Electroresponsiveness of medial entorhinal cortex layer III neurons in vitro

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Neuroscience Vol. 81, No. 4, pp. 937–950, 1997 Copyright ? 1997 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/97 $17.00+0.00 S0306-4522(97)00263-7

ELECTRORESPONSIVENESS OF MEDIAL ENTORHINAL CORTEX LAYER III NEURONS IN VITRO C. T. DICKSON, A. R. MENA and A. ALONSO* Department of Neurology and Neurosurgery, McGill University and Montreal Neurological Institute, Montreal, Quebec, Canada H3A 2B4 Abstract––The entorhinal cortex funnels sensory information from the entire cortical mantle into the hippocampal formation via the perforant path. A major component of this pathway originates from the stellate cells in layer II and terminates on the dentate granule cells to activate the hippocampal trisynaptic circuit. In addition, there is also a significant, albeit less characterized, component of the perforant path that originates in entorhinal layer III pyramidal cells and terminates directly in area CA1. As a step in understanding the functional role of this monosynaptic component of the perforant path, we undertook the electrophysiological characterization of entorhinal layer III neurons in an in vitro rat brain slice preparation using intracellular recording techniques with sharp micropipettes and under current-clamp conditions. Cells were also intracellularly injected with biocytin to assess their pyramidal cell morphology. Layer III pyramidal cells did not display either the rhythmic subthreshold membrane potential oscillations nor spike-cluster discharge that characterizes the spiny stellate cells from layer II. In contrast, layer III pyramidal cells displayed a robust tendency towards spontaneous activity in the form of regular tonic discharge. Analysis of the voltage–current relations also demonstrated, in these neurons, a rather linear membrane voltage behaviour in the subthreshold range with the exception of pronounced inward rectification in the depolarizing direction. Depolarizing inward rectification was unaffected by Ca2+conductance block but was abolished by voltage-gated Na+-conductance block with tetrodotoxin, suggesting that a persistent Na+-conductance provides much of the inward current sustaining tonic discharge. In addition, in the presence of tetrodotoxin, an intermediate threshold (2"50 mV) Ca2+dependent rebound potential was also observed which could constitute another pacemaker mechanism. A high-threshold Ca2+-conductance was also found to contribute to the action potential as judged by the decrease in spike duration towards the peak observed during Ca2+-conductance block. On the other hand, Ca2+-conductance block increased spike duration at the base and abolished the monophasic spike afterhyperpolarization. Analysis of the input–output relations revealed firing properties similar to those of regularly spiking neocortical cells. Current–pulse driven spike trains displayed moderate adaptation and were followed by a Ca2+-dependent slow afterhyperpolarization. In summary, the intrinsic electroresponsiveness of entorhinal layer III pyramidal cells suggest that these neurons may perform a rather high-fidelity transfer function of incoming neocortical sensory information directly to the CA1 hippocampal subfield. The pronounced excitability of layer III cells, due to both Na+ and Ca2+ conductances, may also be related to their tendency towards degeneration in epilepsy. ? 1997 IBRO. Published by Elsevier Science Ltd. Key words: brain slice, intracellular, pyramidal cells, perforant path, epilepsy, rat.

The entorhinal cortex (EC) is a unique anatomical structure, as previously recognized by Ramon y Cajal,60 due to its position at the interface between the neocortex and the hippocampal formation. The cortico-hippocampus-cortico circuit in which the EC is crucial, is a major contributor to basic cognitive functions such as memory,5,44,66,70,87 and mental states such as motivation and attention.52 Additional evidence of the functional relevance of the EC is the *To whom correspondence should be addressed. Abbreviations: AHP, afterhyperpolarizing potential; EC, entorhinal cortex; f–I, frequency–intensity; f–t, frequency–time; gNap, persistent sodium conductance; INap, persistent sodium current; ISI, inter-spike interval; MSL, maximal spike latency; Ri, apparent input resistance; sAHP, slow afterhyperpolarizing potential; TTX, tetrodotoxin; V–I, voltage–current; Vm, membrane potential.

correlation of the onset of neurological diseases, such as Alzheimer’s disease9,11,35,37,80 and schizophrenia8,40 with the degeneration of EC neurons in selective layers. Furthermore, strong evidence suggests that the EC plays a major role in the expression of mesial temporal lobe epilepsy, though it is unclear whether this role is generative, regulative, or both.23,24,34,42,59,61,69,79,81,82 In this respect, particular attention has been paid to layer III of the EC in recent years after the realization that this layer degenerates with the most early onset in mesial temporal lobe epilepsy.27,28 While the stellate cells from layer II of the EC give rise to the perforant path projection component that terminates upon the dentate gyrus and CA3,62,63,76 pyramidal cells from EC layer III project to CA1.75,83 The importance of the perforant path input upon the dentate gyrus as a major drive for the intrinsic

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hippocampal circuitry has long been recognized. In contrast, the functionality of the layer III to CA1 projection has been questioned for some time, though recent studies have clearly demonstrated that indeed this pathway is functional. In particular, both in vivo86 and in vitro18,26 studies have shown shortlatency excitatory responses in the CA1 region to perforant path stimulation. Also, activation of the EC can generate inhibitory postsynaptic potentials in CA1 pyramidal cells,31,67 via a feedforward mechanism.13,15,43,46 Moreover, there is good evidence that the EC layer III projection upon CA1 pyramidal cells contributes significantly to the production of theta rhythm in the hippocampus. First, EC layer III neurons discharge rhythmically in bursts of action potentials during theta rhythm.1,25 Second, EC lesions result in an altered current–source density distribution of theta activity along the somatodendritic surface of CA1 pyramidal cells.12,14 In relation to the genesis of rhythmic activity in the hippocampus, a recent study by Charpak et al.,17 using multiple-site extracellular and intracellular recording in the guinea-pig demonstrated that the EC layer III to CA1 pathway is also involved in the genesis of fast (30–60 Hz) CA1 oscillations. It thus appears that EC layer III plays an important role in the genesis of coherent oscillatory activity in the hippocampal system. In order to gain an understanding of the specific functional role(s) of the EC and the way it transfers neocortical information towards the hippocampus, it is necessary to decipher the electrophysiological properties of its neurons. Surprisingly, while the electrophysiology of EC layer II neurons is rather well understood,2,41,43 much less is known with respect to the cells in layer III. Here we report investigations on the intrinsic electroresponsiveness of pyramidal cells from layer III of the EC in a rat brain slice preparation. We focus on the medial entorhinal cortex since it is better known electrophysiologically, and because it is that portion of the EC that is preferentially damaged in epilepsy.27 Some of the material presented in this paper has been previously published as a preliminary report.56

EXPERIMENTAL PROCEDURES

Horizontal brain slices of the retrohippocampal area of Long–Evans rats (125–250 g: Charles River) were prepared as previously described.2 Briefly, animals were anaesthetized by i.p. injection of Nembutal (50 mg/kg), and decapitated using a standard guillotine. The brain was rapidly removed, and placed in a cold (4–6)C) oxygenated Ringer solution containing (in mM) 124 NaCl, 5 KCl, 1.2 KH2PO4, 2.4 CaCl2, 2.6 MgSO4, 26 NaHCO3, and 10 glucose. The pH of the Ringer solution was maintained at 7.4 by saturation with 95% O2/5% CO2. Slices were cut at 350 µm using a Vibratome and normally included the entorhinal cortex (medial and lateral), the hippocampal formation, and part of the perirhinal cortex. Following sectioning, slices were submerged in oxygenated Ringer solution and incubated for at least 2 h.

Slices were transferred one at a time, to an interface recording chamber which was constantly superfused with the oxygenated Ringer solution. Layer III of the medial EC was readily recognized in this preparation by means of transillumination and visualization through a dissecting microscope. The basic electrophysiological properties of EC layer III cells were obtained under current-clamp using standard intracellular recording techniques. Impaling was conducted with micropipettes pulled to a fine tip and filled with 2–3 M potassium acetate (tip resistance 60–140 MÙ), or 1–2% biocytin in 2–3 M potassium acetate in those experiments conducted to identify the morphological characteristics of cells. Signals were amplified by an Axoclamp 2A in bridge mode, digitized by a Neuro-Corder, visualized on-line on a digital storage oscilloscope and stored on VHS tape for subsequent analysis on a 386-based computer. Tetrodotoxin (TTX) was added directly to the superfusing Ringer solution from a stock (200 µM) solution. Cadmium (CdCl2:200 µM) was dissolved in Ringer solution without phosphate and sulphate salts, containing (in mM): 126 NaCl, 5 KCl, 2.4 CaCl2, 2.6 MgCl2, 26 NaHCO3, and 10 glucose. Cobalt (CoCl2:2 mM) was added to a similar Ringer solution with half the added calcium (CaCl2: 1.2 mM). Electrophysiological parameters were measured as follows: Apparent input resistance (Ri) was calculated from the voltage deflection (mV) measured in response to injections of small amplitude (0.1 to 0.2 nA) hyperpolarizing square-wave current pulses. The time constant (ô) was defined as the time required for the voltage trace to reach 63% of its peak deflection in response to a 0.1 nA hyperpolarizing pulse. Spike threshold was determined from traces in which a minimum current pulse step was required to trigger a single spike. The peak value of the spike was the voltage level attained at the positive peak (inflection point) of the spike. Spike amplitude was calculated as the voltage difference between the peak and threshold voltages. Spike duration was measured across the voltage crossings at the threshold level. All action potential parameters were measured using a high (<0.02 ms: 50 kHz) sampling rate. The amplitude of the afterhyperpolarization (AHP) was measured as the voltage difference between the threshold value and the maximum value of the AHP following a spike. The maximum spike latency (MSL) was measured as the maximum latency to firing for a given threshold depolarizing current pulse of 300–500 ms duration. Means were calculated as arithmetic averages and reported with standard deviations (mean&S.D.). To determine the cellular morphology of the medial EC-III neurons, some of the cells recorded were filled with biocytin. Slices with filled cells were fixed in 4% paraformaldehyde for at least 2 h, and then transferred to a 30% sucrose solution and left overnight. The slices were then resectioned at 50–100 µm using a freezing microtome, and biocytin was revealed using avidin–biotin–horseradish peroxidase reaction. The slices were then mounted on glass slides, dehydrated, and coverslipped. RESULTS

Neurons reported in this paper (52) had a stable resting membrane potential negative to "50 mV, an input resistance larger than 45 MÙ, and a spike amplitude greater than 60 mV. In this group, we could identify only one type of cell according to both morphological and electrophysiological criteria. Biocytin was intracellularly injected in 23 of the EC layer III cells and in 18 of these cases was successfully revealed. As in the case illustrated in Fig. 1A, all revealed cells displayed a pyramidal cell morphology with spiny dendrites. The cell soma measured on

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Fig. 1. Morphological and basic electrophysiological characteristics of medial EC layer III neurons. (A) Photomicrograph of a biocytin-filled medial EC layer III neuron revealed with avidin–biotin–peroxidase complex. The cell, located in the most superficial aspect of layer III, has a characteristic pyramidal morphology with a triangular-shaped cell body, the apex of which is directed towards the pial surface (top of picture) and a moderately profuse basal dendritic tree. The axon can be seen emanating from the basal aspect of the cell body and travels towards the deeper layers (arrowhead) before bifurcating and sending a collateral parallel to the pial surface which branches further within layer III. A thick apical dendrite emerges from the apex of the cell and divides into two branches, both of which are directed superficially, perpendicular to the cellular layers. They extend through layer II and into layer I where they ramify. Spines can be observed on all dendrites. Scale bar=50 µm. (B) An example of low-frequency tonic firing at rest observed in a large proportion of these cells. (C) A high sweep speed trace of the first action potential shown in B. Note that action potential decays monotonically followed by a monophasic afterhyperpolarization.

average 14.5&1.4 µm. The basal dendritic tree was always profuse with a mediolateral extension of about 214.3&12.3 µm and was typically constrained to the limits of layer III. One primary apical dendrite, frequently dividing in two close to the soma, ascended vertically towards layer I and then branched profusely towards the superficial aspect of layer II and within layer I. In some cases (n=6) the axon could be followed to the angular bundle. In general terms, the electrophysiological properties of the medial EC layer III neurons that were recorded were similar to those described for neocortical regular-spiking neurons.19,54,56 In addition to the pyramidal cells reported in this study, we also occasionally impaled fast spiking cells in layer III. Unfortunately, long-lasting recordings were not made and have not been further considered. Layer

III pyramidal cells presented clear differences with respect to the intrinsic electroresponsiveness of EC layer II projection cells. First, layer III pyramidal cells never displayed the persistent rhythmic subthreshold membrane potential oscillations typical of the layer II stellate cells, nor did they manifest pronounced time-dependent inward rectification that also characterizes the stellate cells as well as the pyramidal-like cells from layer II.2 Second, layer III cells had apparent input resistances (76.9&20.1 MÙ) about double than those reported for layer II neurons.2 Third, while layer II neurons are typically silent at rest, a large proportion (58%) of layer III neurons displayed spontaneous activity consisting of a regular tonic discharge at 22 Hz (Fig. 1B). Finally, the average duration of action potentials of layer III cells was longer (2.2&0.7 ms) than that previously

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Total Spont. (n) active(n) 52

Vr(mV)

Ri(MÙ)

ô(ms)

Spike Spike Spike Spikedur. sAHPamp. thres.(mV) peak(mV) amp.(mV) (ms) (mV)

30(58%) "61.4&3.0 76.9&20.1 16.7&4.7 "45.6&3.4 27.3&6.2 73.0&6.2 2.2&0.7

6.0&2.0

MSL (ms) 207.3&50.7

All values (except n) are means&S.D. for 52 cells. Total, number of cells recorded; Spont. active, number of cells showing spontaneous spiking; Vr, resting membrane potential (in those cells not showing spontaneous activity: n=22); Ri, apparent input resistance; ô, apparent membrane time constant; Spike thres., spike threshold; Spike peak, spike peak value; Spike amp., spike amplitude; Spike dur., spike duration; sAHP amp., amplitude of slow afterhyperpolarization; MSL, maximal spike latency. See Experimental Procedures for details on measurements.

reported for the layer II stellate (1.3&0.2 ms) and pyramidal-like (1.8&0.4 ms) cells2 and was followed by a monophasic AHP (Fig. 1C) instead of a fast AHP–medium AHP sequence as seen in layer II neurons. Table 1 summarizes the mean value of several passive membrane properties of layer III cells. The following sections describe in detail the voltage– current relations as well as the repetitive firing properties that also set these neurons apart from those of layer II. Voltage–current relations The study of the voltage–current (V–I) relationship of layer III pyramidal cells was approached using injection of square-current pulses. The potentials generated by hyperpolarizing and depolarizing current pulses for a typical cell resting at "61 mV are shown in Fig. 2A. The membrane response had smooth charging curves in the hyperpolarizing direction except for pulses larger than 0.5 nA where there was a break in the trajectory after a few (5–10) ms. Despite a lack of marked repolarization following this break, the membrane potential always transiently overshot the resting level upon termination of the hyperpolarizing current pulses. These rebound potentials, due largely to the activation of a noninactivating Na+ conductance (see below, Fig. 5), typically fired the cell with a delay which decreased with increasing amplitude of hyperpolarization. Small depolarizing current pulses also generated a membrane response with a rather smooth asymptotic trajectory. With slightly larger current pulses, the initial response was followed by a rising membrane depolarization that culminated in the generation of an action potential. The V–I relationship for the cell illustrated in Fig. 2A is plotted in Fig. 2B. In all layer III neurons, as in this example, the V–I curve displayed a pronounced concavity at the positive end which is indicative of robust inward rectification in the depolarizing direction. Inward rectification was clear when comparing the amplitude of the voltage responses evoked by a small depolarizing current pulse with that evoked by an equal hyperpolarizing current pulse, which was always smaller (Fig. 2B inset). Because the V–I curve of layer III neurons is non-linear, the cell’s apparent input resistance is

markedly voltage-dependent. This property is made evident by exploration of the V–I relationship with application of a small hyperpolarizing current pulse as the resting membrane potential was altered by injection of d.c. current. As in the case illustrated in Fig. 3, in all neurons examined the amplitude of the voltage response increased with depolarization and decreased with hyperpolarization. Thus, apparent input resistance was found to increase or decrease when the resting membrane potential was shifted in the positive or negative direction, respectively (Fig. 3, control). In many brain neurons, inward rectification in the depolarizing direction has been shown to be generated by a TTX-sensitive low-threshold noninactivating Na+-conductance (gNap).16,19,45,71,74 To determine whether layer III cells also possessed this conductance, Ri versus voltage plots were examined before and during bath application of TTX (1 µM). In all neurons tested (Fig. 3; n=7), inward rectification in the depolarizing direction was selectively blocked by TTX. To examine the possibility that an inward Ca2+-current may also contribute to subthreshold inward rectification we also constructed the Ri versus voltage plots before and during bath application of Co2+ or Cd2+. As demonstrated in Fig. 4, block of Ca2+-conductances with inorganic cations did not appear to have a significant effect on the subthreshold Ri versus voltage relationship, in this, and all other cells recorded (n=5). In consequence, inward rectification in the depolarizing direction appears to reflect the regenerative nature of an inward Na+-current. In fact, as Fig. 5 illustrates, the rebound potentials typically observed upon termination of hyperpolarizing pulses from rest (about "60 mV) (panel A) were also likely to be due to the activation of the same gNap that causes inward rectification since they were always abolished by TTX (n=7, panels B and C). However, when hyperpolarizing pulses were applied from (or positive to) a membrane potential level of "50 mV, TTX-insensitive rebound potentials were observed in most cases (86%; six of the seven cases) (Fig. 5D and E). Since these type of TTX-insensitive regenerative events are typically generated by Ca2+-conductances3,50 we tested the effect of the inorganic Ca2+-channel blocker Co2+ (2 µM). As Fig. 5E–G illustrates, Co2+ reversibly blocked the

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Fig. 2. Voltage–current relationship in a medial EC layer III neuron. (A) Responses to injection of depolarizing and hyperpolarizing current pulses applied from resting membrane potential. Note that a low-amplitude square pulse (0.04 nA) in the depolarizing direction evokes an active membrane response, and that large-amplitude square pulses (>0.4 nA) in the hyperpolarizing direction result in rebound potentials following the end of the pulse. (B) Plot of membrane potential (Vm) vs current injected from the data shown in A. Note that the plot is concave at depolarizing levels (positive to "60 mV), thus indicating robust inward rectification in the depolarizing direction. Inset shows voltage responses to low-amplitude subthreshold depolarizing and hyperpolarizing current pulses of the same amplitude in a different cell. Note that the voltage deflection is greater in the depolarizing direction, directly demonstrating inward rectification.

TTX-insensitive rebound potentials in all cases tested (n=4). Repetitive firing For a more complete understanding of the input/ output relations of layer III neurons and to compare with the known properties of projection neurons from layer II,2 we analysed in detail the firing responses of layer III neurons to 2400 ms depolar-

izing current pulses of increasing amplitude (Fig. 6). Larger current steps always resulted in faster firing rates, and firing frequency always decreased with time (adapted) (Fig. 6A). Frequency–current (f–I) plots were constructed for early and late interspike intervals (ISIs) (Fig. 6B). All f–I plots showed a monotonic graded increase in firing frequency with increasing current intensity. Early and late ISI typically showed a linear or bilinear relationship between current and firing frequency. For the first ISI, the

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Fig. 3. Effect of Na+ conductance block on the apparent input resistance (Ri) versus voltage relationship in medial EC layer III neurons. (A) Voltage responses to a hyperpolarizing constant current pulse ("0.1 nA) applied at depolarized (top) and hyperpolarized (bottom) membrane potential levels in control Ringer (left panel) and Ringer containing 1 µM TTX (right panel). Note that the apparent input resistance is markedly voltage-dependent in control, but not in 1 µM TTX Ringer. (B) From data shown in A, a plot of the apparent input resistance as a function of membrane potential in control Ringer solution (squares /) and in the presence of 1 µM tetrodotoxin (TTX) (circles -). Na+ conductance block by TTX abolished depolarizing inward rectification as shown by the lack of increase of Ri at potentials greater than "65 mV.

mean f–I curve slope was 256&51 Hz/nA (n=6). Reflecting adaptation, the f–I slope for the second ISI decreased to 162&14 Hz/nA (ratio first to second ISI f–I slope: 1.58). The f–I slope for subsequent ISI declined at an increasingly smaller rate and tended to stabilize after the sixth ISI (Fig. 6B). To further characterize adaptation, plots of instantaneous firing rate versus time (f–t) were also constructed (Fig. 6C). For small current steps (0.2– 0.3 nA) there was minimal decline in firing rate. However, for larger current steps there was an obvious decline in the firing rate up to the 4th or 5th ISI at which point the firing rate tended to stabilize. In no case did firing cease during the current pulse, as is typically observed in layer II stellate cells which display a more pronounced adaptation.2 For current pulses §0.4 nA the f–t plot was well fitted by a biexponential function with a fast time constant of

10–30 ms (14.5&8.7) and a slow time constant longer than 100 ms (217&106) (n=7). The action potentials activated during current– pulse triggered spike-trains displayed conspicuous waveform changes as the train preceded (Fig. 7). First, there was a progressive and pronounced (200%) increase in spike duration during the initial four to six spikes and then usually a subsequent much smaller decrease that remained for the duration of the spike-train (Fig. 7B and C). Second, concomitant with the increase in spike duration, there was also a parallel decrease in spike amplitude and rate of rise (Fig. 7B). Finally, spike threshold always increased substantially during the current pulse. This increase reached 4–6 mV for current steps of 1.0 nA. Paralleling the moderate degree of spike-train adaptation, current–pulse triggered spike-trains were followed by a relatively small slow afterhyper-

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Fig. 4. Effect of Ca2+ conductance block on the apparent input resistance (Ri) versus voltage relationship in medial EC layer III neurons. (A) Voltage responses to the injection of a hyperpolarizing constant current pulse ("0.05 nA) applied at a depolarized (top) and hyperpolarized (bottom) membrane potential levels in control Ringer solution (right panel) and after Ca2+ conductance block with 2 mM CoCl2 (Co2+). (B) Plot of the Ri as a function of membrane potential in control Ringer solution (squares /) and in the presence of Co2+ (circles -) from data shown in A. Note that Ca2+ conductance block has no effect on the depolarizing inward rectification.

polarization (sAHP) (Fig. 8A). A plot of the relationship between sAHP amplitude and current intensity in Fig. 8C demonstrates that increasing the intensity of the pulse, and thus the number of spikes, increased the amplitude of the sAHP (filled squares). Similarly, the larger the intensity of the current pulse the longer the sAHP duration (Fig. 8C, filled circles). As in other neurons, progressive Ca2+ influx during spike trains is probably responsible for the generation of the sAHP via the activation of a Ca2+-dependent K+ conductance.7,21,36 To test whether this rule applied to layer III neurons, Ca2+ channels were blocked by bath applications of Cd2+ (200 µM) or Co2+ (2 mM) (n=6). Fig. 8B illustrates the response of the same cell as in Fig. 8A to an equal intensity current pulse as in Fig. 8A during Ca2+-channel block. Note that the sAHP was completely abolished by this manipula-

tion. Fig. 8D illustrates in the same cell the effects of Ca2+-conductance block on the single-spike waveform. As in this case, and in all other neurons examined, Cd2+ or Co2+ had no significant effect on the rate of rise of the spike, but produced an increase and decrease of the spike rate of fall towards the peak and base, respectively. These changes resulted in a average decrease of 13&5% in spike duration as measured at one third amplitude from the peak and an average increase of 26&17% in spike duration at the base. Concomitantly, the amplitude of the spike AHP was decreased on average by 48&30% (n=5). DISCUSSION

Pyramidal cells of layer III of the EC give rise to the so-called monosynaptic component of the

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Fig. 5. Effect of Na+ and Ca2+ conductance block on the rebound potential. (A–C) Voltage response to a hyperpolarizing constant current pulse ("0.2 nA) from a resting potential of "60 mV before (A) and after (B) bath application of TTX (1 µM). (C) Superimposition of traces in A and B. The rebound potential was virtually abolished after application of TTX. (D and E) Voltage responses to hyperpolarizing constant current pulses (from "0.1 to "0.5) applied at a membrane potential of "60 mV (D) and "50 mV (E) during TTX perfusion. Note the appearance of rebound potentials at the end of the hyperpolarizing pulses when the membrane potential is depolarized to "50 mV. (F) Blockage of Ca2+ conductances by the addition of CoCl2 (Co2+:2 mM) depressed the rebound potentials at this depolarized level. (G) Following a 20 min washout of CoCl2, the rebound potentials returned.

perforant path that terminates in a wellcircumscribed and topographically organized manner upon the CA1 pyramidal cells.6,75,83,84 The results of the present study indicate that EC layer III neurons do not have the complex electroresponsiveness that characterize the stellate cells from layer II.2,4 Layer III neurons do not display either sustained subthreshold membrane potential oscillations nor do they discharge in spike-clusters. In contrast, the electrophysiology of layer III cells relates more to that described for regularly spiking neocortical neurons.20,54,55 In this respect, layer III pyramidal cells appear to be well suited to serve as relays for a high-fidelity transfer of incoming information from the neocortex to the hippocampus. A characteristic of layer III neurons in our in vitro situation was their pronounced tendency towards displaying low frequency tonic discharge. Many of the cells fired action potentials at the resting membrane potential, and in the rest of the cases small d.c. depolarizing current injection produced tonic firing. This tendency to produce spontaneous activity was probably facilitated by the relatively high K+ concentration (6.2 mM) in our Ringer solution. However, layer II neurons recorded in the same Ringer solution do not display spontaneous activity and are less excitable.2 Importantly, analysis of the voltage– current relationship of layer III neurons demon-

strated a very pronounced inward rectification in the depolarizing direction which was unaffected by Ca2+conductance block but was abolished with TTX. These data suggest that depolarizing inward rectification is probably caused by a low-threshold persistent sodium current (INap).4,22,48,72 This current probably provides much of the depolarizing pacemaker mechanism responsible for the spontaneous activity of the cells. Similar phenomena have been observed in several other neurons, both in cortical32,47,73 as well as in subcortical structures.38,39,48,51 Activation of INap in EC layer III neurons was also responsible for the production of rebound potentials and firing at the termination of hyperpolarizing current pulses. We also observed, however, that membrane hyperpolarization from relatively depolarized potentials (2"50 mV) triggered Ca2+-dependent rebound potentials. These are likely to be generated by low- or intermediate threshold Ca2+ conductances49,57 which should also contribute to the pacemaker drive of the cells. Interestingly, layer II neurons do not typically show such prominent Ca2+-dependent electroresponsiveness,45 unless pharmacological manipulations are applied. Our analysis of the firing frequency versus injected current relationships in layer III cells revealed behaviours similar to those described in other cortical

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Fig. 6. Firing frequency vs injected current in an medial EC layer III neuron. (A) Increased firing frequency response to injection of constant current pulses of increasing amplitude (from left to right: 0.2, 0.4, and 0.9 nA). Spike frequency adaptation is not observed with low, appears small with moderate and is seen for the first five to six spikes only during large amplitude current pulses. (B) Plot of f–I relationship for early (1, 2, 3, and 4) and late (6, 8, and 10) inter-spike intervals (ISIs) from data shown in (A. C) Plot of instantaneous firing rate vs time for three different steps of injected current for the same data as shown in A. At low amplitudes (0.2 nA) of current injection, little to no adaptation is evident as witnessed by the lack of fast decay of the curve. At higher levels (0.4 to 0.9 nA) a biexponential decay can be seen. The instantaneous frequency decays rapidly for the first four to five spikes and then decays very slowly for the duration of the pulse.

projection neurons.53,55,73 As compared to layer II stellate cells, however, layer III pyramidal cells displayed a much less pronounced frequency adaptation.2 This again indicates that EC layer III neurons may function more as relays than do stellate cells in layer II. It is worth noting, however, that a very recent report has described an additional group of non-spiny layer III pyramidal cells that do display significant spike-frequency adaptation33 and may thus not be as efficient relay cells as the only group we observed. Even though the action potential of layer III neurons did not manifest a prominent ‘‘shoulder’’ on the falling phase, Ca2+-conductance block decreased spike duration at the top, and concomitantly increased spike duration at the base and blocked the spike AHP. This suggests that substantial highthreshold inward Ca2+ current prolongs the spike duration towards the top and that this process leads to the activation of a Ca2+-dependent K+ outward current that generates the spike AHP as demonstrated in many other neurons.10,65,68,77,85 In EC layer II neurons as well as in other cortical cells, however, Ca2+-conductance block does not decrease spike duration at the top.45,65,68,77 This suggests a particularly prominent spike driven Ca2+-influx in EC layer III neurons. On the other hand, a rise in intracellular Ca2+ concentration during spike trains

appeared also responsible for the sAHP that followed them since the sAHP was also abolished by Ca2+conductance block.7,21,36 Modulation of the sAHP by neurotransmitters such as acetylcholine65,78 may aid in the development of bursting activity that layer III neurons display in vivo during periods, for example, of theta rhythmicity1,25 Functional implications EC layer III conducts the traffic of afferent neocortical information towards the CA1 hippocampal subfield.76,83 In contrast to the proven relevance of the EC layer II-dentate gyrus projection, there has been a lack of consensus regarding the physiological significance of the EC layer III-CA1 projection (see Ref. 15 for recent review). However, recent studies have presented convincing evidence that perforant path activation can produce in CA1 pyramidal cells either short-latency excitatory postsynaptic responses18 or inhibitory postsynaptic potentials probably due to the activation of CA1 interneurons.31,67 Perhaps more significantly, current source density analysis of theta activity in the CA1 region combined with lesion experiments has demonstrated a direct influence of the EC input in the generation of the field activity by the pyramidal cells.12,14 A similar observation has been done with

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Fig. 7. Spike and interspike trajectories during current–pulse triggered spike-trains in a medial EC layer III neuron. (A) Spike train triggered by a 250 ms current pulse of 1.0 nA. (B) Superimposition of the 1st, 2nd, 3rd, 6th, and 18th spike of the train in A. The traces have been aligned to match spike thresholds. There is a systematic decrease in spike amplitude and an increase in the duration of spikes for subsequent spikes in the train. (C) Plots of spike duration vs spike number during individual trains evoked by current pulses of increasing amplitude. Note that the spike duration in subsequent spikes of the spike-train shows an increase during the initial four to six spikes and then a subsequent smaller decrease that proceeded for the duration of the spike-train.

regard to the fast (40 Hz) CA1 hippocampal oscillations.17 EC layer III thus provides both theta and gamma oscillatory drive to the CA1 subfield. Indeed, during both theta and fast oscillations EC layer III neurons fire rhythmically.1,17,25 In contrast to EC layer II neurons,2 EC layer III pyramidal cells lack the manifestation of intrinsic oscillatory properties. This suggests that the oscillatory dynamics of EC layer III neurons are probably primarily determined by circuit mechanisms rather than by the intrinsic properties of individual pyramidal neurons. Regardless of the relevance of EC layer III in the generation of synchronized activity, the fact that the EC layer III projection to CA1 pyramidal cells is anatomically well circumscribed and topographically organized, suggests that afferent neocortical sensory representations are well mapped into the hippocampus.15 Our finding that the input–output relations of layer III neurons are quite linear is in line with the idea that layer III neurons function as relays, providing the CA1 region of the hippocampus with a high-fidelity copy of perhaps the same cortical information that has been subjected to more complex processing via EC layer II and the trisynaptic pathway. Synchroniz-

ation implemented via oscillatory dynamics in both the trisynaptic and monosynaptic components of the EC-hippocampal projection may be an important mechanism by which CA1 pyramidal cells may extract the precise information to construct, for example, high spatial resolution of the environment.58 Recent studies have shown that neurons in layer III of the EC degenerate preferentially in human temporal lobe epilepsy28 as well as in various rat models of temporal lobe epilepsy.27 The reasons for this particular vulnerability are unknown. It could be related to an excessive rise in intracellular-free Ca2+ concentration triggered by massive N-methyl-aspartate receptor activation.29,30,64 In fact, depolarizations recorded in these cells during epileptiform field activity in the slice are of larger amplitude and of longer duration on average than those recorded in layer II neurons.24a However, the sensitivity of these cells may also be related to Ca2+-entry through voltage-gated Ca2+-currents that in these neurons appear prominent as judged from their contribution to the action potential. Both of these possibilities may work in tandem in the course of neurodegeneration.

Entorhinal layer III pyramidal cells

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Fig. 8. Characteristics of the slow afterhyperpolarizing potential (sAHP) evoked following current–pulse triggered spike trains in MEC layer III neurons. (A and B) sAHP after a 1.0 nA current pulse in control Ringer (A) and in the presence of 200 µM Cd2+ (B). Following Cd2+ application, the sAHP was totally abolished. (C) Plot of peak amplitude and duration of the sAHP vs pulse intensity in control condition. Note that the amplitude and the duration of the sAHP increases as a function of the amplitude of the pulse. (D) Superimposition of action potentials traces in control and in Cd2+. Inset: same traces at a fast sweep speed. Note that during blockade of Ca2+ conductances, the spike duration decreased at the top while it increased at the base, and that the spike afterhyperpolarization was significantly diminished.

CONCLUSIONS

In summary, neurons from layer III of the medial EC show relatively linear input/output properties in terms of their current–voltage relationship and firing properties. These characteristics render them as highfidelity relays of neocortical input to the hippocampus. Although they do not show the intrinsic

theta rhythmicity of EC layer II stellates, they may function rhythmically in this state through rhythmic synaptic input in vivo. Their high level of excitability and contribution of Ca2+-entry during the action potential may lend a clue to the selective neurodegeneration seen in this layer in both clinical and experimental epilepsy.

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