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Department of Physiology and Neuroscience, New York University Medical School, New York, New York
Submitted 5 April 2005; accepted in final form 25 May 2005
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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-glycyrrhetinic acid (18-GA), a pharmacological gap junction blocker. Under our experimental conditions, 18-GA had no adverse effects on intrinsic electroresponsive properties of IO neurons, other than the block of gap junction-dependent dye coupling and the resulting change in cells' passive properties. Application of 18-GA did not abolish single cell oscillations. Pharmacologically uncoupled IO neurons continued to oscillate with a frequency and amplitude that were similar to those recorded in control conditions. However, these oscillations were no longer synchronized across a population of IO neurons. Our optical recordings did not detect any clusters of synchronous oscillatory activity in the presence of the blocker. These results indicate that gap junctions are not necessary for generating subthreshold oscillations, rather, they are required for clustering of coherent oscillatory activity in the IO. The findings support the view that oscillatory properties of single IO neurons endow the system with important reset dynamics, while gap junctions are mainly required for synchronized neuronal ensemble activity. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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; Sotelo et al. 1974) formed by connexin 36 proteins (Condorelli et al. 1998, 2000; Rash et al. 2000). The distribution and properties of gap junction coupling in the IO have been extensively studied (Benardo and Foster 1986; Devor and Yarom 2002; De Zeeuw et al. 1995; Leznik et al. 2002; Llinás and Yarom 1981b, 1986; Llinás et al. 1974; Ruigrok et al. 1990; Sotelo et al. 1974). It is now established that one of the functions of gap junction coupling is to synchronize rhythmic firing and subthreshold oscillatory responses among IO neurons (Benardo and Foster 1986; De Zeeuw et al. 2003; Llinás and Yarom 1981a; Llinás et al. 1974; Long et al. 2002).
However, several studies have suggested that electrotonic coupling not only mediates synchrony, but it is also required for the generation of subthreshold neuronal oscillations in the IO (Bleasel and Pettigrew 1992; Lampl and Yarom 1997; Loewenstein et al. 2001; Manor et al. 1997, 2000; Yarom 1991). A recent study of connexin 36 knockout mice has challenged this idea. Electrical coupling and gap junctions are largely abolished in the IO of the connexin 36 knockout mice (De Zeeuw et al. 2003; Long et al. 2002). However, the genetically uncoupled IO neurons still generate sustained subthreshold oscillations of their membrane potential with a similar frequency and amplitude to those recorded from the wild-type animals (De Zeeuw et al. 2003; Long et al. 2002). This result implies that IO oscillations may be a single cell phenomenon, generated by intrinsic conductances of individual IO neurons. Conversely, De Zeeuw et al. (2003) have suggested that oscillations in the connexin 36 knockout mice are qualitatively different from those in the wild-type animals, and these differences are caused by structural and physiological changes in the knockout IO. Single cell oscillations may occur in such knockout cells as the result of compensatory ionic mechanisms not encountered in the normal animal.
Thus the role of gap junctions in generating IO oscillations in wild-type animals is still controversial. To address this issue, we studied the effects of 18-glycyrrhetinic acid (18-GA), a pharmacological gap junction blocker (Davidson and Baumgarten 1988), on the oscillations in the IO. Glycyrrhetinic acid and its derivatives are presumed to inhibit gap junction coupling by disturbing the arrangement of connexons within gap junctional plaques (Davidson and Baumgarten 1988; Goldberg et al. 1996). These uncoupling agents are known to block gap junctions in various cell types, as shown by dye transfer assays and paired cell recordings (Balice-Gordon et al. 1998; Eugenin et al. 1998; Ishimatsu and Williams 1996; Martin et al. 1991; Tordjmann et al. 1997; Travagli et al. 1995; Yamamoto et al. 1998; reviewed in Rozental et al. 2001).
Results presented in this study support the hypothesis that gap junctions are not necessary for generating subthreshold IO oscillations, but they are required for oscillatory synchronization. In our experiments, addition of 18-GA did not block single cell oscillations, whereas the drug prevented clustering of coherent rhythmic activity in the IO. These results indicate that oscillatory properties of uncoupled IO neurons in the knockout are not caused by long-term compensatory changes, but rather, are the single cell properties of normal IO cells.
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METHODS |
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Parasagittal brain stem slices were prepared from Sprague-Dawley rats (postnatal days 10–20) following protocols from previous in vitro studies, with some modifications (Bleasel and Pettigrew 1992; Leznik et al. 2002; Llinás and Yarom 1981a). In brief, animals were anesthetized with 15–20 mg of ketamine and decapitated after loss of limb-withdrawal reflex. The brain stem was isolated and placed in cold high-sucrose artificial cerebrospinal fluid (ACSF) containing (in mM) 248 sucrose, 26 NaHCO3, 1.25 NaH2PO4, 5 KCl, 2 MgSO4, 1 CaCl2, and 10 glucose, aerated with 95% O2-5% CO2 to a final pH of 7.4. Parasagittal slices (300 µm in thickness) were sectioned using a vibratome. The major portion of the IO was contained in two or three slices. Slices were transferred to a holding chamber with normal ACSF containing (in mM) 126 NaCl, 1.25 NaH2PO4, 5 KCl, 2 MgSO4, 2 CaCl2, 26 NaHCO3, and 10 glucose (pH = 7.4). Slices were incubated at room temperature for at least 1 h before use. Experiments were conducted at a bath temperature of 32 ± 1°C. All procedures used in this study were approved by the New York University Medical School Animal Care and Use Committee.
Intracellular recordings
Intracellular recordings were obtained from IO neurons using glass micropipettes filled with 3 M KAcetate (60–100 M
). Electrodes were advanced blindly using a Narashige manipulator. Only cells with a membrane potential negative to –55 mV were kept. Intracellular recordings were amplified with an Axoclamp-2A amplifier (Axon Instruments) and acquired at 10 kHz with a digital oscilloscope (Nicolet 4094, Nicolet Instruments) for off-line computer analysis.
Intracellular data were analyzed using Igor-based software (WaveMetrics). Spike heights were measured from the resting potential to the peak of the spike. The beginning of high-threshold Ca2+ spike (HTS) was defined as the time-point immediately preceding the HTS at which the second derivative of voltage with respect to time equaled zero.
Statistical analyses were performed with a two-tailed unpaired and paired Student's t-test. Differences were considered significant if P < 0.01. Population statistics are presented here as means ± SE.
Recording and analysis of optical signals
Individual slices were stained with the voltage-sensitive dye RH 414 (Molecular Probes, Eugene, OR) dissolved in normal ACSF to a final concentration of 0.025 mg/ml. Slices were incubated for 10 min in the dye solution before being transferred to the interface recording chamber. Optical signals were monitored with a fast CCD camera (HR Deltaron 1700, Fujix) with 128 x 128 pixels of spatial resolution. The total area imaged was 2.7 x 2.7 mm, and each pixel collected light from a surface of about 21 x 21 µm. Images were sampled every 4.8 ms (208 frames/s).
In most imaging experiments, single trial intracellular and optical recordings were simultaneously acquired. To increase the signal-to-noise ratio of the optical responses, optical data were averaged based on the temporal profile of the intracellular recordings. First, oscillatory peaks were detected in the intracellular trace. Two cycles of oscillations were repetitively averaged by matching their depolarizing peaks. Next, optical traces were cut into the same two cycle fragments and averaged using the time reference points from the intracellular data. As a result, the final optical recordings contained two cycles of oscillations averaged three or four times over the oscillatory sequence. Similar averaging procedures have been described elsewhere (Kawahara et al. 1997).
The optical recordings were analyzed using Matlab-based software (The Mathworks). In brief, the recordings were detrended to compensate for bleaching of the dye and slow responses from glia cells (Konnerth et al. 1987; Lev-Ram and Grinvald 1986). The signals were filtered with a three-dimensional moving average (3 x 3 x 3) and with a Gaussian low-pass filter. Changes in membrane potentials were evaluated as 
F/F. The optical signals were displayed using the RGB 256 color scale in such a way that their maximum amplitude equaled the maximum red color intensity of the RGB scale.
Neurobiotin staining
Neurobiotin staining was done following the protocol of Bloomfield et al. (1997), with some modifications. The recording electrode was filled with a 2 M KAcetate solution containing 4% Neurobiotin (Vector Laboratories). After physiological characterization of a cell, Neurobiotin was ionophoresed into the neuron using positive square pulses (3 Hz, 0.8 nA peak-to-peak) for 
30 min. After labeling the last cell in an experiment, the slice was incubated for 1 h and fixed overnight in PBS with 4% paraformaldehyde at 4°C. The labeled slices were washed in PBS and incubated in a methanol/hydrogen peroxide (18%) solution for 1 h to block peroxidase activity in blood vessels. The slices were subsequently reacted with the Elite ABC kit (Vector Laboratories) and 1% Triton X-100 and dissolved in PBS overnight. Next, slices were processed for peroxidase histochemistry using 3,3'-diaminobenzidine (DAB, Sigma). Slices were dehydrated, cleared overnight in methyl salicylate (Senatorov 2002), and mounted for light microscopy.
Pharmacological reagents
Drugs were applied by switching from the control ACSF solution to the one containing a known concentration of drug. The following reagents were used: 18-GA (150 µM), carbenoxolone (100 µM), and picrotoxin (10 µM) (Sigma, St Louis, MO). 18-GA was first dissolved in DMSO and diluted 1,000-fold into ACSF to a final concentration of 150 µM. An incubation of 20 min was allowed for the drugs to exert their effects.
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RESULTS |
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. Neurons outside these parameters were discarded.
18-GA significantly reduces Neurobiotin dye transfer in the IO
Dye transfer assays are often used in slice preparations as a measure of gap junction coupling (Goodenough et al. 1996; Rozental et al. 2001). It has been previously shown that when a marker such as Lucifer yellow (MW = 457) or Neurobiotin (MW = 323) is injected into a single IO neuron, it passes across gap junctions and labels additional cells next to the injection site (Benardo and Foster 1986; Devor and Yarom 2002; De Zeeuw et al. 2003). Thus to confirm that 18-GA blocks gap junction coupling in the IO, a series of Neurobiotin-labeling experiments were conducted in control experiments and after application of 18-GA.
In control injections, Neurobiotin spread across gap junctions and labeled multiple cells in the vicinity of the injected neuron. The number of indirectly stained cells varied from 9 to 16, with an average of 12 ± 1.1 cells (n = 6 injections). In an example shown in Fig. 1A (left), Neurobiotin spread to 12 other IO neurons, indicating extensive coupling in the IO. Note that in accordance with previous results (Devor and Yarom 2002), Neurobiotin was primarily detected in the somas of coupled cells.
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-GA largely reduced dye transfer and therefore inhibited gap junction coupling in the IO. In the presence of the blocker, the number of indirectly stained neurons was 0.2 ± 0.1 (n = 9 injections), a value significantly smaller (P < 0.0001) than that obtained in control conditions (Fig. 1B). Seven of nine Neurobiotin injections showed no evidence of dye coupling (Fig. 1A, right), and the remaining two injections resulted in indirect labeling of one cell (data not shown). Thus these results indicate that 18-GA can be used to pharmacologically disrupt gap junction communication in the IO.
18-GA does not affect the intrinsic electrical properties of IO neurons
To address concerns about possible nonspecific side effects of glycyrrhetinic acid and its derivatives on neuronal physiology (Jahromi et al. 2002; Rozental et al. 2001; Travagli et al. 1995), a series of experiments was conducted to study whether 18-GA affected intrinsic electrical properties of IO cells. This was done by analyzing suprathreshold responses of IO neurons in control conditions and during application of the drug (Fig. 2; Table 1).
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-GA (n = 13 cells). Intracellular recordings from the same cell before and after application of 150 µM 18-GA are superimposed in Fig. 2A. In each case, the suprathreshold response consisted of a fast sodium spike, followed by a dendritic HTS and afterhyperpolarization (Llinás and Yarom 1981a,b). 18-GA only increased the duration of the HTS, indicating that there was a larger invasion of dendritic spikes (Llinás and Yarom 1981b).
Likewise, a 100-ms hyperpolarization of IO neurons produced the same rebound response in control experiments and during administration of 18-GA (n = 15). In both conditions, hyperpolarizing pulses were followed by a somatic low-threshold Ca2+ spike (LTS) that triggered a fast sodium spike at its peak (Fig. 2B). In some experiments (n = 3 cells), 18-GA increased excitability of IO cells at the hyperpolarized levels. In these cases, the rebound LTS occurred during, and not after, membrane hyperpolarization (data not shown). De Zeeuw et al. (2003) recorded the same rebound response in IO neurons of connexin 36 knockout mice, suggesting that it may be caused by the block of gap junctions. However, several of our control cells (n = 4) that generated large subthreshold oscillations of their membrane potential also showed increased excitability at hyperpolarized levels. Thus the observed increase in excitability may not be directly related to the gap junction block, but alternatively, be a function of the strength of subthreshold oscillations.
Because IO neurons are extensively coupled through gap junctions (De Zeeuw et al. 1995; Sotelo et al. 1974), suppression of electrical transmission should affect passive biophysical properties of IO cells (Amitai et al. 2002). In particular, higher membrane resistance and lower capacitance would be expected in uncoupled IO neurons because the loading affect from surrounding cells is removed. As predicted, IO cells showed an increase in input resistance and decrease in membrane capacitance after treatment with 18-GA. On average, there was a 15 ± 2.1% (n = 8 cells) increase in the steady-state resistance and 27 ± 2.0% (n = 8 cells) decrease in capacitance (Table 1). These changes in cells' passive properties were found to be statistically significant when tested with the paired Student's t-test (P < 0.002; Table 1).
In summary, 18-GA had no adverse effects on the intrinsic ionic conductances of IO neurons. The main effect observed was the block of gap junctions, as accessed by a reduction in dye coupling, and the resulting change in the cells' passive electrical properties (Fig. 2B; Table 1). No significant changes in the amplitude of HTS, LTS, or sodium spike amplitude and duration were detected in the presence of 18-GA. Similar results were reported by Placantonakis et al. (2004) who used carbenoxolone, another gap junction blocker derived from glycyrrhetinic acid, in IO slices. Although small increases in spike amplitudes were observed in our experiments when the spikes were recorded from the same cell before and after application of the drug (n = 5 cells), these changes were not found to be statistically significant when tested across a population of control (n = 29) and 18-GA-treated (n = 15) cells (Table 1). Only the duration of HTS was significantly prolonged with 18-GA (P < 0.001; Table 1). This result suggests that there was a larger back-propagation of dendritic spikes in uncoupled IO neurons, probably because of the increase in cells' input resistance.
18-GA does not block subthreshold oscillations in the IO
To test whether electrical coupling is necessary for the generation of oscillatory activity in the IO, subthreshold responses were recorded from IO neurons in the presence of 18-GA. All of the recorded cells in this test (n = 23) continued to generate spontaneous oscillations of their membrane potential after addition of 18-GA. Furthermore, the probability of recording oscillations in a given slice preparation was not affected by the blocker. In animals older than 2 weeks, 
85% of the recorded IO cells manifested spontaneous oscillations in both control experiments and with 18-GA. Similar results were obtained with carbenoxolone, another gap junction antagonist (n = 17 cells, data not shown).
An example of intracellular recordings from an oscillating IO cell before and after 18-GA is shown in Fig. 3A. Addition of 18-GA increased the amplitude and frequency (see power spectra in Fig. 3A) of subthreshold oscillations without affecting the cell's resting membrane potential. Interestingly, there was an inverse relationship between the frequency and amplitude of oscillations in control conditions and the strength of the effect produced by 18-GA (Fig. 3, B and C). The smaller the frequency and amplitude were in control experiments, the larger their change was with the blocker. Accordingly, the effect of 18-GA was less profound when oscillations approached their upper limit for frequency and amplitude in control conditions (Fig. 3, B and C).
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-GA, there was a 22 ± 4.6% increase in oscillation frequency and 7.6 ± 2.3% increase in oscillation amplitude (n = 7 cells). However, statistical analysis of control (n = 31 cells) and uncoupled IO oscillations (n = 23 cells) using the unpaired Student's t-test did not reveal any significant differences in their oscillatory parameters (Table 1). Thus, although 18-GA largely blocked gap junction communication in the IO, it did not inhibit subthreshold IO oscillations. In fact, rather than eliminating subthreshold oscillations, 18-GA had an enhancing effect on them, probably because of increased input resistance of the uncoupled IO neurons. These results imply that gap junctions are not required for IO oscillatory activity and that such oscillations are produced by the intrinsic membrane conductances of single IO neurons.
It is important to note that, although electrotonic coupling is not necessary for the generation and maintenance of IO oscillations, it can clearly affect oscillatory properties of IO cells (Fig. 4). In control conditions, subthreshold oscillations are known to persist over a wide range of membrane potentials, and their oscillatory frequency is independent of the membrane potential of the recorded cell (Lampl and Yarom 1997; Llinás and Yarom 1986). These findings were confirmed in our study. When hyperpolarizing and depolarizing currents were injected into single oscillating cells, oscillatory activity was present within a large range of membrane potential levels (Fig. 4A, n = 6 cells). The amplitude of oscillations varied with membrane polarization, but the oscillatory frequency stayed close to constant (Fig. 4, C and D, control). In contrast, when gap junction coupling was blocked with 18-GA, subthreshold oscillations occurred only at a limited range of voltages (Fig. 4B, n = 8 cells). Oscillations were still present over the physiological range of membrane potentials (from –45 to –70 mV), but further depolarization or hyperpolarization blocked oscillatory activity of IO neurons. Furthermore, both the frequency and amplitude of uncoupled oscillations became dependent on the membrane potential of the recorded cell (Fig. 4, C and D, 18-GA). Thus the different voltage dependent characteristics of control and uncoupled oscillations (Fig. 4, C and D) indicate that gap junctions modulate subthreshold oscillatory activity in the IO by making oscillations less sensitive to membrane potential as far as frequency and amplitude are concerned.
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-GA prevents synchronization of oscillatory activity in the IO
To determine whether gap junction coupling is required for synchronization of oscillatory responses in the IO, spatial profiles of synchronous IO oscillations were recorded in control conditions and in the presence of 18-GA using in vitro high-speed voltage-sensitive dye imaging. The successful imaging of spontaneous oscillations required that several steps to be taken to increase the signal-to-noise ratio of the imaged responses. First, single trial intracellular and optical recordings of IO oscillations were simultaneously acquired. Intracellular recordings provided information about oscillations in individual cells, while optical recordings described patterns of ensemble oscillatory activity in the IO. Because there is a close correspondence between the oscillatory frequencies of single-cell and ensemble oscillations (Leznik et al. 2002), optical recordings were cut into fragments of two or three oscillatory cycles and averaged based on the temporal profile of intracellular recordings (see METHODS). The final imaging recordings consisted of several oscillation cycles, averaged two or three times over the oscillatory sequence. These imaged responses mainly showed synchronous oscillatory activity that was in phase with the intracellularly recorded oscillations.
Examples of simultaneously acquired optical and intracellular recordings of spontaneous oscillations are shown in Fig. 5 (n = 8 slices). In control conditions, oscillatory activity at 3 Hz was detected in both types of recordings (Fig. 5A). Optically recorded ensemble oscillations originated from several spatially separated fluorescent clusters of coherent activity (Fig. 5A, imaging panel). All clusters had the same frequency and phase as the intracellularly recorded single cell oscillations. Note that while additional oscillatory clusters with a different phase and frequency may have been also present in the slice, the experimental design was developed to only detect synchronized oscillatory activity that was in phase with the intracellular recordings.
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-GA blocked formation of oscillatory clusters, despite the fact that single cell oscillations were still present in individual IO neurons (Fig. 5B). Because the imaging responses detected the spatial distribution of synchronous oscillatory activity and these responses were abolished in the presence of 18-GA, we conclude that 18-GA prevented oscillatory temporo-spatial coherence.
We have previously shown that picrotoxin increases the level of coherent oscillatory activity in the IO (Leznik et al. 2002). 18-GA blocked the picrotoxin-induced changes in synchronized oscillatory patterns (Fig. 6, n = 5 slices). Examples of optically recorded oscillatory clusters in control conditions, in the presence of 10 µM picrotoxin, and in the presence of 150 µM 18-GA and 10 µM picrotoxin are shown in Fig. 6. In control experiment, three clusters of synchronous oscillatory activity were observed (Fig. 6A). Application of picrotoxin alone significantly increased the size of fluorescent clusters, indicating that the number of cells oscillating in-phase was increased (Fig. 6B). The picrotoxin-induced effect was blocked by the concurrent application of 18-GA, during which no oscillatory clusters were detected (Fig. 6C). Thus the block of gap junction coupling by 18-GA prevented picrotoxin from exerting its effects. This result confirms the hypothesis that picrotoxin enhances synchronous oscillatory activity in the IO by modulating the level of effective electrotonic coupling between IO cells (Lang et al. 1996; Leznik et al. 2002).
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DISCUSSION |
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-GA, it was shown that gap junctions are not necessary for the generation and maintenance of subthreshold oscillations in individual IO cells. The results also confirmed that gap junction coupling is required for synchronizing the oscillatory responses of groups of IO neurons and for the clustering of coherent rhythmic activity in the IO.
18-GA acutely blocks gap junction-dependent dye coupling without affecting the intrinsic electrical properties of IO neurons
The results from Neurobiotin dye labeling experiments show that 18-GA can be used to disrupt gap junction communication in the IO. In the presence of 18-GA, Neurobiotin dye transfer was significantly reduced (P < 0.0001, Student's t-test), indicating that the drug blocked most of gap junction-dependent coupling in the slice. After 18-GA application, no adverse effects on the intrinsic electrical responses of IO neurons were detected. The main difference observed was the change in the cells' passive electrical properties that accompany the gap junction block. A 15 ± 2.1% increase in steady-state resistance in our experiments (Table 1) is consistent with data from the connexin 36 knockout mice, where genetically uncoupled IO neurons have an 30% higher input resistance than that of wild-type cells (Long et al. 2002). The discrepancy between present results and those from the knockout mice is most probably caused by the secondary changes of dendritic IO neuron morphology as recently reported (De Zeeuw et al. 2003). Such morphological changes were not observed in this study, where dendritic morphology could be followed throughout the gap junction block. This is not unexpected given the comparatively rapid pharmacological effect of gap junction blockers compared with the developmental time-course of a knockout preparation.
Gap junctions are not required for the generation and maintenance of single cell oscillations
18-GA did not block subthreshold oscillatory activity in the IO. In the presence of the drug, pharmacologically uncoupled IO neurons continued to generate sustained oscillations of their membrane potential at a frequency of 1–10 Hz. When oscillations were recorded from the same cell before and after application of 18-GA, small changes in oscillatory frequency and amplitude were observed. The changes were the expected result from the increased input resistance and decreased membrane capacitance produced by the gap junction block. However, these changes were not found to be statistically significant when tested across a population of control and 18-GA–treated cells. Thus these results show that gap junctions are not necessary for the generation of subthreshold oscillations and such oscillatory activity is most likely produced by the intrinsic rhythmic conductances of individual IO cells.
18-GA did not fully block dye coupling in two of the nine Neurobiotin-labeling experiments. The incomplete block of gap junction coupling is similar to results from several other studies (Margineanu and Klitgaard 2001; Martin et al. 1991; Rozental et al. 2001). Because the block of gap junctions by 18-GA is not always complete, it could be argued that the residual gap junction coupling was able to sustain oscillations in some of the IO cells. However, this possibility can be ruled out by the fact that the probability of recording subthreshold oscillations was not affected by the blocker. Approximately 85% of the recorded IO cells generated spontaneous oscillations in both control experiments and with 18-GA. Furthermore, a large reduction of gap junction coupling, as accessed by a significant decrease in Neurobiotin dye transfer in our experiments, was sufficient to block synchronized oscillatory responses in IO neurons.
It is important to note that while gap junctions are not required for the generation of subthreshold oscillations, they are essential for certain aspects of the oscillatory activity of IO neurons. In control conditions, IO oscillations persist over a wide range of membrane potentials, and their frequency is independent of the membrane potential of the recorded cell (Lampl and Yarom 1997; Llinás and Yarom 1986). However, in the presence of 18-GA, subthreshold oscillations occurred only over a limited range of voltages, and both the frequency and the amplitude of oscillations became dependent on the cell's membrane potential. Different voltage-dependences of control and uncoupled oscillations imply that gap junction coupling modulates oscillatory activity in the IO. As suggested by Long et al. (2002), input from a network of electrically coupled cells may maintain oscillations over a range of voltages that tend to inactivate rhythmic conductances of individual IO neurons. Interestingly, gap junction coupling in the IO endows the ensemble oscillatory activity with robustness. In addition, the fact that individual neurons are capable of oscillation gives the oscillatory ensemble temporal agility in phase resetting by afferent input activity (Makarenko and Llinás 1998). These phase reset characteristics of IO oscillations are considered an important property of the organization and timing control of motricity (Llinás et al. 2002).
Gap junction coupling synchronizes rhythmic activity in the IO and regulates the size of synchronously oscillating clusters of cells
Electrotonic coupling through gap junctions is thought to be essential for synchronizing neuronal responses in the IO (Llinás 1974; Llinás et al. 1974; Long et al. 2002; De Zeeuw et al. 2003). Consistent with this idea, application of 18-GA inhibited synchronization of IO oscillations. 18-GA prevented formation of optically registered oscillatory clusters, despite the fact that single cell oscillations were still observed in individual IO cells. Because no synchronized oscillatory activity was detected in the presence of the gap junction blocker, we conclude that electrotonic coupling is essential for generating clusters of coherently oscillating cells.
Given that electrotonic coupling regulates coherent clustering of IO neurons, what determines the extent and spatial parameters of such synchronized oscillatory activity? IO neuronal gap junctions occur within specialized structures known as IO glomeruli, which are locally innervated by excitatory and inhibitory presynaptic terminals in close proximity to the gap junctions (De Zeeuw et al. 1989; King 1976; Sotelo et al. 1974). It has been proposed that activity of the intraglomerular chemical synapses can dynamically regulate the efficacy of electrotonic coupling and therefore the patterns of synchronous activity in the olivocerebellar system (Llinás 1974). In support of this hypothesis, several studies have shown that the distribution of synchronous complex spikes is modulated by the release of GABA and glutamate within the IO (Lang 2001, 2002; Lang et al. 1996; Llinás and Sasaki 1989). For instance, Lang et al. (1996) have shown that intraolivary injections of picrotoxin, a GABAA receptor blocker, significantly increase complex spike synchrony in the cerebellar cortex. We have previously shown that picrotoxin enhances the level of synchronous oscillatory activity in the IO nucleus (Leznik et al. 2002). In this study, the effects of picrotoxin were abolished by the concurrent application of 18-GA, confirming our hypothesis that picrotoxin induces its changes by modulating the effective electrotonic coupling between IO cells. Taken together, these results indicate that the spatial distribution and the extent of synchronous activity in the IO depend on the level of effective electrotonic coupling and therefore on the state of the IO network.
Our data are in general agreement with the results from the experiments on the connexin 36 knockout mice conducted by Long et al. (2002) and De Zeeuw et al. (2003). Both groups showed that genetically uncoupled IO cells show sustained spontaneous oscillations of their membrane potential, but these oscillatory responses are not synchronized. However, De Zeeuw et al. (2003) suggested that subthreshold oscillations in the knockout mice are generated by different mechanisms to those in the wild-type animal because of compensatory changes in the knockout IO. Results presented here challenge that interpretation. In our experiments, pharmacological block of gap junctions, during which no developmental compensatory changes could have occurred, produced similar results to those observed in the knockout mice. Thus our data support the view that individual IO cells generate sustained oscillatory responses because of their intrinsic membrane conductances. Gap junction coupling is mainly required for synchronizing these oscillations across a population of IO neurons.
Finally, the issue of the ultimate functional significance of electrotonic coupling in motor behavior has been challenged on the basis of the lack of obvious motor abnormalities in the connexin 36 knockout animals. This point was recently addressed by several studies (Kistler et al. 2002; Placantonakis et al. 2004). Placantonakis et al. (2004) showed that while macroscopic motricity appeared normal in the connexin 36–deficient animals, a detailed analysis of motor pattern generation showed a 10- to 20-ms degradation in the temporal coordination of muscle firing. Similarly, Kistler et al. (2002) showed that the connexin 36 knockout mice had a significant delay (in the order of tens of milliseconds) in the latency of the ocular optokinetic reflex. While these abnormalities may be not be grossly evident, precise temporal biding that yields motor execution nimbleness, is vital to animal "real life" survival (Carrier 1996).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Address for reprint requests and other correspondence: R. Llinás, New York Univ. Medical School, Dept. of Physiology and Neuroscience, 550 First Ave., MSB 432, New York, NY 10016 (E-mail: llinar01{at}endeavor.med.nyu.edu)
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REFERENCES |
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Balice-Gordon RJ, Bone LJ, and Scherer SS. Functional gap junctions in the schwann cell myelin sheath. J Cell Biol 142: 1095–1104, 1998.
Benardo LS and Foster RE. Oscillatory behavior in inferior olive neurons: mechanism, modulation, cell aggregates. Brain Res Bull 17: 773–784, 1986.[CrossRef][ISI][Medline]
Bleasel AF and Pettigrew AG. Development and properties of spontaneous oscillations of the membrane potential in inferior olivary neurons in the rat. Brain Res Dev Brain Res 65: 43–50, 1992.[CrossRef][Medline]
Bloomfield SA, Xin D, and Osborne T. Light-induced modulation of coupling between AII amacrine cells in the rabbit retina. Vis Neurosci 14: 565–576, 1997.[ISI][Medline]
Carrier DR. Ontogenetic limits on locomotor performance. Physiol Zool 69: 467–488, 1996.
Condorelli DF, Belluardo N, Trovato-Salinaro A, and Mudo G. Expression of Cx36 in mammalian neurons. Brain Res Brain Res Rev 32: 72–85, 2000.[CrossRef][Medline]
Condorelli DF, Parenti R, Spinella F, Trovato Salinaro A, Belluardo N, Cardile V, and Cicirata F. Cloning of a new gap junction gene (Cx36) highly expressed in mammalian brain neurons. Eur J Neurosci 10: 1202–1208, 1998.[CrossRef][ISI][Medline]
Davidson JS and Baumgarten IM. Glycyrrhetinic acid derivatives: a novel class of inhibitors of gap-junctional intercellular communication. Structure-activity relationships. J Pharmacol Exp Ther 246: 1104–1107, 1988.
Devor A and Yarom Y. Electrotonic coupling in the inferior olivary nucleus revealed by simultaneous double patch recordings. J Neurophysiol 87: 3048–3058, 2002.
De Zeeuw CI, Chorev E, Devor A, Manor Y, Van Der Giessen RS, De Jeu MT, Hoogenraad CC, Bijman J, Ruigrok TJ, French P, Jaarsma D, Kistler WM, Meier C, Petrasch-Parwez E, Dermietzel R, Sohl G, Gueldenagel M, Willecke K, and Yarom Y. Deformation of network connectivity in the inferior olive of connexin 36-deficient mice is compensated by morphological and electrophysiological changes at the single neuron level. J Neurosci 23: 4700–4711, 2003.
De Zeeuw CI, Hertzberg EL, and Mugnaini E. The dendritic lamellar body: a new neuronal organelle putatively associated with dendrodendritic gap junctions. J Neurosci 15: 1587–1604, 1995.[Abstract]
De Zeeuw CI, Holstege JC, Ruigrok TJ, and Voogd J. Ultrastructural study of the GABAergic, cerebellar, and mesodiencephalic innervation of the cat medial accessory olive: anterograde tracing combined with immunocytochemistry. J Comp Neurol 284: 12–35, 1989.[CrossRef][ISI][Medline]
Eugenin EA, Gonzalez H, Saez CG, and Saez JC. Gap junctional communication coordinates vasopressin-induced glycogenolysis in rat hepatocytes. Am J Physiol 274: 1109–1116, 1998.
Goldberg GS, Moreno AP, Bechberger JF, Hearn SS, Shivers RR, MacPhee DJ, Zhang YC, and Naus CC. Evidence that disruption of connexon particle arrangements in gap junction plaques is associated with inhibition of gap junctional communication by a glycyrrhetinic acid derivative. Exp Cell Res 222: 48–53, 1996.[CrossRef][ISI][Medline]
Goodenough DA, Goliger JA, and Paul DL. Connexins, connexons, and intercellular communication. Annu Rev Biochem 65: 475–502, 1996.[CrossRef][ISI][Medline]
Ishimatsu M and Williams JT. Synchronous activity in locus coeruleus results from dendritic interactions in pericoerulear regions. J Neurosci 16: 5196–5204, 1996.
Jahromi SS, Wentlandt K, Piran S, and Carlen PL. Anticonvulsant actions of gap junctional blockers in an in vitro seizure model. J Neurophysiol 88: 1893–1902, 2002.
Kawahara S, Toda S, Suzuki Y, Watanabe S, and Kirino Y. Comparative study on neural oscillation in the procerebrum of the terrestrial slugs Incilaria bilineata and Limax marginatus. J Exp Biol 200: 1851–1861, 1997.[Abstract]
King JS. The synaptic cluster (glomerulus) in the inferior olivary nucleus. J Comp Neurol 165: 387–400, 1976.[CrossRef][ISI][Medline]
Kistler WM, De Jeu MT, Elgersma Y, Van Der Giessen RS, Hensbroek R, Luo C, Koekkoek SK, Hoogenraad CC, Hamers FP, Gueldenagel M, Sohl G, Willecke K, and De Zeeuw CI. Analysis of Cx36 knockout does not support tenet that olivary gap junctions are required for complex spike synchronization and normal motor performance. Ann NY Acad Sci 978: 391–404, 2002.
Konnerth A, Obaid AL, and Salzberg BM. Optical recording of electrical activity from parallel fibres and other cell types in skate cerebellar slices in vitro. J Physiol 393: 681–702, 1987.
Lampl I and Yarom Y. Subthreshold oscillations and resonant behavior: two manifestations of the same mechanism. Neuroscience 78: 325–341, 1997.[CrossRef][ISI][Medline]
Lang EJ. Organization of olivocerebellar activity in the absence of excitatory glutamatergic input. J Neurosci 21: 1663–1675, 2001.
Lang EJ. GABAergic and glutamatergic modulation of spontaneous and motor-cortex-evoked complex spike activity. J Neurophysiol 87: 1993–2008, 2002.
Lang EJ, Sugihara I, and Llinás R. GABAergic modulation of complex spike activity by the cerebellar nucleoolivary pathway in rat. J Neurophysiol 76: 255–275, 1996.
Lev-Ram V and Grinvald A. Ca2+- and K+-dependent communication between central nervous system myelinated axons and oligodendrocytes revealed by voltage-sensitive dyes. Proc Natl Acad Sci USA 83: 6651–6655, 1986.
Leznik E, Makarenko V, and Llinás R. Electrotonically mediated oscillatory patterns in neuronal ensembles: an in vitro voltage-dependent dye-imaging study in the inferior olive. J Neurosci 22: 2804–2815, 2002.
Llinás R. Eighteenth Bowditch lecture. Motor aspects of cerebellar control. Physiologist 17: 19–46, 1974.[Medline]
Llinás R, Baker R, and Sotelo C. Electrotonic coupling between neurons in cat inferior olive. J Neurophysiol 37: 560–571, 1974.
Llinás R, Leznik E, and Makarenko VI. On the amazing olivocerebellar system. Ann NY Acad Sci 978: 258–272, 2002.
Llinás R and Sasaki K. The functional organization of the olivo-cerebellar system as examined by multiple Purkinje cell recordings. Eur J Neurosci 1: 587–602, 1989.[CrossRef][ISI][Medline]
Llinás R and Yarom Y. Electrophysiology of mammalian inferior olivary neurones in vitro. Different types of voltage-dependent ionic conductances. J Physiol 315: 549–567, 1981a.
Llinás R and Yarom Y. Properties and distribution of ionic conductances generating electroresponsiveness of mammalian inferior olivary neurones in vitro. J Physiol 315: 569–584, 1981b.
Llinás R and Yarom Y. Oscillatory properties of guinea-pig inferior olivary neurones and their pharmacological modulation: an in vitro study. J Physiol 376: 163–182, 1986.
Loewenstein Y, Yarom Y, and Sompolinsky H. The generation of oscillations in networks of electrically coupled cells. Proc Natl Acad Sci USA 98: 8095–8100, 2001.
Long MA, Deans MR, Paul DL, and Connors BW. Rhythmicity without synchrony in the electrically uncoupled inferior olive. J Neurosci 22: 10898–10905, 2002.
Makarenko V and Llinás R. Experimentally determined chaotic phase synchronization in a neuronal system. Proc Natl Acad Sci USA 95: 15747–15752, 1998.
Manor Y, Rinzel J, Segev I, and Yarom Y. Low-amplitude oscillations in the inferior olive: a model based on electrical coupling of neurons with heterogeneous channel densities. J Neurophysiol 77: 2736–2752, 1997.
Manor Y, Yarom Y, Chorev E, and Devor A. To beat or not to beat: a decision taken at the network level. J Physiol Paris 94: 375–390, 2000.[CrossRef][ISI][Medline]
Margineanu DG and Klitgaard H. Can gap-junction blockade preferentially inhibit neuronal hypersynchrony vs. excitability? Neuropharmacology 41: 377–383, 2001.[CrossRef][ISI][Medline]
Martin W, Zempel G, Hulser D, and Willecke K. Growth inhibition of oncogene-transformed rat fibroblasts by cocultured normal cells: relevance of metabolic cooperation mediated by gap junctions. Cancer Res 51: 5348–5351, 1991.
Placantonakis DG, Bukovsky AA, Zeng XH, Kiem HP, and Welsh JP. Fundamental role of inferior olive connexin 36 in muscle coherence during tremor. Proc Natl Acad Sci USA 101: 7164–7169, 2004.
Rash JE, Staines WA, Yasumura T, Patel D, Furman CS, Stelmack GL, and Nagy JI. Immunogold evidence that neuronal gap junctions in adult rat brain and spinal cord contain connexin-36 but not connexin-32 or connexin-43. Proc Natl Acad Sci USA 97: 7573–7578, 2000.
Rozental R, Srinivas M, and Spray DC. How to close a gap junction channel. Efficacies and potencies of uncoupling agents. Methods Mol Biol 154: 447–476, 2001.[Medline]
Ruigrok TJ, de Zeeuw CI, van der Burg J, and Voogd J. Intracellular labeling of neurons in the medial accessory olive of the cat. I. Physiology and light microscopy. J Comp Neurol 300: 462–477, 1990.[CrossRef][ISI][Medline]
Senatorov VV. Dark-field microscopy visualization of unstained axonal pathways using oil of wintergreen. J Neurosci Methods 113: 59–62, 2002.[CrossRef][ISI][Medline]
Sotelo C, Llinás R, and Baker R. Structural study of inferior olivary nucleus of the cat: morphological correlates of electrotonic coupling. J Neurophysiol 37: 541–559, 1974.
Tordjmann T, Berthon B, Claret M, and Combettes L. Coordinated intercellular calcium waves induced by noradrenaline in rat hepatocytes: dual control by gap junction permeability and agonist. EMBO J 16: 5398–5407, 1997.[CrossRef][ISI][Medline]
Travagli RA, Dunwiddie TV, and Williams JT. Opioid inhibition in locus coeruleus. J Neurophysiol 74: 518–528, 1995.[Abstract]
Yamamoto Y, Fukuta H, Nakahira Y, and Suzuki H. Blockade by 18beta-glycyrrhetinic acid of intercellular electrical coupling in guinea-pig arterioles. J Physiol 511: 501–508, 1998.
Yarom Y. Rhythmogenesis in a hybrid system-interconnecting an olivary neuron to an analog network of coupled oscillators. Neuroscience 44: 263–275, 1991.[CrossRef][ISI][Medline]
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