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
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ABSTRACT |
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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
-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.
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INTRODUCTION |
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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 -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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METHODS |
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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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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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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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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
M. 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 M
.
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.
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RESULTS |
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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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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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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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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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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."
|
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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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
).
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ACKNOWLEDGMENTS |
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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).
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FOOTNOTES |
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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.
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NOTE ADDED IN PROOF |
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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).
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REFERENCES |
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