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Laboratoire de Dynamique des Fluides Complexes, Unité 7506 Centre National de la Recherche Scientifique, Université Louis Pasteur, Institut de Physique, Strasbourg, France
Submitted 19 May 2004; accepted in final form 16 January 2005
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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-methyl-4-carboxyphenylglycine (MCPG) removed the very slow depolarizing component of the EPSP and prevented the sustained firing at very low rate. A metabotropic glutamate receptor (mGluR)-activated calcium sensitive conductance is therefore responsible for a very slow synaptic component associated with firing at very low rate. In addition, our observations suggested that the asynchronous release of glutamate might participate also in the recurring bursting. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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). To persist after a stimulus, the electrical activity has to be sustained in the absence of any external input and to be stimulus-selective. We address the question of the mechanisms sustaining activity once initiated. The electrical activity is usually assumed to be sustained by the propagation of reverberating synaptic excitation through a neural network, thanks to the high efficiency of recurrent synapses ( Wang 2001). Recent experiments ( Egorov et al. 2002; Fransen et al. 2002) and models ( Lisman et al. 1998; Tegner et al. 2002; Wang 1999, 2001) have emphasized the possible role of the intrinsic slow temporal decay of the excitatory postsynaptic potential (EPSP) in the maintenance of a stable persistent state at low physiological frequencies (10–50 Hz). The slow decay time of the EPSP is attributed to the activation of slow synaptic or synaptic-dependant conductance. While negative feedback mechanisms following a spike forbid the short term reinitiation of a spike, refiring after a long time interval requires the activation of a slow depolarizing component. Therefore if neuronal firings are partially synchronous and if synaptic mechanisms are implied, models predict that their decay time needs to exceed the typical time interval between spikes ( Wang 1999, 2001).
In this study, we analyzed synaptic-dependant mechanisms involved in sustaining activity at low firing rates. We used simple model systems of highly synchronous excitatory networks: hippocampal excitatory autapses and pairs. Reverberating processes through large networks were prevented since they were bound to follow a one (or 2) neuron(s) loop. It has been observed previously that neurons undergo great synaptogenesis in culture ( Verderio et al. 1999) and that synapses develop characteristics comparable with synapses in the intact brain ( Wilcox et al. 1994). Autaptic neurons were constrained to connect only to themselves so that each spike leads to a large EPSP ( Bekkers and Stevens 1991; Segal and Furshpan 1990). The activation of all synapses was therefore highly synchronous allowing an easy discrimination of synaptic components according to their kinetics ( Bekkers et al. 1990; Cummings et al. 1996).
Despite the fact that large reverberating pathways were hindered in autaptic neurons and pairs of neurons grown in vitro, bursts lasting for several seconds at low (10–20 Hz) or very low (1–2 Hz) frequencies were observed spontaneously or after a brief stimulation. This sustained firing occurred as bursts of spikes either briefly evoked or spontaneously occurring after a long period of silence. Bursts were very similar to those observed in larger neuronal networks in culture ( Bacci et al. 1999; Segal and Furshpan 1990). They were synaptically driven since no more activity was observed when glutamate synapses were blocked ( Bacci et al. 1999). Here, we analyze the nature and the role of slow synaptic dependant components of the EPSP in sustaining recurring bursting activity at low frequencies in absence of reverberating excitation through large ensemble of neurons.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Pyramidal neurons from rat hippocampus were grown on the substrates according to the protocol derived from Banker ( Goslin and Banker 1991). Patterned coverslips (see next paragraph) were incubated for 5 days in neuron-plating medium containing 10% horse serum (Invitrogen, Carlsbad, CA). Hippocampi from E18 rats embryos were dissociated chemically (0.25% trypsin, 20 min) and mechanically using fire-polished Pasteur pipettes. Neurons plated on the patterned substrates (densities ranging from 1,000 to 10,000 cells/cm2) were maintained in a 5% CO2 atmosphere at 37°C. After 4 h, neuron-plating medium was replaced by a serum-free maintenance medium, and a feeding layer of glial cells was added to each dish. Glial cells proliferation in the culture was stopped by AraC after 2 days (1 µg/ml, Sigma, St. Louis, MO).
Photolithography
The lithography protocol has been detailed previously ( Wyart et al. 2002). In brief, cleaned coverslips were coated with hydrophobic fluorosilane C8H4Cl3F13Si (ABCR, Karlsruhe, Germany) in dichloromethane and n-decane, for one-half an hour, at 4°C. After rinsing in chloroform, the silanized surfaces were spin-coated with a positive photoresist. Each coverslip was pressed against a mask and exposed to UV light. Incubation in a development bath removed the exposed photoresist. The fluorosilane layer (no longer protected by the photoresist) was removed with an H2O plasma, and the glass surface was coated with poly-L-lysine (Sigma P2636, 1 mg/ml for 3 h at 37°C). Unexposed photoresist was washed out with acetone. Patterned domains for autapses have been optimized to obtain on average a single neuron per disk with a large probability of survival. Patterns for pairs consisted in two 60-µm-diam disks connected to each other by a thin line (2–4 µm wide and 60–100 µm long) to guide the growth of the neurites. Masks for lithography were prepared in the laboratory: after a standard metallization procedure using chromium, we obtained typically 100–1,000 patterns on a coverslip.
Electrophysiological recordings
Cell-attached and whole cell patch-clamp recordings were obtained at room temperature from 2- to 4-wk-old cells. All recordings were performed using Axopatch 200B (Axon Instruments, Foster City, CA). Patch pipettes were made of borosilicate tubes (Clarks) and had a resistance of 3–4 M
when filled with the standard pipette solution. In cell-attached recordings, a 5-mV pulse was regularly applied to check that the perforation of the cell membrane did not occur. To monitor the recording characteristics in whole cell experiments, leak resistance was measured periodically during the recordings and ranged between 250 M and 1 G for a given cell. Leak current, monitored in voltage clamp, ranged from 10 to 200 pA. Cells older than 3 wk with a larger surface could sometimes not be clamped in voltage mode and would fire a spike on the edge of the excitatory postsynaptic current (EPSC) in this configuration. We have discarded these cells for our analysis of synaptic properties. Collection of data were interrupted if the recording showed a significant change in leak resistance. Fast and slow capacitance and series resistance compensation were performed in the whole cell mode. Series resistance in whole cell configuration was <10–12 M and was compensated 
60%. Recording data were acquired at 5 kHz in real-time with an Axon Digidata 1320A (Axon Instruments).
Recording solutions
The bath solution contained (in mM) 145 NaCl, 3 KCl, 3 CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES, pH = 7.25, and its osmolarity was adjusted to 315 mOsm. The pipette solution contained (in mM) 9 NaCl, 136.5 KGlu, 17.5 KCl, 0.5 CaCl2, 1 MgCl2, 10 HEPES, and 0.2 EGTA (pH =7.25), and its osmolarity was equal to 310 mOsm. In our conditions, the reversal potential for glutamatergic currents was 0 mV, allowing us to distinguish them from GABAergic currents (reversal potential of –60 mV). Bath solution was superfused locally at 0.5–1 ml/min with a microperfusion tube inlet and outlet from a peristaltic pump. All experiments were performed at fixed temperature (22–25°C).
Drugs
In some experiments, the following transmitter antagonists (from Sigma) were applied in the bath: 100 µM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) for non-N-methyl-D-aspartate receptors (NMDARs); 50–100 µM 2-amino-5-phosphopentanoic acid (APV) and 10 µM MK801 for NMDARs; and 250 µM -methyl-4-carboxyphenylglycine (MCPG) for metabotropic glutamate receptors. EGTA-AM (Molecular Probes, Eugene, OR) was dissolved in 0.5% dimethyl sulfoxide before dilution at 50 µM in the bath solution. Cells were incubated for 15 min. A solution of 1 mM EGTA was also used as a comparison to the EGTA-AM experiments. EGTA is a slow calcium buffer ( Feller et al. 1996) that modifies the shape of a calcium transient by providing a faster initial decay while producing a smaller and slower subsequent phase.
Calcium imaging
Cultures were loaded with 5 µM of the membrane-permeant acetoxymethyl ester of Fura-2 AM (Molecular Probes) for 15 min at room temperature and rinsed for 30 min. A 100-W Xenon lamp filtered at 380 nm ensured the excitation of the probe, and the emission was filtered at 510 nm. Binned images (8 x 8) obtained with a CCD (CoolSnap HQ, Roper Scientific, Duluth, GA) were acquired at 20 Hz, stored, and analyzed using Metamorph to measure the fluorescence intensity variation in a cell body. Each spike in a Fura-2 AM-loaded neuron induced a large calcium entry, associated with a decrease of the fluorescence emission ( Mao et al. 2001). Therefore the variations of fluorescence intensity in the soma reflected the occurrence of spikes with the time resolution of our acquisition system (50 ms). The concentration of Fura-2 in the soma was estimated to be of the order of 50 µM.
Detection of spikes in cell attached recordings
Spikes were detected above a threshold equal at least to three times the peak-to-peak electrical noise of the recording. By combining cell-attached recordings with spike detection by calcium imaging, we checked that no spikes were missed. A limitation of cell attached recordings is the ambiguity to distinguish the signal due to a spike from a signal due to large EPSPs. A large volley of EPSPs arriving very synchronously in the case of an autapse has a rising phase lasting for only a few milliseconds. For high-frequency signals, the cell-attached technique provides a measurement proportional to the derivative of the neuronal membrane potential. Thus large autaptic EPSPs gave rise often to a negative peak in their early phase that is similar to a spike. For this reason, we did not consider higher firing rates than 50 Hz, and we limited our analysis to interspike intervals (ISIs) >20 ms.
Analysis
Statistics on burst duration and on median intraburst frequency were obtained with the criterion of 5 s as the maximal ISI within a burst and after suppression of ISI inferior to 20 ms. ISI distributions were normalized for each cell to compensate for differences in the duration of recordings or in burst frequencies between distinct cells.
ESTIMATION OF THE INTEGRATED CHARGE ASSOCIATED WITH THE AUTAPTIC RESPONSE. We evoked a spike in voltage clamp by 2-ms depolarizing pulses of current at 0.05 Hz to monitor a stable autaptic EPSC. The total integrated charge was estimated by integrating the EPSC from 4 to 600 ms after the evoked spike. To distinguish between the very slow and the slow components of the EPSC, we used also the partial integrated charges corresponding to the integration of the EPSC from 4 to 200 ms and from 200 to 600 ms.
ESTIMATION OF THE FREQUENCY OF ASYNCHRONOUS MINIATURE EVENTS.
Discrete asynchronous miniature EPSCs (mEPSCs) can be detected 200 ms after a spike on the autaptic response. These mEPSCs occur for 
1 s at higher frequency than spontaneous mEPSCs at rest. We estimated their mean frequency in a 500-ms time window beginning 200 ms after a spike. The slow component of the EPSC was fitted to a single exponential that was subtracted to the recording trace before the detection of mEPSCs with the MiniAnalysis software (Synaptosoft). For comparison, we show the mean frequency of miniature events at rest measured in TTX at –60 mV. Results are always presented as means ± SD. The experiments followed European Community guidelines on the care and use of animals (86/609/CEE, CE official journal L358, 18 December 1986), French legislation (decree no. 97/748, 19 October 1987, J. O. République française, 20 October 1987), and the recommendations of the CNRS.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Single isolated neurons having only autaptic synapses were obtained with neuronal cultures on patterned surfaces (see METHODS). This protocol allowed for the proper maturation of cells 5 wk in vitro ( Wyart et al. 2002). Autapses grew most of their neurites along the border of the poly-L-lysine disks (Fig. 1A) and were constrained to connect only to themselves. We studied only excitatory neurons exhibiting highly ramified dendritic trees. Their excitatory nature was confirmed in whole cell voltage clamp by measuring the reversal potential of the autaptic EPSC (0 mV in our conditions, see METHODS). After 10 days in vitro (DIV; 10–31 DIV; age = 19.2 ± 8.5 DIV), spontaneous activity was detected in two-thirds of the neurons (80 among 126 cells) in cell-attached recordings (Fig. 1B) but not in whole cell recordings, probably because of the rapid dialysis of the intracellular components. Activity was also revealed by calcium imaging as large calcium transients occurring spontaneously (Fig. 1, C and D). Calcium transients were always abolished by bath application of TTX (0.5 µM; n = 7; age = 20.1 ± 5.7 DIV, data not shown) indicating that they arose from sodium action potentials. All spontaneously spiking cells fired bursts, i.e., groups of spikes separated by less than a few seconds. Interburst intervals had a widespread distributions with a mean in the order of tens of seconds (97 ± 43 s; Fig. 1B). We set the following 5 s as the maximum ISI to define a burst. Burst detection was usually unambiguous since interburst intervals usually exceeded 10 s (Fig. 1, B and E, top).
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; Wang 1999).
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In the case of cells firing at low frequencies, the EPSC exhibited two components (Fig. 4, A1 and A2). The main component had a decay time of about 10 ms and was suppressed by 100 µM CNQX (data not shown), indicating the activation of AMPA receptors. The second component was reduced by bath application of NMDAR antagonists (MK801, 10 µM with APV, 100 µM; Fig. 4, A1 and A2), revealing the activation of NMDARs: the integrated charge (see METHODS) decreased from 67.3 ± 11.9 to 32.4 ± 11.0 pA·s in presence of the drugs (n = 6, P < 0.05; Fig. 5A3). The application of APV+MK801 in neurons bursting at low frequencies (Fig. 4B1) prevented also the occurrence of bursts (Fig. 4B2). Only single spikes or single pairs of spikes were detected (n = 6, Fig. 4B2). This effect was reversed by washing out the drugs (Fig. 4B3). This shows that the NMDA component of the EPSC (with a decay time of about 100–200 ms) was mainly responsible for sustained firing at 10 Hz within bursts. On the contrary, the AMPA component of the EPSC (with a decay time of about 10 ms) was not lasting long enough to sustain recurring bursting.
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Neurons with sustained activity at very low frequencies exhibited a very slow component of the autaptic response that lasted for several hundreds of milliseconds (both in voltage and current clamp; see Fig. 5, A1 and A2). Application of MK801 and APV (Fig. 5, A1 and A2) modified only slightly the EPSC and the EPSP: the integrated charge (see METHODS) decreased only from 170.3 ± 98.3 to 167.1 ± 93.8 pA·s in presence of the drugs (n = 9, not significant; P < 0.05; Fig. 5A3). Application of the drugs did not abolish either the spontaneous sustained firing (n = 3, Fig. 5, B1–B3). Moreover, firing within the bursts was less regular: ISIs shorter than 5 s exhibited a larger dispersion (P < 0,05; see Fig. 5C) in the presence of MK801-APV (1.6 ± 2.9 s) than under control conditions (1.3 ± 0.7 s). This observation shows that, at very low rates, the NMDA component was important for the regularity of firing, but was not necessary to sustain firing.
Because bursts were associated with large calcium transients (Fig. 1, C and D), we tested if this very slow autaptic component was dependent on the intracellular calcium concentration. Fifteen-minute bath incubation in 50 µM EGTA-AM was sufficient to suppress the slow autaptic response (Fig. 6A1). The same result was obtained using 1 mM EGTA in the pipette intracellular medium (data not shown). The slow component, estimated as the 200- to 600-ms integrated charge (see METHODS), decreased by 85% in EGTA-AM (Fig. 6C), whereas the 0- to 200-ms integrated charge was only lowered by 30% (Fig. 6C). EGTA-AM incubation also modified the maintenance of activity in 3- to 4-wk-old cells (n = 9, Fig. 6A2). In four cells, it prevented bursts to occur, and cells showed only single spikes (data not shown). In five of nine cells, bursts of three to six spikes separated by 200-ms intervals on average were still observed after long periods of silence. In these cells, burst duration decreased remarkably under 1 s (680 ± 130 ms, n = 4, see Fig. 6A3). ISI distribution shifted to lower values; long ISIs (>300 ms) were abolished (Fig. 6A4). Thus it mimicked the ISI distribution of 2-wk-old cells (Figs. 3C and 6A4).
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). Bath application of MCPG (250 µM), a nonselective mGluR antagonist, greatly reduced the slow component of the EPSP (Fig. 6B1). In six cells, the 200- to 600-ms integrated charge was decreased by 61 ± 13% in MCPG (Fig. 6C), whereas the 0- to 200-ms integrated charge was only reduced by 8 ± 3% (Fig. 6C). Therefore the very slow component of the EPSP corresponded to a calcium-sensitive conductance, activated by metabotropic glutamate receptors. We measured moreover its reversal potential (3 ± 4 mV), which was very close from the reversal potential for all cations calculated with the Nernst equation (–2.5 mV) in our conditions ([cations]ext = 148 mM, [cations]in= 163 mM). This indicates that this conductance was a calcium-sensitive nonselective cationic conductance activated by mGluRs. The application of MCPG (250 µM) altered also the maintenance of firing (see Fig. 6B2): cells fired only single spikes or few (2–3) spikes (n = 6), separated by short (<300 ms) intervals. This is consistent with the observation that the autaptic responses of 3- to 4-wk-old cells incubated in EGTA-AM (Fig. 6A1) or with MCPG (Fig. 6B1) and of 2-wk-old cells (Fig. 4A1) had a similar relatively short timescale (about 100 ms) and did not allow sustained firing at very low frequencies (i.e., with ISIs > 500 ms). Presynaptic calcium-dependant mechanisms sustaining recurring firing at very low frequency
Neurons, showing sustained bursting activity at very low frequencies, exhibited, on top of the very slow component of the autaptic response, delayed release of glutamate. Asynchronous miniatures occurred at a very high frequency in a time window of 200 ms to 1 s after a spike (Fig. 7A). The frequency of asynchronous miniature events increased noticeably with the number of DIV (Fig. 7B1) and significantly with the firing frequency, reaching a plateau level after a train of action potentials at 4 Hz (Fig. 7B1). Fifteen-minute bath incubation in 50 µM EGTA-AM was sufficient to decrease the frequency of the asynchronous miniature events associated with the continuous component of the slow autaptic response (Fig. 7B2).
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; Cummings et al. 1996; Feller et al. 1996; Goda and Stevens 1994; Hagler and Goda 2001; Zucker 1999), intervenes in the suprathreshold reactivation of action potentials within a burst. The stochastic nature of these asynchronous events might also explain the irregular firing that was observed with APV + MK801 in the bath. In the case of very low frequency firing, NMDAR activation was not necessary for maintenance of activity in a burst. Our data suggest that a postsynaptic calcium-sensitive nonselective cationic conductance activated by mGluRs and the presynaptic delayed release of glutamate were contributing together to the firing at very low frequency within a burst.
Origin of the long relative refractory period in 3- to 4-wk-old cells
To control spiking in a defined range of frequencies, negative and positive feedback mechanisms are necessary, respectively, to forbid spiking shortly after a spike and to allow reactivation of a spike after a long ISI ( Wang 1999, 2001). In 2-wk-old cells, we observed a classical refractory period (10–20 ms). On the contrary, in 3- to 4-wk-old cells, a relative refractory period lasted for about 150 ms (see Fig. 8). This long refractory period was associated with an enormous (about 50%) decrease of the membrane resistance during the EPSP (see Fig. 8 for the determination of the membrane resistance). This huge shunt was likely responsible for the inhibition of the spike reactivation. It could also explain the fact that 3- to 4-wk-old cells did not fire at low frequencies exactly as 2-wk-old cells when mGluR activation or intracellular calcium increase was inhibited: their large EPSP was responsible for an abnormally long shunt, superior to the time decay of the NMDA component.
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The synaptic mechanisms previously described could consist in specific properties of an autaptic system with only homosynapses (synapses connected to the neuron itself), deprived of heterosynapses (synapses connecting distinct neurons). To test this issue, we carried out similar experiments on pairs of isolated neurons (n = 9) in double cell-attached configuration. Spontaneous activity of 2-wk-old cells in a pair consisted in synchronous bursts with large ISIs separated by long silences (Fig. 9A1) and was suppressed by bath application of CNQX (100 µM). Figure 9A2 shows that any cell could spike first (e.g., cell 1 for the 1st 2 spikes and cell 2 for the last 2 spikes on Fig. 9A2) and systematically activate the other neuron in a 10-ms time window (Fig. 9A2). Presumably, both cells remained silent for a few hundred of milliseconds due to the long relative refractory period. Therefore there was no asynchronous spiking in the paired neuron configuration.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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). We studied experimentally the mechanisms underlying the recurring bursting. Spontaneously or evoked bursts showed the same characteristics, indicating that firing was self-sustained in a burst. Autapses after 2 wk fired bursts of action potentials, with a gradual decrease of the median interspike frequency from 11 (2 wk) to 2 Hz (3–4 wk). The median ISIs (90 and 500 ms, respectively) were always longer than the decay time of the AMPA component of the EPSC. We showed that the activation of these receptors was not sufficient to sustain firing. Instead, we showed that slower synaptic components, dependent on the activation of NMDARs and mGluRs, were necessary for sustained firing at low frequencies. Comparison with previous studies in culture and other preparations
Hippocampal glutamatergic cells in culture ( Bacci et al. 1999; Maeda et al. 1995; Murphy et al. 1992; Opitz et al. 2002; Segal 1991), as in slices ( Garaschuk et al. 1998; Menendez de la Prida and Sanchez-Andres 2000), fire bursts of spikes at low frequency (<10 Hz). Studies on recurring bursting in culture ( Harris et al. 2002; Muramoto et al. 1993; Murphy et al. 1992; Opitz et al. 2002; Robinson et al. 1993; Voigt et al. 2001) have been achieved mainly at high density. Whereas the duration of bursts (2–5 s) is similar in our study, their high frequency (0.2 instead of 0.01 Hz) and regularity of occurrence (referred as "oscillatory" bursting) are distinct. This can be attributed to the higher density of cells (and certainly of synapses; Muramoto et al. 1993) and to the presence of inhibitory cells that modulate firing ( Opitz et al. 2002; Siebler et al. 1993; Voigt et al. 2001). On the contrary, Bacci et al. (1999), as in our study, report that bursts last for a few seconds and are separated by tens of seconds; they show also that spontaneous activity is abolished with CNQX and TTX but not with APV, which makes firing more irregular; the spontaneous activity of autapses is characterized by an interburst interval of about 1 min, and intracellular recordings show that action potentials occur on a very slow depolarizing component. Therefore our model system did not properly exhibit an "oscillatory bursting," since the occurrence frequency of burst differs. However, because bursts duration and intraburst frequencies are similar in all cases, mechanisms for sustained firing are likely to be identical.
Origin of the difference between cells firing at low and very low frequencies
The shift in firing rate between 2- and 3- to 4-wk-old cells could be due to a change in protein expression (for either mGluRs or the channels responsible for the slow inward current). By applying the agonist DHPG from group I mGluRs, we observed (for 2- and 3- to 4-wk-old cells) the induction of a slow inward current (n = 7, data not shown). This suggests that the difference in firing rate does not correspond to a difference in protein expression; it is probably due to the huge increase of the autaptic EPSP with the number of DIV.
Indeed we can draw a simple model (Fig. 10) that explains the sustained firing at low and very low rates. In Fig. 10, we approximated the slow synaptic inward currents and the membrane resistance (Rm). We assumed that their product was proportional to the slow depolarization of the EPSP. Our scheme neglects many negative feedback mechanisms, such as calcium adaptation and potassium channels activation and deactivation, which probably play a role in the rate control in vivo. However, it provides a good explanation of the difference in firing rate with the number of DIV. In this scheme, the rate of the sustained firing is determined by the relative values of the decay time of the slow synaptic component and of the relative refractory period. The refractory period of 2-wk-old cells with a small and fast EPSP (10–20 mV) is 10–20 ms: the cell can fire, because of the NMDA component, 20–200 ms after the previous spike. In 3- to 4-wk-old cells with a large and slow EPSP (60 mV), the shunting effect lasts for about 150 ms: the cell can fire in a time window of 150–500 ms because of the calcium-sensitive cationic conductance and the delayed release of glutamate.
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). The relative refractory period associated with the slow depolarizing conductance is critical for the firing rate control. NMDAR activation necessary for 10-Hz firing
The suppression of bursts by APV + MK801 in 2-wk-old cells indicated that the NMDA component was necessary for sustaining activity at about 10 Hz. This corroborates previous observations in hippocampal cultures ( Bacci et al. 1999; Mangan and Kapur 2004) and in hippocampal slices ( Bonansco and Buno 2003; Dingledine et al. 1986; Lee and Hablitz 1990, 1991; Schneiderman and MacDonald 1987; Wang and Jensen 1996; Williamson and Wheal 1992). At the same time, NMDAR antagonists abolish epileptiform discharges ( Lee and Hablitz 1990, 1991), and NMDA induces rhythmic oscillations ( Bonansco and Buno 2003).
We observed that NMDAR activation was not necessary for sustained firing in 3- to 4-wk-old cells. Similarly, it has been shown to play a weaker role in inducing ictal discharge in adult slices compared with immature ones ( Wang and Jensen 1996). It was also sufficient but not necessary for burst generation in the CA3 region ( Neuman et al. 1988), indicating that other conductance might be involved.
Moreover, we observed that NMDAR activation reinforces regular firing within burst when the calcium-sensitive cationic conductance and the delayed release are playing a key role. Similar observations were reported in experiments performed on hippocampal cultures ( Bacci et al. 1999) and on slices of rat visual cortex ( Harsch and Robinson 2000).
A slow depolarizing conductance activated by mGluRs necessary for 2-Hz firing
The autaptic response of 3- to 4-wk-old cells had a very slow depolarizing component (500 ms to 1 s), attributed to a nonselective cationic calcium-sensitive conductance activated by mGluRs. Similar conductance have been described in the hippocampus ( Congar et al. 1997; Crepel et al. 1994) and in the entorhinal cortex ( Egorov et al. 2002; Fransen et al. 2002). Three arguments indicate that this slow conductance sustained activity within a burst: 1) its kinetics matched long ISIs; 2) application of EGTA-AM or MCPG suppressed long ISIs; and 3) it also decreased the burst duration.
Some arguments indicate that such calcium-sensitive slow conductance operate in other systems. In pairs of excitatory cells, the synaptic response and the firing were sensitive to EGTA-AM and MCPG. In standard culture, a slow depolarizing current has also been noticed in spontaneous current-clamp recordings ( Bacci et al. 1999). In slices, a slow depolarizing current induced by group I mGluRs in CA1 ( Crepel et al. 1994) has been described after high-frequency stimulations ( Congar et al. 1997). Finally, several experiments have emphasized the possible role of a calcium-sensitive cationic current, activated by cholinergic muscarinic receptors, in the graded persistent activity in entorhinal cortex neurons ( Egorov et al. 2002; Fransen et al. 2002). Therefore calcium-sensitive nonspecific cationic conductance might be involved in an ubiquitous manner in self-sustained activity.
Delayed release of glutamate necessary for retriggering a spike
Our data suggest that the delayed release of glutamate ( Cummings et al. 1996; Goda and Stevens 1994; Hagler and Goda 2001; Van der Kloot and Molgo 1993) facilitated the bursting activity. We observed that the asynchronous release of glutamate was widely present in autapses as in pairs of excitatory cells after 3 wk in vitro. When the NMDAR was blocked, cell firing became irregular as if a stochastic mechanism was contributing to firing. Finally, the replacement of extracellular calcium by strontium increased the burst duration and favored long ISIs within a burst. This suggests that delayed release through large asynchronous events could trigger a spike after a long delay (hundreds of milliseconds) within a burst in glutamatergic networks.
Using an in vitro model of synchronous glutamatergic networks, we were able to identify that the nature and temporal dynamics of synaptic or synaptic-dependant conductance play a key role in recurring bursting. Our study reveals new mechanisms that may be involved in the sustained firing of glutamatergic neuronal networks. Further experiments are necessary to show their implication in vivo.
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GRANTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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Address for reprint requests and other correspondence: L. Bourdieu, Laboratoire de Neurobiologie Moléculaire et Cellulaire, UMR CNRS 8544, Ecole Normale Supérieure, 46 rue d'Ulm, 75005 Paris, France (E-mail: laurent.bourdieu{at}ens.fr)
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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F/F) of the fluorescence intensity (F) averaged over the soma reveals large calcium transients. E: spontaneous firing in a cell (top) is abolished by bath application of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 100 µM; bottom). Scale bar on images is 50 µm (A and C).
