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The Journal of Experimental Biology 202, 1301–1309 (1999)
Printed in Great Britain © The Company of Biologists Limited 1999
JEB2092
PHYSIOLOGY OF ELECTROSENSORY LATERAL LINE LOBE NEURONS IN
GNATHONEMUS PETERSII
YOSHIKO SUGAWARA1,*, KIRSTY GRANT2, VICTOR HAN3 AND CURTIS C. BELL3
1Department of Physiology, Teikyo University School of Medicine, Kaga 2-11-1, Itabashi-ku, Tokyo 173-8605, Japan,
2Institut Alfred Fessard, Centre National de la Recherche Scientifique, 91190 Gif-sur-Yvette, France and
3Neurological Sciences Institute, Oregon Health Science University, Portland, OR 97209, USA
*e-mail: [email protected]
Accepted 16 February; published on WWW 21 April 1999
Summary
In mormyrid electric fish, sensory signals from
to the ELL molecular layer in vitro evokes an excitatory
electroreceptors are relayed to secondary sensory neurons
postsynaptic potential (EPSP), generally followed by an
in a cerebellum-like structure known as the electrosensory
inhibitory postsynaptic potential (IPSP), in the efferent
lateral line lobe (ELL). Efferent neurons and interneurons
neurons. In MG cells, the same stimulation evokes an
of the ELL also receive inputs of central origin, including
EPSP, often followed by a small IPSP. Synaptic
electric organ corollary discharge signals, via parallel fibers
transmission at parallel fiber synapses is glutamatergic and
and via fibers from the juxtalobar nucleus. To understand
is mediated via both N-methyl-D-aspartate (NMDA)- and
the cellular mechanisms of the integration of sensory inputs
(AMPA)-type glutamate receptors. The inhibitory
and central inputs in the ELL, the intracellular activity and
component of the parallel fiber response is GABAergic. It
ionic properties of the efferent projection neurons and
is probably mediated via the stellate neurons and the MG
interneurons were examined in an in vitro slice
cells, which are themselves GABAergic interneurons
preparation.
intrinsic to the ELL network.
We focus here on the electrophysiological properties of
A hypothetical neural circuit of the intrinsic connections
the efferent neurons of the ELL network, the large
of the ELL, based on the known morphology of projection
fusiform cells and large ganglion cells, and on a class of γneurons and medium ganglion interneurons, is presented.
aminobutyric acid (GABA)-ergic interneurons known as
This circuit includes an excitatory and an inhibitory
medium ganglion (MG) cells. In response to current
submodule. The excitatory submodule is centered on a
injection through a recording pipette, both types of efferent
large fusiform cell and appears to relay the sensory input
neuron fire a large narrow spike followed by a large
as a positive ‘ON’ image of an object. The inhibitory
hyperpolarizing afterpotential. The MG cells fire a complex
submodule is centered on a large ganglion cell and relays
spike which consists of small narrow spikes and a large
a negative ‘OFF’ image to the next higher level. We suggest
broad spike. Although the forms of the action potentials in
that MG cells exert an inhibitory bias on efferent neuron
efferent neurons and in MG cells are different, all spikes
types and that the ELL network output is modulated by the
are mediated by tetrodotoxin (TTX)-sensitive Na+
dynamically plastic integration of central descending
conductances and spike repolarization is mediated by
signals with sensory input.
tetraethylammonium (TEA+)-sensitive K+ conductances.
In the presence of TEA+, substitution of Ba2+ for Ca2+ in
the bath revealed the presence of a high-voltage-activated
Key words: mormyrid, electric fish, electrosensory lateral line lobe,
corollary discharge, Na+-dependent dendritic action potential,
Ca2+ conductance.
excitatory submodule, inhibitory submodule, Gnathonemus petersii.
Stimulation of parallel fibers conveying descending input
Introduction
Weakly electric mormyrid fish use their electric sense for
electrolocation and electrocommunication by detecting the
electrical current flow through specialized cutaneous
electroreceptors generated by the fish’s own electric organ
discharge (EOD). Three classes of electroreceptor are
present: mormyromasts, which serve in active electric
imaging of the environment; ampullary receptors, which are
sensitive to low-frequency electric events in the water; and
Knollenorgans, whose primary role is communication.
Centrally projecting fibers from electroreceptors terminate in
the electrosensory lateral line lobe (ELL) (Maler, 1973; Bell
and Russell, 1978a). Primary afferents from mormyromast
and ampullary electroreceptors terminate in separate zones of
the cortex of the ELL (Bell et al., 1989), while primary
1302 Y. SUGAWARA AND OTHERS
afferents from Knollenorgan receptors terminate in the
nucleus of the ELL (nELL) (Bell and Grant, 1989).
All three classes of electroreceptor respond to the fish’s own
EOD and to stimuli from the environment (Szabo and
Hagiwara, 1967; Bell and Russell, 1978b; Bell and Szabo,
1986). The self-induced afferent signals are known as
‘reafferent’ signals (von Holst and Mittelstaedt, 1950), and
their significance to the animal is different in each of the three
electrosensory pathways. In the context of the Knollenorgan
and ampullary electroreceptive pathways, reafferent input may
potentially cause interference with the reception of exafferent
electrosensory signals (those originating from the
environment, rather than being a result of the animal’s
‘autostimulation’). For the mormyromast electrosensory
pathway, however, reafferent electrosensory input is of prime
interest to the active imaging of the environment.
Each pathway-specific zone of the ELL also receives
corollary discharge input associated with the motor command
that drives the electric organ (electric organ corollary
discharge, EOCD) (Zipser and Bennett, 1976; Bell, 1982).
Thus, with each EOD, the neurons of the ELL are affected both
by reafferent electrosensory signals and by descending EOCD
signals. The interactions between reafferent and EOCD signals
are different in each of the electrosensory zones of the ELL
(Bell and Szabo, 1986).
Those interactions revealed in studies in vivo are briefly
described as follows. Afferent signals from Knollenorgan
receptors encode information about the timing of brief electrical
signals, such as the EODs of other fish, and evoke excitatory
postsynaptic potentials (EPSPs) in the neurons of the nucleus
of the ELL (Bell et al., 1989). The corollary discharge to the
nELL induces a brief and precisely timed inhibitory
postsynaptic potential (IPSP) in the nELL neurons, which
completely blocks the electrosensory-reafferent-driven EPSP
(Zipser and Bennett, 1976; Russell and Bell, 1978; Szabo et al.,
1979). Complete blockade lasts for only 1–2 ms, but this brief
corollary discharge inhibition is sufficient to block the
reafferent response and to remove the interference that would
have been caused by reafferent input (Bell and Grant, 1989).
Ampullary electroreceptors are specialized for measuring
low-frequency electrical signals (Kalmijn, 1974). Afferent
fibers from ampullary electroreceptors project to the
ventrolateral zone of the ELL (VLZ in Fig. 1). Primary
afferents discharge tonically with a frequency that may be
modulated up or down by external stimuli. The fish’s own EOD
causes an acceleration followed by a deceleration of the tonic
discharge rhythm. A brief EOCD inhibition, such as in the
Knollenorgan region, would not be effective in inhibiting
reafferent ampullary input. The corollary discharge input to the
ampullary zone evokes a more complex, temporally structured
series of excitatory and inhibitory events which selectively
filter the longer-lasting reafferent input (Bell, 1981). Thus,
under stable conditions, the corollary discharge effect
counterbalances or minimizes sensory reafferent inputs.
In the mormyromast electrosensory pathway, reafferent
responses convey actively sought information about the
environment. Mormyromast electroreceptors serve active
electrolocation by measuring the intensity of current flow
induced by the fish’s own EOD. Object images are generated
by the distortions that they impose on the current flow within
the fish’s own electric field. Repetitive association of reafferent
electrosensory signals with the descending EOCD signal
SM
Voltage
LC
Current injection
SD
EGp
PF
MZ
DLZ
Fig. 1. Diagram of a transverse
section of the electrosensory lateral
line lobe (ELL) to show the positions
of the recording and stimulating
electrodes in vitro. DLZ, dorsolateral
zone of ELL; EGp, eminentia
granularis posterior; LC, lobus
caudalis; MZ, medial zone of the
ELL; PF, parallel fibers; VLZ,
ventrolateral zone of the ELL; SD,
SM, recording electrodes in the deep
fiber
and
molecular
layers,
respectively.
VLZ
Afferents from
electroreceptors
Physiology of the mormyrid ELL 1303
modulates the effect of the EOCD-driven input to the ELL
networks. This plasticity follows an anti-Hebbian learning rule
(Hebb, 1949; Bell, 1981, 1990; Bell et al., 1992; Bell and
Grant, 1992), such that the EOCD-driven feedback to the ELL
generates a negative image of the recent reafferent responses
and acts as an adaptive filter of actual or current reafferent
responses (Bell and Grant, 1992; Bell et al., 1993, 1997a). One
plausible site for the adaptive filter is thought to be at the
synapses between parallel fibers and the medium ganglion cells
of the ELL (Bell et al., 1997b).
Our goals are to understand the adaptive filtering of the
electrosensory inputs by descending signals on the basis of the
cellular mechanisms of electrogenesis in the ELL neurons,
focusing particularly on the mormyromast region. In this paper,
we give a brief description of the basic anatomical organization
and the electrophysiological properties of the efferent
projection neurons and GABAergic interneurons of the ELL
cortex. Both in vivo and in vitro intracellular recordings have
revealed that the forms of action potentials and the firing
properties are different in these cell types. On the basis of
evidence obtained from the brain in vivo and from in vitro
slices of the ELL, we will discuss a hypothetical model of
electrosensory processing in the ELL.
Materials and methods
The results presented in Figs 2–4 were obtained by using
patch recording pipettes in the whole-cell configuration in slices
of the ELL. The ELL was excised from Gnathonemus petersii,
anesthetized with MS-222 (Sandoz) (100 mg l−1), and sliced at a
thickness of 300 µm in low-[Na+] ACSF (see below) in which
half the Na+ had been replaced with iso-osmotic sucrose as
described previously (Grant et al., 1998). After incubation for
1–2 h in low-[Na+] ACSF, the slice was placed in the recording
chamber and perfused with standard ACSF. The composition of
standard ACSF was as follows (in mmol l−1): 125 NaCl, 2.5 KCl,
1.25 NaH2PO4, 2.0 CaCl2, 1.6 MgSO4.7H2O, 24 NaHCO3 and
10 glucose, pH 7.2–7.4. The ACSF was bubbled with 95 % O2
and 5 % CO2. Parallel fiber stimulation was applied through a
monopolar tungsten wire electrode in the molecular layer.
Primary afferent fibers were stimulated with a similar electrode
in the deep fiber layer. The responses of ganglion layer neurons
of the ELL were recorded using a patch pipette (resistance 10–20
MΩ) filled with an internal pipette solution which contained (in
mmol l−1): 140 potassium gluconate, 3.0 KCl, 10 Hepes, 10
EGTA, 1.0 CaCl2, 1.0 MgCl2 and 110 µg ml−1 nystatin (Sigma).
The pH was adjusted to 7.3. The response was amplified with a
patch-clamp amplifier (Nihon Koden, CEZ-2300), stored on a
digital tape recorder (Sony, PC204A) and analyzed offline.
The intrinsic network of the ELL: efferent neurons and
medium ganglion cells (GABAergic interneurons)
The ELL cortex has six layers: the molecular, ganglionic,
plexiform, granule, intermediate and deep fiber layers. Primary
afferents from the A and B receptor cells of mormyromast
electroreceptors terminate in the granule layer of the ELL medial
zone (MZ in Fig. 1) and dorsolateral zone (DLZ in Fig. 1)
respectively. Sensory signals transmitted to the granule cells are
relayed to efferent projection neurons and inhibitory interneurons,
including medium ganglion (MG) cells. Large ganglion (LG) cells
and large fusiform (LF) cells were recorded in the ganglionic cell
layer and in the plexiform or granule layers, respectively (Grant
et al., 1996; Meek et al., 1996; Han et al., 1999). The efferent
axons of the LG cells and the LF cells project to the preeminential
nucleus and to the lateral nucleus of the torus semicircularis. The
LF and LG cells, also referred to as E-cells and I-cells, respond
to stimuli in the center of their electroreceptive field with increased
activity and decreased activity, respectively (Bell et al., 1997a).
Medium ganglion, cells are GABAergic interneurons, as shown
by immunohistochemistry (Meek et al., 1996) and pharmacology
(Grant et al., 1998). The cell bodies of the MG cells are found in
the ganglionic cell layer and are of two types: MG1 cells with
basal dendrites confined to the plexiform layer and axonal
terminals in the ganglion, plexiform and granule layers; and MG2
cells with basal dendrites in the granule layer and axonal terminals
confined to the ganglion and plexiform layers (Meek et al., 1996;
Han et al., 1999). The MG cells are 7–10 times as numerous as
the LG cells (Meek et al., 1996; Han et al., 1999).
The EOCD signals from the EOD motor command nucleus
arrive by at least two pathways (Bell et al., 1983). The first arises
via the paratrigeminal command-associated nucleus, which
projects to the eminentia granularis posterior (EGp in Fig. 1).
The granule cells of the EGp in turn give rise to the parallel fibers
that project to the outer two-thirds of the molecular layer of the
ELL (Maler, 1973). The second EOCD pathway arises via the
mesencephalic command-associated nucleus, which projects to
the juxtalobar nucleus via the juxtaleminiscal region (Bell et al.,
1995; Bell and von der Emde, 1995). The cells of the juxtalobar
nucleus in turn project to the mormyromast region of the ELL,
primarily to the granular layer, although some terminals are also
found in the plexiform and ganglion layers (Bell et al., 1995;
Bell and von der Emde, 1995). Thus, integration of
electrosensory signals with the descending EOCD inputs
probably takes place in at least two stages: an early stage, in the
granular layer, with the EOCD inputs from the juxtalobar
nucleus, which convey accurate information on the timing of the
EOD motor command (Bell et al., 1995; Bell and von der Emde,
1995); and a later stage in the molecular layer at the level of the
spiny dendrites of the efferent cells and MG cells, which receive
parallel fiber input. Parallel fibers convey descending signals
from the EGp (Bell et al., 1981, 1995; Bell and von der Emde,
1995; Meek et al., 1996). In the in vitro slice preparation,
integration in the molecular layer of the ELL has been examined
by applying field stimulation to the parallel fiber that mimics
descending input, without the influence of EOCD-driven effects
on the early stage in the granule layer (Fig. 1).
Action potentials in LG, LF and MG neurons in vitro
Intracellular recordings from efferent neurons and
interneurons, subsequently identified by labeling with biocytin,
1304 Y. SUGAWARA AND OTHERS
A
Fig. 2. Action potentials and synaptic
responses of efferent neurons to molecular
layer stimulation and to current injection
through the recording pipette. (A–C) Current
injection through the recording pipette evoked
bursts of large narrow spikes. Note that the
bursting spikes were induced on the
depolarizing phase of an oscillatory membrane
fluctuation (open arrow). Membrane potential -72 mV
was −72 mV. The intensity of the stimulus is
shown to the right of each trace. (C) At high
intensity, the bursts changed to repetitive
D
firing. (A) Molecular layer stimulation (SM)
evoked an excitatory postsynaptic potential
(EPSP) and large narrow spikes, sometimes
followed by an inhibitory postsynaptic
potential (IPSP) (filled arrow). Two
superimposed traces show that the occurrence
of an IPSP suppresses the second and third
spikes. (E) A single sweep showing an IPSP
on an expanded scale.
C
B
100 pA
255 pA
110 pA
20 mV
50 ms
E
10 mV
20 mV
SM
20 ms
showed that the form of action potentials in these two types of
neurons is different in vivo (Bell et al., 1997a) and in vitro
(Grant et al., 1998). LG and LF efferent neurons fire large
narrow spikes. In results obtained by using a sharp
microelectrode in the in vitro slice preparation, Grant et al.
(1998) reported that in LG cells these action potentials were
25–50 mV in amplitude (35.7±2.2 mV, mean ± S.E.M., N=25)
and 1–2 ms in duration; in LF cells, action potentials were
30–60 mV in amplitude (42.2±3.8 mV, N=15) and 1–2 ms in
duration. Large hyperpolarizing afterpotentials follow these
spikes in both LG and LF neurons. There was little difference
between the forms of action potentials and their large
hyperpolarizing afterpotentials in LG and LF efferent neurons.
In contrast, intracellular recordings from MG cells in vitro
showed two types of spikes: a large broad spike of 25–60 mV
in amplitude (49.8±1.7 mV, N=28) and 10–20 ms in duration
with a small after-hyperpolarization, and a small narrow spike
1–10 mV in amplitude (5.8 mV ±0.4 mV, N=7) and 1–2 ms in
duration. Small narrow spikes also occurred in the absence of
the large broad action potentials. Under some conditions, a
medium-sized broad spike, 8–20 mV in amplitude and 4–6 ms
in duration, was also seen in isolation (Bell et al., 1997a; Grant
et al., 1998). This suggests that each component of the complex
spike may have a different origin and a different role in cell
activities. As described in a previous study (Bell et al., 1993),
the expression of plasticity appeared to depend on the
activation of a large broad spike in the postsynaptic neuron.
Two types of MG neuron were distinguished on the basis of
cell morphology, MG1 and MG2 cells. No differences were
observed, however, in the recorded electrophysiological
properties of these two cell types.
Responses around the threshold for firing were examined by
using a patch pipette whose tip resistance was lower than that
of a sharp microelectrode. In the efferent neurons, current
injection through a recording patch pipette evoked a slow
SM
20 ms
depolarizing potential (open arrow in Fig. 2A). Injection of
low-intensity current evoked an oscillatory membrane
fluctuation leading to a large narrow spike or to bursts of
several spikes. With increased current intensity, the number of
spikes in a burst increased and the inter-burst interval
decreased (Fig. 2B). Even stronger stimulus intensity evoked
a repetitive tonic discharge (Fig. 2C). The latency to the first
spike gradually decreased with the increase in current intensity
(Fig. 2A,B). Latency is plotted against current intensity in
Fig. 3C (open squares). However, the initial frequency,
calculated from the interval between the first and the second
spikes of the response train, appeared to be fixed (Fig. 3D;
open squares). Thus, in efferent neurons, current injection
initially evoked an oscillatory membrane depolarization which
then induced a large narrow spike. An increase in stimulus
current intensity evoked an increase in the amplitude of
membrane fluctuation and a shortening of the burst interval.
With large depolarizations of more than approximately
−50 mV, the burst firing pattern changed from bursting to
repetitive firing (Fig. 2C). Therefore, the fixed first interval
suggests that the conductances underlying spike generation in
efferent neurons may be saturated. Further analysis of the ionic
currents involved is required.
Molecular layer stimulation in vitro excites parallel fibers
and evokes short-latency EPSPs in efferent neurons which are
sufficient to trigger large narrow spikes (Fig. 2D). In many
efferent cells, IPSPs follow the initial EPSP with a latency of
2–3 ms or more and may be sufficient to block later spikes of
the response (Fig. 2D,E). Grant et al. (1998) reported that
EPSP–IPSP sequences of this sort were observed in
approximately 48 % of the efferent neurons recorded; an EPSP
only was obtained in 45 % of efferent cells and an IPSP only
was found in 7 % of the efferent neurons.
IPSPs recorded in LG and LF neurons may result from parallel
fiber excitation of GABAergic stellate cells in the molecular
Physiology of the mormyrid ELL 1305
layer, whose axons terminate on the apical dendrites of these
neurons, and from the excitation of neighboring MG neurons,
whose axons terminate on the somata of LG and LF neurons.
In MG cells, current injection through the recording pipette
in vitro evoked both small narrow and large broad spikes
(Fig. 4A–C). The threshold for initiation of small spikes was
always lower than that for the large broad action potentials. The
broad spike thus always arose from a small spike (Fig. 4C,D),
being initiated increasingly close to the peak of the small spike
as current intensity increased. The latency of both spike types
decreased with increasing current intensity (Fig. 3C). At high
current intensity, the frequency of both small narrow and large
broad spikes increased (Fig. 3D), as calculated from the interval
between the first and second spikes. This is in contrast to the
behavior of the large narrow spikes in efferent neurons. Since
the curve showing frequency as a function of current intensity
for small spikes showed a steeper slope (filled triangles and
circles in Fig. 3D) than that for large broad spikes (open circles
and triangles in Fig. 3D), the generation of small spikes seemed
to be sensitive to the stimulus intensity.
In MG cells, parallel fiber stimulation evoked an EPSP from
which several sizes of small spikes and large broad spikes
could arise (Fig. 4D,E). When the tungusten stimulating
electrode was moved a small distance (100 µm) along the
parallel fibers, a small IPSP was evoked at the time that the
second small spike arose (Fig. 4F). This supports the view that
the MG cells also receive inhibitory inputs from the stellate
cells or from neighboring MG cells, as shown in the
morphological study of Meek et al. (1996).
Voltage-dependent channels in efferent neurons and
medium ganglion cells
The addition of 0.3 µmol l−1 tetrodotoxin (TTX) to the bath
perfusion medium abolished all regenerative spikes in both
efferent neurons and medium ganglion cells. This means that
Na+ is the primary carrier of the inward current during the large
narrow spike, the large broad spikes and the small narrow
spikes. In efferent neurons, after suppression of large narrow
spikes by TTX, current injection induced large depolarizing
plateau potentials when Ca2+ was replaced with Ba2+ in the
presence of the K+ channel blocker tetraethylammonium
(TEA+; 25 mmol l−1). In medium ganglion cells, after
replacement of Ca2+ with Ba2+ and in the presence of TEA+,
a long depolarizing potential followed the broad spike. In both
cell types, these large, slow potentials were blocked by the
addition of 0.1 mmol l−1 Cd2+. Since Ba2+ is a potent charge
carrier through Ca2+ channels and Cd2+ suppresses Ca2+
Frequency (Hz)
Latency (ms)
B
A
Fig. 3.
Relationships
First frequency
First frequency
between the intensity of
the
stimulus
current
Latency
Latency
injected
through
the
patch pipette and spike
generation in efferent
and medium ganglion
(MG) cells. (A) Burst
firing of an efferent cell
20 mV
20 mV
in
response
to
a
20
ms
50 ms
suprathreshold
current
pulse shown in Fig. 2A,B.
Large and small open
500
400
squares
show
the
C
350 D
measured
points
for
latency
and
first
300
100
frequency, respectively.
■■
250
(B) Responses of an MG
200
cell to current injection.
Filled symbols indicate
150
10
small spikes and open
100
symbols indicate large
broad spikes. (C) Plot of
50
first spike latency against
l
0
1
stimulus current intensity.
50 100 150 200 250 300 350
0
50 100 150 200 250 300 350
0
Open squares indicate
Current (pA)
Current (pA)
large narrow spikes of an
efferent cell. Circles indicate one MG cell and triangles indicate a second MG cell. The large narrow spikes in the efferent cells had a lower
stimulus threshold than the small narrow spikes or large broad spikes in MG cells. Note that the small narrow spikes in MG cells were evoked
with a lower stimulus intensity than the large broad spikes. (D) Plot of initial frequency (the inverse of first interspike interval) against
injected current intensity for an efferent cell and two MG cells (symbols as in Fig. 3C). Note that the initial frequency of the small spikes in
the MG cells increased with current intensity, while the initial frequency was almost constant in the efferent cell, probably due to an
activation of oscillatory membrane fluctuation.
1306 Y. SUGAWARA AND OTHERS
Fig. 4. Responses of medium
C
B
A
ganglion (MG) neurons to molecular
layer stimulation and to current
injection through the recording
pipette. (A–C) Current injection
through the recording pipette evoked
bursts of small narrow spikes.
Membrane potential was −65 mV.
100 pA
125 pA
175 pA
The intensity of the stimulus is shown
20 mV
to the right of each trace. (A,B) Low- -65 mV
intensity current injection evoked
50 ms
small narrow spikes. (C) An increase
F
E*
D
in current intensity induced repetitive
firing of small narrow spikes and a
large broad spike. (D) Low-intensity
molecular layer stimulation (SM1)
10 mV
10 mV
evoked only a small spike (asterisk).
Higher-intensity stimulation evoked
*
a larger excitatory postsynaptic
20 mV
20 ms
20 ms
potential (EPSP) inducing small
SM1
SM2
narrow spikes and a large broad
20 ms
spike. (E) The response marked with
SM1
an asterisk in D shown on an
expanded scale. (F) When the stimulating tungsten electrode was moved by 100 µm (SM2), low-intensity molecular layer stimulation induced a
small inhibitory postsynaptic potential (IPSP) (arrow). Two traces with and without an IPSP are superimposed.
conductances (Hagiwara and Barley, 1981), these results
suggest that the slow depolarizing potentials in both types of
neuron are induced by activation of a Ca2+ conductance.
However, the stimulus threshold for activation of this Ba2+
spike was approximately 3–4 times greater than that for the
Na+ spike. The high threshold for these slow potentials
suggests the activation of high-voltage-activated Ca2+
channels. The high threshold could also be partly due to a distal
distribution of Ca2+ channels in the apical dendrites.
Ion channel distribution and function
The relative distribution of ion channel densities cannot be
established with the available data. However, it appears that
voltage-sensitive Na+ channels must be present in the axonal,
somatic and proximal dendrite membranes, as in hippocampal
pyramidal neurons (Turner et al., 1991; Jaffe et al., 1992), in
neocortical pyramidal neurons (Regehr et al., 1993; Stuart and
Sakmann, 1994) and in pyramidal neurons of the gymnotid
ELL (Turner et al., 1994). ELL efferent neurons have relatively
large myelinated axons. Large narrow spikes are probably
evoked as propagating spikes in these axons with a low
stimulus threshold. The spike then invades the soma
antidromically and possibly also the apical dendritic tree.
Broad dendritic spikes may be present in the apical dendrites
of efferent neurons but are obscured in somatic recordings by
the presence of K+ channels. The large after-hyperpolarizations
suggest a high density of K+ channels in these efferent neurons.
In MG cells, the small narrow spikes are probably evoked
in the axon and the large broad spikes probably arise in the
soma or proximal dendrites. These suggestions are based on
the following reasoning. (1) The small narrow spike has a
lower threshold than the large broad spike. Action potential
initiation occurs at the point of lowest threshold in the neuron,
usually in the initial segment of the axon (Eccles, 1964). This
has been demonstrated in the neocortical pyramidal neuron
(Stuart and Sakmann, 1994) in which the action potential is
initiated in the axon, leading to a somatic action potential that
actively invades the apical dendritic tree via activation of
dendritic TTX-sensitive Na+ channels. (2) The small size of
the narrow spikes in MG cells may be a simple consequence
of geometry and ionic channel density, as predicted in the
impulse propagation model of Moore et al. (1983). The MG
cell has quite a thin axon, which terminates within the ELL
(Meek et al., 1996). Because of the small diameter of the axon,
its action potential may not generate enough action current to
overcome the abrupt increase in load at the soma and will
propagate only electrotonically to the soma. The depolarization
caused by a small spike is not sufficient to generate a broad
spike, which requires additional excitatory synaptic input. The
broad spike always arises from a small spike because of its
higher threshold. (3) The finding that broad spikes can be
recorded extracellularly quite high in the molecular layer in
vivo (Bell et al., 1997a,b), distant from the cell bodies in the
ganglionic cell layer, also supports the hypothesis that large
broad spikes are propagated into the apical dendrites. The
pairing of postsynaptic broad spikes with EOCD-driven EPSPs
was necessary, in vivo, for the induction of plastic changes in
the response to this synaptic input. Similarly, the occurrence
of the broad spike is necessary for the anti-Hebbian plasticity
at the parallel fiber synapse that has been demonstrated in vitro
(Bell et al., 1997b).
Physiology of the mormyrid ELL 1307
Comparison of responses to ascending input in vivo and in
vitro
Primary afferent activation in vitro is achieved by electrical
stimulation in the deep layers of the ELL (SD in Fig. 1),
whereas in vivo it is achieved by natural electrosensory stimuli.
The following description is based on both in vitro (Grant et
al., 1998) and in vivo (Bell et al., 1997a) results. Comparison
of the two reveals interesting features of the neural network in
the ELL.
Deep fiber layer stimulation in vitro evoked predominantly
IPSPs in efferent neurons. In LF cells, IPSPs were observed
in 88 % of the cells tested, and EPSPs were present in 18 %
of the cells. In LG cells, IPSPs were evoked in 100 % of cells
and EPSPs in 18 % of cells. In the in vivo recordings,
however, the response with the lowest threshold for local
electrosensory stimuli in LF cells was an EPSP, which
evoked a brief burst of spikes at higher intensity. The
minimum latency was 3–4 ms. The LF cell in vivo receives
predominantly excitatory input via granule cells from the
sensory stimulation of the center of the receptive field, and
has been classified as an E-cell (excited cell) (Bell et al.,
1997a). The response with the lowest threshold in LG cells
in vivo was generally an IPSP with a minimum latency of
3–7 ms and a duration of more than 100 ms. The LG cells in
vivo receive predominantly inhibitory input via granule cells
from the sensory stimulation to the center of the receptive
field and correspond to the I-cells (inhibited cell) of the
classification of Bell et al. (1997a).
Deep fiber layer stimulation in vitro also evoked
predominantly IPSPs in MG cells. Of 32 MG cells tested,
IPSPs only were recorded in 47 %, EPSPs only were recorded
in 9 % and an IPSP–EPSP sequence was recorded in 6 %.
Strong deep fiber stimulation in vitro evoked small narrow
spikes and also large broad spikes, similar to those evoked by
parallel fiber stimulation (Grant et al., 1998). In vivo,
electrosensory-evoked IPSPs in most MG cells occurred with
minimal latencies of 4–7 ms.
Thus, there are several differences between the afferent
responses recorded in vivo and in vitro. In particular,
electrosensory stimulation in vivo evoked EPSPs in the LF
cells, whereas electrosensory stimulation in vitro evoked
predominantly IPSPs in these neurons. Although the
connection between granule cells and the efferent or MG
cells is still not completely understood, these differences
may arise because the LF cell receives inputs from both the
excitatory and inhibitory granule cells and from inhibitory
interneurons; the total number of inputs from inhibitory
neurons may be larger than the population of excitatory
granule cell inputs. In vitro particularly, it is possible that
field stimulation in the deep fiber layer does not reproduce
exactly the structure of the projection of peripheral
electroreceptive fields and might stimulate a larger
population of inhibitory afferent neurons, masking the
contribution of the granule cells responsible for the
excitatory ‘center’ of the receptive field.
The functional network of the ELL
The cellular mechanisms underlying the integration in the
ELL are not yet completely understood, but some of the roles
and interactions of a number of different neurons have been
identified. In the following paragraphs, we present a schematic
summary sufficient to explain some of the interactions between
neurons in the ELL. The hypothetical neuronal circuit
presented in Fig. 5 is based on known morphology (Meek et
al., 1996; Grant et al., 1996; Bell et al., 1997a,b; Han et al.,
1999) and electrophysiological properties studied in vivo (Bell
et al., 1997a,b) and in vitro (Grant et al., 1998). However, it
also contains several features that are still hypothetical.
The circuit includes seven types of neuron: two types of
efferent neuron (large ganglion and large fusiform cells); two
types of inhibitory Purkinje-like medium ganglion cells (MG1
and MG2); two types of granule cell (one of which inhibits
MG1 and LG cells and one of which excites large fusiform and
MG2 cells); and stellate cells of the molecular layer.
Parallel
fibers
E-module
I-module
(EOCD)
SC
MG1
MG2
OFF image
Inhibitory
bias
LG
Inhibitory
bias
LF
ON image
Juxtalobar
afferents
(EOCD)
Efferent axons
GCs
GCs
Primary
afferents
Inhibitory cell and synapse
Excitatory cell and synapse
Fig. 5. Schematic representation summarizing sensory integration in
the electrosensory lateral line lobe (ELL). The two types of efferent
neuron (large fusiform and large ganglion cells) are shown within
two separate submodules, the E-module and the I-module. Each
module also has a medium ganglion cell and granule cells. The
medium ganglion cells, which are γ-aminobutyric acid (GABA)ergic neurons, control the activity of the efferent neurons (shown as
‘inhibitory bias’ in the diagram). The descending inputs include
EOCD-conveying parallel fibers in the molecular layer and EOCDconveying fibers from the juxtalobar nucleus in the deep part of the
ELL. The excitatory module transfers a positive ‘ON’ image of
electrosensory inputs to higher levels, whereas the inhibitory module
transfers a negative ‘OFF’ image. LF, large fusiform cell; LG, large
ganglion cell; MG1 and MG2, medium ganglion cells; GCs, granule
cells; SC, stellate cell; EOCD, electric organ corollary discharge
(modified from Han et al., 1999).
1308 Y. SUGAWARA AND OTHERS
Morphological reconstructions suggest that MG1 cells
preferentially inhibit large fusiform cells and that MG2 cells
preferentially inhibit large ganglion cells. MG cells are known
to terminate on other MG cells, but whether the connections
are preferentially reciprocal, with MG1 cells inhibiting MG2
cells and vice versa, is still speculative. The circuit can be
understood as a small module that processes electrosensory
information from a local region of the skin and that is
reproduced repeatedly across the electrosensory maps of the
two mormyromast zones.
EOCD signals are shown entering the ELL via parallel fibers
and via fibers from the juxtalobar nucleus. The diagram in
Fig. 5 greatly simplifies the effects of EOCD signals in the
ELL. The juxtalobar input probably does more than simply
gate the electrosensory input to LF, LG and MG cells at the
early stage of integration. It also probably elicits EPSPs and
IPSPs in these cells via direct or indirect connections. EOCD
signals conveyed via the parallel fibers also integrate
proprioceptive information and past electrosensory history in
the form of a feedback pathway from the preeminential
nucleus. In addition, fibers of the direct preeminential tract
originating from the preeminential nucleus terminate in the
lower molecular layer of the ELL, also bringing feedback from
higher levels of the electrosensory pathway and EOCD-related
information. Thus, the late stage of integration in the molecular
layer involves EOCD signals, but also concerns feedback from
other modalities of sensory information as well as the higher
stages of the electrosensory pathway itself.
Following the scheme in Fig. 5, electrosensory stimuli in the
center of receptive fields will evoke EPSPs in LF and MG2
cells, but will evoke IPSPs in LG and MG1 cells. Cells that are
inhibited in the center of their receptive field will receive
excitation from other sources, such as the EOCD, or from the
periphery of their receptive field. Each efferent cell receives
inhibition from a type of MG cell that responds in an opposite
manner to the same sensory stimulus. Efferent cell responses
will be enhanced by such inhibition. LF cells, for example, will
be excited by a stimulus increase in the center of their receptive
field, and the same stimulus will inhibit neighboring MG1
cells. Inhibition of the MG1 cell will disinhibit the LF cell,
enhancing its excitatory response to the stimulus. The
inhibitory control of the efferent neurons exerted by the MG
cells is shown as ‘inhibitory bias’ in Fig. 5.
The two types of efferent cells and their associated MG cells
and granule cells may be viewed as excitatory and inhibitory
submodules, as indicated by the outlined shaded areas in
Fig. 5. The excitatory submodule (E-module), centered on LF
cells, will convey a positive ‘ON’ image to higher levels of the
electrosensory system, i.e. an image in which increases in
discharge rate signal increases in peripheral stimulus intensity.
In this E-module, additive integration in the LF cell will occur
between EOCD-driven EPSPs and sensory input. The
inhibitory submodule (I-module), centered on large ganglion
cells, will convey a negative ‘OFF’ image to higher levels, i.e.
an image in which increases in discharge rate signal decreases
in peripheral stimulus intensity. In this I-module, subtractive
integration in the LG cell will occur between EOCD-driven
EPSPs and sensory input. The two types of submodule may
thus be compared with the ‘on’ and ‘off’ components of the
visual system.
Conclusion
The mormyromast region of the ELL receives afferent input
from mormyromast electroreceptors and relays it to the next
higher level: the preeminential nucleus and the lateral nucleus
of the torus semicircularis. Electric organ corollary discharges
(EOCD) enter by two pathways: via the juxtalobar nucleus
projection to the granule cell layer of the ELL and via the EGp
and the parallel fiber projection to the molecular layer of the
ELL.
The early stage of integration in the ELL may occur in the
granule cells, which receive the afferent input and EOCDdriven input from the juxtalobar nucleus. The granule cells must
have two types of activities, excitatory and inhibitory,
depending on their connection with the primary afferents. In
response to stimuli at the center of the receptive field, excitatory
granule cells will induce EPSPs in the E-module, centered on
the efferent LF cell, and will induce IPSPs in the I-module.
Stimuli to the periphery of the receptive field will evoke an
opposite response for each module. EOCD-driven descending
inputs evoke EPSPs in the apical dendrites just before the
arrival of the reafferent sensory input. Consequently, in the LF
cell of the E-module, EOCD-driven EPSPs and reafferentdriven EPSPs will be summated, leading to additive
depolarization which induces a propagating large narrow spike.
In the LG cell of the I-module, EOCD-driven input in the apical
dendrite evokes an EPSP, but reafferent input will induce
subtractive IPSPs which may block initiation of the spike. Thus,
the E-module will transfer the positive-going ‘ON’ image, and
the I-module will transfer the negative-going ‘OFF’ image to
the next higher level. The MG cells, which receive sensory
input opposite to that of the efferent cell in each module and
also EOCD-driven EPSPs via their apical dendrites, make
inhibitory synapses with the efferent neuron in their module and
thus control its activity. If plastic changes occur at the EOCDdriven synapses, their effects within the network will be visible
as modulation of the output of efferent neurons.
This research was supported by grants from the del Duca
Foundation and Yamada Science Foundation to Y.S., by
grants from the National Science Foundation and the National
Institute of Mental Health to C.B. and by contract C11*CT920085 from the European Economic Community, a grant from
NATO and funds from the Centre National de Recherche
Scientifique to K.G.
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