1Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario K1H 8M5; and 2Center for Research in Neuroscience, Montreal General Hospital Research Institute, Montreal, Quebec H3G 1A4, H3A 1B1, Canada
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ABSTRACT |
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Berman, Neil, Robert J. Dunn, and Leonard Maler. Function of NMDA Receptors and Persistent Sodium Channels in a Feedback Pathway of the Electrosensory System. J. Neurophysiol. 86: 1612-1621, 2001. Voltage-dependent amplification of ionotropic glutamatergic excitatory postsynaptic potentials (EPSPs) can, in many vertebrate neurons, be due either to the intrinsic voltage dependence of N-methyl-D-aspartate (NMDA) receptors, or voltage-dependent persistent sodium channels expressed on postsynaptic dendrites or somata. In the electrosensory lateral line lobe (ELL) of the gymnotiform fish Apteronotus leptorhynchus, glutamatergic inputs onto pyramidal cell apical dendrites provide a system where both amplification mechanisms are possible. We have now examined the roles for both NMDA receptors and sodium channels in the control of EPSP amplitude at these synapses. An antibody specific for the A. leptorhynchus NR1 subunit reacted strongly with ELL pyramidal cells and were particularly abundant in the spines of pyramidal cell apical dendrites. We have also shown that NMDA receptors contributed strongly to the late phase of EPSPs evoked by stimulation of the feedback fibers terminating on the apical dendritic spines; further, these EPSPs were voltage dependent. Blockade of NMDA receptors did not, however, eliminate the voltage dependence of these EPSPs. Blockade of somatic sodium channels by local somatic ejection of tetrodotoxin (TTX), or inclusion of QX314 (an intracellular sodium channel blocker) in the recording pipette, reduced the evoked EPSPs and completely eliminated their voltage dependence. We therefore conclude that, in the subthreshold range, persistent sodium currents are the main contributor to voltage-dependent boosting of EPSPs, even when they have a large NMDA receptor component.
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INTRODUCTION |
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N-methyl-D-aspartate
(NMDA) receptors are widely expressed in the vertebrate brain
(Monyer et al. 1994) but, because of their voltage
dependence, were hypothesized to provide relatively little synaptic
current at resting membrane potentials (Collingridge and Lester
1989
). It has been recently shown that, at some synapses, NMDA
receptors do contribute to synaptic potentials when the neuron is far
below spike threshold (Armstrong-James et al. 1993
;
Berman et al. 1997
; D'Angelo et al.
1995
; Fleidervish et al. 1998
). At these
synapses, NMDA receptors could contribute to voltage-dependent boosting
of subthreshold excitatory postsynaptic potentials (EPSPs). It has also
been demonstrated that persistent sodium currents, activating below
spike threshold, can augment EPSPs from distal dendritic inputs
(Andreasen and Lambert 1999
; Crill 1996
;
Hirsch and Gilbert 1991
; Lipowsky et al.
1996
; Schwindt and Crill 1995
; Stuart and
Sakmann 1995
; Urban et al. 1998
). These results
raise the question of the relative contribution of NMDA receptors and persistent sodium channels to voltage-dependent boosting of distal inputs. There are important computational consequences hinging on this
problem: NMDA receptors will boost EPSPs at their specific synaptic
site while persistent sodium channels, especially if they are expressed
at the soma, might indiscriminately amplify all excitatory synaptic input.
The electrosensory lateral line lobe (ELL) of the gymnotiform fish,
Apteronotus leptorhynchus, is a layered hindbrain nucleus receiving topographically organized input from electroreceptors on the
fish's body surface (Carr and Maler 1986). The ELL
contains, within seperate laminae, GABAergic interneurons and pyramidal cells; some pyramidal cells have basal dendrites as well as an extensive spiny apical dendritic tree that ramifies in a molecular layer. Electroreceptor afferents terminate on the basal dendrites of
pyramidal cells and granular interneurons. The apical dendritic tree of
pyramidal cells receives feedback input from two sources: a direct
feedback pathway enters the ELL via a compact fiber bundle (stratum
fibrosum or StF) and terminates in the ventral molecular layer (VML).
An indirect feedback pathway emanates from cerebellar granule cells;
parallel fibers (PF) from these granule cells terminate on distal
pyramidal cell dendrites in the dorsal molecular layer (DML) of the ELL.
Electroreceptor afferents, as well as both direct and indirect feedback
pathways, are glutamatergic (Wang and Maler 1994); ELL
pyramidal cells express high levels of the NR1 subunit of the NMDA
receptor (Bottai et al. 1997
), and its molecular layer contains high levels of NMDA receptor binding (Maler and
Monaghan 1991
). We have previously shown that the direct
feedback pathway had a prominent NMDA receptor component and that EPSPs
evoked by stimulation of this pathway are voltage dependent
(Berman et al. 1997
). Pyramidal cells also express
prominent persistent sodium current in their somata and proximal apical
dendrites (Turner et al. 1994
); we have not directly
tested the hypothesis that the voltage dependence at the direct
feedback synapses is due to their NMDA receptors rather than the
persistent sodium channels.
In this paper we have examined the relative contribution of NMDA receptors versus persistent sodium channels to the EPSPs evoked by stimulation of indirect feedback pathway. Our results demonstrate that, although functional NMDA receptors are prominently expressed at synapses of the indirect feedback pathway, somatic persistent sodium channels and not NMDA receptors amplify these distal EPSPs in the subthreshold voltage range.
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METHODS |
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Preparation of antibodies
A glutathione-S-transferase-Apteronotus NMDAR1 (AptNR1)
expression vector was constructed by insertion of the cDNA encoding the
carboxyl terminal 57 amino acids, excluding the alternative splice
cassettes C1, C1' and C1", of the AptNR1 coding sequence (amino acids
844-901) (Bottai et al. 1998) into the GST expression vector pGEX4T1 (Pharmacia, Uppsala, Sweden). The fusion protein was
expressed in Escherichia coli strain DH5-a and purified from inclusion bodies as described by Frangioni and Neel
(1993)
. NMDAR1 polyclonal antibodies were raised in rabbits and
depleted of GST immunoreactivity by adsorption to GST protein bound to
Affigel 10 beads (Biorad, Richmond, CA) and then affinity purified by adsorption to AptNR1 fusion protein bound to Affigel 10 beads.
Immunoblotting
Apteronotus brain proteins were prepared by homogenization of brain tissue in 50 mM Tris/HCl, pH 7.5, 0.25 M sucrose, 25 mM KCl, 5 mM MgCl2, 1 mM phenylmethylsulfonylfluoride at 4°C. The homogenate was clarified by centrifugation (800g, 10 min) and then membranes purified by centrifugation (100,000g, 60 min). Soluble and membrane fractions were separated by electrophoresis on a 7.5% polyacrlamide gel and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA). Membranes were incubated with the antibody (0.45 µg/ml) overnight at 4°C and the immunoreactive proteins detected with horseradish peroxidase coupled secondary antibody and chemiluminescence (NEN Life Sciences, Boston, MA).
Immunohistochemistry
Fish were anesthetized (0.2% 3-aminobenzoic acid ethyl ester, MS-222) and perfused intracardially with 4% paraformaldehyde in phosphate-buffered saline (0.1 M, pH 7.4 PBS) and stored in fixative overnight (4°C). Vibratome sections of ELL (30-40 µM) were collected in chilled PBS, mounted and incubated in blocking buffer (10% normal horse serum, 1% bovine serum albumen, 0.2% Triton-X-100 in PBS; 2 h, RT). Sections were transferred to 0.1 times blocking buffer containing anti-AptNR1 (1:100) for 48 h (4°C). After washing (PBS), the sections were incubated (2-3 h, RT) in biotinylated donkey anti-rabbit antiserum (1:200 in 0.1 times blocking buffer; Amersham, Oakville, Ontario, Canada), washed and incubated in streptavidin-CY3 (1:100 in PBS for 2-3 h, RT; Sigma-Aldrich, St. Louis, MO). Sections were viewed on a Zeiss confocal microscope.
We retrogradely labeled pyramidal cells to mark their basal dendrites.
Fish were anesthetized and crystals of Neurobiotin (Vector
Laboratories, Burlingame, CA) placed into the torus semicircularis (Carr and Maler 1986). After 1 or 2 days survival, the
fish were perfused, sectioned, and incubated in blocking buffer as
described above, followed by streptavidin-CY3 (1:100 in PBS) overnight
(4°C). If strong labeling of pyramidal cell basal dendrites had been achieved, sections were subsequently immunostained for NR1 as above,
using a goat anti-rabbit antibody conjugated to Alexa 488 (1:100 in 0.1 times blocking buffer; Molecular Probes, Eugene, OR).
Electrophysiology
Preparation of ELL slices and electrophysiological techniques
were as previously described (Berman and Maler 1998b,c
;
Berman et al. 1997
). Briefly, fish were anesthetized,
the ELL was removed, and slices (350 µm) were prepared on a
vibratome. Slices were maintained in an interface chamber in artificial
cerebrospinal fluid (ACSF; in mM: 124 NaCl, 3 KCl, 0.75 KH2PO4, 2 CaCl, 2 MgSO4, 24 NaHCO3, and 10 D-glucose) at room temperature (23°C). Intracellular recordings were made in the pyramidal cell layer of the centromedial segment of the ELL with 2 M potassium acetate-filled electrodes (80-120 M
). As previously described (Berman and Maler
1998c
) membrane potentials and input resistance of pyramidal
cells ranged from
65 to
73 mV and 15 to 28 M
, respectively; we
only analyzed recordings where spike height exceeded 70 mV.
Parallel fibers in DML or StF fibers were stimulated with a monopolar
tungsten electrode (50-100 µs, 1-50 V, 20 µs); previously established criteria were used to establish specificity of stimulation (Berman and Maler 1998a,b
; Berman et al.
1997
). Stimulation sites in DML were >500 µM away from the
pyramidal cell layer. Electrical signals were amplified (Axoclamp-2A,
Axon Instruments), filtered (10 kHz cutoff), digitized (ITC-16,
Instrutech, Port Washington, NY), and analyzed off-line
(IgorPro, Wavemetrics, Lake Oswego, OR). All experiments were software
controlled (A/Dvance, McKellar Designs, Vancouver, BC, Canada or Pulse
Control, NIH).
Drugs were pressure applied (Neurophore, Medical Systems, Great Neck,
NY) to ELL slices. 6-Cyano-7-nitroquinoxaline (CNQX, 1 mM dissolved
with dimethyl sulfoxide in ACSF) and
3-[(RS)-2-carboxypiperazine-4-yr1]-propyl-1-phosphonic acid
(CPP, 1 mM in ACSF; Tocris, Ballwin, MO) were applied to the DML, and
tetrodotoxin (TTX, 16 µM in ACSF; Sigma-Aldrich) was applied to the
plexiform and pyramidal cell layer (Turner et al. 1994).
The GABA-A antagonist, SR-95531 (100 µM in ACSF; Tocris), was ejected
over both DML and the pyramidal cell layer. Microdroplets were
delivered several times until a maximal response was obtained.
QX314 (100 mM in 2 M KAc; Sigma-Aldrich) was applied via an intracellular pipette also containing cesium acetate (4 M) and tetraethylammonium (50 µM); this combination will block sodium, calcium, and potassium channels.
Stimulation of DML parallel fibers always evokes EPSPs and may also
evoke inhibitory postsynaptic potentials (IPSPs) of variable amplitude
(Berman and Maler 1998b). Analysis of the amplitude of
the late phase of the EPSP was only done in cases where minimal IPSPs
were evoked by single or tetanic stimulation.
All experimental protocols were approved by the University of Ottawa Animal Care Committee.
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RESULTS |
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Immunoblotting
The NMDA receptor antibody was produced using the carboxyl
terminal, intracellular tail segment of the NMDA NR1 receptor subunit protein, a segment that is present in all forms of the NMDAR1 receptor
protein (Bottai et al. 1998). Because the NR1 subunit is
required to form functional NMDA receptors, this antibody will recognize all functional NMDA receptor complexes. The specificity of
the antibody was demonstrated in a Western blot experiment, where the
antibody recognized a single protein in the crude membrane fraction
derived from A. leptorhynchus brain. No signal was seen in
the corresponding soluble protein fraction. The protein detected (Fig.
1, inset) is likely to
represent the form of the NMDA receptor lacking the alternatively
spliced inserts because our previous measurements of alternative
spliced NMDAR1 mRNAs in A. leptorhynchus brain demonstrated
that most of the NMDAR1 protein lacks these cassettes (Bottai et
al. 1998
). The size of this protein is approximately 110 kDa,
which compares to a predicted size of 99.0 kDa for the AptNMDAR1
subunit in the nonglycosylated form lacking the alternative splice
cassettes; this increased size is presumably due to the glycosylation
of the native protein. These results demonstrate that the antibody is
highly specific for the NMDAR1 receptor protein and therefore suitable
for detecting NMDA receptors in our immunohistochemical studies.
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Immunohistochemistry
The NR1 antibody labeled only neuronal somata and dendrites
throughout the apteronotus brain, with a distribution similar to that
reported after in situ hybridization with an NR1 probe (data not shown)
(Bottai et al. 1997). Labeling was eliminated when
incubation in the primary antibody was omitted, and absent in brain
regions whose cells lack NR1 mRNA and NMDA receptor binding (data not
shown; e.g., cerebellar molecular layer Purkinje cells) (Bottai
et al. 1997
; Maler and Monaghan 1991
).
In the ELL pyramidal cells, somata were intensely labeled as was their
entire apical dendritic tree (Fig. 1A); the basal dendrites of pyramidal cells were less strongly labeled, although the deep neuropil layer, the site where these dendrites ramify, was strongly labeled (Fig. 1, A and B). All pyamidal cell
subtypes, including deep basilar pyramidal cells (Fig. 1B),
were labeled. The deep neuropil contains thin dendrites arising from
both pyramidal cells and granular interneurons, as well as
electroreceptor afferent fiber terminals, and it was not possible, in
this material, to differentially localize the NR1 immunoreactivity.
Somatic dendrites of pyramidal cells (Maler 1979), which
receive only inhibitory input (Berman and Maler 1998c
;
Maler and Mugnaini 1994
; Maler et al.
1981
), were not immunostained in normal (Fig. 1B) or
double-labeled material (not shown), suggesting that apteronotus NR1
protein is selectively transported to appropriate dendritic target sites.
Immunostaining of the entire apical dendritic tree (in both VML and DML) of pyramidal cells was intense, and optical sectioning suggested that it was predominantly associated with surface membrane rather than cytoplasm; although we lack conclusive electron microscopic data, it appears that dendritic spines were especially strongly labeled (Fig. 1C). The association of NR1 with putative dendritic spines was confirmed by double labeling with retrogradely transported neurobiotin (data not shown). These results suggest that both direct feedback fiber (VML) and indirect feedback fiber (parallel fibers to the DML) evoked EPSPs are likely to have a large NMDA receptor component.
Double labeling of pyramidal cells demonstrated that apteronotus NR1 was also associated with their basal bushes (Fig. 1, D1-D3). Much of the NR1 immunoreactivity in the deep neuropil layer was not coextensive with retrogradely labeled pyramidal cell basal dendrites; presumably this immunolabel is associated with the thin dendrites of the labeled granular interneurons.
Granular interneurons (Fig. 1B), ovoid cells, and VML cells
(data not shown) were moderately immunostained; stellate and
polymorphic cells were barely detectable consistent with their
expression of low levels of NR1 mRNA (data not shown) (Bottai et
al. 1997).
Electrophysiology
We recorded from 46 cells within the pyramidal cell layer of the
ELL. Based on their location and electrophysiological characteristics (Berman et al. 1997; Turner et al. 1994
),
all impalements are likely to have been in pyramidal cells; basilar and
nonbasilar pyramidal cells respond in similar fashion to stimulation of
feedback input (Berman and Maler 1998a
,b
), so we did not
make finer distinctions of cell types. We analyzed the amplitude and
not the time course of evoked EPSPs since the latter is very sensitive
to disynaptic inhibition at the soma (Berman and Maler
1998b
); application of GABA-A blockers to the soma
unfortunately causes paroxysmal discharge and therefore cannot be used
to isolate EPSPs.
We have previously characterized EPSPs evoked by parallel fiber
stimulation (Berman and Maler 1998b). As before, evoked
EPSPs typically peaked at 6-8 ms and then decayed slowly to baseline (>100 ms). Both the peak and the late phase of the EPSP were enhanced at depolarized membrane potentials (Fig.
2, A and B); as
previously noted (Berman and Maler 1998b
),
depolarization sometimes caused the appearance of a second peak in the
EPSP (Fig. 3C). Application of
CNQX (with or without CPP, n = 8) blocked most (>90%)
of the parallel fiber-evoked EPSPs (Fig.
4A) (and see Berman and
Maler 1998b
), suggesting that excitatory transmission in the
DML feedback pathway is mediated mainly by ionotropic glutamate
receptors.
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CONTRIBUTION OF NMDA RECEPTORS. Application of CPP (n = 14) had a variable effect on the peak (6-8 ms) of PF-evoked EPSPs (Fig. 2, A and B); no effect was seen in one-half the cases, and on average there was a 27 ± 33% (mean ± SD) reduction in peak amplitude. The effect of CPP was, in every case, pronounced for the late phase of the EPSP (>30 ms; n = 14; Fig. 2, A and B). On average CPP reduced the late phase of the EPSP by 67 ± 27%. However, even after apparent complete blockade of NMDA receptors, PF-evoked EPSP voltage dependence was not altered in the subthreshold potential range (n = 5; Fig. 2D).
Tetanic stimulation of DML parallel fibers produced a characteristic summating compound EPSP followed by a prolonged decay phase that can last in excess of 500 ms (Fig. 2C) (Berman and Maler 1998bCONTRIBUTION OF PERSISTENT SODIUM CURRENTS.
Localized microejection of TTX can be confined to the pyramidal cell
layer of the ELL by ejecting within the plexiform layer (ventral to
pyramidal cells and the location of pyramidal cell efferent axons)
(Turner et al. 1994). Since the StF lies between the
pyramidal cell layer and the DML, we were able to preclude inadvertent
diffusion of TTX to the DML by always monitoring the response of
pyramidal cells to StF stimulation after injections of TTX. TTX
application was continued (2-3 pulses) until intracellular current-evoked spiking (>1 nA) was completely blocked. At this point
the response to StF stimulation was modestly reduced [see Berman et al. (1997)
, for a characterization of the
response of pyramidal cells to StF stimulation]; cases where the
StF-evoked EPSP was reduced by >10% of its initial value were
discarded. We assume that the reduction in StF responses was due to the
diffusion of TTX to the StF and that, as previously shown
(Turner et al. 1994
), it does not diffuse >500 µM to
the site of PF stimulation.
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DISCUSSION |
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The high levels of apteronotus NR1 protein expression in the
apical dendrites of pyramidal cells confirms previous suggestions that
NMDA receptors are prominent in the feedback pathways of ELL
(Maler and Monaghan 1991) and is consistent with the
fact that NMDA receptor currents typically account for ~50% of the EPSP (late phase) evoked by parallel fiber (see above) or direct feedback afferent (Berman et al. 1997
) stimulation.
Although the presence of extrasynaptic NR1 cannot be excluded, our
immumohistochemical results suggest that this protein is mainly located
within dendritic spines of the pyramidal cell apical dendrites and the
nonspiny basal dendritic bush. NR1 immunoreactivity is apparently
lacking in the somatic dendrites of these cells, consistent with their GABAergic input (Maler and Mugnaini 1994
). This
implies a precise targeting mechanism able to selectively direct NMDA
receptors to specific spiny or nonspiny dendritic target sites. NMDA
receptors in rat cortex are associated with members of PSD-95 family of proteins. We have recently characterized this protein family in the
brain of A. leptorhynchus (Lee et al. 2000
)
and demonstrated that several PSD-95 proteins (PSD-93, PSD-95.1, and
SAP-97) are moderately expressed in ELL pyramidal cells; it will be
important to determine whether these proteins are involved in targeting of NMDA receptors or other proteins associated with glutamatergic postsynaptic densities.
The characteristics of the NMDA component of the EPSP evoked by
stimulation of the indirect feedback pathway (parallel fibers, DML) is
similar to those previously reported for the direct feedback pathway
(StF, VML): NMDA receptors produce a very slow EPSP (>500 ms in the
absence of inhibition), but one that is active at relatively hyperpolarized potentials (approximately 70 mV). It appears as if
NMDA receptors might contribute more to the early phase of the EPSP in
the direct compared with the indirect feedback pathway; for the direct
pathway, the peak of the EPSP was typically reduced by 50%
(Berman et al. 1997
), while smaller (27%) or no
reductions were seen for PF stimulation. We cannot, however, exclude
greater cable effects for the more distal indirect feedback pathway.
An NMDA receptor contribution to parallel fiber-evoked EPSPs has also
recently been demonstrated for neurons in the mormyrid ELL
(Grant et al. 1998) and the mammalian dorsal cochlear
nucleus (Manis and Molitor 1996
), suggesting that these
receptors serve an important function in feedback afferents to brain
stem octavolateral nuclei (see also Bell et al. 1997
;
Montgomery et al. 1995
).
All four A. leptorhynchus NR2 subunits have now been
identified and their expression studied by in situ hybridization (R. Finn and R. J. Dunn, unpublished observations): ELL
pyramidal cells contain NR2A, NR2B, and NR2C subunits. The
electrophysiology of pyramidal cell NMDA receptors is most consistent
with the properties of the NR2C subunit. Glutamate stimulation of
co-expressed mammalian NR1 + NR2C subunits produces slow NMDA receptor
currents with a time course similar to that of PF-evoked EPSPs;
further, the presence of the NR2C subunit confers less
Mg2+-dependent channel block, so that an NMDA
receptor containing these subunits can be activated down to
approximately 70 mV (Kutswada et al. 1992
;
Monyer et al. 1992
). Cerebellar granule cells, which contain abundant NR2C, have NMDA receptor activation at relatively hyperpolarized potentials (D'Angelo et al. 1995
), and
it has recently been suggested that, in a subpopulation of rat cortical
cells, NMDA receptor activation at resting membrane potentials is also due to their expressing NR2C subunits (Fleidervish et al.
1998
). It is not known whether this conclusion will generalize
to other vertebrate neurons whose NMDA receptors are activated at
relatively hyperpolarized membrane potentials (Kovalchuk et al.
2000
; Wolszon et al. 1997
).
All mammalian NR1-NR2 subunit combinations exhibit a steep voltage
dependence at potentials positive to approximately 40 mV. It is
therefore likely that the NMDA receptor-mediated component of the
DML-evoked EPSP would have shown voltage dependence had we been able to
depolarize their apical dendrites to this potential. The small
CPP-dependent voltage sensitivity seen at potentials greater than
60
mV (Fig. 4E) is consistent with this idea; further experiments using patch-clamp recordings from apical dendrites will be
required to determine the exact shape of the current-voltage (I-V) curve of the NMDA receptors expressed in ELL pyramidal cells.
Persistent sodium currents have been identified in many types of
neurons (Crill 1996) and have been shown to boost EPSPs. In cortical and hippocampal pyramidal cells, the site of boosting has
been variously attributed to either dendritic (Lipowsky et al.
1996
; Schwindt and Crill 1995
) or somatic
persistent sodium channels (Stuart and Sakmann 1995
); a
recent study of hippocampal pyramidal cells, using methods similar to
ours, has also identified somatic persistent sodium currents as
responsible for amplification of EPSPs from distal dendrites
(Andreasen and Lambert 1999
). It has also been reported
that persistent sodium channels, rather than NMDA receptors, are
responsible for voltage-dependent boosting of EPSPs in neurons of the
visual cortex (Hirsch and Gilbert 1991
). In the studies
cited above, NMDA receptors appeared to make a minor contribution to
EPSPs at resting membrane potentials, and the relative contribution of
these NMDA-R and persistent sodium currents could not be ascertained.
Since both NMDA receptors and persistent sodium channels contribute to
parallel fiber-evoked EPSPs in ELL, we can conclusively identify the
latter as responsible for subthreshold boosting of EPSPs. This
conclusion is consistent with our modeling study of excitatory and
inhibitory feedback input to ELL pyramidal cell apical dendrites
(Berman and Maler 1998b
): when both voltage-dependent
persistent sodium channels and NMDA receptors (kinetics from mammalian
data) were included in our model pyramidal cell, almost all the
voltage-dependent boosting of distal EPSPs was due to the sodium
channels. Our results are also consistent with our more recent detailed
model of an ELL pyramidal cell (Doiron et al. 2001
).
When somatic and proximal dendritic persistent
Na+ channel densities were set so as to account
for the hyperpolarization caused by local (soma or dendrite)
application of TTX (Turner et al. 1994
), distal EPSPs
were boosted to a similar extent to that seen experimentally.
The role of somatic persistent sodium channels, rather than NMDA
receptors, in amplifying EPSPs thus appears to be a general feature of
diverse neuronal types and may therefore subserve an important
computational role(s). There are also persistent sodium channels in the
proximal (although not distal) dendrites of ELL pyramidal cells
(Turner et al. 1994), although, due to the limitations of the TTX ejection techniques, we do not know whether they subserve the same purpose(s) as do the somatic channels.
An important aspect of the somatic localization of persistent sodium
channels is that they will permit interaction of synaptic input
arriving on different dendritic trees. ELL pyramidal cells receive
electroreceptor afferent input to their basal dendrite and feedback
input to their apical dendrites. We have hypothesized that
voltage-dependent conductances may be the biophysical substrate of a
nonlinear amplification of synchronous input to basal and apical
dendrites (supralinear summation due to voltage dependence of the
EPSPs) and that this might underly a sensory "searchlight" (Berman and Maler 1999). The results reported here imply
that somatic persistent sodium channels, and not NMDA receptors, are responsible for the putative searchlight. Experimental and modeling studies of inhibition of ELL pyramidal cells further suggests that
inhibitory input to their soma may act mainly by determining the extent
to which persistent sodium channels can amplify EPSPs (Berman
and Maler 1998b
). This effect is due to the steep voltage dependence of these channels, allowing their ability to amplify EPSPs
to be greatly reduced by relatively small hyperpolarizations; this is
similar to the interaction between persistent sodium channels and IPSPs
recently proposed by Stuart (1999)
. Thus the putative sensory searchlight will be specifically regulated by inhibitory input
terminating on the somata of pyramidal cells.
Since NMDA receptors on ELL pyramidal cell apical dendrites are not
voltage dependent at subthreshold potentials, they will not provide the
nonlinear amplification required for a searchlight mechanism (as
previously proposed) (Berman et al. 1997). We therefore propose two different functions for these NMDA receptors. First, we
hypothesize that, at subthreshold potentials, NMDA receptors will
permit temporal summation of feedback EPSPs; note that, without NMDA
receptors, there would not be a slow phase of the feedback EPSPs to be
amplified by persistent sodium channels. NMDA receptors permit temporal
integration over time intervals >500 ms in comparison to ~100 ms
when only
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)
receptors are activated (see Fig. 2). The functional reason for such
long integration times in the feedback input to ELL pyramidal cells is
not understood at present.
Second, we propose that dendritic NMDA receptors are required for
synaptic plasticity at parallel fiber synapses. Back-propogation of
spikes will depolarize the apical dendrites to potentials of approximately 40 mV (B. Doiron, A. Longtin, and L. Maler, unpublished modeling studies for DML) (Turner et al. 1994
). If, as
is the case of mammalian NMDA receptors, apteronotus NMDA receptors on the apical dendrites are voltage dependent in this range, they will
become maximally activated when feedback synaptic input overlaps spiking; recent studies using Ca2+ imaging have
demonstrated this type of boosting in single spines of cortical
pyramidal cells (Yuste et al. 1999
). Pyramidal cell NMDA
receptors are responsible for synaptic depression at the apteronotus
ELL direct feedback pathway (J. Bastian, personal communication; A.-M.
Oswald and L. Maler, unpublished observations). We therefore
propose that the maximal Ca2+ entry that occurs
during pairing of feedback and action potentials is required for the
anti-Hebbian plasticity of these synapses (see Bastian
1995
, 1996
, 1998
,
1999
). A requirement for NMDA receptors in anti-Hebbian
depression of parallel fiber-evoked EPSPs in apical dendrites of
neurons in the mormyrid ELL has already been demonstrated (Han
et al. 2000
). Thus NMDA receptors may generally be important for anti-Hebbian depression and the cancellation of expected sensory signals in the octavolateral system (Bell et al. 1997
).
In summary we have found that somatic persistent sodium channels are responsible for subthreshold amplification of distal feedback EPSPs; one potential function of this amplification might be to enhance spatially specific peripheral input: the searchlight hypothesis. Although NMDA receptors are abundant at feedback synapses and contribute appreciably to synaptic currents at resting membrane potentials, they are not voltage dependent in the subthreshold range. The function of these NMDA receptors may be for temporal integration of feedback synaptic input and to permit activity-dependent synaptic plasticity.
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ACKNOWLEDGMENTS |
---|
We thank Dr. R. Munger for assistance with confocal microscopy and W. Ellis for assistance with immunostaining.
This work was supported by grants from the Canadian Institutes of Health Research to R. J. Dunn and L. Maler.
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FOOTNOTES |
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Address for reprint requests: L. Maler, Dept. of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada (E-mail: lmaler{at}aix1.uottawa.ca).
Received 13 March 2001; accepted in final form 11 June 2001.
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REFERENCES |
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