Synaptic and Nonsynaptic Contributions to Giant IPSPs and Ectopic Spikes Induced by 4-Aminopyridine in the Hippocampus In Vitro

Roger D. Traub,1 Rea Bibbig,1 Antje Piechotta,2 Reas Draguhn,2 and Dietmar Schmitz3

 1Department of Pharmacology, Division of Neuroscience, University of Birmingham School of Medicine, Edgbaston, Birmingham B15 2TT, United Kingdom;  2Institut für Physiologie der Charité, Humboldt-Universität zu Berlin, 10117 Berlin, Germany; and  3Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California 94143


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Traub, Roger D., Rea Bibbig, Antje Piechotta, Reas Draguhn, and Dietmar Schmitz. Synaptic and Nonsynaptic Contributions to Giant IPSPs and Ectopic Spikes Induced by 4-Aminopyridine in the Hippocampus In Vitro. J. Neurophysiol. 85: 1246-1256, 2001. Hippocampal slices bathed in 4-aminopyridine (4-AP, <= 200 µM) exhibit 1) spontaneous large inhibitory postsynaptic potentials (IPSPs) in pyramidal cells, which occur without the necessity of fast glutamatergic receptors, and which hence are presumed to arise from coordinated firing in populations of interneurons; 2) spikes of variable amplitude, presumed to be of antidromic origin, in some pyramidal cells during the large IPSP; 3) bursts of action potentials in selected populations of interneurons, occurring independently of fast glutamatergic and of GABAA receptors. We have used neuron pairs, and a large network model (3,072 pyramidal cells, 384 interneurons), to examine how these phenomena might be inter-related. Network bursts in electrically coupled interneurons have previously been shown to be possible with dendritic gap junctions, when the dendrites were capable of spike initiation, and when action potentials could cross from cell to cell via gap junctions; recent experimental data showing that dendritic gap junctions between cortical interneurons lead to coupling potentials of only about 0.5 mV argue against this mechanism, however. We now show that axonal gap junctions between interneurons could also lead to network bursts; this concept is consistent with the occurrence of spikelets and partial spikes in at least some interneurons in 4-AP. In our model, spontaneous antidromic action potentials can induce spikelets and action potentials in principal cells during the large IPSP. The probability of observing this type of activity increases significantly when axonal gap junctions also exist between pyramidal cells. Sufficient antidromic activity in the model can lead to epileptiform bursts, independent of alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors, in some principal cells, preceded by IPSPs and spikelets. The model predicts that gap junction blockers should suppress large IPSPs observed in 4-AP and should also reduce the probability of observing antidromic activity, or bursting, in pyramidal cells. Experiments show that, indeed, the gap junction blocking compound carbenoxolone does suppress spontaneous large IPSCs, occurring in 4-AP plus ionotropic glutamate blockers, together with a GABAB receptor blocker; carbenoxolone also suppresses large, fast inward currents, corresponding to ectopic spikes, which occur in 4-AP. Carbenoxolone does not suppress large depolarizing IPSPs induced by tetanic stimulation. We conclude that in 4-AP, axonal gap junctions could, at least in principle, account in part for both the large IPSPs, and for the antidromic activity in pyramidal neurons.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

A number of effects of 4-aminopyridine (4-AP) on networks of hippocampal and neocortical neurons in vitro have been described. These effects include the following:

1) Bursts of action potentials in interneurons, temporally correlated, in principal neurons, with large hyperpolarizing and depolarizing potentials, mediated by GABAA and GABAB receptors. The GABA-dependent potentials in principal neurons are too large to be generated by the firing of single presynaptic interneurons, and therefore populations of interneurons are presumed to be firing with at least approximate synchrony. Ionotropic glutamate receptors do not appear to be necessary for this type of activity. In some interneurons, bursting can occur also during blockade of GABAA receptors, possibly dependent on nonsynaptic interactions. In this case, action potentials appear to originate ectopically (at some distance from the soma), as they can be of abrupt onset, and can occur when the cell is somewhat hyperpolarized. Small action potentials, or "spikelets" (also called fast prepotentials or d-spikes) occur in more hyperpolarized interneurons. In other interneurons, depolarizing GABAA receptor-mediated potentials contribute to the depolarizing envelope underlying burst firing (Aram et al. 1991; Forti and Michelson 1998; Lamsa and Kaila 1997; Michelson and Wong 1991, 1994; Müller and Misgeld 1990, 1991).

2) Synchronized epileptiform bursts, which (in at least some cases) require alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. This activity is notable, as GABAA receptor-mediated inhibition persists in 4-AP and may even be enhanced, at least when measured as a response to a single shock (Avoli et al. 1993; Perreault and Avoli 1991, 1992; Rutecki et al. 1987; Traub et al. 1995).

3) Ectopic action potentials in principal neurons, either solitary (Traub et al. 1995), or clustered during the large GABA receptor-mediated potentials (Avoli et al. 1998). In the latter case (Avoli et al. 1998), action potentials of variable amplitude occur. In CA3 neurons, such ectopic spikes appear to arise along the Schaffer collaterals, perhaps in synaptic terminals (Avoli et al. 1998), as also occurs for the ectopic spikes occurring following tetanic stimulation of the hippocampal slice (Stasheff et al. 1993a,b). A possible mechanism for ectopic spike generation would be block by 4-AP of some fraction of gK in presynaptic terminals, as has been shown to occur in the presynaptic terminal of the calyx of Held (Forsythe 1994), and in presynaptic terminals of cerebellar basket cells (Southan and Robertson 1998). 4-AP increases as well the excitability of certain axons (Kocsis et al. 1983). GABA receptor-dependent effects on axons and/or presynaptic terminals are another possible mechanism that could contribute to generation of ectopic spikes (Alford et al. 1991; Sakatani et al. 1994; Stasheff et al. 1993b).

A purpose of this paper is to try to tie together, in an economical way, the co-occurrence in 4-AP of large inhibitory postsynaptic potentials (IPSPs) with ectopic activity, and to suggest how gap junctions might play a role.

A previous model exists for bursting of interneurons in 4-AP that occurs without chemical synapses and that was hypothesized to depend on gap junctions (Traub 1995). The key physical ideas involved the random spontaneous occurrence of action potentials in axons, and the ability of action potentials to propagate directly from neuron to neuron without chemical synapses. The latter was achieved in the model by having electrically active dendrites (Traub and Miles 1995; see also Martina et al. 2000), along with a large dendritic gap junction conductance (10 nS or higher). That model could account for partial spikes occurring in hyperpolarized interneurons (Michelson and Wong 1994), because spikes could be initiated at some distance from the soma, in the dendrites. While we suggest that the basic principle of this model may still be valid, specific details are suspect given that gap junctional coupling potentials between cortical interneurons appear to be less than ~2 mV (Gibson et al. 1999), and potentials of about 0.5 mV have been recorded (Galarreta and Hestrin 1999), even in cell pairs wherein coupling has been shown to be dendrodendritic (Tamás et al. 2000).

Another means by which collective gap junction-mediated bursting could occur has been shown to be possible, in principle: via gap junctional coupling between axons, again under conditions wherein spikes can cross from one neuron to another (Traub and Bibbig 2000; Traub et al. 1999b). The "drive" in these models also derives from a background of "ectopic" spontaneous antidromic spikes, a reasonable condition to postulate in the presence of 4-AP. The hypothesis of axon-axon gap junctions between principal neurons was suggested by recordings from hippocampal slices bathed in low [Ca2+]o media (Draguhn et al. 1998) and is supported by more recent electrophysiological and anatomical data (D. Schmitz, A. Draguhn, S. Schuchmann, A. Fisahn, E. H. Buhl, R. Dermietzel, U. Heinemann, and R. D. Traub, unpublished data).

Some of these data have been presented in abstract form (Traub et al. 2000b).


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Simulations

The purpose of this model is to advance a set of hypotheses on the origin of large 4-AP-induced IPSPs in pyramidal cells, during which presumed ectopic activity can occur. It is beyond the scope of this work to analyze the biophysical mechanisms underlying ectopic spikes themselves, or how ectopic activity in interneuronal and pyramidal cell networks might come to be temporally correlated (see DISCUSSION).

Simulations were performed of pairs of interneurons, and of larger networks. In each case, the model of a single interneuron was almost identical to that described in Traub and Miles (1995). The sole differences were these: 1) the conductance coupling the axon initial segment to the soma was reduced (in most cases) by 50%. This change allows interneuron somata to produce 10- to 25-mV "partial spikes," as well as full action potentials, in response to axonal spikes; the partial spikes resemble those observed by Benardo (1997) in cortical interneurons exposed to 4-AP and blockers of fast glutamatergic neurotransmission. 2) In addition, in some simulations (but not others), the interneuron dendrites were not as electrically excitable as in the original publication: in such cases, dendritic gNa and delayed rectifier gK densities were multiplied by 0.1. For the simulations presented here, the network effects of this change did not appear to be significant.

Interneurons, either in pair simulations or as part of networks, could interact with one another in any of three ways, alone or in combination: via GABAA receptor-mediated synaptic interactions, or via gap junctions (voltage-independent, nonrectifying) at two possible sites. Gap junctions could be located between homotopic dendritic sites, in compartments centered 85 µm from the soma, or between homotopic axonal sites, in compartments centered 263 µm from the soma. A dendritic gap junction conductance of 1.4 nS produced a DC coupling ratio of 0.15, as measured between somata, while an axonal gap junction conductance of 4.2 nS (which would allow a spike to cross from axon to axon, at least sometimes) produced a DC coupling ratio of only 0.015, also measured between somata. The low somatic coupling ratio, in the axonal case, was the result of the electrical distance of the gap junction from the somata.

For comparison with experimental data on electrically coupled, fast-spiking, cortical interneurons, we quote the following published observations. Gibson et al. (1999) found gap junctions to be at most weakly rectifying and to be voltage independent over an 80-mV range. The mean coupling coefficient was 0.07 ± 0.06 (mean ± SD), and the estimated gap junctional conductance was 1.6 ± 1.3 nS. Galarreta and Hestrin (1999) also found gap junctions to be nonrectifying. Coupling ratios ranged from 0.03 to 0.41, with a mean of 0.064, and the estimated conductance was 0.66 ± 0.18 nS. We suspect that axonal gap junctions might not be detectable experimentally by DC measurements of electrical coupling. Tamás et al. (2000) showed ultrastructurally that gap junctions could occur between the proximal dendrites, or between dendrite and soma, of cortical interneurons. The possibility of strong electrical coupling, perhaps axonal, between hippocampal interneurons is suggested by Fig. 11 of Maccaferri et al. (2000), in which a spike, electrically evoked in one interneuron, can produce a pair of temporally shifted IPSPs in one pyramidal cell, while producing a single IPSP in a different pyramidal cell; this observation indicates that a spike in one interneuron might be able to evoke a spike in a second interneuron.

Figure 1 demonstrates that a combination of dendritic gap junction and GABAA IPSC leads, in simulations, to coupling potentials similar to those observed in cortical interneuron pairs, bathed in normal media (Galarreta and Hestrin 1999; Gibson et al. 1999; Tamás et al. 2000). Axonal gap junctions between simulated interneurons, in contrast, lead [as they do with pyramidal cell simulations (Draguhn et al. 1998)] to sharp potentials with fast upstrokes, sometimes to full action potentials. We refer to these sharp potentials as "spikelets" if the amplitude is <10 mV, and "partial spikes" if the amplitude is 10-25 mV. Sharp potentials of this general appearance can occur in interneurons in the presence of 4-AP (Benardo 1997; Michelson and Wong 1994).



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Fig. 1. Simulation of pairs of model interneurons. Each action potential in cell 1 induces a somatic inhibitory postsynaptic current (IPSC) of 0.2 nS in cell 2, with reversal potential -15 mV relative to rest, and decay time constant 10 ms. In addition, there is a dendritic gap junction (left), or an axonal gap junction (right). The dendritic gap junction was located 85 µm from the soma of each interneuron and had conductance 0.7 nS; the axonal gap junction was located 263 µm from the soma and had conductance 4.2 nS. Each interneuron was held with a -0.05-nA hyperpolarizing current, and cell 1 was induced to fire every 50 ms, with 1-nA, 2-ms somatic depolarizing current pulses. In the case of a dendritic gap junction (left), each spike in cell 1 produced a coupling potential in cell 2, about 1 mV in amplitude. In the case of an axonal gap junction (right), each spike in cell 1 produced a spikelike potential (about 14 mV amplitude) in cell 2.

Apart from gap junctions between interneurons, and aside from some parameter alterations, the network model used in this paper (Fig. 2) is similar to that employed in Traub and Bibbig (2000), which itself was a hybrid of the models described in Traub et al. (1999b,c). In these models, there is an array of pyramidal cells and interneurons, interconnected (randomly or locally randomly) by chemical synapses. The structure of the chemical-synaptic network in this underlying array was described in Traub et al. (1999c). (Some slight differences are listed below.) To this "chemical synaptic" array, we add a pyramidal axon/axon gap junction connectivity, and interneuron gap junction connectivity, details of which are defined further on.



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Fig. 2. Block diagram defining the general structure of the network model. The network contains 3,072 pyramidal cells and 384 interneurons, but in each case a conceptual distinction is drawn between the respective axons and somata/dendrites. Gap junctions occur between some pairs of pyramidal cell axons and between some pairs of interneuron axons. Some interneurons make contacts that stimulate GABAA receptors on the initial segments of pyramidal cell axons (but not of interneuron axons). Interneurons also inhibit, via GABAA receptors, the somata and dendrites of pyramidal cells and of interneurons themselves. alpha -Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), N-methyl-D-aspartate (NMDA) and GABAB receptors are not included in this model, nor are depolarizing GABAA responses. Spontaneous "ectopic" spikes occur in the axons of both pyramidal cells and of interneurons, with frequency that is time dependent.

Briefly, the network consists of 3,072 pyramidal cells and 384 interneurons. Each neuron (pyramidal and inhibitory) is multicompartmental and includes 5 axonal compartments (Traub and Miles 1995; Traub et al. 1994). The pyramidal cells are arranged in a 96 × 32 array and the interneurons in a 96 × 4 array. The interneurons are divided into 4 classes of 96 cells each: "basket cells," inhibiting perisomatic regions; "axo-axonic cells," inhibiting axon initial segments; and 2 types of dendrite-contacting interneuron (for more proximal or more distal dendrites, respectively). We shall call the basket cells and axo-axonic cells "parvalbumin-positive" or "PV+," and the dendrite-contacting interneurons "PV-." The time constant of inhibitory postsynaptic currents (ISPCs) induced by basket cells and axo-axonic cells (in pyramidal cells) was 10 ms, but was 50 ms for dendrite-contacting interneurons (Pearce 1993). Interneurons, other than axo-axonic cells, also synaptically contact other interneurons. The time constant of basket cell IPSCs on interneurons was 5 ms. Synaptic connectivity of pyramidal cells was global in the orginal model, but pyramidal cell connectivity is not germane in the present case, as AMPA and N-methyl-D-aspartate (NMDA) receptors were entirely blocked. Synaptic contacts of interneurons were constrained to take place only to cells at most 500 µm away (25 cell diameters along the long axis of the array). Each pyramidal cell receives input from 80 interneurons; in most simulations, however, for the sake of simplicity, only inputs from PV+ interneurons were used. Each interneuron receives input from 60 other interneurons, including 20 basket cells; the peak conductance produced by PV- interneurons at connections onto other interneurons was 1/10 the peak conductance produced by basket cells. Only synaptically elicited, hyperpolarizing GABAA receptor-mediated chemical synaptic interactions were simulated, not AMPA, NMDA, metabotropic, or GABAB mediated. Although depolarizing GABAA receptor-mediated actions can be prominent in 4-AP, both in pyramidal cells and in interneurons (Avoli et al. 1993; Michelson and Wong 1991; Perreault and Avoli 1991, 1992), such actions add yet another level of complexity, and they were omitted from this study. Further structural details of the model can be found in Traub et al. (1999c). Both pyramidal cells and interneurons received small negative bias currents (from -0.07 to -0.02 nA for pyramidal cells; either constant -0.015 nA, or over the range -0.025 to -0.015 nA for interneurons).

Intrinsic properties of the individual pyramidal neurons were as in Traub et al. (1999c) with the exception that gK(AHP) density was uniformly 0.8 mS/cm2 over the soma/dendrites of pyramidal cells, as in the original paper (Traub et al. 1994). Peak unitary GABAA IPSC values were 0.5 nS on pyramidal cells, 0.2 nS for basket cell-mediated inhibition of interneurons, and 0.02 nS for other IPSCs on interneurons. These values are the "default" values used in this study; when modified in the figures, it will be duly noted. The small GABAA conductance values reflect presumed use-dependent depression of GABA conductance, and release failures, as would be expected to occur during an interneuronal burst (Maccaferri et al. 2000; Tamás et al. 2000).

For pyramidal cells, gap junctions (nonrectifying, voltage-independent) were located between the penultimate axonal compartments (centered 263 µm from the soma) of randomly selected pairs of axons, subject to the constraints that the respective somata were within 200 µm of each other, and no one axon could contact more than four others. (In this paper, "gap junction" is used in a functional sense, to mean "electrical connection between two contiguous structures," rather than in an anatomical sense that refers to a discrete structure formed by a small number of connexin protein molecules. Likewise, "gap junction conductance" here refers to the total conductance of such an electrical connection, not to the unitary conductance of a single anatomical gap junction.) A range of gap junction conductances was used, from 0 to 4.73 nS; a conductance of 4.2 nS would consistently allow action potentials to cross from one axon to the other (Traub et al. 1999b). A conductance of 3.7 nS corresponded to a DC coupling ratio of 0.04, as measured between somata.

The average number of gap junctions lying on a pyramidal cell axon was 1.6. As discussed previously (Traub and Bibbig 2000; Traub et al. 1999b), there are structural implications of this density of gap junctions (Erdös and Rényi 1960): the density lies above the "percolation limit" of 1 gap junction per cell. This means that a "large cluster" will exist, and that all cells not on the large cluster are either isolated or lie on small clusters. (Definitions: a "cluster" is a set of cells, connected together either directly or indirectly, and not lying embedded in any larger connected set. A cluster is "large" if its size is of the same order as the whole system.) Experimental estimates of the number of gap junctions per neuron are not yet available.

Gap junction networks for interneurons had the following characteristics. Dendritic and axonal networks were constructed independently, without spatial constraints. In each case, an interneuron axon contacted, on average, two other axons. Similarly, an interneuron formed, on average, dendritic gap junctions with two other interneurons. Allowed sites for gap junctional contacts are given above. No site (either axonal or dendritic) was allowed to contact more than four other sites, axonal or dendritic, respectively. [For comparison, Benardo (1997) found, with Lucifer yellow or neurobiotin injections into cortical interneurons, that one cell was usually coupled to 0, 1, or 2 others, but possibly up to 5 others.] Following the suggestion of Gibson et al. (1999), gap junctions were only allowed to form between similar sorts of interneurons. We simulated this by allowing gap junctions to form only between pairs of PV+ cells, or between pairs of PV- cells. Interneuron axonal gap junctions had conductances from 0 to 4.2 nS, and interneuron dendritic gap junctions had conductances of 0 to 1.4 nS. As was true for pyramidal cells, interneuron gap junctions were voltage independent and nonrectifying.

As in previous studies (Traub et al. 1999b,c), noise was simulated by the generation of random ectopic spikes, originating from small current pulses applied to the most distal compartment of the five-compartment axons of pyramidal cells and interneurons. The current pulses had Poisson statistics (as approximated with a pseudo-random number generator), independent between different axons. The rates of ectopic spikes were switched abruptly, in pulselike fashion, from 0 to 2 Hz for interneurons, and from 0 to a range of values (up to 10 Hz) for pyramidal cells (see Fig. 3).



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Fig. 3. Overview of the behavior of interneurons and pyramidal cells in the model. Top trace shows the time course for turning on and off the stochastically generated ectopic spikes, in the interneuron and pyramidal cell populations, respectively. In this case, the average ectopic rate for interneurons is 2 Hz per axon, and for pyramidal cells is 1 Hz per axon. The pyramidal cell axon gap junction conductance is 3.7 nS. The middle trace shows a "parvalbumin-positive" interneuron soma that lies on a large cluster. This cell fires a burst of antidromic action potentials, intermixed with partial spikes, about 10 mV in amplitude, also of antidromic origin. The bottom trace shows an inhibitory postsynaptic potential (IPSP) in a pyramidal cell soma, produced by the firing of many interneurons (see Fig. 4). A 2-mV spikelet, of antidromic origin, occurs during the IPSP. Not all pyramidal cells exhibit spikelets, however (see middle left panel of Fig. 8).

Simulation programs saved the following types of data: somatic voltages of selected pyramidal cells (some on the large cluster, some not), and interneurons; voltages in the axon (at the site of the gap junction) of selected pyramidal cells and interneurons, whose somatic potentials were also saved; average signals, consisting of somatic voltages of 224 nearby pyramidal cells or of 28 nearby interneurons; and the total GABAA synaptic conductance to a pyramidal cell, as well as to an interneuron. Figures that illustrate an "axonal" signal use the voltage in the respective axon at the site where gap junctions are allowed to occur.

Programs were written in FORTRAN. Network simulation programs were written in FORTRAN augmented with instructions for a parallel computer. These latter programs were run on an IBM SP2 with 12 nodes (processors); simulations of neuron pairs ran on a single node of the SP2. Some comments on numerical methods are to be found in Traub et al. (1999c). Most simulations were of 550 ms of activity, which took about 3.15 h.

Electrophysiology

SLICE PREPARATION. Horizontal slices containing the hippocampus and entorhinal, perirhinal, and temporal cortices were prepared from 3- to 5-wk-old Wistar rats, as previously described (Schmitz et al. 1995). In brief, the animals were deeply anesthetized with ether and decapitated, and the brain was removed. Tissue blocks containing the temporal cortex and hippocampus were mounted on a Vibratome (Campden Instruments, Loughborough, UK) in a chamber filled with cold (~4°C) artificial cerebrospinal fluid (ACSF) containing (in mM) 129 NaCl, 21 NaHCO3, 3 KCl, 1.25 NaH2PO4, 1.6 CaCl2, 1.8 MgSO4, and 10 glucose, saturated with 95% O2-5% CO2, pH 7.4. For intracellular recordings, horizontal slices were cut at 400-µm thickness and transferred to an interface chamber where they were maintained at 35°C and perfused with ACSF at a rate of 1.5-1.8 ml/min. For patch-clamp recordings, slices were cut at 300-µm thickness and stored in an interface-type storage chamber. We then transferred individual slices to a chamber mounted on an upright microscope with a ×63 water-immersion objective and infrared differential interference contrast (DIC) (Stuart et al. 1993) or infrared gradient contrast optics (Dodt et al. 1999). Slices were then perfused at a rate of 2-3 ml/min at 35°C. The slices were allowed to rest for at least 1 h after the preparation before recording.

ELECTROPHYSIOLOGICAL RECORDINGS. Intracellular electrodes were pulled from borosilicate glass (1.2 mm OD) and filled with 2 M K-acetate. Electrode resistances were 40-60 MOmega . Intracellular recordings were performed in an interface chamber using an SEC10L-amplifier (NPI Instruments, Tamm, Germany). Hyperpolarizing and depolarizing postsynaptic potentials were evoked by electrical stimulation (0.05 ms duration, 5-20 V) via a bipolar insulated stimulation electrode placed in stratum radiatum. For whole cell patch-clamp recordings, we used an EPC-7 amplifier (HEKA) or a SEC 5 l amplifier (NPI) in voltage clamp. Intracellular solution contained (in mM) 135 K-gluconate, 5 KCl, 2 MgATP, 2 Na2ATP, and 10 HEPES, buffered to a pH of 7.2. In some of the recordings 5 mM QX314 was included. When filled with the internal solution, the patch electrodes had resistances of 2-5 MOmega . The signals were filtered at 3 kHz, digitized at 8-10 kHz by an ITC-16 (Instrutech, Port Washington, New York) interface, and subsequently stored on an IBM-compatible computer.

DRUGS AND SOLUTIONS. Drugs were bath-applied at the concentrations indicated. Bicuculline methiodide (5 µM) and 4-AP (200 µM) were both purchased from Sigma (Deisenhofen, Germany). (±)-2-Amino-5-phosphonopentanoic acid (APV, 30 µM) was from Research Biochemicals (Natick, MA). 1,2,3,4-Tetrahydro-6-nitro-2,3-dioxo-benzol[f]quinoxaline-7-sulfonamide (NBQX, 10 µM) was a kind gift from Novo Nordisk (Denmark) and (2S)-3-[[(1S)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl] (phenylmethyl) phosphinic acid (CGP55845A) (2 µM) was a kind gift from Ciba-Geigy (Basel).

DATA ANALYSIS AND STATISTICS. Data were analyzed off-line using Wintida (HEKA, Germany). Data are expressed as means ± SE. Drug effects were analyzed with Student's t-test (Sigmaplot, Jandel, Corte Madera) for paired data and an error probability of P < 0.05 was regarded as significant.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Under the stimulation conditions we used, with cells relatively near to resting potential and excited by ectopic spikes, and with values of dendritic gap junction conductance that produce coupling potentials of about 0.5 mV (as reported in the literature), dendritic gap junctions alone did not lead to cooperative network bursts in the interneuron population. This was true with and without electrically active interneuron dendrites (Martina et al. 2000; Traub and Miles 1995) (not shown). This result stands in contrast to what we reported previously (Traub 1995), because the newer simulations reflect the smaller dendritic gap junctional conductances used in the present study [maximum 1.4 nS, consistent with experimental data of Gibson et al. (1999) and Galarreta and Hestrin (1999)], as compared with the much larger values used in the earlier study (minimum 10 nS). The illustrated simulations below were therefore all performed with axonal gap junctions between interneurons, rather than with dendritic gap junctions.

Dendritic gap junctions between interneurons did, however, exert definite effects under simulation conditions different from those investigated here. For example, when gamma frequency oscillations were evoked, in isolated interneuron networks, using depolarizing currents to the interneurons (e.g., 0.10-0.12 nA), and interneurons synaptically inhibited each other via GABAA receptors (Traub et al. 1996; Whittington et al. 1995), then dendritic gap junctions enhanced the degree of synchrony (not shown). This type of synchrony enhancement will be the subject of a separate study.

Firing patterns of interneurons and pyramidal cells during simulated 4-AP-induced large IPSP

Figure 3 gives an overview of the behavior of the network model. The top traces show the respective time courses of ectopic activity in the axons of pyramidal cells and interneurons. These should be regarded as extrinsic inputs to the model, arbitrarily chosen, and presumed to reflect an action of 4-AP on axons and/or presynaptic terminals. Pyramidal cell ectopic activity starts, in the model, slightly after (10 ms, in this case) interneuron activity, because of the experimental observation that variable-amplitude action potentials occur during, but not usually prior to, the large-amplitude IPSP (Avoli et al. 1998) (see also Fig. 9A).

The middle trace of Fig. 3 illustrates the soma of a PV+ interneuron. Corresponding to the period when ectopic spikes are turned on, this particular cell generates a series of abruptly rising action potentials, as well as partial spikes, without an underlying depolarization. This trace can be compared with some of the recordings of "Type II" interneurons in Michelson and Wong (1994). As the firing of interneurons is collective (Fig. 4), pyramidal cells exhibit a large, temporally correlated, IPSP (bottom trace of Fig. 3). The particular pyramidal neuron illustrated displays a spikelet at the bottom of the IPSP.



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Fig. 4. Behavior of the interneuron network: many interneurons participate in bursting, and firing is temporally organized (partially synchronized). The top trace shows, as a function of time, the total number of "parvalbumin-positive" interneuron axons that are active, arbitrarily defined as being depolarized >= 70 mV, at the site where gap junctions are allowed. Middle and bottom traces are from the same interneuron, a basket cell whose soma was hyperpolarized with -0.1-nA injected current. The axonal trace is from the site of the 2 gap junctions on this cell. Note the partial spikes at the soma, about 10-15 mV in amplitude. The partial spikes are antidromic in origin. The axon also exhibits small amplitude spikes, which are not seen at the soma. R.P., resting potential.

In the conditions simulated (blockade of AMPA and NMDA receptors), the interneurons influence synaptically the pyramidal cells, but not the other way around. One can therefore view the interneurons as an autonomous network, and one can view the pyramidal cells as an autonomous network also, with each neuron receiving a large IPSC that is temporally correlated with IPSCs in other neurons. This point of view simplifies the approach to understanding how the network behaves.

Interneuron axon-axon gap junctions lead to synchronized interneuron network bursts, with partial spikes in hyperpolarized cells

Figure 4 illustrates the cooperative nature of the firing of the interneurons, as the ectopic spikes are "turned on" in interneuron axons. The top trace shows, as a function of time, the number of axons, of PV+ cells, which are depolarized >70 mV from rest; from this trace (and also from the local average of multiple somatic potentials, not shown), one sees that a population burst takes place, with individual action potentials synchronized, at least on average. (Experimentally, the size of the "giant" IPSP in 4-AP indicates that interneuron firing must be cooperative. It is not known, however, so far as we are aware, whether action potentials of different neurons are tightly synchronized.) Recall that the mean ectopic rate in each interneuron is only 2 Hz; yet, the population in this case is firing at over 100 Hz. The population burst represents, then, a novel form of emergent network property, the mathematical properties of which have been studied by Traub et al. (1999b), for pyramidal cells.

The middle and bottom traces of Fig. 4 show, respectively, an axonal site and the soma of a single interneuron, the latter hyperpolarized by passage of current into the soma (-0.1 nA). What is important to note is that the axon (which is minimally hyperpolarized) is capable, under the conditions of the simulation (which allow action potentials to cross from one interneuron axon to another), to fire full action potentials that are driven by firing in its two coupled axons. (Note, however, that not all of this axon's active responses are full action potentials; some are only about 10 mV in amplitude.) The fact that there are two axons coupled to the illustrated axon is not obvious from the simulation, but can be determined by reference to a "network map" constructed by the computer. The full axonal action potentials (but not the small ones) invade the hyperpolarized soma, giving rise to partial spikes (bottom trace). This simulation can be compared also with Michelson and Wong (1994).

Some pyramidal cells exhibit spikelets (corresponding to axonal action potentials) during the simulated large IPSP

Simulations have previously shown how antidromic activity can induce spikelets in pyramidal neurons (Draguhn et al. 1998; Traub et al. 1999b). What is of interest to show here is that antidromic activity can also lead to spikelets during a simulated IPSP (Fig. 5), provided, of course, that the IPSP is not too large. The depth of the pyramidal cell IPSPs in the simulation of Fig. 5 ranges from about 7 to 8 mV below resting potential, depending on the neuron. The underlying IPSC has a peak conductance of 23 nS, comparable to the input conductance of the neuron (27 nS). [Note that Segal (1987) observed a 50% or more fall in input resistance in CA1 pyramidal neurons following application of 4-AP microdrops.] With such a peak IPSC conductance, somatic spikelets could follow full axonal action potentials 1:1 (Fig. 5). When the IPSC peak conductance was increased by 50%, keeping other parameters the same, and this simulation was repeated, the pyramidal cell illustrated exhibited only 3 spikelets, versus the 10 of Fig. 5 (not shown). When the IPSC peak conductance was increased by 100%, the cell still exhibited two spikelets, but both were at the beginning of the IPSP. The reduction in number of spikelets with increasing IPSC peak conductance was caused, in the model, by a reduction in the number of distal axonal action potentials, rather than by failure of proximal axonal spikes to induce spikelets in the soma.



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Fig. 5. Example behavior of the pyramidal cell network. Interneuron activity was as in Fig. 4. The pyramidal cell ectopic rate was 10 Hz per axon, and the gap junction conductance 3.7 nS. Top trace shows the number of pyramidal cell axons depolarized >= 70 mV, at the site where gap junctions are allowed to occur. Activity here is not as rhythmic as for the interneurons (Fig. 4; but see bottom right panel of Fig. 7). The middle and bottom traces are from the same pyramidal cell, which had 2 gap junctions on its axon. These traces demonstrate the antidromic origin of the simulated spikelets.

Some pyramidal cells exhibit full action potentials (antidromic) during the simulated large IPSP

Avoli et al. (1998) illustrated full-sized, as well as smaller, action potentials during the 4-AP-induced large IPSP. It was therefore interesting to see whether full-sized action potentials occurred during simulations of the large IPSP. Figure 6 shows that large action potentials can occur, provided that the frequency of ectopic spikes and the conductance of pyramidal cell axonal gap junctions are both large enough (10 Hz and 4.2 nS, respectively, in the simulation of Fig. 6). All of the action potentials are antidromic, and some have clear inflections, or even notches, on the rising phase (cf. Stasheff et al. 1993a; Fig. 4B of Draguhn et al. 1998). In our simulations, spikelets also occurred in those pyramidal cells that produced full action potentials.



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Fig. 6. Examples of full action potentials generated by 2 different pyramidal cells during the IPSP. Pyramidal cell ectopic rate was 10 Hz per axon, and gap junction conductance 4.2 nS. Insets show the same traces at higher gain. Note the ~2-mV spikelets, and the notches on the leading phase of some of the spikes. R.P., resting potential. Spikes are truncated in the insets.

Extent of spikelet- and action potential firing in pyramidal cells during the large IPSP depends on both the ectopic rate and the pyramidal cell gap junction conductance

The frequency of ectopic spike initiation in individual axons, at the beginning of the large IPSP, is difficult to determine experimentally. Figures 7 and 8 show that, at least over a certain range of parameters, sufficiently many ectopic spikes and sufficiently open gap junctions are both required for enough pyramidal axonal activity to occur, so that ectopic activity is observable in pyramidal cell somata. In our model a pyramidal axonal gap junction conductance of 3.7 nS is near threshold for propagation of spikes from one axon to another: note, in Fig. 7, the slightly greater axonal activity (with a 1-Hz ectopic rate), when the gap junction conductance is 3.7 nS, compared with when it is 0. A gap junction conductance of 4.2 nS is above threshold for spike propagation (from axon to axon): note the large increase in axonal activity when the gap junction conductance increases from 3.7 to 4.2 nS (Fig. 7), particularly with a 1-Hz ectopic rate. With the larger value of gap junction conductance, axonal activity is not only enhanced in extent, but also becomes clearly rhythmic; whether this is true experimentally is not known.



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Fig. 7. The collective behavior of the pyramidal cell axons depends on both the ectopic spike rate and on the gap junction conductance. Each trace shows, as a function of time, the number of pyramidal cell axons in the network that are depolarized >= 70 mV. Data are from 6 different simulations, each using the same time course for the interneuronal activity, and hence producing the same IPSCs in any particular pyramidal cell. The simulations differ, however, in the combination of ectopic rates and axonal gap junction conductance. A 3.7-nS gap junction conductance allows only occasional axonal spikes to cross to other axons, while with a 4.2-nS conductance, such crossing is more probable. The greatest, and most clearly rhythmic, axonal activity occurs with a combination of high ectopic rate and large gap junctional conductance (bottom right).



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Fig. 8. During-IPSP somatic spikelets and action potentials in pyramidal cells depend (in the model) on the collective behavior of the pyramidal cell axons. Data from the same simulations, arranged in the same fashion, as for the previous figure (Fig. 7). Each panel shows superimposed traces from 3 different pyramidal cell somata. Note that some cells can show many more spikelets than others (e.g., bottom left panel), and that enough axonal activity can lead to bursting in some neurons (bottom right panel), preceded by a small hyperpolarization and spikelet activity. Voltage scale is the same for all traces except for the bottom right, which also truncates the action potentials.

Figure 8 shows that ectopic activity in pyramidal cell somata correlates, in a general way, with the activity of the axonal population, but that there are pronounced fluctuations from cell to cell, within a given simulation. These fluctuations are not related, in any obvious way, to the size of the IPSP in different cells. With enough axonal activity (as in the bottom right panel of Figs. 7 and 8), some cells fire bursts of action potentials. Plotting the local average somatic potentials in this simulation, at three different locations, suggests that approximately 20-25% of the pyramidal cells participated in bursting, at two of three locations (not shown). (The reader is reminded that in the simulations in this paper, AMPA and NMDA receptors are not present, so that bursting is a consequence solely of ectopic spikes, gap junctions, and intrinsic membrane properties.)

Carbenoxolone reversibly suppresses large IPSCs that occur in the presence of 4-AP plus ionotropic glutamate blockers, indicating a dependence of the large IPSP/IPSC on gap junctions

We tested experimentally a key prediction of the model: that the generation of interneuron network activity, as reflected in large IPSPs/IPSCs, should be dependent on gap junctions. The first set of experiments was performed in voltage clamp, with slices bathed in 4-AP (200 µM), blockers of ionotropic glutamate receptors (NBQX, 10 µM, and APV, 60 µM), and the GABAB receptor blocker CGP (2 µM). Under such conditions, spontaneous large ("giant") IPSPs/IPSCs are known to occur, dependent on GABA receptors, with hyperpolarizing and depolarizing (respectively, outward and inward current) components (Aram et al. 1991; Avoli et al. 1993; Benardo 1997; Forti and Michelson 1998; Lamsa and Kaila 1997; Michelson and Wong 1994; Müller and Misgeld 1991). In voltage clamp, spontaneous outward/inward current transients occur with a period of tens of seconds (0.017 ± 0.005 Hz, n = 12; duration 2.8 ± 0.3 s; e.g., Fig. 9Aa). In four of eight cases, we also observed large, brief inward currents ("action currents") during the outward current phase, as well as early portions of the inward current phase (Fig. 9Aa slow traces, and insets shown on a faster time scale; in the other 4 cases, QX314, 5 mM, was also present in the electrode). These action currents are presumed to correspond to ectopic spikes. In the presence of carbenoxolone, 100 µM, the large IPSCs were significantly reduced in frequency and amplitude (Fig. 9, A and B), being blocked altogether in 5 of 12 slices. This action of carbenoxolone was at least partly reversible (in 6 of 12 cases; see, e.g., Fig. 9A). In addition, action currents were not observed in carbenoxolone, suggesting that gap junctions contribute to the frequency of ectopic spikes in pyramidal neurons. Carbenoxolone does not appear to have significant effects on the intrinsic properties of hippocampal neurons (Draguhn et al. 1998). It should be noted that, because the experiments were performed in voltage clamp, the form of the traces in Fig. 9 appears different from in our simulations, which were "current clamp."



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Fig. 9. Carbenoxolone, a gap junction blocking compound, suppresses the large IPSCs that occur spontaneously in 4-aminopyridine (4-AP; 200 µM) plus ionotropic glutamate blockers and a GABAB blocker. A: whole cell patch-clamp recording of a CA3 pyramidal cell in voltage clamp. The cell was held at -60 mV. Aa: spontaneous giant IPSCs were induced by 200 µM 4-AP in the presence of 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzol[f]quinoxaline-7-sulfonamide (NBQX; 10 µM), (±)-2-amino-5-phosphonopentanoic acid (APV; 30 µM), and CGP (2 µM). Top and bottom traces show 2 subsequent sequences. Carbenoxolone at a concentration of 100 µM applied for 15 min abolished the activity. Following a prolonged wash out of carbenoxolone, the activity partially recovered. Note the ectopic "action currents" during some of the giant IPSCs, shown in more detail on a more expanded scale. Action currents have been truncated. Ab: current responses of the same CA3 neuron, before and following the application of carbenoxolone, to an 8-mV hyperpolarizing voltage pulse, revealed no intrinsic changes in membrane properties induced by carbenoxolone. B: bar histograms summarize the results of 12 different experiments. In some experiments the activity was completely blocked by carbenoxolone, while in other experiments the activity was strongly reduced.

As a control, we checked that carbenoxolone does not block GABAA receptor-dependent events, particularly depolarizing ones. Following Kaila et al. (1997), depolarizing GABA potentials were evoked by tetanic stimulation (40 pulses at 100 Hz), without 4-AP in the bath, but in the presence of blockers of ionotropic glutamate receptors (NBQX, 10 µM; APV, 60 µM) and GABAB receptors (CGP, 2 µM; Fig. 10). Depolarizing GABA potentials could be evoked when stimulation was given every 4-5 min, with rundown occurring when stimuli were delivered more often. Carbenoxolone (100 µM) had no apparent effect on these depolarizing GABA events.



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Fig. 10. Carbenoxolone has no effect on the large hyperpolarizing/depolarizing potentials, occurring in the presence of ionotropic glutamate blockers plus a GABAB blocker, after strong tetanic stimulation (40 pulses at 100 Hz). Intracellular recordings were performed with conventional sharp microelectrodes. A sequence of hyperpolarizing and then depolarizing GABAA receptor-mediated potentials were elicited by direct electrical stimulation of GABAergic interneurons in the presence of NBQX (10 µM), APV (30 µM), and CGP (2 µM). Stimulation was performed at the time indicated by the bar. Note that carbenoxolone (100 µM) had no effect on the depolarizing GABAA potentials. Bottom plots summarize the results from 5 experiments.


    DISCUSSION
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ABSTRACT
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METHODS
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DISCUSSION
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Axonal gap junctions versus dendritic gap junctions in interneurons

The idea of axonal gap junctions between principal hippocampal neurons was suggested by the electrophysiological recordings of Draguhn et al. (1998), which revealed high-frequency oscillations in principal neurons that persisted in low [Ca2+]o media, but which were suppressed by pharmacological blockade of gap junctions; and during which, intracellular recordings revealed action potentials with notches on the upstroke, as well as presumed coupling potentials (so-called spikelets) with rapid upstroke and slower decay (see also Perez-Velazquez et al. 1997). The form of these spikelets could not be replicated in simulations of pyramidal cell pairs, in which a gap junction was placed between somata or between dendrites, but could be replicated with an axonal gap junction (Draguhn et al. 1998; Traub et al. 1999b). The reason why an axonal gap junction was successful was this: a spike in the axon of the presynaptic cell could evoke, across the junction, an action potential (or sometimes a 10- to 25-mV partial spike) in the axon of the coupled cell; this could occur due to the high-input resistance in the axon, together with a high gNa density. Antidromic conduction of the axonal action potential, or partial spike, could then evoke a response in the soma of the coupled cell: either a spikelet, partial spike, or full action potential, depending on membrane potentials and conductances in the coupled neuron. It was subsequently shown that, in principle, axonal gap junctions with such properties, could (if present in networks of principal neurons at low densities, and in the presence of a low-frequency background of spontaneous action potentials) lead to high-frequency network oscillations (Traub et al. 1999b).

Could such ideas be relevant to the generation of interneuron network bursts in 4-AP, bursts that can occur even when ionotropic glutamate receptors and GABAA receptors are blocked, in which spikes appear ectopic, and wherein hyperpolarization uncovers small amplitude spikes (Michelson and Wong 1994)? An earlier model was proposed for the generation of such interneuron bursts, the essential physical idea of which was that spikes should cross from cell to cell via dendritic gap junctions (Traub 1995). Certainly there is morphological evidence for the occurrence of dendritic gap junctions coupling particular types (for example, parvalbumin-positive) of hippocampal interneurons (Fukuda and Kosaka 2000; Katsumaru et al. 1988; Kosaka 1983a,b). Our earlier model used, however, a value for the gap junctional conductance (10 nS), which is quite large compared with current estimates for putative dendritic gap junctions (e.g., 1.6 ± 1.3 nS) (Gibson et al. 1999). Pair recordings have been obtained from electrically coupled pairs of cortical interneurons, corresponding morphologically (in at least some cases) to dendrodendritic or to dendrosomatic gap junctions (Galarreta and Hestrin 1999; Gibson et al. 1999; Tamás et al. 2000). Most (not all) illustrated coupling potentials have amplitudes of about 0.5 mV, and spike transduction (the ability of a spike in one cell to induce a spike in a coupled cell) has not been reported, to our knowledge, although entraining effects between coupled neurons can be observed. Axonal gap junctions have been reported to exist between members of one class of putative GABAergic neuron, retinal horizontal cells (Johnson and Vardi 1998; Vaney 1993), but not (again, so far as we are aware) between cortical interneurons.

Because of these considerations, and in view of simulations such as those illustrated here (Figs. 3 and 4), we propose a new hypothesis: that, at least in 4-AP, gap junctions are functional between some interneuron axons and can lead to collective bursts under certain conditions. Whether the oscillatory aspects of this collective behavior, as can occur in simulations at >100 Hz (Fig. 4), are relevant experimentally remains to be seen. If interneuronal gap junctions do exist between axons, 4-AP could facilitate not only the initiation of ectopic spikes, but also passage of spikes from one axon to another, via a likely increase in axonal excitability (Debanne et al. 1997; Kocsis et al. 1983). We recognize that such an effect is also conceivable, however, for dendrites, in that 4-AP has been reported to increase the excitability of pyramidal cell dendrites (Hoffman et al. 1997).

Depolarizing GABAA effects between interneurons (not included in the present simulations) are expected to exert additive effects with (hypothesized) axonal gap junctions in promoting interneuron bursts. If, however, the gap junctions play a primary role in promoting interneuronal bursts, then gap junction blockers would be expected to eliminate the large IPSPs induced by 4-AP in pyramidal cells.

Pharmacological evidence that interneuron gap junctions contribute to network bursts

The data in Figs. 9 and 10 indicate that the gap junction blocking compound carbenoxolone suppresses, at least partly, spontaneous large IPSCs occurring in 4-AP plus ionotropic glutamate blockers (plus a GABAB blocker), but does not suppress the large IPSCs evoked by strong tetanic stimulation (Kaila et al. 1997). Evidently, carbenoxolone does not block the GABAA receptor-dependent IPSCs themselves, and the data suggest instead that it is spontaneous interneuron network bursts that have been blocked (partly) by the drug. The experimental evidence is therefore consistent with our basic hypothesis. Unfortunately, with available techniques, we are not able to distinguish between effects of carbenoxolone on dendritic gap junctions, on putative axonal gap junctions, or on both.

Cooperativity between ectopic spikes and gap junctions

Our model suggests that both ectopic spikes and gap junctions are likely to be important, not only for the interneuronal bursting, but also for the occurrence of flurries of spikes and spikelets that sometimes occur in pyramidal cells (Figs. 7, 8, and 9A). The physical reason for this is that axonal gap junctions provide a form of amplification, if the conductance is large enough: the gap junctions can then allow a spontaneously arising axonal spike to cross to many other axons, and hence to invade a large number of somata, rather than just the single parent soma of the neuron whose axon fired spontaneously.

Testing this concept experimentally in pyramidal cells could prove tricky, however, in that the process(es) that presumably link together, in time, putative ectopic spikes in interneurons and in pyramidal cells is not known. (In the model, the simulation program simply forces the ectopic rates to be linked together in a specified way.) One can imagine several possibilities, including the following:

1) Direct gap junctional coupling could occur between axons of the respective populations (designated by the "?" in Fig. 2). Electrical coupling has been found, in rat somatosensory cortex slices, between excitatory cells (spiny stellate neurons) and fusiform interneurons, but the membrane site of coupling is not known (Venance et al. 2000).

2) GABA release, occurring during the collective interneuron burst, excites axons directly (Alford et al. 1991; Sakatani et al. 1994; Stasheff et al. 1993b).

3) Ionic concentration (including pH) changes induced by the interneuron burst (Lamsa and Kaila 1997) might excite axons. Lamsa and Kaila (1997) reported that spontaneous 4-AP-induced GABAergic events could elevate [K+]o by 0.7 ± 0.3 mM, perhaps contributing to axonal excitability; such events also can produce a pH elevation of 0.01-0.08 units. The latter effect would favor the opening of gap junctions (Spray et al. 1981).


    ACKNOWLEDGMENTS

We thank E. H. Buhl and M. A. Whittington for helpful discussions. R. D. Traub is a Wellcome Principal Research Fellow.

This work was supported by the Wellcome Trust and the Deutsche Forschungsgemeinschaft. D. Schmitz was supported by the Emmy-Noether-Programm (DFG).


    FOOTNOTES

Address for reprint requests: R. D. Traub, Dept. of Pharmacology, Division of Neuroscience, University of Birmingham School of Medicine, Vincent Drive, Edgbaston, Birmingham B15 2TT, UK (E-mail: r.d.traub{at}bham.ac.uk).

Received 4 August 2000; accepted in final form 22 November 2000.


    NOTE ADDED IN PROOF

Two abstracts have reported effects of carbenoxolone on 4-AP-induced activity: suppression of ectopic spikes in pyramidal cells induced by 4-AP plus intracellular alkalinization (Gladwell SJ and Jefferys JGR, J Physiol 194P: 523.P, 2000); and suppression of collective interneuron firing in 4-AP plus blockers of ionotropic glutamate receptors and of GABAA receptors (Yang Q and Michelson HB, Soc Neurosci Abstr 26: 354, 2000).


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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