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Neurological Sciences Institute, Oregon Health and Sciences University, Beaverton, Oregon 97006
Submitted 6 March 2003; accepted in final form 21 April 2003
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Various types of descending inputs have been identified in sensory systems,
including: input from higher levels of the same sensory modality as the
structure under study (Bastian
1986
; Brandt and Apkarian
1992; Sillito et al.
1994; Singer
1995); input from central structures that convey information about
other sensory modalities (Jay and Sparks
1984; Meredith and Stein
1983); and corollary discharge input associated with motor
commands (Bell et al. 1992;
Suga and Schlegel 1972;
Toyama et al. 1984;
Zipser and Bennett 1976). The
last type, corollary discharge input, prepares a sensory region for the
reafferent (von Holst and Mittelstaedt
1950) sensory responses that will arrive as a consequence of the
motor action. All three types of descending input are present in the mormyrid
ELL, but corollary discharge effects are particularly prominent and
particularly accessible in ELL. The present study focuses on the interaction
in ELL between peripherally originating electrosensory information and
centrally originating corollary discharge signals associated with the motor
command that drives the electric organ discharge (EOD). The goal is to
understand how the information provided by these two types of inputs is
integrated within the cells and circuitry of ELL.
The mormyrid ELL is a cerebellum-like structure and the first stage in the
central processing of information from electroreceptors. Primary afferent
fibers from electroreceptors terminate in the deeper layers of ELL where they
form a map of the body surface. Purkinje-like cells, known as medium
ganglionic (MG) cells, and efferent cells of ELL are strongly affected by the
afferent input from electroreceptors, the afferent input being relayed to the
basilar dendrites of these cells via ELL granular cells that receive the
afferent input directly. The apical dendrites of MG cells and efferent cells
extend up into a molecular layer where they are contacted by parallel fibers
that originate from an external granule cell mass known as the eminentia
granularis posterior (EGp). As with true cerebellar Purkinje cells in all
ray-finned fish, the MG cells of ELL are inhibitory interneurons that
terminate locally on efferent neurons. Other types of interneurons, such as
granular cells, stellate cells, thick smooth dendrite cells and medium
fusiform cells are also present in ELL (see
Han et al. 1999;
Meek et al. 1996 for a
description of the morphology of ELL cells).
Primary afferent fibers from two types of electroreceptors terminate in the
cortex of ELL (Bell 1990b;
Bell et al. 1989). Afferent
fibers from mormyromast electroreceptors, the type of electroreceptors
responsible for active electrolocation, terminate in the medial and
dorsolateral zones of ELL. Afferent fibers from ampullary electroreceptors,
the type responsible for low-frequency passive electrolocation, terminate in
the ventrolateral zone. The mapping in all three zones is somatotopically
organized. This study is concerned only with the mormyromast zones of ELL.
Some of the electric organ corollary discharge (EOCD) effects in ELL are
plastic and depend on the sensory input that has followed the motor command in
the previous few minutes (Bell and Grant
1992; Bell et al.
1997b). Other EOCD effects appear to be fixed and are not affected
by previous pairing with a sensory stimulus
(Bell and Grant 1992). The
EOCD signals enter ELL via three different pathways: parallel fibers from EGp
(Bell and Szabo 1986;
Maler 1973); fibers from a
higher-order electrosensory nucleus, the nucleus preeminentialis
(Bell et al. 1981); and fibers
from the juxtalobar nucleus (Bell et al.
1981).
A previous study of the mormyrid ELL examined the electrosensory and EOCD
responses of MG cells and efferent cells
(Bell et al. 1997b). This
description was incomplete, however, because the responses of several
morphologically distinct cell types were not determined. Most importantly, two
types of MG cells (MG1 and MG2) can be distinguished morphologically
(Han et al. 1999;
Meek et al. 1996), but no
corresponding difference was established physiologically in the previous
study. The morphology suggested that MG1 cells might be inhibited by
electrosensory stimuli (I cells), whereas MG2 cells might be excited (E
cells), but the previous study found mostly inhibitory receptive fields for MG
cells and the morphology was not good enough to distinguish the two types of
MG cells (Bell et al. 1997b;
Grant et al. 1998).
This first paper in the present series of two papers describes the electrosensory and EOCD responses of cell types, which had been previously described morphologically but not physiologically. These cells include the two types of MG cells, the thick smooth dendrite cells and the medium fusiform cells. New information is also provided concerning the common features of MG cells, the two types of efferent cells, and the properties of two previously undescribed cell types, the interzonal cell and the large thick smooth dendrite cell. The second paper in this series examines the origins of EOCD responses in ELL by recording the responses of morphologically identified ELL cells to electrical stimulation of two of the three sources of EOCD input to ELL, the juxtalobar and preeminential nuclei.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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). All
experiments that were performed in this study adhere to the American
Physiological Society's Guiding Principles in the Care and Use of
Animals and were approved by the Institutional Animal Care and Use
Committee of Oregon Health and Sciences University. Overview
Mormyrid fish of the species Gnathonemus petersii were used in these experiments. Surgery was done under anesthesia and curare was given after the surgery. Curare blocks the effect of electromotoneurons on the electric organ, preventing the EOD; but the motor command signal that would normally elicit an EOD continues to be emitted by the electromotoneurons at a variable rate of 2–5 Hz. Responses of ELL cells to the motor command alone are referred to as EOCD responses. The curare makes it possible to examine the EOCD responses in isolation from the EOD that normally follows the motor command and to control the electrosensory input that the cells receive. Responses to the motor command alone, to electrosensory stimuli alone, and to the motor command plus an electrosensory stimulus delivered at various delays were examined. EOCD plasticity was examined by delivering electrosensory stimuli at a fixed delay following the EOD command signal for 1–3 min and comparing the EOCD responses before and after such pairing.
ELL cells were recorded intracellularly from the medial and dorsolateral
zones of ELL, the two zones that receive input from mormyromast
electroreceptors (Bell and Grant
1989). The cell types are quite similar in the two zones and most
of the recordings were taken from the medial zone, which is larger in size and
more accessible than the dorsolateral zone.
The field potentials evoked by electrosensory stimuli and by the EOCD in
ELL are prominent and change dramatically as a function of depth
(Bell et al. 1992), allowing
one to determine the ELL layer in which the recording electrode was positioned
from the extracellular potentials recorded just outside the cell. The
different layers of ELL are as follows (from the external to the internal
surface of ELL): molecular, ganglionic, plexiform, superficial granular, deep
granular, intermediate, and fiber (Grant
et al. 1996; Meek et al.
1999). In most cases, the field potentials were recorded just
outside a cell after intracellular recording, using the same electrode. In
most cases, the field potentials were averaged and subtracted from averaged
intracellular recordings to determine the true transmembrane potential changes
evoked by electrosensory stimuli and the EOCD.
Surgery
A total of 42 fish with body lengths between 11.5 and 18 cm were used. The skull was exposed under anesthesia (MS 222, 1:25,000), and a plastic rod was cemented to the skull anteriorly to hold the head rigid. The posterior part of the skull was removed, and the underlying valvula cerebelli was reflected laterally to expose the molecular layer of the caudal lobe of the cerebellum and the EGp. The ELL is located just beneath these structures. Curare (d-tubocurarine, 10 µg/cm of body length) was given at the end of the surgery, the anesthetic was removed, and aerated water was passed over the fish's gills for respiration.
Recording, stimulation, and data analysis
The EOD command signal was recorded with an Ag-AgCl silver plate placed
over the electric organ. The command signal lasts 
3 ms and consists of a
small negative wave followed by three larger biphasic waves
(Fig. 2, B and
D, bottom). The latencies of synaptic and spike
responses of ELL neurons to the EOCD were measured with respect to the
negative peak of the first large biphasic wave in the command signal (time
0 or t0 in Bell
et al. 1992). In the absence of curare, the EOD occurs 4.5 ms
after t0.
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Intracellular recordings were made with sharp microelectrodes filled with
2% biocytin in 2 M potassium methyl sulfate (180–250 M
). Biocytin
was injected into recorded cells by passing depolarizing intracellular current
pulses at 1 Hz with a duty cycle of 50% and amplitudes of 1–1.2 nA for
5–12 min.
Electrosensory responses were evoked by means of a bipolar stimulating electrode consisting of two small Ag-AgCl balls 6 mm apart. The electrode was held with the axis of the dipole perpendicular to the skin. Individual electroreceptors can be easily distinguished on the skin surface with an operating microscope, and the stimulating electrode could be placed close to the individual receptors. Brief pulses of current (100 µs, 1.5–100 µA) were delivered through the electrode to activate electroreceptors. All cells were tested with sensory stimuli at the EOD delay of 4.5 ms. Cells were also examined either independently of the motor command or at long delays of 60–100 ms to examine the effect of a sensory stimulus alone.
The latency of the electrosensory response of mormyromast afferent fibers
decreases by 9–11 ms as intensity is increased from threshold intensity
to the intensity that gives a maximum response (Bell
1989,
1990a). The latencies reported
in this study for the electrosensory responses of different ELL cells are
minimal latencies obtained by increasing stimulus intensity until no further
reduction in latency was observed. The current intensity needed to obtain the
minimal latency varied from cell to cell. Use of minimal onset latencies makes
it possible to compare the timing of responses in different cells.
Data were recorded on tape and analyzed off-line with a Cambridge Electronic Design interface and with the same company's software. For statistical comparisons, we used the t-test.
Spikes were sometimes truncated by a linear extrapolation from the
beginning of each spike to the end of the spike to estimate the size of the
underlying excitatory postsynaptic potentials (EPSPs). For truncation
purposes, we determined the beginning of each spike by first determining the
time of a threshold crossing in the voltage record (for broad spikes) or in
the derivative of the voltage record (for small spikes). Aligning and
averaging the spikes about this threshold crossing allowed us to establish the
time relative to the threshold crossing when the spike began and when the
spike ended. The beginning of the spike was 2 ms before the threshold
crossing for broad spikes and 1 ms before the threshold crossing for
small spikes.
Histology
After the experiment, fish were anesthetized in concentrated MS 222
(1:10,000) and perfused through the heart with teleost Ringer solution,
followed by a fixative, consisting of 2% paraformaldehyde and 2%
glutaraldehyde in 0.1 M phosphate buffer. The brains were postfixed overnight
and cryoprotected with 20% sucrose. Cryostat sections (50 µm) were reacted
with avidin-biotin complex and diamino-benzidine to reveal the biocytin. The
procedure was the same as that used by Han et al.
(1999) except that
CoCl2 (0.02%) and ammonium nickel sulfate (0.02%) were added to the
bath during development to enhance the reaction. Sections were mounted on
slides and counterstained with Richardson's stain. Reconstructions of cells
were made with a camera lucida attachment to the microscope.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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The Purkinje-like MG cells are a major cell type of ELL and are probably of
central importance to its function. Synaptic plasticity has been demonstrated
at the synapse between parallel fibers and the apical dendrites of MG cells
(Bell et al. 1997c;
Han et al. 2000), and this
plasticity can explain the adaptive sensory processing that occurs in ELL
(Bell et al. 1993,
1997b;
Grant et al. 1998). Two
morphologically distinct types of MG cells, MG1 and MG2, have been identified.
The two cell types have similar apical dendrites, but their basal dendrites
and axonal arbors terminate in different layers of ELL
(Meek et al. 1996;
Han et al. 1999)
(Fig. 1). The dendritic
morphology suggested the hypothesis that MG2 cells, with basal dendrites in
the region of termination of primary afferent fibers, might be excited by
electrosensory stimuli, whereas MG1 cells, with basal dendrites external to
the region of afferent termination, might be inhibited by electrosensory
stimuli (Han et al. 1999;
Meek et al. 1996). We tested
this hypothesis in the present experiments and also established some
additional, previously undescribed, features of MG cells.
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Seven MG1 and four MG2 cells were identified morphologically in this study. An additional four cells were morphologically identified as MG cells, but the basal dendrites and axons were not sufficiently stained to identify the cells further as MG1 or MG2 cells. We first describe the physiological properties that are shared by MG1 and MG2 cells and then describe the differences between the two cell types.
TWO TYPES OF SPIKES IN MG CELLS. Previous in vivo
(Bell et al. 1997b) and in
vitro studies (Grant et al.
1998) showed that MG cells have two types of spikes, a large broad
spike and a small narrow spike (Fig. 2,
A, C, and D). MG cells are the only cells in ELL
with large broad spikes (Bell et al.
1997b; Grant et al.
1998), and the presence of these spikes can therefore be used to
identify MG cells electrophysiologically. We identified 62 cells as MG cells
by this physiological feature, including the 15 MG cells identified
morphologically. The large broad spikes ranged from 20 to 67 mV in amplitude
(44.3 mV ± 9.7 mV (mean ± SD), n = 48) and 8–23
ms in duration (14 ± 3.4 ms, n = 48). The small narrow spikes
ranged from 1.6 to 13.5 mV (6.6 ± 3.1 mV, n = 49) and were
2–6.2 ms (3.9 ± 0.9 ms, n = 49) in duration.
EOCD-evoked field potentials recorded extracellularly immediately after the
intracellular recordings allowed us to estimate the ELL layer from which the
recordings were taken. Broad spikes of similar amplitudes and durations were
recorded in both the outer and inner halves of the molecular layer, suggesting
that the broad spikes are propagated actively into the apical dendrites of MG
cells. This result is consistent with other evidence for such propagation
obtained in in vitro slices (Grant et al.
1998). In contrast, the small spikes of MG cells were either
absent in the outer molecular layer or of lower amplitude than in the inner
molecular layer or ganglionic cell layer (data not shown), consistent with the
hypothesis that the small spikes are axon spikes that are not actively
propagated into the soma or dendrites of MG cells
(Grant et al. 1998).
EOCD RESPONSES. The EOCD evoked an EPSP in most MG cells (Fig. 2, A and B). The EPSPs had amplitudes of 0.6–6 mV (4.7 ± 4.9 mV, n = 54), durations of 45–100 ms (n = 54), and latencies following t0 of 5.2–13.2 ms (8.5 ± 1.7 ms, n = 35). The EPSP usually evoked a burst of small spikes in which the first spike of the burst was more sharply time-locked to the command signal than later spikes (Fig. 2A). In some cells, the EOCD EPSP evoked only a single small spike (Fig. 2B) or no spike at all (middle, Fig. 4A).
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The effect of the EOCD on broad spikes could not be easily determined in
most MG cells because of the low probability of these spikes. Broad spikes
occurred spontaneously in 17 MG cells, however, allowing the EOCD responses to
be observed. In these cells, the EOCD usually evoked only an inhibition of
broad spikes (11/17 cells; Fig.
2C). The inhibition, as indicated by a consistent pause
in spontaneous broad spike activity, started at 20 ms after
t0 and lasted between 10 and 80 ms. In some cells, the
EOCD had an initial excitatory effect, evoking a time-locked broad spike
between 12 and 18 ms after t0 (6 of 17 cells;
Fig. 2D). The
time-locked broad spike of these cells was followed by a brief period in which
no broad spikes occurred. This brief period without broad spikes was not due
to refractoriness because it was also present in those sweeps in which the
time-locked broad spike did not occur. Thus the brief initial excitation of
broad spikes in some MG cells was followed by an inhibition.
The inhibition of broad spikes was particularly striking in that it occurred during the time of the EOCD-evoked EPSP and its accompanying burst of small spikes (Fig. 2, C and D). The presence of two such opposing EOCD effects in MG cells was further indicated by the finding that some cells showed an EOCD-evoked EPSP at one time during the recording and an EOCD-evoked inhbitory postsynaptic potential (IPSP) several minutes later. Transformation from an EPSP to an IPSP often occurred after several minutes of injecting current (1–1.2 nA) into a cell for morphological identification (Fig. 3A). The shape of the EPSP and the IPSP were not identical, usually the EPSP lasted longer (Fig. 3A, 2nd trace). In some cases, the postinjection IPSP changed back into an EPSP (Fig. 3A, 3rd trace) over several additional minutes of recording. The differences between depolarizing and hyperpolarizing EOCD responses were large and were not accompanied by any apparent changes in membrane potential. Thus, it is unlikely that the EOCD responses are due to an IPSP alone, that is, an IPSP which is either depolarizing or hyperpolarizing depending on the membrane potential and the IPSP reversal potential. Moreover, our electrodes did not contain any chloride ions so that the current injection would not have altered the chloride concentration in the cell and the reversal potential for IPSPs.
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Evidence for simultaneous EOCD excitation and EOCD inhibition was also obtained in one cell in which depolarizing the cell from resting potential with increasing amounts of injected current caused a progressive reduction of the EPSP and appearance of an IPSP which was shorter in duration (Fig. 3B, compare a and d). The different effects of the EOCD on broad spikes and small spikes indicates different initiation sites for the two types of spikes, with EOCD-driven inhibition having a stronger effect on broad spike initiation and EOCD-driven excitation having a stronger effect on small spike initiation.
The delivery of intracellular current pulses to evoke broad spikes at different delays after the command signal also revealed the EOCD-evoked inhibition of broad spikes. The current required to evoke broad spikes at a short delay of 20 ms was consistently greater than that required to evoke spikes at other delays (Fig. 3C). The effect was observed in four of the five cells tested.
RESPONSES TO ELECTROSENSORY STIMULI. Although the EOCD responses of MG1 and MG2 cells were the same, the responses to electrosensory stimuli were quite different. Our hypothesis that MG1 cells are inhibited by electrosensory stimuli and MG2 cells are excited was confirmed.
All seven of the morphologically identified MG1 cells responded with an
IPSP to electrosensory stimuli delivered within a restricted skin region. The
IPSP was often larger when the stimulus was given at the EOD delay of 4.5 ms
after t0 of the EOD motor command
(Fig. 4A,
bottom) than when it was given at long delays or independently
(Fig. 4A,
top). Thus the EOCD facilitates the inhibitory effect of
electrosensory stimuli on MG1 cells, when such stimuli are given at the time
of the EOD. The enhanced IPSP was sometimes followed by an increased
excitation (Fig. 4A,
bottom) although no EPSC was observed to the sensory stimulus alone
(Fig. 4A,
top). The excitation following the IPSP could be due to an alteration
in the input to the cell through the circuitry of ELL or to an intrinsic
postinhibitory rebound of the postsynaptic membrane, such as that which occurs
when T type calcium channels are present
(Carbone and Lux 1984). In
some cells, delivery of the sensory stimulus at the EOD delay markedly
depressed the EOCD evoked excitation without evoking an actual
hyperpolarization (Fig.
4B). An additional 10 MG cells, identified as such by the
occurrence of a broad spike, responded to electrosensory stimulus with an IPSP
and showed similar EOCD facilitation of the inhibitory sensory effect or
suppression of the EOCD EPSP, just like the seven morphologically identified
MG1 cells. We classified all 17 cells as MG1 cells.
Low-intensity electrosensory stimuli close to threshold-evoked IPSPs from three to six neighboring electroreceptors in MG1 cells. Increases in stimulus intensity caused an increase in IPSP amplitude and a decrease in latency. Thresholds were between 2.5 and 6 µA (4.1 ± 1.1, n = 10). Minimum latencies at the highest stimulus intensities were between 2.8 and 5.1 ms (3.9 ± 0.8 ms; n = 13). No excitatory responses were observed when the stimulus electrode was placed just outside the cluster of receptors where stimulation caused an IPSP. Thus there was no evidence of an opponent, excitatory surround outside the region of inhibition.
Electrosensory responses of MG2 cells were quite different from those of MG1 cells. The difference was particularly striking when stimuli were given at the EOD delay. In all four of the morphologically identified MG2 cells, the excitatory response to the EOCD plus an electrosensory stimulus was considerably greater than the response to the EOCD alone (Fig. 4, C and D). This enhancing effect of an electrosensory stimulus on the EOCD response contrasted with the suppressive effect of such a stimulus in MG1 cells. An additional seven MG cells showed the same enhancing effect of an electrosensory stimulus as observed in the four morphologically identified cells. We classified all 11 cells as MG2 cells. We conclude that electrosensory stimuli given at the time of the EOD have an inhibitory effect on MG1 cells (I cells) and an excitatory effect on MG2 cells (E cells).
The sensory responses of MG2 cells were more difficult to determine than those of MG1 cells. The thresholds were more variable and often much higher than those of MG1 cells (range: 6–50 µA, 24 ± 13 µA, n = 10). The responses to sensory stimuli given independently of the command were smaller and more varied than those of MG1 cells. Electrosensory stimuli given independently of the command evoked a small EPSP in six cells (Fig. 4C, top), a broad spike with no clear underlying EPSP in two cells, and a small IPSP-EPSP in three cells (Fig. 4D, top). The minimum latencies of sensory responses were between 4.9 and 12.9 ms (9.6 ± 2.9 ms). We expected that MG1 and MG2 cells would have similar sensitivities to electrosensory stimuli. The finding of higher thresholds and greater variability of electrosensory responses in MG2 cells than in MG1 cells was unexpected therefore and the reason for these differences is not known.
The occurrence of EPSPs in some MG2 cells and IPSPs in other MG2 cells in response to stimuli given independently of the command suggests the possibility of receptive fields with center-surround organization. But no evidence for such organization was obtained. Individual cells showed only EPSP responses or only IPSP-EPSP responses regardless of the location of the stimulus on the skin. The variations in electrosensory responses of MG2 cells point to the complexity of information transfer through the ELL granular layer from afferents to the basilar dendrites of MG2 cells.
PLASTICITY OF THE COROLLARY DISCHARGE RESPONSE. The responses of
MG cells to the EOCD have been previously shown to be plastic. The EOCD
responses could be altered by pairing the EOD motor command for a few seconds
to a few minutes with intracellular current injections that evoke broad spikes
(Bell et al. 1997b) or with
electrosensory stimuli in the receptive field of the cell
(Bell et al. 1997b). We tested
the effects of pairing the EOD motor command with electrosensory stimuli to
compare plasticity in MG1 and MG2 cells.
EOCD excitation of MG1 cells was plastic. EOCD excitation after 2–3 min of pairing with an inhibitory electrosensory stimulus given at the EOD delay was stronger than EOCD excitation before the pairing. After pairing, the EOCD evoked a larger EPSP, more spikes, or spikes at a shorter delay (Fig. 5). Both broad and small spikes were affected. The plasticity could also be observed during the pairing period as a gradual decrease in the inhibitory response to the combined effects of the EOCD and a sensory stimulus (note smaller IPSP in EOCD+SS end than in EOCD+SS start in Fig. 5A). Plasticity was observed in seven MG1 cells out of the eight tested.
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EOCD excitation of MG2 cells was also plastic, but the plasticity was less marked than in MG1 cells. In seven of eight cells tested, the number of spikes in the EOCD response showed a slight increase after 2–3 min of pairing with an excitatory sensory stimulus (Fig. 6). In three cells, this increase in spike number was significant (P < 0.001). When the pairing with a sensory stimulus evoked a broad spike (3 of 7 cases), the amplitude of the underlying EOCD EPSP was reduced significantly (P < 0.05) (Fig. 6A; compare EPSP in EOCD before trace with EPSP in EOCD after trace). This apparently paradoxical result, of a decrease in EPSP size with an increase in the number of evoked spikes, was not accompanied by a change in the recorded membrane potential. The increased spike number could be due to a change in the intrinsic excitability of the cell, but this possibility was not tested directly. When no broad spike was evoked during the pairing, the EOCD EPSP after the pairing either remained the same or was slightly increased (4 of 7 cases; Fig. 6C). These latter amplitude changes were not significant.
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Thick smooth dendrite cells
Thick smooth dendrite (TSD) cells have been previously described
morphologically (Han et al.
1999; Meek et al.
1996), and in vitro studies have shown that they respond to
parallel fiber stimulation with an EPSP and to stimulation in the deep layers
of ELL with an IPSP (Grant et al.
1998). However, the physiological responses of TSD cells to the
EOCD or to electrosensory stimuli have not been determined. Seven cells were
morphologically identified in this study after intracellular recording.
TSD cells are non-GABAergic interneurons with cell bodies in the ganglionic
or plexiform layer (Han et al.
1999; Meek et al.
1996). The dendritic morphology is unusual for a vertebrate neuron
in that one thin dendrite arises from the cell body and leads to one
(Fig. 7A) or two
(C) remarkably thick branches in the molecular layer that
in turn give rise to additional thinner branches. The molecular layer
dendrites are confined to the lower half of this layer. Some of the finer
dendritic branches are recurrent and descend back into the ganglionic and
plexiform layers. The extent of the apical dendrite varied between 140 and 400
µm in the mediolateral direction and 150 and 500 µm in the rostrocaudal
direction in the cells that we examined morphologically.
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The axons of TSD cells descend from the soma to branch in the granular
layers. The axonal arbors extend 500 µm in the transverse direction
and 400 µm in the rostrocaudal direction. Axonal swellings, which could be
synaptic terminals, are present in both the deep and superficial granular
layers and in the intermediate layer. Although the axonal arbor covers a large
area, the branching is sparse and the number of swellings is <50. The
extent of the axonal arbor of TSD cells found in our study was larger than
that found in a previous in vitro study of ELL cells
(Han et al. 1999) due most
probably to the severing of axonal branches during preparation of the in vitro
slices.
We supplemented our physiological findings from the seven morphologically identified TSD cells with recordings from an additional six cells that had the same physiological properties. The EOCD elicited a large, stereotyped EPSP (4–12 mV) in TSD cells that evoked a brief burst of one to six small spikes (2–9.5 mV; 6 ± 2 mV, n = 13; Fig. 7, B and D). The spike responses, especially the first spike, showed minimal temporal variability in relation to the time of the command signal. The latency of the EOCD EPSP following the command was very short, (2.5–4.2 ms in different cells; 3.5 ± 0.9 ms, n = 13), shorter than that in any other cell. EPSP durations were between 30 and 62 ms in TSD cells. The EOCD-evoked excitation, in form of synaptic responses and spikes, was the only activity that we observed in these cells in the absence of electrosensory stimuli.
Electrosensory stimuli given independently of the command evoked an EPSP-IPSP (Fig. 7D) in 9 of the 10 cells tested, of which 6 were morphologically identified. Only one cell responded with a pure IPSP (Fig. 7B). Minimal latencies (obtained at near maximal stimulus intensity) of the sensory responses were short, between 1.9 and 2.9 ms (2.4 ± 0.3 ms). Even high-stimulus amplitudes never evoked a spike in response to the sensory stimulus. Receptive fields were small, consisting of one to three electroreceptors. Stimuli outside this cluster of receptors did not elicit any responses, and thus there was no indication of an opponent surround to the receptive field.
Somewhat surprisingly, electrosensory stimuli given at the EOD delay caused a consistent and strong inhibition of the EOCD-evoked EPSP and spike burst (Fig. 7, B and D, bottom) even though stimuli given independently of the command usually evoked EPSP-IPSP sequences. We determined the net response of a sensory stimulus at the time of the EOCD by subtracting the response to the EOCD alone from the response to the EOCD plus a sensory stimulus. The net response of eight cells was a pure IPSP. Five cells showed an EPSP-IPSP sequence as a net response, but only the IPSP component in these cells was enhanced in comparison to the response evoked by an independent sensory stimulus. Thus the interaction between the EOCD and electrosensory inputs was markedly nonlinear for TSD cells. The electrosensory stimulus blocked later spikes of the EOCD-evoked burst but did not block the first spike, even at the strongest stimulus intensities.
EOCD excitation of TSD cells appeared to be plastic in the two cells tested. The EOCD EPSP was larger and evoked more spikes after 2 min of pairing with a sensory stimulus (Fig. 8).
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A previous extracellular study of ELL described cells with a short-latency,
stereotyped burst response to the EOCD that was inhibited by a well-localized
sensory stimulus (Bell and Grant
1992). Such cells were referred to in the previous study as
I1 cells. The close similarities with TSD cells indicate that the
previously described I1 cells were almost certainly TSD cells.
Medium fusiform cells
Medium fusiform cells have been previously described morphologically, and
an in vitro study have shown that they respond to parallel fiber stimulation
with an EPSP and also to stimulation of the deep layers of ELL with an EPSP.
These cells were previously referred to as "small fusiform cells"
(Han et al. 1999;
Meek 1993) but are now
referred to as "medium fusiform cells" because a different and
still smaller fusiform cell has been identified morphologically in ELL (J.
Meek, personal communication). Four medium fusiform cells were morphologically
identified in this study after intracellular recording.
Medium fusiform cells are GABAergic interneurons with cell bodies in the
granular layer and a thick apical dendrite that extends up into the deep
molecular layer where most of the dendritic tree is located
(Fig. 9A)
(Han et al. 1999;
Meek 1993). The apical
dendrite gives off some branches to the granular, plexiform and ganglionic
layer as it ascends, and some thin basilar dendrites arise from the cell body
in the granular layer. The apical dendritic arbor extends further in the
rostrocaudal direction (150–300 µm) than in the transverse
(80–140 µm). As with the TSD cells, our in vivo material yielded a
more complete description of the axonal arbor of medium fusiform cells than
was possible in previous morphological studies using the Golgi technique
(Meek et al. 1996) or
intracellular labeling in in vitro slices
(Han et al. 1999). The axonal
arbor of medium fusiform cells is similar to that of the TSD cell. The axon
exits from the base of the soma and sends branches into the superficial
granular, deep granular and intermediate layers. The arbor extends a large
distance (250–650 µm) in both the transverse and rostrocaudal
directions, but the branching is sparse, and the number of swellings or
presumed terminals is small.
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We supplemented our physiological findings from the four morphologically
identified cells with recordings from an additional three cells that had the
same physiological properties. The EOCD elicited a brief (20–37 ms)
stereotyped EPSP at a latency of 4.6–6.4 (5.5 ± 0.7) ms after the
command signal (Fig. 9B; middle in
C and D). This EOCD response resembled the
response recorded inside primary afferents that is presumed to be due to EOCD
input to granular cells and that is observed inside the afferents because of
the electrical synapses that the afferents make on granular cells (see
following text) (Bell 1990a;
Bell et al. 1997b). The EPSP
often gave rise to a single spike (Fig.
9B). The spike had amplitudes of 12.3–36 mV (22.7
± 8.5 mV) and a prominent afterhyperpolarization of 4–10.2 ms
(7.5 ± 2.2).
Electrosensory stimuli given independently of the command evoked a brief EPSP with a short minimal latency of 2–2.8 ms (2.4 ± 0.2 ms) in the medium fusiform cells (Fig. 9, C and D, top). For electrosensory stimulation, all cells with a spike in the EOCD response were hyperpolarized to reveal the underlying EPSP. When the electrosensory stimuli were given at the time of the EOD, the electrosensory EPSPs and EOCD EPSPs summed, and the summed excitation could elicit a spike (Fig. 9D, bottom). Increasing the stimulus amplitude never evoked more than one spike. The summation of the electrosensory and EOCD EPSPs was linear, in contrast to the nonlinear summation of these two signals in MG and TSD cells. The response to the two signals given together was the same as the sum of the two independent responses (Fig. 9, C and D, inset). No EOCD plasticity was observed in these cells. The EOCD response after pairing with an electrosensory stimulus was the same as the EOCD response before pairing.
Large fusiform cells
Large fusiform cells are one of the two types of efferent cells in ELL.
These cells have been examined previously both in vivo
(Bell et al. 1997b) and in
vitro (Grant et al. 1998). The
in vivo study showed that electrosensory stimuli delivered at the time of the
EOD to the centers of the receptive fields of these cells are excitatory (E
cells) and that the response to the EOCD is markedly plastic, being strongly
affected by a period of pairing with an electrosensory stimulus. Our
recordings from these cells confirm the previous findings and provide some
additional information about these large fusiform cells.
The cell bodies of large fusiform cells are in the granular layer or at the
border between the plexiform and granular layers
(Grant et al. 1996)
(Fig. 10A). Basilar
dendrites are in the granular layers and apical dendrites extend throughout
the molecular layer. Four large fusiform cells were intracellularly recorded
and morphologically identified in this study. The axons could be followed all
the way into the lateral lemniscus, confirming that they were efferent
cells.
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We supplemented the physiological findings from the four morphologically
identified cells with recordings from seven additional cells that had similar
physiological properties. Spikes ranged from 16 to 60 mV and were followed by
an after-hyperpolarization. The EOCD evoked an IPSP in two of the
morphologically identified cells and in four of the cells that were not
identified (Fig.
10B). The onset of the IPSP varied between 6.9 and 17.3
ms (11.9 ± 3.6 ms). The IPSPs were sometimes preceded by a small EPSP
that evoked a spike. The EOCD evoked EPSPs in two of the morphologically
identified cells and three of the cells that were not identified
(Fig. 10C). EPSP
onsets were between 6.3 and 6.5 ms. A previous study found only EOCD-evoked
IPSPs in these cells (Bell et al.
1997b). Large fusiform cells showed either EPSPs or IPSPs in
response to the EOCD. None of the cells showed an EPSPs at one time and IPSPs
at another. Thus although it is possible that the EOCD simultaneously evokes
both inhibition and excitation in large fusiform cells as suggested for MG
cells, with the relative strengths of these two inputs varying from cell to
cell, we have as yet no evidence for such a possibility.
The EOCD alone could elicit EPSPs or IPSPs, but the response to electrosensory stimuli given at the time of the EOD was always excitatory. Electrosensory stimuli given independently of the command elicited EPSPs that could trigger spike trains at higher stimulus amplitudes. Minimal latencies of the EPSPs ranged from 3.7 to 4.4 ms (4 ± 0.3 ms). The excitatory response to a sensory stimulus was greatly facilitated when the stimulus was given at the EOD delay. Stimuli that were ineffective when given independently could elicit vigorous responses when given at the EOD delay (Fig. 10, B and C). Thus the interaction between EOCD and electrosensory inputs was markedly nonlinear in large fusiform cells. This nonlinear interaction is probably due to the local circuitry and the presence of an interneuron between the primary afferent input and the large fusiform cell, an interneuron that is excited by both primary afferent input and by the EOCD (see DISCUSSION).
EOCD responses were clearly plastic in large fusiform cells; both in cells with an EOCD-evoked EPSP and in cells with an EOCD-evoked IPSP. Pairing with an excitatory sensory stimulus for 2–3 min caused a reduction in the EOCD-evoked EPSPs (in all 3 of the 3 cells tested; Fig. 12A) or an increase in the EOCD-evoked IPSPs (in 1 of the 2 cells tested; Fig. 12B).
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Large ganglionic cells
Large ganglionic cells are the second type of efferent cell in ELL. These
cells have also been examined previously both in vivo
(Bell et al. 1997b) and in
vitro (Grant et al. 1998). The
in vivo study showed that electrosensory stimuli delivered at the time of the
EOD to the centers of the receptive fields of these cells are inhibitory (I
cells) and that the response to the EOCD is markedly plastic, being strongly
affected by a period of pairing with an electrosensory stimulus. Our results
concerning large ganglionic cells are described here for comparison with other
cell types and as confirmation of previous findings. Four large ganglionic
cells were intracellularly recorded and morphologically identified in this
study.
The cell bodies of large ganglionic cells are in the ganglionic layer.
Their basilar dendrites are in the plexiform layer and their apical dendrites
extend into the molecular layer. (Grant et
al. 1996) (Fig.
11A). The axons of large ganglionic cells, like those of
large fusiform cells, could be followed into the lateral lemniscus, confirming
that they were efferent cells.
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We supplemented our physiological findings from the four morphologically identified cells with recordings from eight additional cells that had similar physiological properties. The EOCD evoked only minimal responses from large ganglionic cells, provided that no electrosensory stimulus had been paired with the EOCD during the preceding 4 or 5 min. A small EPSP was evoked in some cells at a latency of 7.8–11.6 ms, and this EPSP sometimes triggered spikes (Fig. 11C, middle). Spikes ranged in amplitude from 21 to 40 mV and were followed by an afterhyperpolarization.
Electrosensory stimuli delivered independently of the command evoked long-lasting IPSPs with minimal latencies between 2.4 and 3.6 ms (3.2 ± 0.5 ms). These IPSPs were markedly facilitated when delivered at the EOD delay (Fig. 11, B and C). Thus the interaction between these two signals was clearly nonlinear as in MG, TSD, and large fusiform cells.
EOCD responses were clearly plastic in large ganglionic cells. Pairing with an inhibitory sensory stimulus for 2–3 min resulted in a decrease in the IPSP amplitude during the pairing and an EOCD-evoked burst of spikes after the electrosensory stimulus was turned off (Fig. 12C). An increase in EOCD excitation was observed even after pairing periods as short as 10 s.
Interzonal cell
A previous anatomical study with tracer substances showed that the two
mormyromast zones of ELL, the medial and dorsolateral zones, are mutually
interconnected (Bell et al.
1981). Cells of the medial zone project to the dorsolateral zone
and vice versa. But the cells of origin of these projections were not
morphologically described, and their physiology was unknown. In this study, we
recorded and stained a cell in the medial zone that projected to the
dorsolateral zone.
The cell body was in the deep plexiform layer (Fig. 13A). A single apical dendrite ascended into the molecular layer, and three basal dendrites descended into the superficial granular layer. The apical dendrite thickened as it ascended and branched repeatedly to form an arbor in the inner half of the molecular layer with some recurrent branches being given off to descend back toward the ganglionic and plexiform layers. All of the dendrites were without spines. The axon of this interzonal cell descended into the deep granular layer where it branched, forming a rather sparse arbor that extended 285 µm in the transverse direction and 200 µm in the rostrocaudal direction. Individual branches ended in distinct clusters of presumed terminals in the deep granular layer. One axonal branch descended further into the deep fiber layer and continued 400 µm rostral and lateral to enter the dorsolateral zone of ELL where it branched to terminate in the superficial and deep granular layers of that zone (Fig. 13A, inset). The terminal arbor in the dorsolateral zone was more extensive than that in the medial zone. The axonal branch to the dorsolateral zone terminates 400 µm rostral to the location of the cell body in the medial zone. This termination region and the region of the medial zone in which the cell body is located correspond somatotopically; that is, both regions receive input from the same point on the skin surface(the rostral limit of the dorsolateral zone extends beyond the rostral limit of the medial zone).
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The EOCD evoked a large EPSP with a latency of 7.7 ms
(Fig. 13B,
middle) in this interzonal cell. The EPSP had two peaks with a small
spike occurring on top of the first peak. The second peak was followed by a
slowly declining depolarization lasting 70 ms. Electrosensory stimuli
given independently of the command evoked a complex response consisting of an
initial EPSP with a minimal latency of 2.7 ms, followed by an IPSP with
multiple and variable peaks (Fig.
13B, top). Responses could be evoked from a
cluster of five neighboring receptors using low-intensity stimuli. Delivery of
the electrosensory stimuli at the EOD delay caused a decrease in the second
peak of the EOCD EPSP. Pairing with the electrosensory stimulus for 2 min did
not affect the EOCD response.
The morphology of the interzonal cell is similar to that of the TSD cell, but there are also important differences. The interzonal cell had several basal dendrites in addition to the one apical dendrite, but the TSD cells did not have any basal dendrites, and none of the TSD cells had axonal branches terminating in the other mormyromast zone. The physiology was also somewhat similar to TSD cells, both cells showing an EPSP to the command that was significantly reduced by an electrosensory stimulus given at the time of the EOD. But the EOCD EPSP of the medium fusiform cell was longer in latency (7.7 ms for the interzonal cell vs. 2.5–4.2 ms for the TSD cells) and electrosensory stimuli elicited a larger and more variable IPSP in the interzonal cell. Given at the time of the EOD, the electrosensory stimulus also affected the first spike of the EOCD response, which was unaffected in TSD cells. These anatomical and physiological differences indicate that the interzonal and TSD cells are distinct cell types.
Large TSD cell
We recorded one example of a new cell type that had not been previously described, either morphologically or physiologically. The cell body (14 x 24 µm) was located in the ganglionic layer (Fig. 14A). Three thin apical dendrites extended dorsally from the cell body into the molecular layer where they branched and became considerably thicker. The thick dendrites extended throughout the molecular layer in contrast to the dendrites of TSD cells that were restricted to the inner molecular layer. The dendrites did not have spines. As the thick dendrites approached the outer margin of the molecular layer, they gave off thin branches that penetrated into the preeminential tract, a tract that separates ELL from EGp and conveys fibers from nucleus preeminentialis to ELL and EGp. Some thin dendritic branches were recurrent and extended back into the ganglionic and plexiform layers. The axon of the large TSD cell descended from the cell body down into the deep granular layer where it branched extensively (480 µm in mediolateral and 650 µm in rostrocaudal direction), sending branches into the intermediate, deep granular, superficial granular, and plexiform layers (Fig. 14A).
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The EOCD evoked an EPSP in this cell with a latency of 8.8 ms. The EPSP in turn triggered a burst of 2–3 spikes (Fig. 14C, top). The amplitude of the spikes was between 37 and 38 mV. The individual spikes were followed by large afterhyperpolarizations. Electrosensory stimuli given independently of the command evoked an EPSP with a minimum latency of 6.5 ms (Fig. 14B, top). Increasing stimulus intensity evoked a single spike (Fig. 14B, bottom). Further increases in intensity reduced the latency of the spike but did not yield more spikes. When given at the time of the EOD, the sensory stimulus decreased the number of spikes from 2–3 to 1–2, but the spikes were evoked at a shorter latency and were more time-locked to the command signal (Fig. 14, C, CD+SS, and D). Pairing the EOCD with an electrosensory stimulus for 1 min resulted in a response to the EOCD alone after pairing that had a shorter latency and a larger number of spikes than the EOCD response before pairing (Fig. 14, C and D), suggesting the presence of EOCD plasticity.
The physiology of the large TSD cell is somewhat similar to that of the ordinary TSD cell, but there are also important differences. The EOCD EPSP is longer in latency and smaller in amplitude, and the spikes are larger in the large TSD cell than in the ordinary TSD cell, and electrosensory stimuli alone could evoke spikes in the large TSD cell. These physiological differences together with the morphological differences indicate that the large TSD cell is a distinct cell type.
Primary afferents
Previous studies have shown that synaptic potentials are present in
intracellular recordings from mormyromast afferent fibers terminating in ELL
(Bell 1990a). The synaptic
potentials are evoked by the EOCD and by stimulation of electroreceptors close
to the electroreceptor that gives rise to the recorded afferent fiber (as
indicated by an incoming spike). The synaptic potentials represent synaptic
input to ELL granular cells that is recorded inside the afferent fiber via the
electrical synapses between afferents and granular cells
(Bell et al. 1989). Our
intracellular recordings from primary afferent fibers are described briefly
here for purposes of comparison with EOCD and electrosensory responses in
other ELL cells.
We recorded from seven morphologically identified primary mormyromast
afferent fibers. An additional 38 recordings were determined to be afferent
fibers on the basis of the characteristic spike responses to electrosensory
stimuli and the characteristic synaptic responses to both the EOCD and
electrosensory stimuli (Bell
1990a). The EOCD evoked brief EPSPs in these cells, which ranged
in latency from 4.7 to 6 ms (5.4 ± 0.5). Minimal latencies for spikes
from the periphery in response to electrosensory stimuli ranged from 1.1 to
3.4 ms (2.4 ± 0.5 ms, Fig.
15, trace labeled aff).
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DISCUSSION |
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