Function of NMDA Receptors and Persistent Sodium Channels in a Feedback Pathway of the Electrosensory System

Neil Berman,1 Robert J. Dunn,2 and Leonard Maler1

 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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 MOmega ). 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 MOmega , 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.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1. Inset: the AptNMDAR1antibody recognized a single protein in the brain membrane fraction from apteronotus. Protein samples from the soluble fraction (S, 28 µg) and membrane fraction (M, 36 µg) were fractionated on a 7.5% polyacrylamide gel and probed with the AptNMDAR1 antibody. Molecular weight standards are indicated on the left. A: immunostaining of the electrosensory lateral line lobe (ELL; centromedial and centrolateral segments) with AptNMDAR1 antibody. Pyramidal cell somata were strongly labeled, as were their apical dendrites extending through the stratum fibrosum and ramifying throughout the molecular layer. Basal dendrites of pyramidal cells were less strongly immunoreactive and were seen to extend through the plexiform layer. Strong labeling was also seen in the deep neuropil layer. Scale bar, 100 µm. B: immunolabel was seen in the somata of a basilar pyramidal cell and its basal dendrite (arrowhead); note that immunoreactivity was seen within the cytoplasm of the soma and basal dendrite. A deep basilar pyramidal cell is also labeled in the granule cell layer (asterisk). Weak immunoreactivity was seen in granular interneurons (arrow points to a granular interneuron), while the the deep neuropil was intensely stained. Scale bar, 20 µm. C: immunostaining of pyramidal cell apical dendrites. Note that the immunolabel was weak within the cytoplasm of the dendrites (asterisk) but was intense at their surface; the dendrite indicated with an asterisk was seen to branch at the juncture of ventral (below branch point) and dorsal (above branch point) components of the molecular layer. A single dendrite in the center of the image was seen to run throughout its vertical extent. The bottom half of this dendrite (below arrow 1) was optically sectioned so as to graze the dendritic surface, revealing the punctate distribution of immunoreactivity. The top half of this dendrite (above arrow 1) was optically sectioned through its center, revealing that the punctate immunoreactivity is probably associated with dendritic spines (arrow 1). Putative immunolabeled spines were typically found associated with both longitudinally (arrow 2) and cross-sectioned dendrites (arrow 3; the cross-sectioned dendrite is to the right of the indicated spine). Scale bar, 10 µm. D1: FILL: 2 basal dendrites (one in the center and one at the right edge of the image) were revealed by retrograde labeling with neurobiotin. This confocal image shows part of their dense basal bush ramifying in the deep neuropil layer. D2: NR1: Dense immunoreactivity for NR1 in the same confocal plane. D3: MERGE: merging of the images in D1 and D2 reveals that some (although not all) of the NR1 immunoreactivity is associated with fine dendritic processes of the basal dendrites. Scale bar, 20 µm. DNL, deep neuropil layer; GrL, granular cell layer; Mol, molecular layer; PlL, plexiform layer; PCL, pyramidal cell layer; StF, stratum fibrosum.

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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Fig. 2. A: excitatory postsynaptic potentials (EPSPs; single sweeps in this and subsequent figures) evoked by dorsal molecular layer (DML) stimulation in current clamp at 2 holding potentials (-65.5 and -72 mV) had a slow decay phase; both the peak and late phase (40 ms) were voltage dependent and increase by ~0.5 mV with depolarization (2.5-3 mV and 0.7-1.3 mV, respectively; compare black traces in top and bottom recordings). Application of 3-[(RS)-2-carboxypiperazine-4-yr1]-propyl-1-phosphonic acid (CPP; gray traces in this figure) selectively eliminated the late phase of the EPSP (at -65.5 mV, the reduction was ~1 mV; at -72 mV, the reduction was ~0.5 mV) but did not eliminate its voltage sensitivity (the late phase increases by 0.71 mV; compare gray traces in top and bottom recordings). Stimulus artifacts were removed in this and subsequent figures. B: in a different slice DML stimulation at subthreshold potentials evoked a voltage-dependent EPSP with a small slow CPP-sensitive component (duration ~300 ms). C: tetanic stimulation (same cell as B) evoked a summating EPSP with a prominent late phase (>800 ms) that is blocked by CPP. Action potentials were truncated in this and subsequent figures. D: voltage sensitivity of the late phase of the EPSP (same case as B; measured at 50 ms); although CPP reduced the EPSP, it did not alter its voltage dependence.



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Fig. 3. A: both the peak and late phase of DML-evoked EPSP were markedly reduced by somatic application of TTX (light gray trace throughout this figure; same cell as in Fig. 2B). B: application of CPP (dark gray trace) reduced only the late phase of a DML-evoked EPSP; subsequent somatic application of TTX reduced the peak and eliminated the late phase of the EPSP. C: DML stimulation at subthreshold potentials evokes a voltage-dependent EPSP; both peak and late phase were reduced by somatic TTX. Note the emergence of a second peak of the EPSP at the most depolarized membrane potential (-60.2 mV) and the elimination of this peak by TTX. D: plot of EPSP (data in C; measured at the time of the peak of the EPSP in the -72-mV trace) illustrates voltage dependence at potentials greater than -68 mV; the voltage dependence was blocked by TTX. Similar voltage dependence was also found at latencies >40 ms. E: tetanic stimulation evoked a summating compound EPSP (arrow) with a slow decay phase; asterisk indicates brief voltage-dependent depolarizations characteristic of these cells (Mathieson and Maler 1987). Application of TTX eliminated the slow phase of the EPSP as well as the transient voltage fluctuations.



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Fig. 4. A: in a relatively depolarized cell, parallel fiber (PF) stimulation evoked an EPSP followed by a plateau potential. TTX (light gray trace) reduced the peak of the EPSP and eliminated the plateau potential; subsequent application of CNQX + CPP (dark gray trace) greatly reduced the EPSP. B: tetanic stimulation (same cell as A) caused a plateau potential lasting >2 s; the compound EPSP during stimulation was reduced by TTX, and the plateau potential was completely blocked. C: DML-evoked EPSPs with intracellular QX314 (+TEA and Cs2+) were brief (black trace). Application of SR-95531 (dark gray trace) increased the peak and late phase of the EPSP; CPP (light gray trace) then caused a small reduction of the late phase of the EPSP. D: tetanic stimulation (same case as C) caused a rapidly decaying compound EPSP (black trace). SR-95531 greatly increased the EPSP but only caused a slight slowing of its decay. Addition of CPP blocked the late phase of the compound EPSP resulting in faster decay. E: same case as C. In the presence of SR-95531 EPSPs were voltage independent below -62 mV, although a small degree of voltage dependence was seen at potentials greater than -60 mV (asterisk); this was eliminated by CPP.

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 1998b). Application of CPP always greatly reduced the amplitude and duration of the decay phase (n = 10; Fig. 2C).

CONTRIBUTION 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.

TTX application always immediately reduced both the peak and late phase of the PF-evoked EPSP (n = 11, Fig. 3A): the mean reduction in peak amplitude was 32 ± 24%, while that of the late phase of the EPSP was on average reduced by 47 ± 30%. The effect of TTX was independent of NMDA receptor activation, since it was also obtained subsequent to the application of CPP (Fig. 3B; similar results were also obtained with the opposite order of drug application, not shown). Note that, in Fig. 3B, CPP has no effect on the peak of the EPSP, while TTX reduced it substantially.

As already demonstrated (Fig. 2, A, C, and D), the evoked EPSP was voltage dependent at membrane potentials greater than -68 mV (Fig. 3, C and D). Subsequent to TTX application, all voltage dependence of the EPSP was eliminated (n = 6; Fig. 3D). While it can be argued that the reduction in EPSP height might be due to inadvertent diffusion of TTX to the DML, the elimination of voltage dependence cannot be interpreted in this way and supports our hypothesis that persistent sodium channels contribute to PF-evoked EPSPs.

The late decay phase of the tetanus-evoked compound EPSP was also greatly reduced by somatic application of TTX (Fig. 3E: the EPSP was reduced by ~3.8 mV at 200 ms). The contribution of somatic sodium currents to this reduction cannot be rigorously estimated; it is likely that NMDA receptors are voltage dependent at suprathreshold membrane potentials (see Fig. 4E) and that blocking action potentials therefore also reduces the amplitude of the NMDA receptor component of the EPSP (see Fig. 2C).

When the resting membrane potential of impaled cells was relatively depolarized (>60 mV), stimulation of DML could occasionally induce a plateau potential that was sensitive to TTX (n = 2; Fig. 4A); subsequent application of CNQX + CPP completely eliminated the EPSP. Tetanic stimulation in this case resulted in a plateau potential that lasted >1 s and caused spiking; again, somatically applied TTX completely blocked the noninactivating current responsible for the plateau potential, suggesting that it is due to sodium channels. Note that, after TTX application, the compound EPSP attains a depolarization >4 mV, yet fails to evoke a plateau potential; in contrast, for single stimuli, the plateau potential could be evoked by depolarizations <2 mV. This strongly suggests that the effect of TTX application on EPSP are due to blockade of somatic sodium channels and not distal parallel fibers.

Previous experiments have demonstrated that persistent sodium channels are also present on the proximal apical dendrites of ELL pyramidal cells (Turner et al. 1994), and ejection of TTX onto the pyramidal cell somata would not block these dendritic channels. It was therefore puzzling that all EPSP voltage dependence was eliminated by somatic application of TTX. There are several likely reasons for this result. 1) We injected current at the soma and expect substantial current attenuation in the apical dendrites, leading to a reduced range of depolarization. 2) There is a lower density of Na+ channels on the apical dendrites (Turner et al. 1994), and this should result in lower persistent Na+ currents. 3) A recent modeling study of ELL pyramidal cells (Doiron et al. 2001) has demonstrated nonlinear positive feedback between somatic and dendritic persistent Na+ currents. Blocking somatic Na+ greatly reduced EPSP voltage dependence, and subsequent blockade of dendritic Na+ had little additional effect; the same result was also obtained by blocking dendritic Na+ first. Thus, although the blockade of distal EPSP voltage dependence by somatic TTX application is to be expected, it is also likely that dendritic Na+ channels contribute to boosting synaptic inputs to the apical dendrite (see Doiron et al. 2001).

We therefore attempted to block both somatic and dendritic sodium channels with injection of intracellular QX314; we initiated parallel fiber stimulation and data collection after failure of current-evoked action potentials. Although QX314 rapidly blocked action potentials, we could see a progressive decline in the amplitude of the evoked EPSP (over a few minutes); this is presumably due to the slow diffusion of the drug into the extensive dendritic tree of pyramidal cells. We could maintain stable recordings (>20 min) with QX314-containing pipettes in only four cells, and, because of the short impalement time, we cannot be certain that we have completely blocked all dendritic sodium channels. QX314 is expected to block calcium and potassium (Alreja and Aghajanian 1994; Andrade 1991; Oda et al. 1992; Talbot and Sayer 1996) as well as sodium channels, and we included additional potassium channel blockers in the pipette to reduce the contribution of potassium channels to the repolarization of EPSPs.

EPSPs recorded with QX314-containing pipettes decayed rapidly (n = 4; Fig. 4C). Application of the GABA-A antagonist SR-95531 enhanced the evoked EPSP (both peak and late phase, see Berman and Maler 1998b), and CPP subsequently selectively reduced its late phase (Fig. 4C). Tetanic stimulation evoked compound EPSPs with rapid decay; although SR-95531 greatly increased the EPSP, it caused only a slight prolongation of its decay phase, and this was eliminated by subsequent application of CPP (Fig. 4D). In these cases we were able to depolarize pyramidal cells to ~54 mV; in the absence of inhibition (SR-95531), there appeared to be a relatively minor voltage dependence of the EPSP at potentials greater than -60 mV, and this was eliminated by CPP (Fig. 4E; n = 4 for lack of subthreshold voltage dependence with QX314-containing pipettes; n = 2 for the effect of CPP + QX314). Although our sample size is inadequate for quantitative comparisons with control cases, the QX314 experiments are consistent with our hypothesis that persistent sodium channels contribute to the voltage dependence of DML-evoked EPSPs; they cannot, however, rule out a possible contribution of calcium channels to EPSP voltage dependence. These experiments further suggest that, at more depolarized potentials, the NMDA receptor component of these EPSPs might also exhibit voltage dependence; although this would be expected from the properties of mammalian NMDA receptors, experiments using direct dendritic recordings from ELL pyramidal cells will be required to confirm this possibility.

It is not possible to use TTX to determine the extent of sodium current amplification of the direct feedback pathway to the ELL ventral molecular layer, since its fiber tract (StF) lies adjacent to the pyramidal cell layer. We have previously shown that StF-evoked EPSPs are voltage dependent and that they have a large NMDA receptor component (Berman et al. 1997). In agreement with the results reported above, following application of CPP, there was no diminution of the voltage dependence at these synapses; further, when QX314-containing pipettes were used, voltage dependence of this pathway was eliminated (not shown). We conclude that, for both glutamatergic feedback pathways to the molecular layer of the ELL, their subthreshold voltage dependence is likely due to persistent sodium channels rather than NMDA receptors.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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.


    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.


    FOOTNOTES

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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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society