Network and Intrinsic Contributions to Carbachol-Induced Oscillations in the Rat Subiculum

Margherita D'Antuono,1,3 Hiroto Kawasaki,1,2 Carmela Palmieri,1,4 and Massimo Avoli1,3

 1Montreal Neurological Institute and Department of Neurology and Neurosurgery, McGill University, Montreal, Quebec H3A 2B4, Canada;  2Neurological Institute, Tokyo Women's Medical College, Tokyo 162, Japan;  3Istituto di Ricovero e Cura a Carattere Scientifico Neuromed, 86077 Pozzilli (Isernia); and  4Ospedale San Paolo, Università degli Studi di Milano, 20142 Milan, Italy


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D'Antuono, Margherita, Hiroto Kawasaki, Carmela Palmieri, and Massimo Avoli. Network and Intrinsic Contributions to Carbachol-Induced Oscillations in the Rat Subiculum. J. Neurophysiol. 86: 1164-1178, 2001. Low-frequency network oscillations occur in several areas of the limbic system where they contribute to synaptic plasticity and mnemonic functions that are in turn modulated by cholinergic mechanisms. Here we used slices of the rat subiculum (a limbic area involved in cognitive functions) to establish how network and single neuron (intrinsic) membrane mechanisms participate to the rhythmic oscillations elicited by the cholinergic agent carbachol (CCh, 50-100 µM). We have found that CCh-induced network oscillations (intraoscillatory frequency = 5-16 Hz) are abolished by an antagonist of non-N-methyl-D-aspartate (NMDA) glutamatergic receptors (n = 6 slices) but persist during blockade of GABA receptors (n = 16). In addition, during application of glutamate and GABA receptor antagonists, single subicular cells generate burst oscillations at 2.1-6.8 Hz when depolarized with steady current injection. These intrinsic burst oscillations disappear during application of a Ca2+ channel blocker (n = 6 cells), intracellular Ca2+ chelation (n = 6), or replacement of extracellular Na+ (n = 4) but persist in recordings made with electrodes containing a blocker of voltage-gated Na+ channels (n = 7). These procedures cause similar effects on CCh-induced depolarizing plateau potentials that are contributed by a Ca2+-activated nonselective cationic conductance (ICAN). Network and intrinsic oscillations along with depolarizing plateau potentials were abolished by the muscarinic receptor antagonist atropine. In conclusion, our findings demonstrate that low-frequency oscillations in the rat subiculum rely on the muscarinic receptor-dependent activation of an intrinsic oscillatory mechanism that is presumably contributed by ICAN and are integrated within the network via non-NMDA receptor-mediated transmission.


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INTRODUCTION
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Rhythmic, low-frequency (i.e., 4-12 Hz) network oscillations are recorded in vivo and in vitro from many regions of the limbic cortex (Alonso and Garcia-Austt 1987; Bland 1986; Bland and Colom 1993; Buzsaki and Eidelberg 1983; Dickson et al. 1995; Fischer et al. 1999; MacVicar and Tse 1989). These oscillations, referred to as theta rhythm when studied in vivo, may contribute to long-lasting changes in synaptic efficacy and thus to mnemonic functions (Buzsaki et al. 1994; Huerta and Lisman 1993; Larson and Lynch 1986; Pavlides et al. 1988; Singer 1993). A common way to induce in vitro low-frequency oscillations relies on the use of cholinergic agents (Chapman and Lacaille 1999; Konopacki et al. 1988; MacVicar and Tse 1989; McMahon et al. 1998; Osehobo and Andrew 1993). Interestingly, one type of theta recorded in vivo is sensitive to treatments that antagonize muscarinic transmission (Kramis et al. 1975). Cholinergic mechanisms are indeed involved in learning and memory (Ashe and Weimberger 1991; Dunnett and Fibiger 1993; Winkler et al. 1995) as well as in the phatophysiogenesis of epileptic discharges (Nagao et al. 1996; Turski et al. 1989).

It is believed that low-frequency network oscillations result from specific oscillatory properties of the neuron membrane that are implemented within the network through the interaction of excitatory and inhibitory synaptic mechanisms (Bland and Colom 1993; Chapman and Lacaille 1999; Klink and Alonso 1997a; McMahon et al. 1998). It is, however, unclear how the membrane oscillations are processed within the network since they are often subthreshold for action potential discharge. Moreover, the ionic mechanisms responsible for the intrinsic oscillatory behavior remain poorly defined. For instance, Na+ electrogenesis contributes to subthreshold oscillations (Klink and Alonso 1993; Mattia et al. 1997; Pape and Driesang 1998), but activation of muscarinic receptors leads to a decrease of Na+ currents (Cantrell et al. 1996; Mittmann and Alzheimer 1998).

In this study, we have used slices of the rat subiculum to establish how network and single neuron (intrinsic) membrane properties contribute to the low-frequency, rhythmic oscillations elicited by the nonhydrolizable cholinergic agent carbachol (CCh). The subiculum represents a gating point for most of the information entering and leaving the hippocampus proper (Amaral and Witter 1989; Lopes da Silva et al. 1990), and it may be involved in cognitive functions such as spatial learning (Barnes et al. 1990; Sharp and Green 1994) as well as in the spread of seizure activity within the limbic system (Lothman et al. 1991). Muscarinic receptor activation in the subiculum leads to the appearance of depolarizing plateau potentials that are caused by a Ca2+-activated nonselective cation conductance (ICAN) (Kawasaki et al. 1999; cf. Caesar et al. 1993; Fraser and MacVicar 1996; Klink and Alonso 1997a,b; Schwindt et al. 1988). Here we report that subicular neurons can generate synchronous oscillations at 5-16 Hz in the presence of CCh and that these network oscillations depend on the function of non-N-methyl-D-aspartate (NMDA) glutamatergic receptors, while they persist with blockade of GABA receptor-mediated transmission. In addition, we have found that during blockade of glutamatergic (and GABAergic) transmission, single subicular cells can produce voltage-dependent rhythmic burst oscillations at 2.1-6.8 Hz that may be contributed by an ICAN-like mechanism. Finally, both network and intrinsic oscillations are abolished by the muscarinic receptor antagonist atropine.


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Brain slices were obtained from male Sprague-Dawley rats (200-300 g), following standard procedures (Kawasaki et al. 1999; Nagao et al. 1996). Animals were anesthetized with halothane and decapitated. After removing the brain, transverse slices (400-450 µm) were cut with a vibroslice apparatus. Slices containing the whole subicular complex were transferred to a tissue chamber and kept at an interface between oxygenated artificial cerebrospinal fluid (ACSF) and humidified gassed (95% O2-5% CO2) atmosphere (33 ± 1°C). ACSF composition was (in mM) 124 NaCl, 2 KCl, 1.25 KH2PO4, 2 MgSO4, 2 CaCl2, 26 NaHCO3, and 10 glucose (pH 7.4). The following drugs were bath applied: atropine (0.5-1 µM), bicuculline methiodide (BMI, 10 µM), CCh (50-100 µM, most often 70 µM), 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX, 10 µM), 3-((±)-2-carboxypiperazin-4-yl)-propyl-l-phosphonic acid (CPP, 20 µM), P3-amino-propyl, P-diethoxymethylphosphonic acid (CGP-35348, 1 mM), CoCl2 (2 mM), (D-Ala2-N-Me-Phe, Gly-ol)enkephalin (DAGO, 10 µM), (+)-alpha-methyl-4-carboxy-phenylglycine (MCPG, 1 mM), picrotoxin (50-100 µM), tetrodotoxin (TTX, 1 µM), and D-tubocurarine (D-TC, 1 µM). When CoCl2 was used KH2PO4 was omitted, MgSO4 was replaced with MgCl2, and KCl was increased from 2 to 3.25 mM. In some experiments, NaCl in the ACSF was replaced by equimolar choline. Chemicals were acquired from Tocris Cookson (Langford, UK) or Sigma (St. Louis, MO), while CGP-35348 was a kind gift of Novartis (Basel).

Field potential recordings were made in the subiculum with ACSF-filled glass pipettes (tip resistance = 2-6 MOmega ) that were connected with a DC amplifier. Sharp-electrode intracellular recordings were performed with pipettes filled with one of the following solutions: 3 or 4 M K-acetate (tip resistance = 70-120 MOmega ); 3 M K-acetate + 0.2 M of the Ca2+ chelator bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA; tip resistance = 70-120 MOmega ); or 3 M K-acetate + 50 mM 2-(tri-methyl-amino)-N-(2-6-dimethyl-phenyl)-acetamide (QX-314, a kind gift of Astra, Toronto, ON). Intracellular signals were fed to a high-impedance amplifier with internal bridge circuit for intracellular current injection. The bridge was monitored throughout the experiment and adjusted as required. Whenever necessary the resting membrane potential (RMP) was kept at a given value by injecting steady intracellular current. In some experiments, surgical cuts were made with a micromanipulator-mounted knife to isolate the subiculum, while a bipolar, stainless steel electrode was used to deliver extracellular stimuli (90 µs, <= 2 µA) at a site that was close (300-800 µm) to the recorded neuron. By doing so, we established whether the stimulus-induced synaptic responses contained any excitatory component, and thus we assessed the efficacy of the block exerted on ionotropic excitatory transmission by CNQX and CPP (see Fig. 7B).

Extracellular and intracellular signals were displayed on an oscilloscope as well as on a Gould pen chart recorder or a Gould WindoGraf recorder. They were also stored on a video cassette recorder and/or fed to a computer interface (Digidata 1200B, Axon Instruments) for subsequent analysis made with the software pClamp8 (Axon Instruments). Average power spectra of the field potential and/or intracellular signals (duration = 7-10 s) were calculated by using the Hamming's equation with the software Clampfit8 (Axon Instruments). Power spectra of the intracellular activity generated by subicular cells were obtained after filtering the signals with a low-pass filter set to 100 Hz.

Subicular cells included in this study responded to brief (10-120 ms) intracellular pulses of depolarizing current (<0.7 nA) by generating bursts of action potentials. Hence they could be identified as bursting-firing cells according to the criteria reported in previous studies (Mattia et al. 1993, 1997; Stewart and Wong 1993; Taube 1993). The fundamental electrophysiological parameters of these cells were measured as follows: RMP after cell withdrawal; input resistance (Ri) from the maximum voltage change in response to a hyperpolarizing current pulse (100-200 ms, < -0.5 nA); and action potential amplitude from the baseline. The electrophysiological properties recorded in normal ACSF in a representative group of subicular cells were RMP = -64.1 ± 9.0 mV (n = 26), Ri = 45.6 ± 9.9 MOmega (n = 26), and action potential amplitude = 70.4 ± 3.2 mV (n = 23). Quantitative results throughout this paper are expressed as means ± SD, and n indicates the cell number. Results were compared with the paired Student's test and were considered significantly different if P < 0.05.


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Field potential and intracellular characteristics of carbachol-induced network oscillations

Spontaneous field potential oscillations at 5-16 Hz appeared after ~20 min of continuous application of ACSF containing CCh. These oscillations could persist throughout the experiment (<= 5 h) and were organized in series of waves riding a DC negative shift, 1-4 mV in amplitude. The oscillatory sequences occurred at intervals of 2.5-18 s (8.1 ± 3.2 Hz, n = 25), each lasting 0.8-8.3 s (3.6 ± 1.6 s, n = 25; Figs. 1A and 2A, field). The plot in Fig. 1B shows that the duration and the interval of occurrence of the oscillatory sequences recorded in different slices were directly correlated. As illustrated in Fig. 1C, these field oscillations became of larger amplitude when the recording electrode was positioned at 0.8-1.5 mm from the pia and changed in polarity when obtained near the pial surface, thus suggesting a current sink between these two recording sites. Moreover, they continued to occur spontaneously in the subicular cortex after cutting the connections with either the hippocampus proper (n = 5; Fig. 1D) or the entorhinal cortex (n = 7; not illustrated). Hence these data demonstrated that neuron networks in the subiculum responded per se to CCh by producing low-frequency field potential oscillations similar to those reported in other limbic structures maintained in vitro (Dickson and Alonso 1997; Konopacki et al. 1988; MacVicar and Tse 1989).



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Fig. 1. Spontaneous, network oscillations induced by carbachol (CCh, 70 µM) in the rat subiculum. A: simultaneous intracellular (-68 mV) and field potential (field) recordings show the regular occurrence of sequences of oscillations at 5.5-12 Hz, occurring at intervals of 5-9 s, each lasting 2.2-2.6 s. Note the close relation between intracellular and field potential signals. B: plot of the durations vs. intervals of occurrence of the sequences of oscillations analyzed in the subiculum of 25 slices; values for each experiment are the mean of the measurements obtained from 5 to 10 sequences. C: field potentials recorded at 3 different locations in the subiculum during continuous application of CCh. The position of the recording electrodes is shown in the inset. Note that the negative-going field potential oscillations induced by CCh are of larger amplitude when recorded with electrode 3, which was positioned 1.4 mm from the pia. Note also the change in polarity in the field potential traces obtained with electrode 1. D: field potential oscillations recorded in the subiculum before (control) and after cutting the connections with the CA1 area of the hippocampus proper (after cut). Note that the field potential oscillations continue to occur after the cut; the extension of the cut in this experiment is shown in the inset.



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Fig. 2. A: intracellular (-68 mV) and field potential (field) recordings obtained in the subiculum in the presence of CCh (100 µM) show a close correlation between the 2 signals during a sequence of spontaneous oscillations. Note that at this membrane potential (-68 mV) ~50% of the intracellular oscillations trigger bursts of action potentials (down-arrow ) as well as that the frequency of the oscillations at both intracellular and field potential level decreases during progression of the sequence. A lapse of ~450 ms occurs between the 2 samples. B: power spectra of the field potential oscillations recorded during the first 1.5 s (CCh start) and at the end (CCh end) of the CCh-induced oscillatory sequences. Note that the peak frequency drops from 15.1 to 12.0 Hz. C: superimposed intracellular (1) and field potential (2) recordings obtained at the beginning (a) and at the end (b) of the sequence. In both cases, traces were aligned by using the negative peak of the field potential waves (black-triangle). Note that there is a decrease in rhythmicity for both field potential and intracellular signals as the sequence progresses toward the end.

Intracellular recordings made from subicular bursting neurons in the presence of CCh revealed that the field potential oscillations were associated with sequences of rhythmic depolarizations that reached amplitudes of 8-24 mV and could trigger either single action potentials or action potential bursts (Figs. 1A and 2A). The frequency of the field potential oscillations (and of the concomitant rhythmic depolarizations) within each sequence was higher at onset (10.8 ± 4.6 Hz, values obtained from 14 experiments during the initial 1 s) as compared with what seen at the end (7.3 ± 1.6 Hz, values calculated in the same slices during the last 1 s, P < 0.02). Such a change in frequency could be also appreciated by obtaining the power spectra of the field potential occurring at the beginning and at the end of each oscillatory sequence (Fig. 2B). Moreover, the intracellular oscillations occurring at the beginning of each sequence were characterized by a higher degree of correlation with the field potentials as well as by a more pronounced rhythmicity (Fig. 2C).

Changing the membrane potential with injection of hyperpolarizing or depolarizing current in nine subicular cells recorded from six slices, did not influence the occurrence of the sequences of rhythmic oscillations or their pattern rhythmicity, further demonstrating that the oscillations resulted from neuron network interactions. This procedure, however, modified the amplitude of the rhythmic depolarizations occurring during each oscillatory sequence. As illustrated in Fig. 3A (middle), <50% of the rhythmic depolarizations triggered action potentials when the membrane was slightly hyperpolarized from an RMP = -66 mV. Further hyperpolarization abolished all action potential firing while increasing the amplitude of the rhythmic depolarizations (Fig. 3A, bottom). Progressive hyperpolarization of the membrane also revealed a steady depolarizing envelope that occurred throughout the sequence of field potential oscillations. Conversely, depolarizing the neuron membrane with steady injection of positive current caused sustained firing of action potentials during the rhythmic oscillations; this action potential discharge appeared to obscure the periods of firing silence occurring between the rhythmic depolarizations (Fig. 3A, top).



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Fig. 3. Rhythmic oscillations induced by CCh (70 µM) in the subiculum recorded at different membrane potentials produced by steady injection of hyperpolarizing or depolarizing current. A: intracellular (top) and field potential (field) recordings were obtained at 3 different membrane potentials as indicated on the left of each pair of traces. Intracellular pulses of hyperpolarizing (-53 and -78 mV samples) and depolarizing (-70 mV samples) pulses were injected throughout the recordings. B: superimposed intracellular (1) and field potential (2) recordings obtained at the 3 different membrane potentials shown in A during the middle portion of the oscillatory sequence; in all cases the traces were aligned by using the negative peak of the field potential waves (black-triangle). Note in both A and B that hyperpolarizing the membrane potential aborts action potential firing, increases the amplitude of the rhythmic depolarizations and discloses a steady depolarizing envelope that occurs throughout the oscillation sequence. Note also that the steady membrane depolarization causes sustained firing that occurred in association with the rhythmic oscillations as well as rhythmic burst oscillations that are not mirrored by any concomitant field potential (up-arrow  in A).

The relation between field potential and intracellular activity at the different membrane potentials is further shown in the samples illustrated in Fig. 3B where traces were superimposed by using the negative peaks of the field potential oscillations. By bringing the membrane potential to depolarized values, we could also record rhythmic intracellular oscillations at 3-6 Hz that were often associated with action potential bursts and were not mirrored by any field potential activity (Fig. 3A, up-arrow ).

Pharmacological features of the of CCh-induced network oscillations

Both field potential and intracellular oscillations induced by CCh were unaffected by bath application of the NMDA receptor antagonist CPP (10 µM, n = 5; not shown) but disappeared during application of the non-NMDA receptor antagonist CNQX (10 µM, n = 6; Fig. 4A). Under these conditions, intracellular rhythmic depolarizations continued to occur when the membrane potential was set at slightly depolarized levels (Fig. 4A, bottom, up-arrow ). These intrinsic oscillations, which were capable of triggering single action potentials or action potential bursts, had a lower frequency (i.e., 3.5-7 Hz) than those associated with field potential activity before CNQX application (i.e., 8-16 Hz). As shown in Fig. 4A (top, up-arrow ), "asynchronous," burst oscillations were also seen under control conditions when the membrane was depolarized with injection of intracellular current. The power spectra illustrated in Fig. 4B were obtained from the intracellular activity corresponding to the asynchronous oscillations (a) and to the network oscillations (b) recorded under control conditions as well as to the asynchronous oscillations seen during CNQX (c).



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Fig. 4. A: effects induced by the non-N-methyl-D-aspartate (NMDA) receptor antagonist 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX, 10 µM) on the spontaneous oscillations induced by CCh (70 µM) and analyzed with intracellular (-58 mV) and field potential (field) recordings in the subiculum. Note that under control conditions both asynchronous oscillations, at times associated with action potential bursts (up-arrow  in the top also identified as a), and network oscillations (identified as b) are seen. During CNQX application the network oscillations disappear, while intracellular rhythmic depolarizations continue to occur when the membrane potential is set at slightly depolarized levels (up-arrow  in the bottom sample, also identified as c). Note also that the intrinsic oscillations recorded under control conditions and during application of CNQX have a lower frequency than those associated with field potential activity under control conditions. The resting membrane potential (RMP) of this neuron was -65 mV, and it was set to -58 mV to observe the occasional generation of intrinsic rhythmic membrane oscillations. B: power spectra of the intrinsic (a) and network-driven (b) oscillations recorded intracellularly under control conditions as well as of the intrinsic oscillations generated during application of CCh and CNQX (c). Note that the peaks of the power spectra of the intrinsic oscillations recorded under control and CNQX application are characterized by similar frequencies that are lower than what seen with the power function of the network-driven intracellular oscillations obtained in control.

CCh-induced rhythmic oscillations continued to occur during concomitant application of the GABAA and GABAB receptor antagonists BMI (10 µM) and CGP 35348 (1 mM; n = 11; Fig. 5). The oscillations recorded in the presence of BMI and CGP 35348 were of longer duration and had longer intervals of occurrence (P < 0.02; Fig. 5, B and C). Moreover, the longer duration was characterized by a larger amount of low oscillation frequency as compared with what seen under control conditions (P < 0.05; Fig. 5A). Since BMI may influence Ca2+-dependent K+ conductance (Debarbieux et al. 1998), we also analyzed in five slices the effects induced on the network oscillations by application of the GABAA receptor antagonist picrotoxin (50-100 µM) along with the GABAB receptor antagonist CGP 35348 (1 mM). As shown in Fig. 6A, CCh-induced oscillations continued to occur in the presence of picrotoxin. However, in contrast to what observed with BMI, picrotoxin decreased the duration (P < 0.02) and increased the interval of occurrence (P < 0.05) of the oscillatory sequences (Fig. 6C, a and b). Moreover, picrotoxin did not change significantly the frequency of the oscillations occurring within each sequence (Fig. 6, B and Ca). Activation of µ-opioid receptors by DAGO (10 µM; n = 4), a procedure that presumably blocks GABA release (Capogna et al. 1993) did not cause any consistent change in the oscillatory pattern (not illustrated).



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Fig. 5. Effects induced by GABAA [bicuculline methiodide (BMI), 10 µM] and GABAB [P3-amino-propyl, P-diethoxymethylphosphonic acid (CGP 35348), 1 mM] receptor antagonists on the CCh-induced network oscillations. A: intracellular (-70 mV) and field potential (field) recordings obtained during application of CCh (70 µM) and after further addition of BMI and CGP 35348. Note that the sequence of rhythmic oscillations recorded during blockade of GABA receptors is longer than under control conditions as well as that there is an increase in slow oscillatory activity. At the intracellular level these changes are mirrored by an increased amount of action potential bursts. B and C: summary of the changes induced by BMI and CGP 35348 on the duration, frequency, and interval of occurrence of the CCh-induced network oscillations. Differences were statistically significant in all cases.



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Fig. 6. Effects induced by GABAA (picrotoxin, 50 µM) and GABAB (CGP 35348, 1 mM) receptor antagonists on the CCh-induced network oscillations. A: field potential recordings obtained during application of CCh (70 µM) and after further addition of picrotoxin + CGP 35348. Note that the sequence of rhythmic oscillations recorded during blockade of GABA receptors is shorter than in control and characterized by a similar intraoscillatory frequency. B: power spectra of the field potential oscillations recorded under control conditions (CCh) and after addition of picrotoxin and CGP 35348. Note that the 2 power spectra have similar frequency peaks. C: summary of the changes induced by picrotoxin and CGP 35348 on the duration and frequency (a) as well as on the interval of occurrence (b) of the CCh-induced network oscillations analyzed in 5 experiments. Differences were statistically for the values obtained by measuring the duration and the interval of occurrence.

The field potential rhythmic oscillations induced by CCh were abolished by application of the muscarinic receptor antagonist atropine (0.5 µM, n = 5; not illustrated) but were not influenced by the nicotinic receptor antagonist D-TC (1 µM, n = 4; not illustrated) or by the metabotropic glutamate receptor antagonist MCPG (1 mM; n = 3; not illustrated).

Voltage-dependent burst oscillations induced by CCh during blockade of excitatory transmission

The ability of subicular cells to generate "asynchronous" membrane oscillations in the presence of CCh during steady membrane depolarization and the occurrence of a similar oscillatory pattern during application of CNQX suggested to us that intrinsic membrane mechanisms might contribute to the oscillations expressed by the subicular network under control conditions. Hence we studied the ability of subicular bursting cells to generate oscillations during application of CCh and excitatory amino acid receptor antagonists. We assumed that under these conditions, ionotropic excitatory synaptic transmission was not operant since only isolated inhibitory postsynaptic potentials (IPSPs) were induced by extracellular focal stimuli (Fig. 7B).



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Fig. 7. Recurrent bursting induced by CCh in a subicular bursting neuron during blockade of ionotropic glutamatergic transmission. In A1, under control conditions [CNQX and 3-((±)-2-carboxypiperazin-4-yl)-propyl-l-phosphonic acid (CPP) in the medium], subthreshold membrane oscillations develop with increasing levels of depolarization (*, a truncated action potential). These oscillations are depressed by CCh (100 µM), but further depolarization (~3 mV) induces a sustained pattern of bursting that is also shown at a different time base in A2. B: stimulus-induced synaptic responses recorded in control medium containing CNQX and CPP. Note that at RMP, the response consists of a hyperpolarizing potential that is not preceded by any depolarizing component; during intracellular injection of hyperpolarizing current pulses this inhibitory postsynaptic potential (IPSP) inverts in polarity at -75 mV. C: frequency and amplitude plots of the subthreshold oscillations recorded in 8 cells in control and CCh (70-100 µM) containing medium. Data were obtained by analyzing under control conditions and during CCh application the membrane oscillations generated during steady depolarizations to potential levels that in control induced oscillations without action potential discharge (e.g., -62 mV in A1).

First, we analyzed the effects induced by CCh on the intrinsic oscillations generated by 26 subicular neurons during blockade of excitatory transmission (cf. Mattia et al. 1997). As illustrated in Fig. 7A1 (left), steady depolarizing commands positive to -65 mV made subicular neurons recorded during application of CNQX and CPP, generate persistent subthreshold membrane oscillations at 4.8-6.3 Hz (Mattia et al. 1997). These oscillations were voltage dependent since further depolarization to approximately -60 mV caused an increase in their amplitude, and the occasional occurrence of single action potentials (Fig. 7A1, *). Subicular neurons also generated clusters of single action potentials when the membrane potential was brought to values more positive than the level dominated by the subthreshold oscillations (not illustrated) (but see Mattia et al. 1997).

CCh addition caused a small, marginally significant (5.4 ± 3.3 mV, n = 24), steady depolarization of the RMP, a nonsignificant increase of the membrane input resistance measured at membrane values equal to control RMPs (from 45.6 ± 9.9 to 48.5 ± 12.0 MOmega , n = 26) and a decrease of the afterhyperpolarizations (not shown) (but cf. Kawasaki et al. 1999). Moreover during CCh application, steady depolarization to membrane values similar to those used under control conditions, elicited subthreshold membrane oscillations that were at times lower in frequency and significantly smaller in amplitude than in control (Fig. 7, A1 and C). However, under these conditions, further membrane depolarization caused the appearance of a series of action potential bursts that repeated at 3.1 ± 0.7 Hz (n = 31; range = 2.1-6.8 Hz) and were separated by hyperpolarizations with amplitudes <= 18 mV and durations ranging 100-280 ms (Fig. 7A1, right, and A2).

The spontaneous recurrent bursts recorded in ACSF containing CCh + CCP + CNQX could be initiated by a discharge of action potentials (Fig. 7A2), by a series of membrane oscillations (Fig. 8A), or by a depolarizing plateau potential triggered by a brief depolarizing pulse of intracellular current (not illustrated). Recurrent burst oscillations could repeat for several seconds until the action potential firing became tonic. Such transition in firing modality was accompanied by disappearance of the interburst hyperpolarizations. Isolated hyperpolarizing potentials (amplitude = 4-8 mV, duration = 80-170 ms) that were not immediately preceded by any action potential, and thus represented presumptive spontaneous IPSPs, were also seen at RMP or during the spontaneous recent bursts (Fig. 8A, up-arrow ).



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Fig. 8. A: CCh-induced bursting recorded during blockade of ionotropic glutamatergic transmission gradually develops from a subthreshold oscillatory pattern during steady membrane depolarization to -50 mV. Note that the recurrent bursting pattern gradually progresses toward tonic firing that is later abolished by a manual hyperpolarizing command. Note also the presumptive spontaneous IPSP that occurs during tonic firing (up-arrow ). The concentration of CCh in this experiment was 70 µM. B: lack of effects induced by GABAA (BMI, 10 µM) and GABAB (CGP 35348, 1 mM) receptor antagonists on recurrent bursting induced by intracellular current injection during application of CCh (100 µM) and ionotropic excitatory amino acid receptor antagonists. C: recurrent bursting induced by CCh (70 µM) is not elicited by a depolarizing command during application of the muscarinic receptor antagonist atropine (1 µM); under this pharmacological procedure, tonic firing is generated even during sustained depolarization of the membrane achieved by injecting large amplitude of depolarizing current.

Pharmacological features of the voltage-dependent burst oscillations induced by CCh

The ability of subicular cells to generate recurrent burst oscillations in the presence of CNQX and CPP indicates that this type of CCh-induced neuron activity is not contributed by the activation of ionotropic excitatory amino acid receptors. We also established whether inhibitory conductances participated to these intrinsic, recurrent burst oscillations by bath applying the GABAA and GABAB receptor antagonists BMI (10 µM) and CGP-35348 (1 mM) to medium containing CCh + CCP + CNQX (n = 4). As shown in Fig. 8B, blockade of these GABA receptors did not influence the CCh-induced bursting oscillations but abolished both stimulus-induced and spontaneous hyperpolarizing potentials (not illustrated).

Recurrent burst oscillations induced by application of medium containing either CCh + CCP + CNQX or CCh + CPP + CNQX + BMI + CGP 35348 were abolished by the muscarinic receptor antagonist atropine (1 µM, n = 7; Fig. 8C). By contrast, they were not affected by treatment with either D-TC (1 µM, n = 4) or MCPG (1 mM, n = 3; not illustrated).

Ca2+ dependence of the voltage-dependent burst oscillations induced by CCh

CCh-induced recurrent bursting was not seen during application of the Ca2+ channel antagonists Co2+ (2 mM, n = 6), which abolished stimulus-induced synaptic responses (not illustrated). As shown in Fig. 9A, steady depolarization of the neuron membrane to values more positive that those capable of inducing bursting oscillations under control conditions (i.e., in medium containing CNQX + CPP + CCh) caused the appearance of subthreshold oscillations that had shape and frequency similar to those seen before (4.8-6 Hz) application of CCh. In addition, bath application of Co2+ abolished the ability of subicular cells treated with CCh to generate depolarizing plateau potentials (not shown) (but see Kawasaki et al. 1999).



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Fig. 9. A: effects of the Ca2+ channel blocker Co2+ on the burst oscillations recorded during application of CCh (100 µM) and excitatory amino acid receptor antagonists. Note that during CCh a small, manual depolarizing command (up-arrow ) elicits bursting pattern that terminates when the membrane potential is brought to a level that is more hyperpolarized than before induction of the burst (down-arrow down-arrow ). This procedure was preceded by removal of the current required for eliciting the recurrent burst (down-arrow ). During Co2+, the cell does not generate bursting even when the membrane potential is brought to -48 mV, but subthreshold oscillations and action potential clusters occur. B: intracellular recordings obtained with an electrode filled with K-acetate + BAPTA in the presence of CCh (70 µM), CNQX and CPP. Shortly after impalement (5 min) recurrent burst oscillations (a) and a depolarizing plateau potential (b) are elicited by steady current injection (down-arrow , up-arrow ) or by an intracellular depolarizing current pulse (0.6 nA, 20 ms, ). Later (+19 min), no recurrent burst oscillations or depolarizing plateau potentials are elicited by similar procedures. C: summary of the oscillations frequency values recorded during different experimental procedures. Neuron numbers were 4, 6, and 6 for control, CCh, and Co2+ treatment, respectively; values of the oscillations obtained during application of CCh were arbitrarily restricted to those associated with bursting. Values obtained with K-acetate + BAPTA-filled electrodes (n = 4) were grouped according with the time after impalement: , samples obtained during the initial 8 min of recording; , values obtained 15-40 min after impalement.

Next we used electrodes filled with K-acetate and the Ca2+ chelator BAPTA to analyze the intrinsic, recurrent bursting induced by application of CCh and excitatory amino acid receptor antagonists (n = 6). During the first (<= 12) minutes of intracellular recording, cells recorded with BAPTA-filled electrodes generated both bursting oscillations during steady membrane depolarization and depolarizing plateau potentials in response to brief depolarizing pulses (Fig. 9B, +5 min). Later (i.e., >15 min after impalement), a progressive blockade of both types of activity ensued. Under these conditions, subicular cells that were steadily depolarized could oscillate at frequencies (i.e., 5.9 ± 09 Hz, n = 5) within the range of those seen before CCh application. In addition, brief pulses of intracellular depolarizing current only induced action potential bursts (Fig. 9Bb). The plot in Fig. 9C summarizes the frequency values of the oscillations recorded under different conditions that were aimed at reducing Ca2+ activity during application of CCh.

Na+-mediated conductances and carbachol-induced intrinsic burst oscillations

The burst oscillations recorded in medium containing CCh and excitatory amino acid receptor antagonists were abolished by addition of the Na+ channel blocker TTX (1 µM, n = 5). As reported in previous studies performed in the subiculum (Kawasaki et al. 1999; Mattia et al. 1993, 1997), the effects induced by TTX were characterized by blockade of all regenerative activity including plateau potentials and action potential bursts induced by brief depolarizing current pulses (not illustrated). These results suggested that the TTX-induced effects could reflect the inability of depolarizing commands to influence neuronal membrane compartments (e.g., dendrites) that were remote to the recording site (see also Fraser and MacVicar 1996). Hence, we used QX-314-filled electrodes to analyze the involvement of voltage-gated, Na+ electrogenesis in CCh-induced intrinsic burst oscillations. Intracellular application of QX-314 (or of other lidocaine quaternary derivatives) blocks voltage-gated Na+ channels and also depresses K+ conductances (Andreasen and Hablitz 1993; Mulle et al. 1985), thus allowing a better electrotonic somatodendritic transfer.

As illustrated in Fig. 10, the recurrent burst oscillations induced by CCh changed in shape during intracellular recordings performed with QX-314-filled electrodes (n = 7). In particular, fast action potential burst riding over the oscillations were replaced over time by slow events representing presumptive Ca2+ spikes. In spite of these changes, subicular cells recorded with QX-314-filled electrodes continued to generate slow oscillatory events in response to steady depolarizing commands (Fig. 10Aa). In addition the frequency of the CCh-induced oscillatory pattern was not significantly changed (Fig. 10Aa). Finally, under these recording conditions brief pulses of depolarizing current continued to induce depolarizing plateau potentials (Fig. 10Ab).



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Fig. 10. Involvement of Na+ in the CCh-induced recurrent burst oscillations and depolarizing plateau potentials. A: intracellular recordings made with a K-acetate + 2-(tri-methyl-amino)-N-(2-6-dimethyl-phenyl)-acetamide (QX-314)-filled electrode. In a, recurrent burst oscillations were induced by steady depolarizing manual command, while in b, depolarizing plateau potentials were elicited by brief depolarizing current pulse (0.6-1 nA, 20-50 ms, ). Time after impalement is provided on the left of each trace. CCh concentration in this experiment was 100 µM. Note that progressive diffusion of QX-314 blocks fast action potentials and discloses slow events representing presumptive Ca2+ spikes. In spite of these changes, both recurrent slow oscillations and depolarizing plateau potentials continue to occur. By contrast, extracellular Na+ replacement with choline abolishes both recurrent oscillations and depolarizing plateau potentials. Responses induced by depolarizing current pulses in the 18- and 39-min panels were superimposed. B: summary of the results obtained with QX-314-filled electrodes before, during, and after Na+ replacement. Values obtained under control conditions were segregated according with the time of impalement: control 1 (3-10 min) and control 2 (10-15 min). Note that choline substitution results in blockade of recurrent oscillations and depolarizing plateau potentials.

In four of the seven subicular cells recorded with QX-314-filled electrodes, we analyzed the effects induced by extracellular Na+ replacement on CCh-induced oscillations and depolarizing plateau potentials. As shown in Fig. 10A, application of choline-containing medium abolished the slow membrane oscillations elicited by steady membrane depolarizations (a) along with the depolarizing pulse-triggered plateau potentials (b). A quantitative summary of the results obtained with QX-314-filled electrodes before, during, and after Na+ replacement is illustrated in Fig. 10B. It is evident from this plot that CCh-induced oscillations and depolarizing plateau potentials in subicular neurons were changed in a similar fashion by extracellular Na+ replacement.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We have found that bath application of CCh to slices of the rat subiculum induces network-driven oscillations that occur at 5-16 Hz and are caused by the activation of muscarinic receptors. This type of activity depends on the function of non-NMDA glutamatergic receptor synchronizing mechanisms and is enhanced rather than abolished, by blockade of GABAA and GABAB receptors. In addition, we have discovered that during CCh treatment subicular cells generate voltage-dependent rhythmic burst oscillations that repeat at 2.1-6.8 Hz even when ionotropic excitatory transmission is abolished. This CCh-induced burst activity also depends on muscarinic receptor activation and is presumably contributed by a nonselective cationic conductance that is instrumental in the generation of depolarizing plateau potentials as well.

CCh-induced, network-driven oscillations in the subiculum

Rhythmic oscillations of neuron populations can be recorded in vitro from limbic structures (Dickson and Alonso 1997; Fischer et al. 1999; Konopacki et al. 1988; MacVicar and Tse 1989; Osehobo and Andrew 1993) as well as from the neocortex (Lukatch and Maciver 1997) during application of cholinergic agents. The field potential characteristics of the oscillations recorded in the rat subiculum share many similarities with those reported in these previous studies, including intraoscillation frequency, organization of the pattern in sequences lasting <= 8 s and sensitivity to the muscarinic receptor antagonist atropine. In addition, these oscillations continue to occur during application of nicotinic or metabotropic glutamate receptor antagonists (Taylor et al. 1995).

The oscillations induced in the subiculum by CCh are synaptically driven and rely on glutamatergic transmission mediated through non-NMDA receptors. This conclusion rests on electrophysiological (i.e., amplitude changes during injection of hyperpolarizing or depolarizing current) and pharmacological evidence (i.e., effects of selective receptor antagonists), and it is in line with experimental findings obtained in both hippocampus (MacVicar and Tse 1989) and entorhinal cortex (Dickson and Alonso 1997) as well as with computer-simulation data (Traub et al. 1992). Moreover, the glutamatergic network underlying these rhythmic oscillations resides in the subiculum as suggested by their persistence following surgical isolation of this structure from the hippocampus proper or the entorhinal cortex. Interestingly, the intracellular rhythmic depolarizations induced by CCh were rarely associated with action potential bursts, and firing was aborted by small membrane hyperpolarizations. Hence, as proposed in the modeling study of Traub et al. (1992), subicular CCh-triggered network oscillations are presumably generated within a limited range of increased excitatory synaptic conductance.

A further similarity of our findings with some published studies (MacVicar and Tse 1989; Traub et al. 1992) resides in the ability of network oscillations to occur during blockade of GABA receptor-mediated inhibition or activation of µ-opioid receptor, which leads to inhibition of GABA release from interneuron terminals (Capogna et al. 1993). It should be also mentioned that CCh-induced network oscillations occur in neocortical slices only after bicuculline application (Lukatch and Maciver 1997) and that intra-hippocampal injection of CCh + bicuculline elicits theta-like oscillations in vivo (Colom et al. 1991). However, these previous data, as well as our own findings, are at odds with studies indicating that GABAA receptor function may be necessary for maintaining the repetitive structure of CCh-induced oscillations (White et al. 2000; Williams and Kauer 1997). In addition, different classes of hippocampal interneurons have been shown to pace CCh-induced rhythmic activity in pyramidal cells (Chapman and Lacaille 1999; McMahon et al. 1998). It is unlikely that our pharmacological procedures failed to block GABAA and GABAB receptors in the subiculum network. Rather our findings, along with evidence obtained in experiments in which theta-like oscillations were recorded in the presence of GABAA receptor antagonists (Colom et al. 1991; Lukatch and Maciver 1997; MacVicar and Tse 1989), suggest that IPSPs resulting from interneuron activity are not essential for the occurrence of CCh-induced network-driven oscillations.

Mechanisms underlying CCh-induced intrinsic oscillatory bursts

CCh application during blockade of ionotropic glutamatergic transmission reduces the subthreshold oscillations generated by subicular neurons (cf. Klink and Alonso 1997a,b), presumably through a reduction of voltage-gated Na+ currents (Cantrell et al. 1996). Indeed, Mittmann and Alzheimer (1998) have demonstrated that muscarinic activation inhibits a persistent Na+ current in rat neocortical pyramidal cells. However, in our experiments, this effect is accompanied by the appearance of voltage-dependent burst oscillations that recur at 2.1-6.8 Hz and are dependent on the activation of muscarinic receptors. Similar asynchronous oscillatory activity could also occur during steady membrane potential depolarization in slices that generated CCh-induced network oscillations.

It has been proposed that action potential bursts play a role in synaptic plasticity and information processing (Lisman 1997). In our case the voltage-dependent intrinsic burst rhythm may represent a strong factor in establishing network oscillatory activity in the subiculum when synaptic excitation is operant. Moreover the different frequency that characterizes intrinsic and network oscillations suggests that excitatory (and inhibitory) interactions within subicular networks shape the oscillatory pattern at a higher rate. In keeping with this view, the initial portion of the network oscillations was characterized by a higher frequency and by a higher degree of synchronization between intracellular and field potential signals.

Muscarinic receptor-dependent, intrinsic burst oscillations were abolished by the voltage-gated Ca2+ channels blocker Co2+, indicating that they depend on Ca2+ entry. In addition, they progressively disappeared when recordings were made with intracellular electrodes containing the Ca2+ chelator BAPTA. This evidence indicates that the intrinsic burst oscillations induced by CCh in the subiculum may be dependent on voltage-gated Ca2+ entry as well as on Ca2+ release from intracellular stores. This conclusion is in line with findings showing that muscarinic activation leads to Ca2+ mobilization from intracellular stores via an intracellular messenger cascade (Berridge and Irvine 1989; McKinney 1993).

We have also found that the intrinsic burst oscillations induced by CCh are abolished by the voltage-gated Na+ channel blocker TTX. However, this effect is unlikely to reflect the dependence of CCh-induced oscillations on voltage-gated Na+ conductances. Rather it rests on the inability of subicular bursting cells, which are endowed of pronounced voltage-gated Na+ electrogenesis (Mattia et al. 1993, 1997), to produce in the presence of TTX regenerative events capable of depolarizing sites (e.g., the dendrites) remote to the soma. In line with this view, subicular cells recorded with QX-314-filled electrodes generated intrinsic voltage-gated oscillations associated with slow, presumptive Ca2+ spikes, while fast action potentials were blocked. QX-314 abolishes voltage-gated Na+ channel activity but also reduces K+ currents and increases the input resistance of the recorded neuron (Andreasen and Hablitz 1993; Mulle et al. 1985), thus allowing depolarizing commands delivered within the soma to influence dendritic compartments. Hence, while action potential bursts depend on voltage-gated Na+ electrogenesis, our data indicate that these conductances are not required for the generation of slow rhythmic oscillations recorded in subicular neurons during muscarinic receptor activation.

By contrast, intrinsic, slow oscillatory activity induced by CCh was not triggered by depolarizing subicular cells during replacement of extracellular Na+ with choline. In these experiments as well, we used QX-314-filled electrodes to depolarize the neuron membrane in regions that were presumably remote to the recording site. Moreover, during extracellular Na+ replacement, oscillations were not triggered even by injecting very large depolarizing currents. Therefore a TTX-insensitive influx of Na+ along with other cations such as Ca2+ is necessary for the occurrence of the muscarinic receptor-dependent, intrinsic, slow oscillations. Overall this evidence suggests that the intrinsic burst oscillations generated by subicular neurons in medium containing CCh and glutamatergic antagonists may be due to a Ca2+ activated, nonselective cationic conductance (also termed ICAN). This mechanism is responsible for the occurrence of depolarizing afterpotentials and depolarizing plateau potentials in subicular (Kawasaki et al. 1999), hippocampal (Caeser et al. 1993; Fraser and MacVicar 1996), entorhinal (Klink and Alonso 1997a,b), and neocortical (Haj-Dahmane and Andrade 1996; Schwindt et al. 1988) neurons. In line with this view, we have found that block of the CCh-induced intrinsic burst oscillations by Co2+, choline or during recordings with BAPTA-filled electrodes is paralleled by a similar effect on the depolarizing plateau potentials generated in response to brief pulses of depolarizing current.

Functional relevance of the low frequency oscillatory rhythms induced by CCh

It is debated whether the oscillatory activity induced by pharmacological (mostly cholinergic) manipulations in brain slices maintained in vitro represents an in vitro model of the electroencephalographic theta rhythm recorded in limbic areas during specific behavioral states (Traub et al. 1992). Regardless of the possible similarities that may exist between in vitro and in vivo low-frequency oscillations, there is general agreement on the contribution played by neuron membrane oscillations in synchronizing neuron populations, in implementing functional co-operativity and thus in ensuring synaptic plasticity (Buzsaki et al. 1994; Huerta and Lisman 1993; Larson and Lynch 1986; Pavlides et al. 1988). Hence the cellular and membrane mechanisms identified in this study may contribute to muscarinic receptor-dependent oscillatory behavior involved in synaptic plasticity. In addition, these activity patterns may be also related to epileptogenesis, in particular to the occurrence of seizures in patients suffering of temporal lobe epilepsy. Such a conclusion has been put forward in the entorhinal cortex (Dickson and Alonso 1997) and may also apply to the subiculum given its strategic position within the limbic system.


    ACKNOWLEDGMENTS

We thank Drs. D. Fraser, D. Paré, and B. McVicar for critical comments and helpful discussion and T. Papadopoulos for editorial assistance.

This work was supported by grants from the Canadian Institutes of Health Research (MT-8109), the Savoy Foundation, and the Fragile X Research Foundation of Canada.


    FOOTNOTES

Address for reprint requests: M. Avoli, 3801 University St., Montreal, Quebec H3A 2B4, Canada (E-mail: mavoli{at}po-box.mcgill.ca).

Received 28 September 2000; accepted in final form 16 May 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society




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