B and C). Averaged across these experiments (n = 4), the htIPSC integrated over time in 1 mM Ni2+ and 150 µM Cd2+ [0.01 nA · s (SD 0.01)] versus in 1 mM Ni2+-containing saline [0.49 nA · s (SD 0.33) was significantly smaller: P = 0.027204; Bonferroni test (one-way ANOVA)]. The block of IPSCs evoked by high-threshold depolarization by Cd2+ confirms that this synaptic response is ICaHT dependent (Fig. 10, B and C). The absence of any effect of 1 mM Ni2+ on this htIPSC compared with Control (Fig. 10, A and B) indicates that Ni2+ at least at 1 mM is a selective blocker of LVA Ca currents and does not have any sizable effect on synaptic transmission evoked by HVA Ca current, and thus on HVA ICa.
Cd2+ increases background fluorescence in heart interneurons and this effect has been attributed to Cd2+ influx into cells (Ivanov and Calabrese 2000). Our current data confirm these findings (Fig. 10, D and E). Ni2+ had no significant effect on background fluorescence [the changes in background fluorescence in 1 mM Ni2+-containing saline compared with Control vs. changes in 1 mM Ni2+ and 150 µM Cd2+-containing saline compared with Control were significantly different: P = 0.016158; Bonferroni test (one-way ANOVA)], but did significantly increase the amplitude of fluorescent signal in response to depolarization [the changes in fluorescence evoked by high-threshold depolarization in 1 mM Ni2+-containing saline compared with Control vs. changes in 1 mM Ni2+ and 150 µM Cd2+-containing saline compared with Control were significantly different: P = 0.015574; Bonferroni test (one-way ANOVA)] (Fig. 10, B, D, E1, and E2), indicating that Ni2+ can enter the cell during depolarization and after cessation of depolarization can somehow be bound up or eliminated/extruded from the cytoplasm. Like almost all other Ca indicators, Calcium Orange can bind a range of di- and trivalent cations with corresponding changes in the fluorescent signal; the ratio of the change in fluorescence of Ca Orange to 5 µM Ni2+ versus 100 µM Ca2+ is 71/96 (Haugland 1996).
Partial blockade of LVA Ca current with 1 mM Ni2+ affects modulation of spike-mediated synaptic transmission between heart interneurons
In leech heart interneurons, spike-mediated synaptic transmission (smIPSC) is independent of previous spike activity (Nicholls and Wallace 1978a,b) and is modulated by changes in presynaptic background Ca2+ concentration, which depends in turn on LVA Ca currents (Ivanov and Calabrese 2003). Here we wanted to determine the effect of blockade of LVA Ca currents by 1 mM Ni2+ on modulation of smIPSCs. Isolated third or fourth segmental ganglia were bathed in 5 mM Ca2+/20 mM Mg2+ saline to prevent spontaneous spiking activity (Nichols and Wallace 1978a). The presynaptic cell was current clamped at –55 to –50 mV, and the postsynaptic cell was voltage clamped at –40 mV. During a 2-s subthreshold depolarizing current step, a train of brief (6-ms) suprathreshold current pulses was superimposed to evoke presynaptic spikes; suprathreshold current pulses were also applied before and after the step. Postsynaptic responses were recorded as graded and spike-mediated IPSCs. After control recordings of cells activity were made, ganglia were superfused for 3–6 min with 1 mM Ni2+ added to the saline, tested, and then superfused for another 3–6 min with both 1 mM Ni2+ and 150 µM Cd2+ added to the saline and tested again (Fig. 11A). In some experiments, immediately after control recordings, ganglia were superfused for 3–6 min with only 150 µM Cd2+ added to the saline (Fig. 11B). Under control condition, typical postsynaptic responses with clearly distinguishable graded and spike-mediated components were recorded (Fig. 11, A1 and B1); the time course of the modulation in spike-mediated synaptic transmission reproduced our previous findings (Fig. 11A4a, Control) where changes in smIPSC amplitude were attributed to changes in intracellular Ca2+ concentration (Ivanov and Calabrese 2003). Addition of 1 mM Ni2+ blocked gIPSCs and slightly delayed the buildup of synaptic modulation (P = 0.000108; repeated-measures ANOVA) (Fig. 11, A2 and A4, 1 mM Ni2+). The time constant for a single exponential fit to the rise of the amplitude of smIPSCs equaled 324.8 ms in control saline and 499.5 ms in 1 mM Ni2+-containing saline. In addition, the time from the beginning of the first high-threshold depolarization to the maximal smIPSC achieved was significantly shorter in control versus 1 mM Ni2+-containing saline [P = 0.00103; Bonferroni test (one-way ANOVA)] (Fig. 11A, 4a and 4b). Addition of 150 µM Cd2+ (both in the absence and in the presence of 1 mM Ni2+) completely blocked smIPSCs (Fig. 11, A3 and B2). gIPSCs were only moderately affected by addition of 150 µM Cd2+ in the absence of 1 mM Ni2+ (Fig. 11B2), but were completely blocked when both 1 mM Ni2+ and 150 µM Cd2+ were added to the saline (Fig. 11A3).
These results further corroborate our previous work (Ivanov and Calabrese 2000; Lu et al. 1997), where we showed that Cd2+ is a highly selective blocker of HVA Ca currents in leech heart interneurons, and confirms the critical role of ICaHT in spike-mediated transmission (Lu et al. 1997; Simon et al. 1994). Changes in the time course of synaptic plasticity produced by 1 mM Ni2+ likely result from a reduction in intracellular background Ca2+ caused by partial block of LVA Ca current by 1 mM Ni2+, with a resultant delay in binding of Ca2+ to the modulatory Ca binding site (Ivanov and Calabrese 2003). This effect of 1 mM Ni2+ on the time course of synaptic plasticity resembles the effect of the slow Ca2+ chelator EGTA (Ivanov and Calabrese 2003).
|
|
DISCUSSION |
|---|
|
; Ivanov and Calabrese 2000; Lu et al. 1997; Olsen and Calabrese 1996; Pan 2000; Pan et al. 2001; Uhrenholt and Nedergaard 2005), expanding the view that LVA Ca channels are localized mainly in dendrites and serve mainly to drive spiking activity or generate the pacemaker activity supporting bursting activity (Erickson et al. 1993; Fisher and Bourque 2001; Huguenard 1996; McCormick and Bal 1997; Yunker 2003a). In some cases the involvement of LVA Ca channels in synaptic transmission may be masked by much stronger HVA Ca channel-dependent synaptic transmission and thus underestimated. Therefore the characterization of the involvement of LVA Ca channels in synaptic transmission in leech heart interneurons is important not only for understanding heartbeat central pattern generator but also for defining a role of LVA Ca channels in synaptic transmission that is potentially applicable to other systems. In the current study, we were able to partially separate the effects of ICaF and ICaS on inhibitory synaptic transmission between heart interneurons, describe the power relation between LVA currents and the strength of synaptic transmission, and more precisely evaluate the effects of HVA and LVA Ca currents on inhibitory synaptic transmission between heart interneurons.
Comparison of LVA Ca channels in leech heart interneurons and known cloned T-type Ca channels
Similar to its effect on T-type Ca channels in other systems (Huguenard 1996; Lee et al. 1999a; Perez-Reyes 2003; Yunker 2003b; but see Zamponi et al. 1996), we found that Ni2+ in relatively high concentration (1 mM) is an effective blocker of ICaF and ICaS LVA Ca currents in heart interneurons. Cloning and expression studies (Perez-Reyes 2003) have identified three cloned T-type Ca channels (pore-forming subunits): 
1G (Cav3.1), 1H (Cav3.2), and 1I (Cav3.3). All these channels are Ni2+ sensitive, but Cav3.1 and Cav3.3 are 20 times less sensitive than Cav3.2 to Ni2+ (Lacinová 2000; Lee et al. 1999a; Perez-Reyes 2003). Ni2+ shifts I–V curves to more depolarized potentials and Ni2+ block is reduced at more depolarized test potentials (Lee et al. 1999a,b; Perez-Reyes 2003). The low sensitivity to Ni2+ (effective concentration 1 mM) of both ICaF and ICaS (Fig. 8) and the observation that Ni2+ block was less effective at higher test potentials (Fig. 9) make these channels similar to Cav3.1 and Cav3.3.
Kinetics also indicate similarity of ICaF and ICaS to the cloned T-type Ca channels; the time constants of activation and inactivation of Cav3.1 and Cav3.3 (6 and 30 ms, and 30 and 137 ms, respectively) (Klöckner et al. 1999) are in the same range as ICaF and ICaS, respectively (Angstadt and Calabrese 1991). The time course of recovery of ICaF current from inactivation described here is also on the same scale as that in T-type currents (Klöckner et al. 1999). All these comparisons suggest that the ICaF channel of heart interneurons is similar to Cav3.1, and the ICaS channel to Cav3.3.
Comparison of the properties and functional role of low-threshold Ca channels of heart interneurons and T-type Ca channels of retinal bipolar cells
The properties of LVA Ca currents in heart interneurons (Angstadt and Calabrese 1991) and in vertebrate retinal bipolar cells, especially in rod bipolar cells (Pan 2000), are very similar. In both heart interneurons and bipolar cells, LVA Ca currents consist of two components (fast and slow), have similar kinetics, and are blocked by Ni2+ in the millimolar range. In bipolar cells, LVA channels are localized in synaptic terminals and are responsible for tonic (asynchronous, low-threshold) synaptic transmission (Pan 2001). The similarity between these two very different cell types extends further; the HVA Ca channels that are responsible for ultrafast synaptic transmission in retinal bipolar cells are of the L-type, and the HVA Ca channels in heart interneurons appear to be similar, being highly sensitive to Cd2+ (Lu et al. 1997). Moreover, Ma and Pan (2003) found that T-type channels appear to be essential for the initiation of the spontaneous pacemaker-like activity recorded in the majority of bipolar cells. Similarly in heart interneurons, LVA Ca channels generate the plateau potentials that drive bursts of action potentials (Arbas and Calabrese 1987; Olsen and Calabrese 1996). Thus the main types of Ca channels in vertebrate retinal bipolar cells and in heart interneurons are similar and share corresponding physiological/functional properties.
Localization of LVA Ca channels at release sites and their involvement in graded synaptic transmission between heart interneurons
Graded synaptic transmission between heart interneurons mediated by LVA currents (ICaS and ICaF) is delayed and partially suppressed by intracellular EGTA (Ivanov and Calabrese 2003), suggesting that these channels are located relatively far from the release trigger compared with HVA channels, because spike-meditated transmission is unaffected by EGTA. The difference between ICaF and ICaS in mediating transmitter release (Fig. 1), where the rapid initial transfer of a smaller amount of charge by ICaF (i.e., smaller Ca2+ entry) evokes a larger postsynaptic response than the delayed transfer of a larger amount of charge by ICaS (i.e., larger Ca2+ entry) is explained most easily, if ICaF and ICaS channels share the same release sites. The massive transmitter release mediated by Ca2+ entering by ICaF channels appears to result from the involvement of multichannel Ca2+ domains of (probably) clustered ICaF channels in this fast graded inhibitory synaptic transmission (Fig. 6). The initial ICaF-evoked transmitter release partially depletes the readily releasable pool of synaptic vesicles that is common for Ca2+ entering by both LVA Ca currents, without sufficient replenishment by vesicles from reserve pools for depolarizations of the order of 2–5 s. The very long time lag between the peaking of ICaS and of the gIPSCs (Fig. 1) argues against cooperativity among ICaS channels in mediating transmitter release and suggests that single (individual) ICaS channels are distributed uniformly and not specifically in the vicinity of release sites, possibly exerting their effect through radial diffusion of Ca2+ (Augustine 2001; Neher 1998). The possibility that ICaS might evoke transmitter release at sites different from the ICaF-controlled release sites seems unlikely because the release sites from which ICaF releases transmitter are depleted by Ca2+ entering as ICaS (Fig. 5). Apparently, only a fraction of Ca2+ delivered by ICaS during prolonged depolarization is involved in transmitter release, possibly attributable to depletion of the pool of readily releasable vesicles by ICaF, and possibly arising from strong Ca2+ buffering in proximity of the release trigger.
After a short (1- to 200-ms) low-threshold presynaptic depolarization, the replenishment of the readily releasable vesicle pool is rate limiting for recovery of graded synaptic transmission, not recovery of ICaF from inactivation that proceeds more rapidly (Figs. 4 and 5). The recovery of graded synaptic transmission after prolonged (3- to 5-s) low-threshold presynaptic depolarization occurs simultaneously with the recovery of ICaF (with its complete deinactivation time of 8–12 s) (Fig. 4). For intermediate durations (0.5- to 3-s depolarizations) recovery of ICaF outstrips the recovery of graded transmission, indicating that ICaS depletes a pool of readily releasable vesicles normally available for release by ICaF. The factors that contribute to the replenishment of the readily releasable pool by mobilization of vesicles from a reserve pool, vesicle recycling, or priming reluctant vesicles (Congar and Trudeau 2002; Harata et al. 2001; Kelly 1993; Neher 1998; Neher and Zucker 1993; Schneggenburger et al. 2002; Voets 2000; Wang and Kaczmarek 1998; Wang and Zucker 1998; Wu and Borst 1999) remain to be determined in heart interneurons.
Overall our results indicate that ICaF and ICaS channels evoke transmitter release from the same release sites. ICaF channels are probably clustered near release sites but not intimately associated with the release trigger. Multichannel Ca2+ domains and a rapid increase in intracellular Ca2+ concentration (Figs. 1, 2, 6, and 7) produce fast and massive synaptic release by ICaF. In contrast, ICaS channels appear to be distributed more uniformly and not specifically in the vicinity of release sites, and they probably interact with the release trigger by radial diffusion of Ca2+.
The versatile intimate interrelations between fast and slow LVA Ca channels in regulating graded transmitter release together with the continuously changing dependency of graded synaptic transmission on the availability of synaptic vesicles and intracellular Ca2+ (Figs. 2 and 3)—demonstrated here as well as the previously described modulation (homosynaptic enhancement) of spike-mediated transmission mediated by Ca2+ entering by LVA Ca channels (see Fig. 12 in Ivanov and Calabrese 2003)—form the basis for a unique dynamics of reciprocally inhibitory synaptic transmission between heart interneurons that are further explored in a companion paper.
|
|
GRANTS |
|---|
|
|
|
FOOTNOTES |
|---|
Address for reprint requests and other correspondence: A. I. Ivanov, Department of Biology, Emory University, 1510 Clifton Road N.E., Atlanta, GA 30322 (E-mail: Andrei.Ivanov{at}emory.edu)
|
|
REFERENCES |
|---|
|
Arbas EA and Calabrese RL. Ionic conductances underlying the activity of interneurons that control heartbeat in the medicinal leech. J Neurosci 7: 3945–3952, 1987.[Abstract]
Augustine GJ. How does calcium trigger neurotransmitter release? Curr Opin Neurobiol 11: 320–326, 2001.[CrossRef][Web of Science][Medline]
Augustine GJ, Adler EM, and Charlton MP. The calcium signal for transmitter secretion from presynaptic nerve terminals. Ann NY Acad Sci 635: 365–381, 1991.[Web of Science][Medline]
Bertram R, Smith GD, and Sherman A. Modeling study of the effects of overlapping Ca2+ microdomains on neurotransmitter release. Biophys J 76: 735–750, 1999.[Web of Science][Medline]
Bollmann JH and Sakmann B. Control of synaptic strength and timing by the release-site Ca2+ signal. Nat Neurosci 8: 426–434, 2005.[Web of Science][Medline]
Borst JGG and Sakmann B. Effect of changes in action potential shape on calcium currents and transmitter release in a calyx-type synapse of the rat auditory brainstem. Philos Trans R Soc Lond B Biol Sci 354: 347–355, 1999.
Calabrese RL. Cellular, synaptic, network, and modulatory mechanisms involved in rhythm generation. Curr Opin Neurobiol 8: 710–717, 1998.[CrossRef][Web of Science][Medline]
Congar P and Trudeau LE. Perturbation of synaptic vesicle delivery during neurotransmitter release triggered independently of calcium influx. J Physiol 542: 779–793, 2002.
Dodge FA Jr and Rahamimoff R. Co-operative action a calcium ions in transmitter release at the neuromuscular junction. J Physiol 193: 419–432, 1967.
Erickson KR, Ronnekleiv OK, and Kelly MJ. Role of a T-type calcium current in supporting a depolarizing potential, damped oscillations, and phasic firing in vasopressinergic guinea pig supraoptic neurons. Neuroendocrinology 57: 789–800, 1993.[Web of Science][Medline]
Fedchyshin MJ and Wang LY. Developmental transformation of the release modality at the calyx of Held synapse. J Neurosci 25: 4131–4140, 2005.
Felmy F, Neher E, and Schneggenburger R. The timing of phasic transmitter release is Ca2+-dependent and lacks a direct influence of presynaptic membrane potential. Proc Natl Acad Sci USA 100: 15200–15205, 2003.
Fisher TE and Bourque CW. The function of Ca2+ channel subtypes in exocytotic secretion: new perspectives from synaptic and non-synaptic release. Prog Biophys Mol Biol 77: 269–303, 2001.[CrossRef][Web of Science][Medline]
Frazier CJ, Serrano JR, George EG, Yu X, Viswanathan A, Perez-Reyes E, and Jones SW. Gating kinetics of the 1I T-type calcium channel. J Gen Physiol 118: 457–470, 2001.
Gentile L and Stanley EF. A unified model of presynaptic release site gating by calcium channel domains. Eur J Neurosci 21: 278–282, 2005.[CrossRef][Web of Science][Medline]
Hagiwara S, Ozawa S, and Sand O. Voltage clamp analysis of two inward current mechanisms in the egg cell membrane of a starfish. J Gen Physiol 65: 617–644, 1975.
Harata N, Pyle JL, Aravanis AM, Mozhayeve M, Kavalali ET, and Tsien RW. Limited numbers of recycling vesicles in small CNS nerve terminals: implications for neural signaling and vesicular cycling. Trends Neurosci 24: 637–643, 2001.[CrossRef][Web of Science][Medline]
Haugland RP. Handbook of Fluorescent Probes and Research Chemicals (6th ed.). Eugene, OR: Molecular Probes, 1996.
Huguenard JR. Low-threshold calcium currents in central nervous system neurons. Annu Rev Physiol 58: 329–348, 1996.[CrossRef][Web of Science][Medline]
Huguenard JR and Prince DA. A novel T-type current underlies prolonged Ca2+-dependent burst firing in GABAergic neurons of rat thalamic reticular nucleus. J Neurosci 12: 3804–3817, 1992.[Abstract]
Ivanov AI and Calabrese RL. Intracellular Ca2+ dynamics during spontaneous and evoked activity of leech heart interneurons: low-threshold Ca currents and graded synaptic transmission. J Neurosci 20: 4930–4943, 2000.
Ivanov AI and Calabrese RL. Modulation of spike-mediated synaptic transmission by presynaptic background Ca2+ in leech heart interneurons. J Neurosci 23: 1206–1218, 2003.
Jeziorski MC, Greenberg RM, and Anderson PAV. The molecular biology of invertebrate voltage-gated Ca2+ channels. J Exp Biol 203: 841–856, 2000.[Abstract]
Kelly RB. Storage and release of neurotransmitters. Neuron 10, Suppl.: 43–53, 1993.[CrossRef][Web of Science][Medline]
Kleinhaus AL and Angstadt JD. Diversity and modulation of ionic conductances in leech neurons. J Neurobiol 27: 419–433, 1995.[CrossRef][Web of Science][Medline]
Klöckner U, Lee JH, Cribbs LL, Daud A, Hescheler J, Pereverzev A, Perez-Reyes E, and Schneider T. Comparison of the Ca2+ currents induced by expression of three cloned 1 subunits, 1G, 1H and 1I, of low-voltage-activated T-type Ca2+ channels. Eur J Neurosci 11: 4171–4178, 1999.[CrossRef][Web of Science][Medline]
Lacinová L, Klugbauer N, and Hofmann F. Regulation of the calcium channel 1G subunit by divalent cations and organic blockers. Neuropharmacology 39: 1254–1266, 2000.[CrossRef][Web of Science][Medline]
Lee JH, Daud AN, Cribbs LL, Lacerda AE, Pereverzev A, Klöckner U, Schneider T, and Perez-Reyes E. Cloning and expression of a novel member of the low voltage-activated T-type calcium channel family. J Neurosci 19: 1912–1921, 1999b.
Lee JH, Gomora JC, Cribbs LL, and Perez-Reyes E. Nickel block of three cloned T-type calcium channels: low concentrations selectively block 1H. Biophys J 77: 3034–3042, 1999a.[Web of Science][Medline]
Lin JW and Faber DS. Modulation of synaptic delay during synaptic plasticity. Trends Neurosci 25: 449–455, 2002.[CrossRef][Web of Science][Medline]
Lu J, Dalton JF IV, Stokes DR, and Calabrese RL. Functional role of Ca2+ currents in graded and spike-mediated synaptic transmission between leech heart interneurons. J Neurophysiol 77: 1779–1794, 1997.
Ma YP and Pan ZH. Spontaneous regenerative activity in mammalian retinal bipolar cells: roles of multiple subtypes of voltage-dependent Ca2+ channels. Vis Neurosci 20: 131–139, 2003.[CrossRef][Web of Science][Medline]
McCormick DA and Bal T. Sleep and arousal: thalamocortical mechanisms. Annu Rev Neurosci 20: 185–215, 1997.[CrossRef][Web of Science][Medline]
Neher E. Vesicle pools and Ca2+ microdomains: new tools for understanding their roles in neurotransmitter release. Neuron 20: 389–399, 1998.[CrossRef][Web of Science][Medline]
Neher E and Zucker RS. Multiple calcium-dependent processes related to secretion in bovine chromaffine cells. Neuron 10: 21–30, 1993.[CrossRef][Web of Science][Medline]
Nicholls JG and Baylor DA. Specific modalities and receptive fields of sensory neurons in the central nervous system of the leech. J Neurophysiol 31: 740–756, 1968.
Nicholls J and Wallace BG. Modulation of transmission at an inhibitory synapse in the central nervous system of the leech. J Physiol 281: 157–170, 1978a.
Nicholls J and Wallace BG. Quantal analysis of transmitter release at an inhibitory synapse in the central nervous system of the leech. J Physiol 281: 171–185, 1978b.
Olsen ØH and Calabrese RL. Activation of intrinsic and synaptic currents in leech heart interneurons by realistic waveforms. J Neurosci 16: 4958–4970, 1996.
Pan ZH. Differential expression of high- and two types of low-voltage-activated calcium currents in rod and cone bipolar cells of the rat retina. J Neurophysiol 83: 513–527, 2000.
Pan ZH. Voltage-activated Ca2+ channels and ionotropic GABA receptors localized at axon terminals of mammalian retinal bipolar cells. Vis Neurosci 18: 279–288, 2001.[CrossRef][Web of Science][Medline]
Pan ZH, Hu HJ, Perring P, and Angrade R. T-type Ca2+ channels mediate neurotransmitter release in retinal bipolar cells. Neuron 32: 89–98, 2001.[CrossRef][Web of Science][Medline]
Park JY, Kang HW, Jeong SW, and Lee JH. Multiple structural elements contribute to the slow kinetics of the Cav3.3 T-type channel. J Biol Chem 279: 21707–21713, 2004.
Perez-Reyes E. Molecular physiology of low-voltage-activated T-type calcium channels. Physiol Rev 83: 117–161, 2003.
Schneggenburger R, Sakaba T, and Neher E. Vesicle pools and short-term synaptic depression: lessons from a large synapse. Trends Neurosci 25: 206–212, 2002.[CrossRef][Web of Science][Medline]
Simmons PJ. Presynaptic depolarization rate controls transmission at an invertebrate synapse. Neuron 35: 749–758, 2002.[CrossRef][Web of Science][Medline]
Simon TW, Schmidt J, and Calabrese RL. Modulation of high-threshold transmission between heart interneurons of the medicinal leech by FMRF-NH2. J Neurophysiol 71: 454–66, 1994.
Staras K, Gyõori J, and Kemenes G. Voltage-gated ionic currents in an identified modulatory cell type controlling molluscan feeding. Eur J Neurosci 15: 109–119, 2002.[CrossRef][Web of Science][Medline]
Talley EM, Cribbs LL, Lee JH, and Peres-Reyes E. Differential distribution of three members of a gene family encoding low voltage-activated (T-type) calcium channels. J Neurosci 19: 1895–1911, 1999.
Tarasenko AN, Kostyuk PG, Eremin AV, and Isaev DS. Two types of low-voltage-activated Ca2+ channels in neurons of rat laterodorsal thalamic nucleus. J Physiol 499: 77–86, 1997.
Thomson AM. Molecular frequency filters at central synapses. Prog Neurobiol 62: 159–196, 2000.[CrossRef][Web of Science][Medline]
Thomson AM. Presynaptic frequency- and pattern-dependent filtering. J Comput Neurosci 15: 159–202, 2003.[CrossRef][Web of Science][Medline]
Uhrenholt TR and Nedergaard OA. Involvement of different calcium channels in the depolarization-evoked release of noradrenaline from sympathetic neurons in rabbit carotid artery. Basic Clin Pharmacol Toxicol 97: 109–114, 2005.[CrossRef][Web of Science][Medline]
Voets T. Dissection of three Ca2+-dependent steps leading to secretion in chromaffin cells from mouse adrenal slices. Neuron 28: 537–545, 2000.[CrossRef][Web of Science][Medline]
Wang C and Zucker RS. Regulation of synaptic vesicle recycling by calcium and serotonin. Neuron 21: 155–167, 1998.[CrossRef][Web of Science][Medline]
Wang LY and Kaczmarek LK. High-frequency firing helps replenish the readily releasable pool of synaptic vesicles. Nature 394: 384–388, 1998.[CrossRef][Medline]
Wicher D and Penzlin H. Ca2+ currents in central insect neurons: electrophysiological and pharmacological properties. J Neurophysiol 77: 186–199, 1997.
Wu LG and Borst JG. The reduced release probability of releasable vesicles during recovery from short-term synaptic depression. Neuron 23: 821–832, 1999.[CrossRef][Web of Science][Medline]
Yunker AMR. Low-voltage-activated ("T-type") calcium channels in review. J Bioenerg Biomembr 35: 533–575, 2003a.[CrossRef][Web of Science][Medline]
Yunker AMR. Modulation and pharmacology of low-voltage-activated ("T-type") calcium channels. J Bioenerg Biomembr 35: 577–598, 2003b.[CrossRef][Web of Science][Medline]
Zamponi GW, Bourinet E, and Snutch TP. Nickel block of a family of neuronal calcium channels: subtype- and subunit-dependent action at multiple sites. J Membr Biol 151: 77–90, 1996.[CrossRef][Web of Science][Medline]
Zühlke RD, Pitt GS, Deisseroth K, Tsien RW, and Reuter H. Calmodulin supports both inactivation and facilitation of L-type calcium channels. Nature 399: 159–162, 1999.[CrossRef][Medline]
This article has been cited by other articles:
|
|
 |
|
  A. Husch, M. Paehler, D. Fusca, L. Paeger, and P. Kloppenburg Distinct Electrophysiological Properties in Subtypes of Nonspiking Olfactory Local Interneurons Correlate With Their Cell Type-Specific Ca2+ Current Profiles J Neurophysiol, November 1, 2009; 102(5): 2834 - 2845. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Ch. Ludwar, C. G. Evans, J. Jing, and E. C. Cropper Two Distinct Mechanisms Mediate Potentiating Effects of Depolarization on Synaptic Transmission J Neurophysiol, September 1, 2009; 102(3): 1976 - 1983. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Husch, M. Paehler, D. Fusca, L. Paeger, and P. Kloppenburg Calcium Current Diversity in Physiologically Different Local Interneuron Types of the Antennal Lobe J. Neurosci., January 21, 2009; 29(3): 716 - 726. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. V. Olypher and R. L. Calabrese Using Constraints on Neuronal Activity to Reveal Compensatory Changes in Neuronal Parameters J Neurophysiol, December 1, 2007; 98(6): 3749 - 3758. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. J. Norris, A. L. Weaver, A. Wenning, P. S. Garcia, and R. L. Calabrese A Central Pattern Generator Producing Alternative Outputs: Pattern, Strength, and Dynamics of Premotor Synaptic Input to Leech Heart Motor Neurons J Neurophysiol, November 1, 2007; 98(5): 2992 - 3005. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. J. Haedo and J. Golowasch Ionic Mechanism Underlying Recovery of Rhythmic Activity in Adult Isolated Neurons J Neurophysiol, October 1, 2006; 96(4): 1860 - 1876. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. I. Ivanov and R. L. Calabrese Spike-Mediated and Graded Inhibitory Synaptic Transmission Between Leech Interneurons: Evidence for Shared Release Sites J Neurophysiol, July 1, 2006; 96(1): 235 - 251. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
TOP
ABSTRACT
INTRODUCTION
Control and