Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada
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Berman, Neil J., James Plant, Ray W. Turner, and Leonard Maler. Excitatory amino acid receptors at a feedback pathway in the electrosensory system: implications for the searchlight hypothesis. J. Neurophysiol. 78: 1869-1881, 1997. The electrosensory lateral line lobe (ELL) of the South American gymnotiform fish Apteronotus leptorhynchus has a laminar structure: electroreceptor afferents terminate ventrally whereas feedback input distributes to a superficial molecular layer containing the dendrites of the ELL principle (pyramidal) cells. There are two feedback pathways: a direct feedback projection that enters the ELL via a myelinated tract (stratum fibrosum, StF) and terminates in the ventral molecular layer (VML) and an indirect projection that enters as parallel fibers and terminates in the dorsal molecular layer. It has been proposed that the direct feedback pathway serves as a "searchlight" mechanism. This study characterizes StF synaptic transmission to determine whether the physiology of the direct feedback projection is consistent with this hypothesis. We used field and intracellular recordings from the ELL to investigate synaptic transmission of the StF in an in vitro slice preparation. Stimulation of the StF produced field potentials with a maximal negativity confined to a narrow band of tissue dorsal to the StF. Current source density analysis revealed two current sinks: an early sink within the StF and a later sink that corresponded to the anatomically defined VML. Field potential recordings from VML demonstrated that stimulation of the StF evoked an excitatory postsynaptic potential (EPSP) that peaked at a latency of 4-7 ms with a slow decay (~50 ms) to baseline. Intracellular recordings from pyramidal cells revealed that StF-evoked EPSPs consisted of at least two components: a fast gap junction mediated EPSP (peak 1.2-1.8 ms) and a chemical synaptic potential (peak 4-7 ms) with a slow decay phase (~50 ms). The amplitudes of the peak and decay phases of the chemical EPSP were increased by depolarizing current injection. Pharmacological studies demonstrated that the chemical EPSP was mainly due to ionotropic glutamate receptors with bothN-methyl-D-aspartate (NMDA) and non-NMDA components. NMDA receptors contributed substantially to both the early and late phase of the EPSP, whereas non-NMDA receptors contributed mainly to the early phase. Stimulation of the StF at physiological rates (100-200 Hz, 100 ms) produced an augmenting depolarization of the membrane potential of pyramidal cells. Temporal summation and a voltage-dependent enhancement of later EPSPs in the stimulus train permitted the compound EPSP to reach spike threshold. The nonlinear behavior of StF synaptic potentials is appropriate for the putative role of the direct feedback pathway as part of a searchlight mechanism allowing these fish to increase the electrodetectability of scanned objects.
Vertebrate sensory transmission consists of ascending pathways leading from receptors to higher "integrative" regions and eventually to motor areas of the brain. At each level, local neuronal interactions generate receptive fields that characteristically increase in selectivity and complexity at succeeding levels of the neuraxis. Sensory systems also have extensive feedback projections and long-range horizontal interactions. These connections may permit regions outside the cell's classic receptive field to modulate responses to receptive field input and may underlie "higher level" effects such as attention and adaptation to sustained input.
Preparation of ELL slices
A total of 87 weakly electric fish of the species Apteronotus leptoryhchus (Brown Ghost Knife Fish) were used. Transverse slices of the ELL were prepared as previously described (Mathieson and Maler 1988
Stimulation of StF
Stimulating electrodes were either bipolar (65 µm nichrome wire) or a monopolar tungsten electrode (<5-µm tip diameter). The stimulating electrode was positioned at the dorsal aspect of the StF within the medial segment to prevent direct electrical activation of centromedial segment interneurons, pyramidal cell efferent axons within the plexiform layer (Turner et al. 1994 Drug application
Drugs were applied using two methods. 1) Bath applications for field potential experiments. After 1 h of normal ACSF, perfusate was switched for >1 h to either 4 mM Mn2+-ACSF solution [containing (in mM) 129 NaCl, 10 D-glucose, 3.25 KCl, 0.2 CaCl2, 11.4 tris(hydroxymethyl)aminomethane, and 20 N-2-hydroxyethylpiperazine-N Recording of synaptic potentials
FIELD POTENTIAL RECORDING.
Potentials recorded by glass micropipettes (1 M NaCl; 2-10 M ANALYSIS OF FIELD POTENTIALS.
Under each stimulus condition, 10-15 consecutive StF-evoked field potentials were averaged. Field potentials were mapped along the dendro-somatic axis of ELL pyramidal cells (dashed line in Fig. 1) by two methods. 1) StF field potentials were recorded from positions corresponding to visually identifiable anatomic structures within the ELL slice preparation, allowing for the construction of low-resolution spatial maps of StF-evoked field potentials (n = 8). 2) High-resolution maps constructed by recording field potentials (50 µm depth) every 25 µm along most of the ELL pyramidal cell axis (n = 3) were used for one-dimensional current source density (CSD) analysis. Standard methods for CSD analysis in slices were followed (Bode-Greul et al. 1987 INTRACELLULAR RECORDING.
Intracellular recording pipettes (3 M potassium acetate, 70-200 M The ELL is a laminar structure with separate layers for electrosensory afferent (deep fiber layer) and feedback input (VML, DML). The majority of efferent neurons are within the pyramidal cell layer, whereas most interneurons are located in the granular cell layer. The StF is a myelinated fiber tract the unmyelinated terminal fibers of which branch densely in the VML, which occupies ~100-150 µm (15-20%) of the total molecular layer width (measured along the pyramidal cell dendritic axis: dashed line, Fig. 1). Pyramidal cell axons run in the plexiform layer to exit at the medial aspect of the ELL. As seen in Fig. 1, the dendritic axis of pyramidal cells is orthogonal to the ELL cellular and fiber laminae (Maler 1979 StF-evoked EPSPs
FIELD POTENTIAL RECORDINGS.
Laminar profiles of StF-evoked field potentials were constructed, and CSD analysis carried out to examine the site for termination and the nature of synaptic inputs activated by StF stimulation (Fig. 2). The largest field potential response evoked by StF stimulation always was recorded in the VML as a biphasic potential consisting of a short-duration positivity followed by a rapid negative-going potential (peak latency 3.2 ± 0.56 ms, PHARMACOLOGY OF STF-EVOKED FIELD POTENTIAL.
The above identification of StF-evoked responses was further tested using blocking agents of synaptic transmission (Fig. 3). The StF-evoked VML potential was decreased significantly by bath application of 4 mM Mn2+ in low Ca2+ (0.2 mM)-containing medium, decreasing from 3.0 ± 1.5 to1.73 ± 1 mV (P < 0.01; n = 6; Fig. 3A). The negative peak of the remaining triphasic potential had a latency of 2.55 ± 0.73 ms and was the expected response for a fiber volley in StF afferent fibers (Fig. 3A; note that subsequent experiments described below indicated an additional contribution by an electrotonic synaptic component). Subtraction of the remaining response in Fig. 3A revealed a long-lasting, synaptically mediated component of the StF-evoked response with a peak latency of 4.3 ± 1.3 ms (n = 6), a value similar to the peak latency of EPSPs recorded with intradendritic impalements (see below) and significantly greater than the latency of the fiber volley (P < 0.05). These data indicated that the StF fiber volley slightly overlapped the early component of the field synaptic response, contributing to this aspect of field potential recordings in normal medium. Nevertheless, there was sufficient separation between presynaptic fiber and synaptic potentials to pharmacologically distinguish an early and late phase of the EPSP (see below).
Intracellular recording
Recordings were obtained from >100 somatic and 13 proximal apical dendritic penetrations of pyramidal cells (somatic vs. dendritic recordings were distinguished using criteria established by Turner et al. 1994
INTRACELLULAR RECORDING: PHARMACOLOGY.
Consistent with the results from the extracellular studies, pressure application of APV or CPP into the region of the VML (n = 44) consistently reduced the earliest chemical component of the StF-evoked EPSPs by 40-50% (Fig. 8A). The effect on the slow component of the evoked responses was variable and ranged from no effect to complete blockade; Fig. 8A illustrates a typical case with a reduction of the late phase of the EPSP. These results therefore suggest that ~50% of the peak EPSP (<10 ms) can be accounted for by NMDA-receptor activation. The slow voltage-dependent component of StF-evoked EPSPs also is mediated partially by NMDA receptors, although its lability and overlap with IPSPs makes it difficult to quantify the contribution of NMDA receptors.
In this study, we have shown that the direct feedback pathway from the rhombencephalic n. praeminentialis remains intact in ELL brain slices. Although the StF is close to pyramidal cell efferent fibers in the plexiform layer, correct placement of the stimulating electrode and limiting stimulation voltages prevented antidromic activation. Physiological (CSD) and anatomic estimates of the thickness of the VML coincide, suggesting that StF stimulation did not recruit DML fibers. The interpretation of our results presented below therefore is based on specific activation of the StF, the direct feedback projection to the ELL.
Relating in vitro properties of Stf-evoked EPSPs to electroreception
The ELL is connected reciprocally and topographically to the n. praeminentialis (Maler et al. 1982
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
) and electrolocation (Bastian 1986a
). Distortions in the fish's EOD are detected by electroreceptors, which project to the medullary electrosensory lobe (ELL) (Carr and Maler 1986
). The ELL is a laminar structure and consists of four segments (medial, centromedial, centrolateral, and lateral segments) optimized for processing of different features of the electrosensory input (Maler 1989
; Shumway 1989
). Pyramidal cells are the principle output neurons of the ELL and project topographically to the midbrain (torus semicircularis) and the isthmic nucleus praeminentialis (Pd) (Carr and Maler 1986
). The ascending electrosensory projections have been mapped in detail up to sensorimotor interfaces in the optic tectum and diencephalon (tectum: Bastian 1982
; diencephalon: Heiligenberg 1991
; Rose and Heligenberg 1988
), and recordings from periphery to diencephalon reveal the elaboration of receptive fields leading to outputs suitable for driving motor responses (Heiligenberg 1991
).
; Sas and Maler 1983
, 1987
): stellate, multipolar, and tufted cells. Stellate cells project directly and topographically back to the ventral molecular layer (VML) of the ELL (direct feedback) via a myelinated fiber bundle (tractus stratum fibrosum, tSF; within the ELL this tract is designated the stratum fibrosum or StF) and terminates densely in the ventral-most portion of the ELL molecular layer (VML; Fig. 1) (Maler 1979
; Maler et al. 1981b
). Multipolar and tufted cells project to cerebellar granule cells overlying the ELL, and these cells in turn project parallel fibers to the dorsal molecular layer (DML) of the ELL (indirect feedback). There is also a direct inhibitory feedback projection from GABAergic bipolar cells in the hilar region of Pd, via fibers lying at the ventral aspect of tSF, to pyramidal cell somata (Maler and Mugnaini 1994
).
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FIG. 1.
Sites for recording and stimulating the stratum fibrosum (StF) projection in an electrosensory lateral line lobe (ELL) transverse section (level T-7) (Maler et al. 1991 ). Injection of wheat germ agglutinin (WGA)-horseradish peroxidase in isthmic nucleus praeminentialis (Pd) anterogradely labeled the stratum fibrosum, which terminated in the ventral molecular layer (VML) and retrogradely labeled pyramidal cells (Maler 1979
; Maler et al. 1992). One basilar pyramidal cell (right of - - -) shows the orthogonal orientation (- - -) of their dendritic arbors to the ELL laminae. Inset: simultaneous recordings obtained from a pyramidal cell proximal apical dendrite (bottom traces) and the mid-VML field potential (top traces) in the centromedial segment during low- (thick trace) and high-intensity (thin traces) StF stimulation in the medial segment (*). AS, antidromic spike; BS, brain stem; EGp, eminentia granularis posterior (cerebellar granule cells); CLS, centrolateral segment; CMS, centromedial segment; DFL, deep fiber layer; DML, dorsal molecular layer; DNL, deep neuropil layer; GCL, granule cell layer; LS, lateral segment; MS, medial segment; PA, primary (electroreceptor) afferents; pf, parallel fiber; PCL, pyramidal cell layer; and PlL, plexiform layer.
) and indirect (Bastian and Bratton 1990
) feedback pathways. Stellate cells have small receptive fields with phasic responses and appear to code for the movement of objects across their receptive fields; their response properties and the topographic nature of their feedback projections prompted Bratton and Bastian (1990)
to hypothesize that the direct feedback pathway was involved in focusing the electrosensory system, i.e., a "searchlight" mechanism (Crick 1984
; see also Maler and Mugnaini 1993
, 1994
). The indirect feedback pathway appears to regulate more global changes in electrosensory processing (Bastian 1986b
,c
; Bastian and Bratton 1990
).
). Studies using receptor binding (Maler and Monaghan 1991
), in vivo pharmacological analysis (Bastian 1993
), and in situ hybridization (Bottai et al. 1995
-1997
) have suggested that both N-methyl-D-aspartate (NMDA) and
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are associated with the excitatory feedback input to the ELL.
METHODS
Abstract
Introduction
Methods
Results
Discussion
References
; Turner et al. 1994
). Fish were anesthetized (MS-222), transferred to a foam-rubber-lined holder, and respirated with 11-14 ml/min of oxygenated water containing anesthetic during surgery. The brain stem was transected, glued to an aluminum block and 350-500 µm transverse ELL slices cut on a Vibratome into oxygenated ice-cold artificial cerebrospinal fluid [ACSF, which contained (in mM) 124 NaCl, 24 NaHCO4, 10D-glucose 1.25 KH2PO4, 2 KCl, 2 MgSO4, and 2 CaCl2; all chemicals from Sigma unless otherwise noted].
) and served as a landmark for the placement of stimulating and recording electrodes. Recordings were obtained exclusively from the centromedial segment of the ELL (Figs. 1 and 2).
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FIG. 2.
Laminar profile and current source density (CSD) analysis of StF-evoked currents. A: schematic diagram of a tissue slice at the level of the ELL. Recordings were made in the centromedial segment along a track parallel to the pyramidal cell soma-dendritic axis (···). Refer to Fig. 1 for abbreviations. B: field potentials recorded at several key sites from a laminar profile of StF-evoked responses with distance noted with respect to the StF recording site; , stimulus time (in this and subsequent figures, artifacts were digitally suppressed). The VML response (- - -) is displayed at half the gain of other potentials (4 mV on calibration bar). *, peak negativity in StF (presumed fiber volley). C: superimposed CSD measurements from 3 locations shown in B. D: a spatial profile of CSD over the pyramidal cell axis at a latency corresponding to the peak of the negative-going field potential in VML shown in B. Profile is aligned with a schematic of a basilar pyramidal cell, shown with StF afferents contacting its apical dendrites in the VML.
) orGABAergic axons from Pd bipolar cells (Maler and Mugnaini 1994
). Stimulus timing was computer-controlled (pClamp, Axon Instruments, CA; A/Dvance, McKellar Designs, BC; Master-8, AMPI, Israel) and delivered via constant voltage or current stimulus isolation units (Digitimer, UK; 10-80 V or 30-800 µA;0.1-0.5 Hz; tetanic stimulation: 100-200 Hz for 100 ms).
-2-ethanesulfonic acid] or Mg2+-free ACSF. 2) Micro-droplet applications: Drugs were applied by brief pressure ejection (<10 psi; 80-190 ms) from pipettes (3- to 7-µm tips) placed at the surface of the slice centered in the VML dorsal to the recording site (Turner et al. 1994
) (NeuroPhore BH-2, Medical Systems, NY).
) were filtered (DC-10 kHz), amplified (Axoclamp-2A, Axon Instruments), digitized (10-25 kHz), and stored on disk for off-line analysis (CLAMPAN, Axon Instruments; A/Dvance; IgorPro, Wavemetrics, OR).
; Richardson et al. 1987
; Taube and Schwartkroin 1988
) using the equation (Bode-Greul et al. 1987
) d2(P)/dz2 = P(z + n*
z)
2*P(z) + P(z
n*
z)/(n*
z)2, where P is the evoked field potential, z is the spatial location,
z is the sampling interval (25 µm), and n is the integration grid (n = 3).
) were advanced into the CMS pyramidal cell layer using a microdrive (Burleigh Instruments, NY). Recordings from pyramidal cell somata or proximal apical dendrites (Turner et al. 1994
) were amplified by an Axoclamp 2A preamplifier, filtered (DC-10 or 25 kHz) and digitized for off-line analysis. Results are given as means ± SD, and statistical analysis is by Student's t-test.
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
); this geometric arrangement allows field potential recordings of activity evoked by stimulation of input or output fiber tracts.
). The second response represents synaptic depolarization of pyramidal cell apical dendrites in the VML (see below). These identifying characteristics allowed stimulus intensity in all subsequent experiments to be set at a level that selectively activated StF afferent inputs.
3.02 ± 1.50 mV; n = 44) with a long duration (>30 ms; Fig. 2B). The initial positivity in the VML was correlated temporally with a sharp, short-duration negativity in the StF (1.7 ± 0.30 ms; n = 35; Fig. 2B, StF trace, *). The next negative peak in the StF response was lower in amplitude and longer lasting, decaying to baseline over ~20 ms. A small broad field positivity was recorded at the level of the pyramidal cell body layer and the proximal DML with a peak latency slightly delayed to that recorded in the VML (4-7 ms; Fig. 2B, PCL trace, *). In the pyramidal cell body layer, single-unit spike discharge was sometimes superimposed on the peak of the positivity (not shown). Much smaller potentials were recorded in the mid-DML and granular cell body layers, most often as a triphasic potential or monophasic positivity, respectively; no field potentials could be detected in the deep fiber layer (DFL) or distal DML.
; Maler et al. 1982
) and that our stimulus did not substantially activate the excitatory parallel fiber input in the adjacent DML. The primary StF response is one of excitatory synaptic drive, with the shortest latency current sink located in the StF, followed by that in the VML. This is consistent with anatomic reports that StF fibers branch sharply to course dorsally and form synaptic contacts with pyramidal cell apical dendrites in the StF and then VML (Maler 1979
; Maler et al. 1981a
). This ventro-dorsal axonal projection and synaptic termination pattern may account for the coincidence of an initial sharp current sink in the StF with a sharp current source in the VML. The latter then may arise in part as a passive current source for active spike discharge and/or synaptic depolarizations of dendritic membrane in the more ventral StF (see below).
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FIG. 3.
Pharmacology of StF-evoked extracellular field potentials in the VML. Ai: superimposed recordings of the VML field potential under control (1, con) conditions and in the presence of 4 mM Mn2+ and 0.2 mM Ca2+ medium (2, Mn2+) to block synaptic transmission. Aii: synaptic component as the difference between the control and test potentials (1 and 2). B-D: superimposed recordings of the VML field potential under control (con) conditions and after exposure to kynurenic acid (B, Kyn), 3-((RS)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP; C), and 1 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; D). Note the partial reduction by kynurenic acid and CPP of both an early (B and C; <10 ms) and late phase (*) of the field response, whereas CNQX (D) completely blocks the synaptic response. E: superimposed recordings of the VML field potential under control (con) conditions and after exposure to nominally Mg2+-free medium for 1 h. Note the pronounced augmentation of both the peak and late phase of the synaptic response by low Mg2+ medium. F: superimposed recordings of a VML field potential augmented under 0 Mg2+ medium and test responses to subsequent application of CNQX and CNQX with CPP. Under these conditions, CNQX only partially blocks the synaptic response, and the remaining component was blocked almost entirely by CPP. Field recordings recovered to near control levels after washout (n = 2) of CNQX, kynurenic acid, or Mg2+-free medium after 1-2 h.
) and application of EAA agonists and antagonists in vivo (Bastian 1993
) indicated that EAA receptors are present in the ELL molecular layer. Bastian (1993)
in particular demonstrated that pressure ejection of the glutamate receptor agonists AMPA or NMDA in the ELL molecular layer increased the excitability of pyramidal cells (although his injections were not specifically restricted to either the DML or VML). He also was able to antagonize the response to these agonists with EAA antagonists [6,7-dinitroquinoxaline-2,3-dione (DNQX), APV]. We therefore tested several antagonists of ionotropic EAA receptors to identify the contribution of glutamate receptor subtypes to the StF EPSP.
also reported that, although APV was a selective antagonist of pyramidal cell responses to NMDA in vivo, DNQX (a non-NMDA receptor antagonist similar to CNQX) also antagonized the response of ELL pyramidal cells to both AMPA and NMDA.
). Resting membrane potential (RMP) and input resistance (Rinput) were similar in somatic (RMP =
72.3 ± 7.9 mV, n = 40; Rinput =18.8 ± 7.6 M
, n = 42) and dendritic recordings (RMP =
72.6 ± 8.7 mV; Rinput = 20.1 ± 8.7 M
).
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FIG. 4.
Intracellular electrophysiology of pyramidal cell responses to StF stimulation. A: responses to a series of graded stimulus intensities in a different cell before (top trace in each set, Control) and after application of CNQX and CPP (bottom trace in each set). Amplitudes of 2 clearly separable peaks (1, 1.1 ms; 2, 5.6 ms) are plotted in B. Traces are connected by dashed line where artifact has been blanked (this and subsequent figures). B: early peak (top) scaled with stimulus intensity but was not significantly affected by CNQX and CPP. Later peak (bottom) similarly scaled with stimulus intensity but was strongly sensitive to CNQX and CPP. C: somatic excitatory postsynaptic potentials (EPSPs) with multiple peaks ( ) recorded while cell was current clamped to various indicated prestimulus (vertical - - -) membrane potentials. Late phase of the EPSP evoked at depolarized potentials lasted 40-60 ms. Note that, in this case, at depolarized potentials (
66 and
62 mV) there is a clear separation of the EPSP into early (3 and 6 ms; arrows 1 and 2) and late (arrow 3) peaks. D: traces (
78,
66, and
62 mV) from C overlaid to show clearly the enhancement of EPSPs at depolarized potentials. E: amplitude plots of EPSPs in C vs. prestimulus membrane potential. Earliest (3 ms) EPSP is insensitive to membrane potential, whereas the later components of the EPSP show strong inward rectification near and above resting membrane potential (
68 mV). F: membrane potential during the EPSPs in C-E can be reset by the occurrence of a spike; note that the potential decays to resting levels (- - -,
62 mV) after a spike (2 of the 3 current injections evoked spikes).
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FIG. 6.
Intracellular electrophysiology of StF-evoked EPSPs. A: single shock evoked EPSP. Note initial fast EPSP, followed by a slowly decaying tail depolarization (65 ms duration). B: same cell as A, but with train (10 pulses, 100 Hz) stimulation. Summating EPSPs during the train evoke several spikes (truncated), followed by a slow tail that lasts for hundreds of milliseconds after the last stimulus. When cell is depolarized (inset, 0.2 nA, 800 ms), the slow tail evokes repetitive spikes. C: different cell: summation of train stimulation-evoked EPSPs caused the slow depolarizing wave (shaded region, top). Examination of the individual EPSPs (bottom trace) shows that the profile of each EPSP is similar, but the initiation of each EPSP rides on the depolarized tail of the previous EPSP, causing significant summation. Markers indicate stimulus timing. D: effect of spiking on the depolarizing wave underlying the train response. With (gray trace) or without (dark trace) a spike the membrane potential followed a similar trajectory during repetitive StF stimulation. Same cell as A and B.
). Because the main EPSP peaked considerably later than the early electrotonic component, our measurements below reflect predominantly chemical transmission in the VML.
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FIG. 8.
Pharmacology of the StF-evoked EPSPs recorded intracellularly.Effects of -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)and N-methyl-D-aspartate (NMDA) antagonists on the EPSPs evoked by single and train pulses. A: staggered drug applications showing NMDA and non-NMDA components of the EPSP. DL-2-amino-5-phosphonopentanoic acid (APV) blocked about half of the EPSP peak amplitude and the tail depolarization. Subsequent addition of CNQX (APV + CNQX) blocked the remaining potential, leaving a small depolarized "hump" (*) followed by a hyperpolarization. Artifact blanked for clarity. B: train-evoked depolarizations with spike rates plotted in inset. Train stimulation (20 pulses, 200 Hz) evoked a strong excitatory response (Con), spiking (Con, inset), and depolarization, which outlasted the stimulus. APV reduced the evoked spike rate (inset) and blocked a major portion of the depolarization after the train. CNQX further reduced the slow depolarizing wave, but its major effect was to further reduce the excitatory response (and eliminate spiking, inset) during the train. These recordings also illustrate that the large inhibitory postsynaptic potentials (IPSPs) evoked by StF stimulation are blocked only partially by CNQX; a detailed analysis of the various components of these IPSPs will be presented elsewhere. Traces are averages of 3 trials.
68 mV typically produced a dramatic increase in the amplitude of the peak and late phase (>10-ms latency) of the EPSP, with the peak shifting to the late phase as it became enhanced at depolarized levels (Fig. 4, C-E; see also Fig. 5B). The combination of these factors could result in a plateau potential that persisted for
60 ms. Because we did not block IPSPs that are evoked by StF stimulation (Berman and Maler, unpublished observations), this may underestimate the duration of the plateau potential. At depolarized levels (more than -65 mV), action potentials often were evoked at a latency of 15-20 ms (Fig. 4F). The action potential also reset the membrane potential, again suggesting that the StF-evoked EPSP is highly voltage sensitive.
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FIG. 5.
Paired pulse facilitation (PPF) of StF synaptic potentials. A: field recordings in VML (12 overlaid traces). After the initial stimulus (first EPSP), a second stimulus was delivered at 10-ms intervals 120 ms. Inset: summary (n = 10) of the decay of PPF from 180% at 10 ms to baseline at intervals >120 ms. - - -, prestimulus baseline and control (first) EPSP peak. B: intracellular recording with paired pulses at an interval of 40 ms. At hyperpolarized potentials (
79 mV, lower dashed line), the EPSP decayed to 0.6 mV above baseline by the time of the second stimulus onset and the second EPSP is 1.2 mV greater than the first (PPF = 0.6 mV). At resting membrane potential (RMP;
68 mV) or depolarized potentials (not shown), the second EPSP was evoked on the late phase of the first EPSP (1.9 mV > prestimulus baseline); the increase in the second EPSP (2.1 mV) can be attributed mainly to summation with the late phase of the first EPSP. Note that in this recording both the early and late phaseof the EPSP are voltage sensitive, and there is only one clear peak. - - -,prestimulus baseline and control EPSP peak for hyperpolarized case.
; Maler et al. 1982
; Sas and Maler 1983
); Bratton and Bastian (1990)
have demonstrated that, with physiological sensory input, Pd stellate cells discharge in short bursts (~100 ms) at rates of 50-200 Hz. We therefore attempted to mimic natural stimulus patterns in vitro. Field potential recordings revealed an apparent strong paired pulse facilitation (PPF; Fig. 5A) at a 10-ms interpulse interval (180%); this facilitation declined to baseline by 120 ms. Intracellular recording also revealed PPF (Fig. 5B). Because this could be seen at hyperpolarized membrane potentials after the EPSP had almost returned to baseline (40 ms after stimulus onset), it suggests that there may be classic presynaptic PPF (Zucker 1989
) at StF synapses. At rest or when the cell is depolarized (Fig. 5B), the facilitation of the peak of the second EPSP mostly can be accounted for by simple summation of the second EPSP with the decaying late phase of the first EPSP.
) also may contribute to this observed effect.
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FIG. 7.
Voltage dependence of train-evoked EPSPs. A: current injection ( 0.3-0.1 nA) during tetanic stimulation of StF (100 Hz, 10 pulses, shaded region). Note characteristic ramp of summating EPSPs at membrane potentials ranging from
74 to
65 (rest) mV; at rest 3 spikes are initiated by the StF-evoked EPSPs. Shaded region expanded in B and C. B: when normalized for prestimulus membrane potential, the
74 and
71 mV traces, including EPSPs, superimpose; at these membrane potentials, the ramp is due entirely to temporal summation of the slow tail of the EPSPs. At
68 mV (thick trace) individual EPSPs still superimpose, but the underlying depolarizing wave shows inward rectification. C: at
65 mV (thick trace), the peaks of individual fast EPSPs show inward rectification. Three EPSPs evoke spikes (truncated) with prominent afterhyperpolarizations. Stimulus timing is indicated (
).
) and will be described elsewhere (Berman and Maler, unpublished observations). As already shown in field recordings, when applied first, CNQX blocked the StF-evoked EPSP, and subsequent application of CPP or APV had no further effect (n = 6). The small depolarization that remained in some cases after application of CPP + CNQX (e.g., Fig. 8A) was resistant to further block by Mn2+ (not shown) and therefore presumably reflects an electrotonic component of the EPSP. We attempted to assess whether depolarizing the cell after application of CNQX would reveal a voltage-dependent NMDA component. However, depolarization induced numerous spontaneous transient depolarizations with a time course similar to that of StF-evoked EPSPs (as reported by Mathieson and Maler 1988
); this precluded visualization of a possible residual NMDA component of the EPSP.
; Turner et al. 1994
) because the EPSP decayed rapidly (<5 ms, Fig. 8A, *). The long posttetanic depolarization was diminished greatly by APV or CPP (Fig. 8B). Subsequent CNQX applications eliminated the remaining slow depolarization (Fig. 8B).
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
). The role of the electrotonic component of the StF-evoked EPSP is unclear, and the discussion below focuses on the larger chemical component of the StF-evoked EPSP.
), the presence of NMDA and AMPA binding sites in the VML [kainic acid binding sites are not present in the VML (Maler and Monaghan 1991
)], and Bastian's (1993) demonstration that pyramidal cells discharge vigorously in response to application of AMPA and NMDA in vivo. The CNQX-sensitive component of the EPSP had a rapid onset (latency to peak of ~3 ms) and decay and was not voltage sensitive, consistent with the physiology of the mammalian AMPA receptor.
-1997)
recently have cloned the A. leptorhynchus NMDA receptor (NR1 subunit) and demonstrated by in situ hybridization that NR1 mRNA is enriched highly in ELL pyramidal cells, including their proximal apical dendrites.
; Edmonds et al. 1995
; Forsythe and Westbrook 1988
; Hestrin et al. 1992
). 3) The NMDA channels contributing to the StF-evoked EPSP appear to conduct at more negative membrane potentials (less than -60 mV) than the hippocampal NMDA receptor, which requires depolarization to more than
60 mV before it contributes appreciable synaptic current (Hestrin et al. 1992
).
; see also Drejer and Honore 1988
). Indeed NMDA receptor (NR1 subunits) of ELL pyramidal cells contain a 5
end insertion (Bottai et al. 1996
) that has been shown to confer marked CNQX sensitivity to mammalian NMDA receptors (Hollmann et al. 1993
). Additionally we have shown that the CNQX block of NMDA receptors in the ELL is due partly to the voltage sensitivity of the NMDA receptor, so that depolarization by the non-NMDA-receptor component of the EPSP is required to relieve their Mg2+ block; similar results have been reported in the hippocampal slice (Blake et al. 1988
).
). Recent kinetic experiments, however, have demonstrated that NMDA channels open, on average, ~10 ms after agonist binding (Dzubay and Jahr 1996
); this is only slightly longer than the apparent peak of the NMDA-receptor component of the StF-evoked EPSP. In addition, fast (<10 ms) NMDA-receptor-mediated transmission has been demonstrated in mammalian sensory systems: retino-geniculate EPSPs (Esguerra et al. 1992
), somatosensory cortex (Armstrong-James et al. 1993
), and visual cortex (Shirokawa et al. 1989
). Electrophysiological analysis of cloned NMDA receptors further demonstrates that combinations of NR1 with different NR2 subunits can result in functional receptors with markedly different kinetics (Monyer et al. 1992
).
). This is likely due to the activation of ELL inhibitory interneurons because application of
-aminobutyric acid-A (GABAA) antagonists prolongs the EPSPs to >100 ms (Berman and Maler, unpublished observations). Stimulus trains do cause prolonged EPSPs (>200 ms in cases where inhibition is weak, e.g., Fig. 6B), which are APV and CPP sensitive, suggesting that the physiology of the NMDA receptors associated with the StF input is similar to that reported for mammalian neurons.
). This suggests that voltage-dependent ion channels also might contribute to the late phase of the StF evoked EPSPs (see Hirsch and Gilbert 1991
). Because QX-314 can block both Na+ and Ca2+ inward currents (Talbot and Sayer 1996
), the channels contributing to the voltage-sensitive late phase still must be elucidated.
). However, recent in vivo studies have suggested that NMDA receptors contribute to the response of cortical neurons to even weak sensory input (Armstrong-James et al. 1993
; Fox et al. 1990
; Kwon et al. 1992
), suggesting that these receptors may operate at near RMPs. Biophysical analysis of cloned NMDA receptors also has demonstrated a wide variation in susceptibility to Mg2+ blockade, and that some subunit combinations can pass appreciable current at less than
70 mV (Kutswada et al. 1992
).
). The NR1 subunit of the NMDA receptor of A. leptorhynchus is highly homologous to the mammalian NR1 subunit (Bottai et al. 1995
-1997
); it therefore will be important to determine which specific combinations of NR1 splice variants and NR2 subunits determine the voltage threshold, time to peak, and time constant of decay of NMDA receptors within the VML.
500 ms in duration after tetanic stimulation) dependent on NMDA receptors and perhaps on voltage-sensitive ion channels as well. The frequency and duration of our tetanic stimuli mimic natural firing patterns of StF (Bratton and Bastian 1990
).
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FIG. 9.
A: summary diagram of the relevant contributions of gap junction and different ionotropic excitatory amino acid receptors to the StF-evoked EPSP. A small electrotonic EPSP (Gap) precedes a CNQX-sensitive, voltage-insensitive EPSP mediated by an AMPA receptor component. NMDA receptors contribute to both the early peak and the late phase of the EPSP; the voltage sensitivity of these components is indicated by the double arrows. B: summary of how the receptive field sizes of pyramidal and stellate cells, time delays of reciprocal ELL-Pd connections, and voltage sensitivity of StF-evoked EPSP might contribute to the hypothesized searchlight function of the StF feedback pathway. As the fish scans past an object, changes in the firing rate of electroreceptors first will drive ELL pyramidal cells with receptive fields on the left (a). The axons of these cells travel in the lateral lemniscus to terminate on stellate cells of the contralateral Pd. Stellate cells have larger receptive fields than ELL pyramidal cells; in this diagram, all the indicated pyramidal cells are supposed to project to the Pd stellate cell. The stellate cell is activated phasically and emits a burst of spikes; this activity reaches the ELL pyramidal cell with receptive field (RF) "b" (gray) with a delay due to slow conduction in the tractus stratum fibrosum and synaptic delay; these delays are matched to the scan rate of the fish. Feedback input therefore arrives at pyramidal cell with RF b at the same time as does electroreceptor input generated by the object entering its receptive field.
) of this feedback pathway. At least four mechanisms may be involved in the typical ramp-like response to tetanic stimulation: presynaptic facilitation may increase the amplitude of succeeding EPSPs; temporal summation of the late phase of the EPSP will generate a rising depolarization; the voltage sensitivity of peak and late phase of the EPSP increase the response to later stimuli in the train and thus contribute to the ramp-like responses; and IPSPs can attenuate the response to later EPSPs in the train (Berman and Maler, unpublished observations). It is clear that the response to StF stimulation can be regulated dynamically at many potential sites (see below) and that it will take more detailed physiological studies as well as modeling to understand transmission at this feedback synapse.
, 1991
; Sas and Maler 1983
); the connections are bilateral in both directions with contralateral projections dominating. Because connections are excitatory in both directions, this represents an example of positive feedback. Bratton and Bastian (1990)
have recorded from stellate cells in Pd, the neurons that give rise to the StF (Sas and Maler 1983
). Stellate cells respond, with high sensitivity and gain, to AM (AMs <16 Hz) of the EOD (contralateral electroreceptors). A. leptorhynchus scans its environment with stereotyped movements, the velocity of which (10-15 cm/s) (Lannoo and Lannoo 1992
) would be expected to generate AMs of <10 Hz (Bastian 1981
). As expected from these considerations, stellate cells respond vigorously and phasically to movement of objects over their receptive fields on the contralateral side of the fish's body (Bratton and Bastian 1990
). On the basis of these data, Bratton and Bastian hypothesized that stellate cells are optimized to detect small moving objects. Taking into account the excitatory reciprocal connectivity between ELL and Pd, they further hypothesized that the StF feedback pathway might act as a searchlight to enhance the response to salient features of the environment. A recent behavioral study in fact has shown that lesions of Pd do affect the electrodetection of objects (Green 1996
).
with regard to the reciprocal connections between mammalian thalamus and cortex. This theory contained three essential ingredients: reciprocal excitatory connectivity, a relatively diffuse parallel inhibitory feedback system that can keep the feedback excitation confined to a limited spatial domain, and a nonlinearity in the responsiveness of the lower order cells (thalamic relay neurons in Crick's thesis) that amplifies stronger inputs. The identification of a diffuse inhibitory (GABAergic) feedback pathway from cells in medial Pd to ELL pyramidal cells suggests that the second of Crick's criteria also is met by the direct feedback system to the ELL (Maler and Mugnaini 1993
, 1994
). As discussed below, the data presented in this paper suggest that the third criteria is met as well.
) or by indirect feedback input to the DML. The latter alternative is certainly plausible, however, other than its involvement in gain control (Bastian 1986b
,c
), little is known about the physiology of the indirect feedback pathway. We therefore hypothesize that when electroreceptor (primary afferents) and StF feedback inputs arrive concurrently at a pyramidal cell, the StF input is enhanced greatly and therefore is very effective at bringing that cell above spike threshold. From this viewpoint, it is the voltage dependence of the NMDA-receptor component of StF synapses that is critical for their function; a similar proposal has been made for the role of NMDA receptors associated with corticothalamic feedback fibers (to lateral geniculate nucleus) (Esguerra and Sur 1992
). The spatial aspects of this theory are discussed below (Fig. 9).
found that the proximal apical dendrites of pyramidal cells conduct Na+ spikes and that inward current associated with these spikes sourced back to the soma where they appeared as depolarizing afterpotentials that could generate the spike bursts seen in these cells in vitro (Turner et al. 1996
). Gabbiani et al. (1996)
recently have shown that ELL pyramidal cells in vivo can signal the occurrence of temporal electrosensory "features" by spike bursts. They also suggest that the feature extraction occurs in the proximal dendrites rather than the soma (Gabbiani et al. 1996
). It is thus possible that StF-evoked EPSPs, in addition to directly triggering spikes, can enhance antidromic spike invasion of the proximal apical dendrite of ELL pyramidal cells and thus increase the probability of burst initiation. A recent study (Magee and Johnston 1997
) has demonstrated in hippocampal pyramidal cells that dendritic depolarization due to EPSPs can in fact increase the amplitude of antidromic dendritic action potentials. In this case, the slow summating component of the StF-evoked EPSP might increase the electrodetectability of moving objects for hundreds of milliseconds by enabling primary afferent input to trigger spike bursts.
; Bratton and Bastian 1990
); the greater size of the stellate cell's receptive field is presumably due to convergence of pyramidal cell input (Maler et al. 1982
). Because pyramidal and stellate cells are connected reciprocally (Maler et al. 1982
), a stellate cell's receptive field will extend ~3.5 mm beyond that of its concentrically matched pyramidal cell. This suggests that an object moving past electroreceptors can cause Pd stellate cells to discharge and that these stellate cells can in turn excite pyramidal cells that have not yet been activated by that object (Fig. 9). The relative spread of the ELL pyramidal cell and Pd stellate cell reciprocal connections will determine how far ahead the descending input will prime ELL pyramidal cells the receptive fields of which lie in the object's future trajectory. Further details on the precision of this connectivity is required to accurately quantify the scope of the searchlight.
have noted that Pd stellate cells respond to electrosensory inputs with a long latency (11 vs. 5.2 ms for ELL pyramidal cells). The tSF projection from Pd to ELL is ~3,500 µm long (estimated from the atlas of Maler et al. 1991
). Our data suggests a delay of ~17 ms for the Pd-ELL projection (conduction velocity of 0.31 m/s produces a conduction delay of 11 ms and a delay to EPSP peak of 6 ms). Data from Bastian suggest a shorter delay of 10 ms. Thus activation of ELL pyramidal cell with RF "a" (Fig. 9) will produce a feedback EPSP in the pyramidal cell with RF "b" after ~20-28 ms. If the fish is scanning at 10 cm/s (Lannoo and Lannoo 1992
), it will traverse ~2-3 mm in this period, placing the object over RF b. The duration of the late EPSP, under this hypothesis, would determine the minimal scanning rate. Therefore, primary afferent input to a pyramidal cell may coincide with feedback input from stellate cells representing adjacent regions of skin; given the voltage dependence of the StF-evoked EPSP discussed above, this will result in large feedback EPSPs and an enhancement of the response to the moving object. These considerations suggest that this feedback system will create a traveling beam (searchlight) of enhanced responsiveness in pyramidal cells, priming them to detect scanned objects. Correlative biophysical and systems studies of the StF feedback pathway may elucidate the cellular basis of an elementary form of spatially and temporally localized attention.
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ACKNOWLEDGEMENTS |
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We thank Dr. Rob Dunn for insightful discussion on the molecular biology of NMDA receptors and W. Ellis for technical support.
This work was supported by Medical Research Council grants to L. Maler and R. Turner, who is a Medical Research Council and Alberta Heritage Foundation for Medical Research Scholar. J. Plant was supported by a National Science and Engineering Research Council Fellowship.
Present addresses: J. Plant, Dept. of Psychology, University of Victoria, PO Box 3050, Victoria, British Columbia V8W 3P5; R. W. Turner, Dept. of Anatomy, University of Calgary, Calgary, Alberta T2N 1N4, Canada.
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FOOTNOTES |
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Address for reprint requests: L. Maler, Dept. of Anatomy and Neurobiology, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada.
Received 7 April 1997; accepted in final form 18 June 1997.
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REFERENCES |
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