Dendritic spike back propagation in the electrosensory lobe of Gnathonemus petersii
1 Laboratory of Neuroscience, University of the Republic, Montevideo,
Uruguay
2 Unité de Neurosciences Intégratives et Computationnelles,
CNRS, 91198 Gif-sur-Yvette, France
3 Center for Sound Communication, Institute of Biology, University of
Southern Denmark, DK-5230 Odense M, Denmark
4 Department of Biomathematics, University of the Republic, Montevideo,
Uruguay
* Author for correspondence at address 1 (e-mail: leonel{at}biomat.fcien.edu.uy)
Accepted 26 October 2004
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Summary |
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The electrosensory lobe has a strict layered organization that makes the preparation suitable for one dimension current source density analysis. Using this technique in an `in vitro' interface slice preparation, we found that following either parallel fiber stimulation or an orthogonal field stimulus, a sink appeared in the ganglionic layer and propagated into the molecular layer. Intracellular records from MG somata showed these stimuli evoked broad action potentials whose timing corresponds to this sink. TTX application in the deep fiber layer blocked the synaptically evoked ganglionic layer field potential and the `N3' wave of the outer molecular layer field potential simultaneously, while the molecular layer `N1' and `N2' waves corresponding to synaptic activation of the apical dendrites remained intact. These results confirm the hypothesis that the broad spikes of MG cells originate in the soma and propagate through the molecular layer in the apical dendritic tree, and suggest the possibility that this backpropagation may contribute to `boosting' of the synaptic response in distal apical dendrites in certain circumstances.
Key words: current source density, electric fish, backpropagating dendritic spike, electrosensory lobe, cerebellum-like network, Gnathonemus petersii.
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Introduction |
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The electrosensory lobe (ELL) of mormyrid electric fish is a
cerebellar-like, layered structure with a very regular cytoarchitectural
organization (Grant et al.,
1996b; Meek, 1994
;
Meek et al., 1996
), in which
it is possible to study such complex integration in a meaningful functional
context, and to clarify the computational roles of dendritic, somatic and
axonal compartments. In this structure, incoming electrosensory activity is
integrated with descending information driven by the electric organ corollary
discharge (EOCD; Bell, 1981
,
1982
,
1989
;
Zipser and Bennett, 1976
).
Electroreceptor primary afferent (PA) terminal arborizations are mapped
topographically in the granular cell layer of ELL. Corollary discharge
information related to the electromotor command enters through three separate
pathways that also integrate central feedback of past electrosensory history,
proprioceptive information and probably other descending signals. Of these
three pathways, we will consider here only that entering through parallel
fibers arising from granule cells of the overlying cerebellar Eminentia
Granularis posterior (EGp), whose axons are distributed through the molecular
layer of ELL. Parallel fibers synapse with the apical dendrites of several
distinct populations of neurons whose cell bodies are situated in the
ganglionic, plexiform and granular cell layers of ELL. These include
Purkinje-like
-amino butyric acid (GABA)-ergic interneurons (medium
ganglionic layer cells: MG) and efferent projection neurons (large ganglionic
layer neurons, LG; large fusiform neurons, LF).
MG neurons account for about 70% of the ELL ganglionic layer neuron
population (Meek et al.,
1996). These cells are easily recognized electrophysiologically by
their characteristically broad action potentials (5-10 ms in vitro;
up to 15 ms in vivo). Broad action potentials can be evoked in
vivo by either corollary discharge input or electrosensory input, and
in vitro by parallel fiber stimulation. Repetitive generation of
broad spikes, for instance as might happen when the fish uses high speed
electromotor scanning of the environment, has been associated in vivo
with spike timing-dependent depression of synaptic responses to corollary
discharge input (Bell et al.,
1993
). In vitro, associative pairing of parallel fiber
EPSPs with post-synaptic broad action potentials has revealed a spike
timing-dependent, anti-Hebbian plasticity rule: when the post-synaptic broad
spike occurred within a window up to 60-80 ms following the presynaptic
activation, the response to parallel fiber became depressed, whereas when a
postsynaptic spike was evoked at other delays the synaptic response was
frequently potentiated (Bell et al.,
1997c
,
1999
;
Grant et al., 1996a
;
Han et al., 2000a
).
Given the extreme sensitivity of these plastic changes to the relative
timing of presynaptic activation and postsynaptic broad spike generation
(Bell et al., 1997c), it is
important to know how and where broad spikes originate, how they propagate
within the cell and what might be the consequences in terms of dendritic
integration. Since MG cells are inhibitory interneurons, plasticity at
parallel fiber synapses probably plays a central role in the temporal `gating'
of sensory reafference, and in the adaptive properties of corollary discharge
driven, active filtering and sensory-motor coordination.
Previous work (Grant et al.,
1998) has suggested the possibility that the broad spikes
characteristic of MG neurons propagate back into the apical dendrites in the
molecular layer. Rather similar broad action potentials have been described in
Purkinje neuron dendrites of the cerebellum
(Llinas and Nicholson, 1971
)
and there is a growing body of more recent in vitro evidence that the
dendritic membrane can have electro-responsive properties, and actively
supports propagation of action potentials in dendritic compartments in several
other types of neurons (Regehr et al.,
1992
,
1993
;
Stuart and Sakmann, 1994
;
Spruston et al., 1995
;
Buzsáki and Kandel,
1998
; Larkum et al.,
2001
; Golding et al.,
2002
). Of particular related interest are studies of the
electrosensory lobe of the gymnotiform fish Apteronotus
leptorhynchus, where Turner et al.
(1994
) have demonstrated the
presence of Na+ channels in the apical dendrites of pyramidal
projection neurons and have shown that oscillatory bursting behavior is
controlled through conditional backpropagation of dendritic spikes
(Turner et al., 2002
;
Noonan et al., 2003
).
The laminar organization of the ELL is clearly visible in the in
vitro slice and makes this a suitable preparation to study the generation
and propagation of broad action potentials in MG neurons
(Grant et al., 1998).
Stimulation of parallel fibers mimics EGp input to ELL and permits us to infer
some rules for the local synaptic activation and postsynaptic cell responses.
The synaptic responses evoked by parallel fiber stimulation can be recorded
intracellularly at the level of the soma but have not been recorded
intracellularly at the level of the distal apical dendrites where parallel
fiber synapses occur. However, extracellular field potentials can be recorded
easily across the layers of ELL and provide an alternative method of study.
The layered organization of the ELL and the geometry and orientation of the
principal cell types constitute an open field arrangement and the synchronous
activation produced by their inputs, whether natural (driven by reafferent
sensory input and the EOCD), or artificial (in response to electrical
stimulation of the parallel fibers or the deep fiber layer), allow us to apply
one-dimensional current source density (CSD) analysis to field potential
recordings. The macroscopic pattern of current sinks and sources obtained with
this method, produced by synchronous activity of local cell populations,
together with anatomical data and intracellular recordings, are interpreted
here to give insight into neuron behavior in response to specific input
activation.
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Materials and methods |
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Slice preparation
Fish were deeply anesthetized by immersion in a cold aerated solution of
tricaine methane sulfonate (MS 222 Sandoz, Schönenwerd, Switzerland) at a
concentration of 100 mg l-1. The skull was opened, and the brain
was irrigated with ice-cold artificial cerebrospinal fluid (ACSF; for
composition, see below). The valvula was retracted laterally, a vertical cut
was made in the transverse plane immediately rostral to the ELL, the spinal
cord was sectioned immediately caudal to the ELL, and the caudal brainstem
block containing the ELL was removed. The brainstem block was transferred to
ice-cold ACSF for 60 s to harden it a little, and then the rostral cut surface
was glued to a vibrating microtome block (Leica VT1000M, Nußloch,
Germany) with cyanoacrylate glue, with the dorsal surface of the ELL facing
the blade. The brain block was lightly supported by a coating of 16% gelatin
dissolved in ACSF. 300-400 µm thick slices were cut in the transverse plane
under ice-cold ACSF, using a sapphire blade (Delaware Diamond Knives,
Wilmington, DE, USA). Slices were retrieved with a wide bore Pasteur pipette
and transferred to a holding bath where they were kept submerged at room
temperature, supported on small squares of Kodak lens paper that served to
minimize direct handling. The ACSF used up to this point was almost Na-free,
with sucrose replacing NaCl to reduce excitotoxic shock caused by the slicing
(Aghajanian and Rasmussen,
1989). The composition of this low sodium ACSF was as follows (in
mmol l-1): NaCl 0, KCl 2.0, KH2PO4 1.25,
NaHCO3 24, CaCl2 2.6, MgSO4.7H20
1.6, glucose 20, and sucrose 213. The slices were transferred to an
interface-recording chamber and superfused for 30 min with an ACSF solution
containing a 1:1 mixture of low sodium ACSF and `normal' ACSF (see below),
before changing to 100% normal ACSF. The composition of the normal ACSF was as
follows (in mmol l-1): NaCl 124, KCl 2.0,
KH2PO4 1.25, NaHCO3 24, CaCl2 2.6,
MgSO4.7H20 1.6, and glucose 20 (osmolarity: 290 mOsm.
Both Na-free and normal ACSF were bubbled with 95% O2 and 5%
CO2, bringing the pH to 7.2-7.4. The slices were supported in the
recording chamber on several thickness of Kodak lens tissue and superfused
with normal ACSF at room temperature (22-25°C), at a rate of 1-3 ml
min-1 by gravity flow.
Stimulation
Parallel fibers were stimulated using monopolar tungsten electrodes (AM
Systems, Sequim, WA, USA) whose tips had been lightly scraped on a sharpening
stone to reduce tip resistance and then plated with gold to reduce electrode
polarization. Stimulating current was delivered between a single such
electrode in the tissue (negative pole) and a second similar electrode or
silver wire (positive pole) in the bath outside the slice close to the
external margin of EGp. The molecular, ganglionic, plexiform and superficial
granule cell layers of ELL can be readily distinguished in the living slice
under the operating microscope, allowing for accurate placing of stimulating
and recording electrodes in these layers. Stimulating electrodes were placed
in the outer half of the molecular layer, where parallel fibers run orthogonal
to the apical dendrites of ganglionic layer neurons
(Fig. 1). Stimuli were constant
current pulses with a duration of 0.1 ms and amplitudes of 5-60 µA.
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Recording
The different zones and layers of ELL can be distinguished in the slice
with the aid of a dissecting microscope. Most recordings were made in the
medial zone of ELL because of its large size, but some were from the
dorsolateral zone.
Intracellular recordings
Intracellular recordings were made with sharp glass micropipettes filled
with 2 mol l-1 potassium methyl sulfate containing 2% biocytin
(Molecular Probes, Eugene, OR, USA; Sigma, St Louis, MO, USA). Tip resistances
were 150-200 M. Based on previous studies, MG layer neurons could be
distinguished by electrophysiological criteria alone, by the distinctive
large, broad action potential that is present only in this cell type
(Bell et al., 1997b
; and see
below).
Recorded neurons were labeled by intracellular iontophoresis of biocytin using DC current (tip positive or negative) of 0.2-0.5 nA for at least 5 min. Slices were fixed for 1 h in 2% paraformaldehyde and 2% glutaraldehyde in 0.1 mol l-1 phosphate buffer, pH 7, and labeled neurons were later revealed using the standard ABC process (Vector Laboratories, Burlingame, CA, USA).
Field potential recordings
Field potential recordings were made using pipettes filled with 3.0 mol
l-1 NaCl having resistances of 3-6 M. Recordings were made
serially along a line perpendicular to the orientation of the layers of ELL,
from the outer molecular layer to the intermediate layer, in conditions as
constant as possible, as illustrated in
Fig. 1A. An example of a series
of recordings obtained at different levels through the molecular layer in
response to parallel fiber stimulation is shown in
Fig. 1B.Recording sites were
separated by steps of 15, 25, 30 or 50 µm
x in Equation 3
below) depending on the brain size and, in some experiments, to check if the
resolution of current source density analysis (see below) improved with
smaller steps. On some occasions, different step sizes were tried in the same
slice and it was found that decreasing the spatial step between recording
points to less than 25-30 µm did not improve the resolution of the method.
At each site, the recording electrode was placed at a depth of 40 µm from
the surface of the slice. Responses were tested with an inter-stimulus
interval of 2 s and 20-30 responses were averaged for each recording point in
order to minimize the influence of response variability and improve the
signal-to-noise ratio. Before calculating current source density, a digital
low-pass filter was applied to further reduce noise
(Richardson et al., 1987
).
Current source density analysis (CSD)
When the tissue has no particular geometry, the current source density
(S) at time t for a given point of a rectangular coordinate
system is calculated as:
![]() | (1) |
where x,
y and
z are
the conductivity tensors for the three x, y, z spatial dimensions
respectively, and V(t) is the extracellular voltage.
However, the ELL complies with what is called an `open field' geometrical
arrangement (Hubbard et al.,
1969
; Johnston and Miao-Sin
Wu, 1995
): it is a layered structure with the principal axis of
majority of cells oriented perpendicular to the plane of the layers. If this
array of neurons is activated by a synchronous synaptic input (i.e. parallel
fiber stimulation) a dipole is established between the apical dendrites and
the somas. Currents that run in the directions parallel to the layers cancel
out, and the problem is reduced to one spatial dimension, as is shown in
Equation 2 (Haberly and Shepherd,
1973
; Mitzdorf,
1985
; Richardson et al.,
1987
):
![]() | (2) |
The empirical process of calculation of the CSD from field potential (FP)
recordings is given in Equation 3:
![]() | (3) |
The waveforms defined by the symbols Va, Vo and Vb are shown in Fig. 1C.
CSDs are represented as color maps to facilitate visualization of the continuity of processes in time and space. In each color map (see for example Fig. 2) green represents zero; colors from green to red indicate sinks (inward currents), and colors from green to blue represent sources (outward currents).
Since CSD was calculated in one dimension and the value of the
extracellular conductivity was not determined, CSD waveforms cannot be
considered absolute quantitative estimates of current density. Units are
therefore not provided on CSD profiles. Rather, CSD estimates are used to
compare the qualitative characteristics of sink-source relationships in ELL
slices. Based on similar reasoning, in order to make some patterns more
evident, we applied an arc-tangent function to the CSD data. This function has
a sigmoid shape, having asymptotes -/2 and
/2 when the variable tends
to -
and +
, respectively, and is equal to 0 when the variable is
0. This procedure amplifies low amplitude events while those of large
amplitude saturate. This operation is symmetrical with respect to 0.
Pharmacology
In order to produce a selective block of sodium-dependent spikes in neuron
somata in the ganglionic layer, in some experiments, a drop of tetrodotoxin
(TTX; 0.5-1.0 mmol l-1) was applied by pressure injection into the
surface of the slice from a glass micropipette. The micropipette was
positioned about 25 µm below the slice surface in the deep fiber layer. The
TTX took several minutes to diffuse from the injection site, reaching the
ganglionic layer before the molecular layer.
Data analysis
Data were recorded using an Axoclamp 2B amplifier (Axon Instruments, Union
City, CA, USA) and stored on the hard disk of a computer via a
Labmaster interface (Scientific Solutions, North Chelmsford, MA, USA) and
Acquis1 software written by Gerard Sadoc (C.N.R.S., Gif sur Yvette, France).
Quantitative measurements were made using Acquis1 or Matlab (MathWorks,
Natick, MA, USA) software and plotted using Matlab.
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Results |
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In the second section of the Results, intracellular recordings of responses to molecular layer stimulation are presented in order to compare synaptic and single cell spiking events with the population events revealed by CSD analysis. Finally, pharmacological tools were used to investigate the identities of phenomena described.
Field potentials and CSD analysis
CSD calculated from sequentially recorded field potentials make it possible
to observe processes that are time-locked to a triggering event (in this case
the parallel fiber stimulus), provided that the field potentials remain
relatively constant over the process of sequential recording of the whole
series. Field potentials were large enough to be observed throughout the
different layers of ELL and were recorded at successive points passing through
the layers, in the same plane as the apical dendritic arborization of
ganglionic layer neurons. The shape of field potentials evoked in ELL was not
affected by the averaging process and low-pass filtering. We first describe
the typical CSD patterns obtained with parallel fiber stimulation (single and
paired stimulation pulses), and then responses evoked by trans-ELL field
stimulation. The effects of multiple stimulation protocols are described for
both types of stimulation and differences and similarities are discussed.
Molecular layer stimulation
Fig. 2 shows the CSD pattern
obtained in response to low intensity molecular layer stimulation, plotted as
a color map. Field potential recordings are superimposed on the CSD color map.
The CSD abscissa shows time in ms and the ordinate represents distance from
the starting recording point in the granular layer of ELL. An MG neuron is
drawn to the same scale to facilitate location of events observed in the CSD
and field potential plots in relation to the cell compartments. The
stimulation artifact has been removed. Red areas are current sinks, i.e.
current flowing into the cell. Blue areas are current sources: i.e. current
flowing out of the cell. Green, as is shown in the color bar, represents the
zero value, when no current flows either in or out of the cells.
The earliest event following the stimulation artifact in the field
potential records is a small negative wave (N1) with a delay
shorter than 1 ms. Wave N1 occurs `on beam' to the stimulation site
between 400 mm and 800 mm in the figure. Grant et al.
(1998) suggested that this
wave corresponds to the parallel fiber action potential. Parallel fibers are
oriented in the same transverse plane as the layers of ELL so their activation
does not fit the open field condition and, consequently, this activity can not
be analyzed reliably with the one dimensional CSD technique. However, since
N1 amplitude is maximum in front of the stimulation electrode and
decays to both sides (up and down in Fig.
2A), a positive concavity (i.e. the yellow-orange spot coinciding
with N1 in Fig. 2A)
will appear when the second derivative is calculated. This may also be
produced in part by activity in the presynaptic terminal
(Mitzdorf, 1985
).
Beginning at around 3 ms, also on the same beam, a second negative wave
(N2) can be observed; this reaches a minimum at approximately 6 ms.
Grant et al. (1998) postulated
that this wave is generated by the EPSP produced by the parallel fibers in
apical dendrites of ELL cells. In the CSD plot, a sink can be seen at the same
delay, also peaking around 6 ms. The red spot is flanked by two blue spots
that correspond to the current sources generated by the central sink. This
sink with its companion sources represents the most intense and longest
activity observed.
The time course of this process is illustrated in
Fig. 2B. The rise time of the
current sink peak was 2 ms and this then decayed as a second order exponential
function. Time constants for decay of the response (red dotted line in
Fig. 2B) were:
1=2.2 ms and
2=93.5 ms. This profile is very
similar to that of the parallel fiber EPSP observed with somatic intracellular
recordings (see fig. 11a in Grant et al.,
1998
).
The spatial distribution of the sources is tightly related to the length
constant of the activated cells (Mitzdorf,
1985). The manner in which current spreads away from the point of
synaptic activation is governed by the electrotonic properties of the cells,
and from this it is possible to estimate the mean length constant of apical
dendrites. The term length constant is generally used to describe a passive
steady-state response property, and may not be entirely applicable to the
present context since small active events may be occurring locally in the
dendrites, linked to synaptic activation, that are below the resolution of
field potential recordings. However, we may derive some information about what
is going on at the population level from spatial plots of CSD and even though
active conductances are involved, this `length constant' is a measure of how
far a depolarization propagates. Fig.
3A shows the CSD corresponding to the timing and spatial
distribution of N2 in a gray-scale color map.
Fig. 3B shows the spatial
profile of current densities at a given time t after the stimulus
(shown by the dotted black line in Fig.
3A), corresponding to the peak of synaptic activation. The `length
constant' of the active cells can be estimated from the spatial decay of the
sources from their peak amplitude. In the inset of
Fig. 3, an exponential function
was fit to the distal tail of the current profile across the layers, giving a
length constant of 34.24±7.06 mm. In different slices this value varied
but was never larger than 90 µm (mean 60 µm,
S.D.=23, N=7). Since in every case an
exponential function fits the experimental results, it can be assumed that in
this slice preparation, the process is mainly passive. The range of estimated
length constants is rather short compared with values calculated for neurons
in some mammalian structures and suggests non-compact cells that would allow
mainly spatially restricted interactions within the dendritic arborization.
This may be important for the integrative properties and the consequences of
synaptic plasticity in ELL neurons.
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Around 9 ms after the beginning of the parallel fiber stimulation artifact, another sink appears at the level of the proximal apical dendrites in the ganglionic layer (Fig. 2A). This sink peaks in the range 7-10 ms and lasts 4-10 ms. It is preceded and followed by a source. This tri-phasic negative/positive/negative sequence can be better appreciated in Fig. 2B, which shows the evolution of current density with time, in the molecular layer and in the ganglionic layer. This pattern is typically produced by an active process. First, excitatory inputs in the apical dendrites spread passively producing a source in the ganglionic layer. The resulting excitation of the ganglionic layer then generates a sink at this level and finally, excitation propagates back into the molecular layer (see also Fig. 4), generating a new source in the ganglionic layer. Note that the simultaneous source associated with the ganglionic layer sink is located asymmetrically, appearing principally in the plexiform and granular layers (see lower blue spot in Fig. 2A, centered at 10 ms and 20 mm). This means that the current tends to leave cells through their basal dendrites, although it is possible that some current produced by the molecular layer, parallel fiber-evoked synaptic sink may also contribute to this source in the deeper layers.
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Paired pulse stimulation in the molecular layer
Paired pulse facilitation (ppf) of the field potentials evoked by ELL
molecular layer stimulation is commonly observed. This involves both the
parallel fiber EPSP-related component of the field potential (N2)
and also a third negative wave that has been called N3
(Fig. 4A;
Grant et al., 1998). The
N3 wave is not always present; its amplitude and morphology are
rather variable and, in some cases there are more complicated patterns of
response, including additional waves. In this subsection we investigate field
potential responses to paired pulses applying CSD analysis.
In Fig. 4A a CSD color plot is shown with the corresponding field potential recordings superimposed, in response to paired pulse stimulation of the molecular layer with an inter-stimulus delay of 50 ms. An MG cell outline is presented on the left, to aid relating events to cell compartments. The response evoked by the first stimulus was similar to those described previously but is different in two ways, probably due to a more intense stimulation of the parallel fibers. The first of these differences is that the sink originating in the ganglionic layer is very well defined in this preparation and extends, with increasing latency, outward through the proximal molecular layer. This suggests an active process that propagates in the direction of the molecular layer from its starting point in the ganglionic layer. This excitation coincides in delay and duration with the broad spikes seen in intracellular recordings in response to parallel fiber stimulation, and shows that in certain circumstances, it is possible to obtain a synchronized population response originating in the region of the soma layer, that backpropagates into the apical dendritic arborization following a single stimulus. The second noticeable difference is the presence of a second positive peak (sink) that appears in the molecular layer sink, approximately 10 ms later than the initial parallel fiber-evoked peak and at the same distance from the soma. This second peak has a lower amplitude than the first (yellow in the color scale axis while the preceding peak is dark red). and coincides with the N3 wave of the field potentials (arrow in Fig. 4A).
In response to the second stimulation pulse both field potential negative peaks, N2 and N3, are larger, and N3 can now be traced down as far as the ganglionic layer. The latency of N3 decreases gradually as the recording site approaches the ganglionic layer. In the underlying CSD color map, a sink clearly follows the N3 wave through the layers, repeating the pattern of latency change. This suggests an active process propagating from the ganglionic layer towards the distal region of the apical dendrites, that spreads with a velocity of approximately 0.05-0.07 m s-1. The reduction in sink amplitude in the intermediate part of its trajectory (between 300-500 mm on the ordinate) could be due to the superposition with the source of the population excitatory postsynaptic potential (EPSP).
In order to better appreciate the characteristics of this process we
subtracted the response evoked by the first parallel fiber stimulus from the
response evoked by the second one (Fig.
4B). This operation highlights the components that are increased
(or decreased) by the double stimulus. Sinks that are facilitated appear in
red. This showed that the initial synaptic response to the second parallel
fiber stimulus was potentiated (probably the result of presynaptic
potentiation; Grant et al.,
1998). Backpropagation of the sink initiated at the ganglionic
layer also became more visible using paired pulse parallel fiber stimulation
protocols.
In Fig. 4B, this sink can be
followed clearly (white dotted line) until it reaches the level of the
molecular layer synaptic sink. While the backpropagating sink was reduced
where it crossed the synaptic source, it was markedly increased when it joined
the synaptic sink. Although this effect was not quantified, the apparent
increase of the back propagated event seems to be greater than expected from
linear summation with the potentiated EPSP, suggesting a boosting of the
backpropagated dendritic spike by the EPSP, similar to that described, for
example, in hippocampus (Migliore et al.,
1999; Watanabe et al.,
2002
).
Trans-ELL field stimulation, single pulses
When a brief (1 ms) electric field stimulus applied across the layers of
ELL was used to activate the network (see Materials and methods and
Fig. 1A: Stim2), we
observed a different pattern of response, illustrated in
Fig. 5. Using this stimulation,
Fig. 5B shows inversion of the
stimulus current peak (black dotted line and arrows) somewhere between the
ganglionic and granular layers; thus, current enters the cells all along the
apical dendritic tree (hyperpolarizing at this level) and leaves the cells
(producing depolarization) close to the ventral pole, at the level of the axon
initial segment and the basal dendrites. This stimulus pattern is a
consequence of the intrinsic organization of the ELL and is a reflection of
the distribution of membrane resistance of the neurons whose dendrites compose
the highly oriented network.
|
Field potential recordings are superimposed over the color map representing the calculated CSD. Field potential traces corresponding to the granular and plexiform layers, situated at the level of the soma and basal dendrites of the MG cell profile on the left, presented a sharp negative wave that lasted about 5 ms, followed by a positive wave of smaller amplitude that lasted more than 10 ms. This pattern gradually inverted as the recording site moved outwards through the ganglionic and molecular layers. Molecular layer field potentials were composed of an initial large positive wave, immediately after the stimulation artifact, followed by a smaller negative wave whose latency increased linearly with increasing distance towards the outer molecular layer. Both positive and negative field potentials lasted about 5 ms. In the CSD color map representation it can be seen that a well-defined sink coincides with the field potential negative wave. Nevertheless, the sink duration is shorter than the negative wave (2-3 ms), reflecting the more precise time resolution of this technique. The sink increases in amplitude from the deep granular layer to the ganglionic layer, where it reaches a maximum, and then decreases again in the molecular layer.
Fig. 5B shows the time function of the CSD at three different levels (indicated by white dotted lines in Fig. 5A), to better appreciate its changing shape. Another interesting point is that this sink produced by trans-ELL field stimulation originates more deeply than the sink evoked by molecular layer stimulation. In the case of field stimulation illustrated in Fig. 5A, the corresponding sources can be observed on both sides of the sink. The source that is generated distal to the active zone is well defined and continuous, but the proximal source is divided in two by the prolongation of the sink at the ganglionic layer and is mainly concentrated in the plexiform and granular layers. The propagation velocity of the sink out into the molecular layer was calculated as 0.09 m s-1 from this data.
Multiple molecular layer and field stimulation
To compare the responses obtained with each type of stimulus in similar
conditions, molecular layer and field stimuli were applied alternately, in the
same slice. The experiment was done with a multiple pulse protocol to observe
whether or not there was potentiation in the two cases. The top panel of
Fig. 6A shows the CSD of the
response to a sequence of three molecular layer stimuli; the bottom panel
shows the CSD of the responses to a sequence of three field stimuli. In both
cases the basic CSD patterns are as described previously. In response to
molecular layer stimulation (Fig.
6A, top) potentiation is very clear, even between the second and
the third stimuli, exhibiting a build-up of the response to the train of
stimuli. Potentiation is clear in both components of the response: the
synaptic event and the back propagated event. In contrast, responses obtained
with field stimulation showed no potentiation
(Fig. 6A, bottom). On the
contrary, successive stimuli produced progressively reduced responses. For the
examples shown in Fig. 6, the
calculated velocities of backpropagated processes were 0.07 m s-1
for the molecular layer stimulation-evoked event and 0.09 m s-1 for
the field stimulation-evoked event.
|
In Fig. 6B a magnified view of the third response to molecular layer stimulation is shown to highlight some response features that were observed in approximately half of the experiments carried out (4 out of 9 experiments). (1) In some cases a sink appeared midway between the site of synaptic entry and the ganglionic layer response (arrow in Fig. 6B). This yellow spot could be an active response of the proximal dendrites produced by the EPSP. (2) The second variation with respect to the patterns described above was observed when the ganglionic sink was in fact composed of two distinct sinks occurring at the same depth, one a little earlier than the other (arrowheads in Fig. 6B). Both had a tendency to propagate back into the molecular layer, but it was always the case that when these two sinks appeared together, that the earlier one was thinner, more elongated, and seemed to propagate outward faster. There may be several explanations for this behavior. It is possible that the two sinks correspond to the spiking activity of different cell populations, e.g. synchronized LG cells and then MG cells. Alternatively, since MG cells are by far the most numerous and probably contribute most to CSD images, part of this population may fire an early spike and the remainder may first be inhibited by the stimulus and then fire on the delayed parallel fiber EPSP. Intracellular records show that MG neurons do not fire two broad action potentials in response to single parallel fiber stimulation.
Intracellular recordings
Responses to molecular layer stimulation were recorded intracellularly in
different cell types. Our purpose was to establish a relationship between the
population events illustrated in the field potential study above and the
behavior of individual cells, and for this reason only recordings obtained
from MG cells are presented here. MG neurons are by far the most numerous cell
type in the ganglionic layer of ELL and their apical dendritic trees account
for probably more than 80% of the dendritic component of the molecular layer.
It is therefore likely that MG cell responses will weight the field potentials
evoked by molecular layer stimulation proportionally more than those of other
cell types contributing to the collective evoked responses.
Fig. 7 illustrates responses recorded in an MG cell. A single parallel fiber stimulus of weak intensity repeated at long intervals produced only a sub-threshold post-synaptic EPSP. But when the same stimulus was repeated at intervals in the range of those used by the fish in highly attentive electromotor scanning (50 ms), the probability of evoking first small narrow spikes, together with occasional medium sized spikes (Fig. 7, arrowhead), and then full sized broad spikes, quickly increased. Traces from top to bottom are examples of responses obtained applying increasing number of stimulation pulses to the parallel fibers. In addition to the typical MG cell spiking responses mentioned before, is interesting to note long-lasting depolarization that builds up in response to repeated stimuli separated by 50 ms intervals, and also the train of small spikes lasting several hundred milliseconds that is superimposed on this depolarization.
|
The inset in Fig. 7 shows a graph of the probability of evoking a broad spike plotted as a function of the number of stimuli applied; this reaches a value of 1 after four stimuli separated by 50 ms intervals. (In this example, probability started at zero because the intensity of the individual pulses was chosen so that a single stimulus did not produce a broad spike. With higher stimulation intensities a single stimulus could evoke a broad spike.) Note that with three stimuli, the protocol used for extracellular recording experiments, the probability of obtaining a broad spike response was in the order of 0.75. Although the timing of broad spikes was somewhat variable, most reached their peak amplitude around 10 ms after the beginning of the stimulus artifact, which coincides precisely with the peak of the ganglionic layer sink described in previous section.
The pharmacological separation of synaptic events and active postsynaptic responses using TTX
To discriminate between the synaptic component of the response obtained
with molecular layer stimulation and the second negative peak of the response,
and to determine if the latter is truly an active process backpropagating from
the ganglionic and plexiform layers, across the molecular layer of the ELL, an
experiment was designed to separate the two components
(Fig. 8). While recording
simultaneously the evoked responses in the distal molecular layer and in the
ganglionic layer, a micro-injection of the sodium channel blocker TTX was made
just below the slice surface in the deep fiber layer. Since the diffusion of
TTX within the slice is a transient process we could not use CSD analysis to
study this phenomenon, and instead, interpretation of the results depends on
the established relation between N3 and the presumed backpropagated
process. Field potentials recorded simultaneously in the molecular layer and
the ganglionic layer are represented as color maps against time from TTX
application, at the top right and bottom right of
Fig. 8, respectively.
Initially, the characteristic N2 and N3 wave patterns
were observed in the molecular layer and correspond to the two horizontally
running blue zones in the top panel (N1 was masked by the
stimulation artifact). The doubled peaked positive wave typical of the
ganglionic layer is shown in the bottom panel of
Fig. 8. In this representation,
the abscissa represents time between successive sweep records and the ordinate
shows time within a sweep. The stimulation artifact was masked and its place
appears in white in the figure.
|
The TTX took a little more than 45 s to diffuse from the application point in the deep fiber layer to the recording point in the ganglionic layer. At this time the response disappeared completely in the plexiform and ganglionic layer level (Fig. 8, bottom). This was accompanied by the simultaneous disappearance of the N3 wave in the distal molecular layer (the lower horizontal blue region seen in Fig. 8, top), although no changes were yet visible in the N2 wave. Approximately 1.5 min later, when the TTX reached the distal molecular region, N2 also disappeared. This experiment demonstrates the origin of the events that produce the N3 wave at the distal molecular layer, showing that it is generated in the ganglionicplexiform layers and depends on the activation of sodium channels.
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Discussion |
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In response to molecular layer stimulation, the first large event that
appears in CSD plots is clearly related to the N2 wave of the field
potential, and most probably corresponds to the EPSP produced by the molecular
layer stimulation of the parallel fibers, which has both (AMPA)- and
N-methyl-D-aspartate (NMDA)-receptor mediated components.
However, several studies using rather similar stimulation paradigms in slices
of the hippocampus and the neocortex, have suggested that it is also possible
that such stimulation may evoke local active processes initiated in the
dendrites and that these components cannot always be discriminated in field
potentials (Turner et al.,
1984,
1991
;
Stuart and Hausser, 2001
).
While CSD analysis cannot discriminate spiking and synaptic events either,
this technique gives an average of the principal events most likely to occur,
their spatial distribution and temporal progression through the network. From
this we can say that although it is possible that responses to synchronous
molecular layer stimulation may also include local dendritic spikes, these
probably do not propagate towards the soma because in this case a downward
propagating sink should appear. Although the relationship between
N2 and parallel fiber synaptic activation was already suggested
from previous electrophysiological studies
(Grant et al., 1998
), the CSD
analysis applied here shows the time course of this process more clearly and
reproduces that of EPSPs recorded intracellularly quite precisely, thus
allowing a more accurate characterization of the global synaptic input
following parallel fiber stimulation.
The synaptic sink is immediately flanked by its sources, meaning that the
current leaves the cells in the immediate vicinity of the synaptic input. We
found that the spatial decay of the `tail' sources were well fit by an
exponential function. This indicates that spatial decay of current after
synaptic activation is mainly passive. An estimate of a population `length
constant' can be made as a measure of these passive properties. From the
present results, very short values were estimated for these `length
constants', compared with the usual values obtained in other preparations. For
hippocampal pyramidal cells, for example
(Turner, 1984), expressing the
length of dendrites in units of space constants gives estimated values between
0.32 and 0.91, whereas our estimations for ELL MG cell apical dendrites are
4-6 space constants. This suggests that the cells may have the possibility of
generating local domains for synaptic interactions with parallel fibers,
decoupled from other synapses nearby. Pushing this reasoning further, this
could allow specific plastic modulation of the weights of individual, or
restricted groups of parallel fiber synapses.
The negative wave N3 visible in field potential recordings can
be traced up from the ganglionic to the molecular layer. From comparison of
field potential data with the timing of events measured intra- and
extracellularly, a relationship was postulated between the N3 wave
and intracellular broad spikes (Grant et
al., 1998). However, as stated before, relationships between field
potentials and cellular events are uncertain and further proof was required to
be sure of this interpretation.
The regular narrow spikes fired by most neurons are difficult to see with CSD analysis since, being brief, they require extreme synchronization in order for their sum to produce visible current sources and sinks. However, the MG cells, which constitute at least 70% of the ganglionic layer soma population and probably give rise to a still larger proportion of the apical dendrites contained in the molecular layer, fire consistently broad action potentials. Because of their long duration, the sinks/sources in individual cells produced by broad spikes would tend to add together sufficiently to produce events visible by the CSD analysis method, even if their generation were not completely synchronous. Confirming this supposition, CSD analyses show that following synaptic activation, a second sink always appears in the ganglionic layer, peaking approximately 10 ms later than the beginning of the stimulation artifact. Broad spikes appear in intracellular recordings with approximately the same latency range. In many cases (depending of the stimulus strength and/or number of stimulating pulses), this sink travels from the ganglionicplexiform region towards the apical dendrites. This CSD event is clearly related to the N3 wave of the field potentials, although it is sometimes present in the absence of a well defined N3 field potential. These results provide further support to the hypothesis of broad spike propagation into the apical dendritic tree. This is confirmed by the abolition of the N3 wave concomitantly with the disappearance of activity at the ganglionic level when TTX is applied to the deep fiber layer of the slice.
An active process with similar characteristics can also be produced with field stimulation, showing that this phenomenon does not necessarily depend on parallel fiber synaptic activation in all circumstances. Although there is overall similarity, some differences exist between the synaptic-evoked and field-evoked processes. In particular, synaptic-evoked processes are more complex, sometimes showing decomposition into two distinct phenomena that could be due to differential synaptic activation of two cell populations. Another difference is seen in responses to repetitive stimulation. When the late backpropagating response is evoked by a train of parallel fiber stimuli, it builds up in a manner that parallels the increase in the molecular layer synaptic related event. The increase in synaptic efficacy leads to more effective triggering of the dendritic population spike and is probably the result of both presynaptic potentiation and prolonged postsynaptic depolarization.
In contrast, when a backpropagating event is evoked directly by a trans-ELL
field stimulus, a decrease in the amplitude of successive responses to a train
of stimuli was observed. This resembles paired pulse depression that has been
described for the granular layer field potential response to stimulation in
the intermediate layer. This depression is sensitive to bicuculline and has
been interpreted as the result of lateral inhibition in the inner layers of
the ELL (Han et al., 2000b),
mediated by large GABA-ergic inhibitory interneurons (large myelinated
interneurons: LMI; Meek et al.,
2001
). The dendrites of these inhibitory neurons do not extend
into the molecular layer and these cells would not be activated by parallel
fiber stimulation, thus explaining the different response behavior to
repetitive stimulation at these different sites.
The backpropagation velocity of dendritic spikes through the molecular
layer was relatively slow: approx. 0.05-0.09 m s-1 in ELL, compared
with, for example, 0.67 m s-1 estimated for backpropagated spikes
in rat cortex by Buszáki and Kandel (1998). The small but consistent
differences in the velocity of the backpropagated events in ELL estimated from
the two stimulation procedures used here, might be explained by the
long-lasting sub-threshold depolarization that builds up postsynaptically
during repetitive molecular layer stimulation
(Fig. 7), possibly facilitating
dendritic spike propagation. The short `length constant' estimated from source
decays can explain this, and at the same time suggests the need for an active
process as a means of intracellular signaling to communicate between distant
locations. Changes in the propagation velocity as were reported in
Apteronotus (Turner et al.,
1994; Lemon and Turner,
2000
), underlying complex dynamics in the cell activation
(conditional backpropagation, ghostbursting), have not been observed in this
preparation although small changes could exist that are below the resolution
level of the method used here.
Further indirect evidence that the propagated CSD event described here corresponds to the backpropagation of the broad action potential comes from the experiment where it was shown that the probability of evoking a broad spike increased monotonically with the number of parallel fiber stimulation pulses. Increasing the number of stimuli increased the probability of evoking a broad spike in individual cells, and the increase in global population activity will be reflected in the population spike seen in the CSD analysis.
The conduction of the signal from the synaptic location to the triggering
point for this backpropagated phenomenon seems to be mainly passive, but on
some occasions a spot of activation was seen as the synaptic potential was
translated down through the molecular layer towards the soma in the ganglionic
layer. It is possible that these `hot spots' occur where apical dendrites
converge at their proximal point of origin and that at the population level,
they represent the initiation and propagation of dendritic spikes towards the
soma. This is also a possible explanation for the medium-sized spikes that can
be recorded in the soma (see arrowhead in second trace in
Fig. 7). The typical geometry
of MG cells shows 10-20 very spiny distal apical dendrites that arise from 3-6
short primary dendrites, which in turn arise from a small soma, 10-12 mm in
diameter (Grant et al., 1996b;
Meek et al., 1996
). This could
provide the necessary anatomical substrate for summation of signals spreading
through distal apical dendrites towards the soma, reaching the threshold for
broad spike generation somewhere near the soma: in the primary dendrites, in
the soma itself, or at the axon hillock. The local initiation of dendritic
regenerative events and their partial or full propagation towards the soma has
been described in different types of neurons in a number of studies (e.g.
Regehr et al., 1993
;
Schwindt and Crill, 1997
;
Stuart et al., 1997
;
Martina et al., 2000
;
Kloosterman et al., 2001
).
Previous pharmacological studies
(Sugawara et al., 1999) have
shown that broad spikes are a Na+-dependent phenomenon and that
they resist removal of extracellular calcium. It is therefore supposed that
voltage-dependent Na+ channels exist in the dendrites and the
possibility that there are dendritic action potential initiation sites cannot
be ruled out. This has also been suggested by Turner et al.
(1994
) who, using an antibody
directed against sodium channels, have described punctate regions of
immunolabel separated by nonlabeled expanses of membrane, over the entire
extent of basal dendrites and also the apical dendritic tree, in the gymnotid
electrosensory lobe.
Repetitive parallel fiber stimulation produced both increasing amplitude of
the population EPSP and an increase in the population backpropagated event.
Coincidence of these events may in fact result in amplification of the
backpropagating dendritic response and boosting of synaptically evoked
activity in distal dendrites, as suggested by the result illustrated in
Fig. 4B. Thus the response
properties of molecular layer dendrites could depend in a nonlinear manner on
the frequency and intensity of parallel fiber input, involving both pre- and
post-synaptic mechanisms. A similar boosting of the backpropagated event has
been described in hippocampus and related to enhanced excitability produced by
the inactivation of A-type potassium channels. The consequent long-lasting
depolarization has been shown to be important for the induction of long-term
potentiation (LTP) (Magee and Johnston,
1997; Watanabe et al.,
2002
; Frick et al.,
2004
).
In ELL, paired pulse, or short interval repetitive stimulation at frequencies in the order of those used in the natural EOD rhythm of the fish, increased both the probability of full spike backpropagation and MG cell output (spikelets; Fig. 7) producing short term facilitation. This type of change in dendritic response properties may be expressed in certain well identified behavior patterns associated with increased sensory attention, e.g. high speed regular electric organ discharge (EOD) observed during active exploration, or the novelty response, in which there is a transient increase in EOD in response to a sudden change in the environment.
This appears to be different from the spike timing-dependent anti-Hebbian
plasticity that has been demonstrated at parallel fiber synapses, whose
expression also depends on the presence of postsynaptic broad spikes
(Grant et al., 1996a; Bell et
al., 1997c
,
1999
;
Han et al., 2000a
). In this
case, repetitive association between synaptic activation and the postsynaptic
generation of broad spikes leads to synaptic depression. It has been suggested
that this plasticity plays a role in updating corollary discharge-driven
feedback signals representing central predictions of the sensory world,
documented in vivo (Bell et al.,
1997a
).
These different mechanisms for the modulation of dendritic integration in MG cells may play different functional roles. Since MG cells are inhibitory interneurons, most likely reciprocally interconnected as well as being pre-synaptic to output neurons, the resultant effects of their activity on output neurons will depend on a delicate balance of the interplay of inhibition and disinhibition. The transition from short-term facilitation to synaptic depression is not yet well understood and better knowledge of the mechanisms involved would have interesting computational implications for information processing in the basic microcircuit of the ELL, as well as in other cerebellum-like or cortical structures in which sensory processing depends greatly on descending feedback. To arrive at a full understanding of the mechanisms of adaptive filtering processes, these functional properties of MG interneurons would need to be considered in terms of the dynamics of local circuits responding to the dual activation of both sensory afferents and descending corollary discharge.
Conclusion
MG interneurons are by far the most abundant type of neurons in the
ganglionic layer of ELL and it is at synapses between parallel fibers with
these neurons that spike timing-dependent plasticity is most readily
expressed. Because these neurons are inhibitory and provide a major source of
input to the somatic region of efferent neurons, and possibly also
reciprocally inhibit each other, it is likely that plastic modulation of their
integrative properties will play a central role in signal processing in ELL.
Backpropagated broad spikes could provide a mechanism to relate activity in
compartments proximal to the soma (sensory inputs, deep path of the corollary
discharge input, axonal spikes) with distal input arriving via
parallel fibers. Linked to the mechanism of synaptic plasticity, dendritic
backpropagation would thus provide a vector for the dynamically changing
relationship between descending and sensory inputs.
This work was partially supported by the European Commission (contracts CI1*-CT92-0085 and IST-2001-34712), an ECOS grant (U97B03) to K.G. and R.B., and a grant from the CSIC - Universidad de la República, Uruguay, to R.B and L.G.
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