INTRODUCTION

A delayed inward current that is activated by membrane hyperpolarization has been found and characterized in cardiac cells (DiFrancesco 1986;Yanagihara and Irisawa 1980) and in a variety of neuronal cells including photoreceptors, hippocampal CA1 pyramidal cells, thalamic relay, spinal cord, and neocortical neurons (Hestrin 1987; Maccaferri et al. 1993;Mayer and Westbrook 1983; Pape and McCormick 1989; Solomon and Nerbonne 1993a). For neuronal cells, this current was termedI_h, for hyperpolarization-activated current (Pape 1996). TheI_h is carried by the monovalent cations Na⁺ and K⁺, showed a delayed onset and a slow activation, and could be blocked by extracellular Cs⁺ but not by Ba²⁺ (reviewed in Pape 1996). In neuronal cells, I_h was thought to be involved in rhythm generation and in the determination of resting membrane potential (Maccaferri et al. 1993;McCormick and Pape 1990a; Soltesz et al. 1991). Four different ion channels related to these currents were recently identified (Ludwig et al. 1998;Santoro et al. 1998, Seifert et al. 1999).

The existence of a hyperpolarization-activated inward current has also been suggested in Cajal-Retzius cells because these cells show a hyperpolarization-activated voltage sag (Zhou and Hablitz 1996a). Cajal-Retzius cells are thought to be the first cell type appearing in the developing neocortex (Bayer and Altman 1991). They play an important role in the establishment of the cortical lamination (for review, see Frotscher 1998). Most, if not all, of the Cajal-Retzius cells disappear later in development most probably by apoptosis (Derer and Derer 1990; Meyer and Gonzales-Hernandez 1993;Naqui et al. 1999; but see Martin et al. 1999; Parnavelas and Edmunds 1983). Cajal-Retzius cells display the typical electrophysiological properties of immature neurons: they have a relatively positive resting membrane potential (RMP), a high input resistance, and slow action potentials with a high threshold (Hestrin and Armstrong 1996;Zhou and Hablitz 1996a). The relatively positive RMP may be involved in the susceptibility of Cajal-Retzius neurons for neuronal cell death (Mienville and Pesold 1999).

Because hyperpolarization-activated inward currents were thought to play an important role in the determination of RMP, we analyzed the properties of the hyperpolarization-activated inward current in Cajal-Retzius cells in detail to elucidate the physiological relevance of these currents. We demonstrate that Cajal-Retzius cells express a prominent hyperpolarization-activated inward current that resembles the properties of I_h and that this current does not contribute to the high RMP of these cells. In addition we show for the first time that the neuronalI_h is regulated by extracellular small anions.

METHODS

Slice preparation

Neonatal Wistar rats (postnatal day 0–3) were deeply anesthetized by hypothermia and decapitated. The brain was quickly removed and stored for 1–2 min in ice-cold artificial cerebrospinal fluid (ACSF). The hemispheres were dissected at the midline, and the pia was removed carefully, using fine tweezers. Tangential neocortical slices (maximum thickness, 400 μm) of both hemispheres were cut on a vibratome (Pelco 101, TPI, St. Louis, MO) using a purpose build holder, which allows to take tangential slices from various regions of the neocortex. The slices were subsequently mounted on fine tissue paper (Kodak Lens Paper) to enable the reconstruction of their original orientation in the neocortex and were transferred to an incubation chamber filled with equilibrated ACSF at 32°C in which they recovered for ≥1 h before recording began.

Identification of cells

The cells of the superficial layer of the tangential neocortical slices were visualized using infrared DIC-videomicroscopy (Dodt and Zieglgänsberger 1990). Cajal-Retzius cells were identified by their unique morphology and electrophysiological properties. Only cells located near the surface of the slice with an unambiguous root like appearance and one thick tapered process (see Fig. 1A) were chosen for experimental examination. Cells were excluded from analysis if their electrophysiological properties did not fit the results reported for Cajal-Retzius cells (RMP between −35 and −75 mV, input resistance >600 MΩ, slow action potentials with prominent afterhyperpolarization, action potential duration at half-maximal amplitude >5 ms; compare to Hestrin and Armstrong 1996;Zhou and Hablitz 1996a).

Experimental setup and procedure

The DIC-videomicroscopic setup consisted of an upright microscope with differential interference contrast optics (Axioskop, Zeiss, Jena, Germany), an infrared filter (KMZ 50–2, λ_1/2 = 750 nm, width 57 nm, Schott, Mainz, Germany), and a CCD-camera (C5405, Hamamatsu, Japan). The video image was contrast enhanced by a video processor (C 2400, Hamamatsu), visualized on a video-monitor, and digitized on-line using a frame grabber card (Screen machine II, Fast, Munich, Germany).

Whole cell patch-clamp recordings were performed according to the procedure described by Stuart et al. (1993). Patch pipettes were pulled from borosillicate tubing (2.0-mm OD, 1.16-mm ID; Science Products, Hofheim, Germany) using a vertical puller (PP-83, Narishige, Tokyo). The patch pipettes were connected to the headstage of a discontinuous voltage-clamp/current-clamp amplifier (SEC05L, NPI, Tamm, Germany). Signals were amplified, low-pass filtered at 3 kHz, visualized on an oscilloscope (TDS210, Tektronix, Beaverton, OR), digitized on-line by an AD/DA-board (ITC-16, Heka, Lamprecht, Germany), recorded and processed with the software WINTIDA 4.11 (Heka), and stored on a personal computer. The bathing solution was connected to ground via a chlorided silver wire except for all Cl⁻ substitution experiments, where a agar bridge (3 M KCl/5% agar) was used. The agar bridge reduced the potential changes induced by the Cl⁻-substitutes to <3 mV. The agar bridge was also used to determine the liquid junction potential of the gluconate-based pipette solution, which amounted to 9.6 ± 2.6 mV (mean ± SD; n = 7).

The slices were transferred into a submerged recording chamber (volume ca. 1 ml) mounted on the fixed stage of the microscope and were superfused with ACSF at a rate of 1–2 ml/min. All experiments were performed at 32°C. Gentle pressure was applied to advance the electrode to the surface of the cell. After obtaining a stable seal of >1 GΩ, the seal was broken by suction. As soon as the whole cell configuration was established, the RMP was recorded and the intrinsic membrane properties were analyzed under current-clamp conditions. For the determination of the input resistance, the current voltage relation, and the active membrane properties, hyperpolarizing and depolarizing current pulses were injected from a holding potential of −60 mV. The input resistance was calculated from a membrane hyperpolarization induced by a current pulse according to Ohm's law. The spike amplitude was measured from the spike threshold and the spike width was determined at the half-maximal spike amplitude.

Histochemical procedure

In all experiments 0.5% biocytin (Sigma, Deissenhofen, Germany) was added to the pipette solution to label the cells on which a whole cell configuration was established. To stain the labeled cells, slices were processed by a modification of the technique described byHorikawa and Armstrong (1988). Slices were fixed in a 4% paraformaldehyde solution for ≥24 h subsequently to the experiment, rinsed, and were incubated 60 min with 0.5% H₂O₂ and 0.8% Triton-X to inhibit endogenous peroxidases. An overnight incubation with an avidin-coupled peroxidase (ABC kit, Vectorlabs, Burlingame, CA) was followed by a preincubation in 0.5 mM diaminobenzidine and a subsequent reaction in diaminobenzidine and 0.015% H₂O₂. Staining was intensified by a treatment with 0.15% OsO₄. The slice was then rinsed, dehydrated slowly through alcohol and propylenoxide, and embedded in Durcopan (Fluka, Buchs, Switzerland).

Solutions

ACSF consisted of (in mM) 124 NaCl, 26 NaHCO₃, 1.25 NaH₂PO₅, 1.8 MgCl₂, 1.6 CaCl₂, 3 KCl, and 20 glucose and was equilibrated with 95% O₂-5% CO₂. The pH of this solution was 7.4 pH units and the osmolarity was 336 mOsm. In experiments in which the membrane was depolarized to potentials above −30 mV, 1 μM tetrodotoxin (TTX), 20 mM tetraethylammonium (TEA), 6 mM 4-aminopyridine (4-AP), 300 μM Cd²⁺, and 100 μM Ni²⁺ were added to the bathing solution to block voltage-gated Na⁺ currents, K⁺ currents, and low- and high-voltage-activated Ca²⁺ currents, respectively. TEA, Ni²⁺, Cd²⁺, Cs⁺, and Ba²⁺ were added to the saline solutions as chloride salts, Ni²⁺, Cd²⁺, Cs⁺, and Ba²⁺ without osmotic compensation, while TEA substituted 20 mM Na⁺. In Na⁺-free solutions, NaCl and NaHCO₃ were replaced by an equimolar amount of choline chloride and choline bicarbonate, respectively, while altered extracellular K⁺ concentrations ([K⁺]_e) were compensated by variation of the extracellular Na⁺concentration ([Na⁺]_e). In Cl⁻-free solutions, Cl⁻ was substituted with I⁻, Br⁻, NO₃⁻, gluconate, isethionate, or methylsulfate. In detail these solutions consisted of (in mM) Br⁻ substituted: 124 NaBr, 3 KBr, 1.6 CaBr₂, 1.8 MgBr₂; I⁻ substituted: 124 NaI, 3 KI, 1.6 CaBr₂, 1.8 MgBr₂; NO₃⁻ substituted: 124 NaNO₃, 3 KNO₃, 1.6 Ca(NO₃)₂, 1.8 Mg(NO₃)₂; gluconate substituted: 124 Na-gluconate, 3 K-gluconate, 1.6 Ca-gluconate, 1.8 Mg-gluconate; Isethionate substituted: 124 Na-isethionate, 3 K-gluconate, 1.6 Ca-gluconate, 1.8 Mg-gluconate; and methylsulfate substituted: 124 Na-methylsulfate, 3 K-methylsulfate, 1.6 Ca-gluconate, 1.8 Mg-gluconate. Different extracellular Cl⁻concentrations ([Cl⁻]_e) were obtained by adding ACSF to gluconate-substituted Cl⁻-free solution. The pipette solution contained (in mM) 117 K-gluconate, 13 KCl, 1 CaCl₂, 2 MgCl₂, 11 EGTA, 10 HEPES, 2 Na₂-ATP, and 0.5 Na-GTP, pH adjusted to 7.4 with KOH and osmolarity to 306 mOsm with sucrose. In some experiments, 5 mM lidocaine N-ethyl bromide (QX-314) was added to the pipette solution. QX-314 and TTX were purchased from RBI (Natic, MA), Na-isethionate and all nitrate salts from Fluka, Na-methylsulfate, MgBr₂, and CaBr₂ from Aldrich (Milwaukee, WI), K-methylsulfate from ICN (Aurora, OH), and all other substances from Sigma.

Statistics

If not otherwise noted, all values were expressed as means ± SD. For statistical analysis the Student's t-test was used, results were designated significant at a level ofP < 0.05.

RESULTS

Properties of the examined cells

Cajal-Retzius cells (n = 109) from 33 animals were used for this investigation. Thirty-six of the 109 investigated cells could be stained with biocytin. In the other cases, the cells were probably removed from the slice because they were detached with the electrode upon withdrawal (compare to Hestrin and Armstrong 1996; Zhou and Hablitz 1996a). All of these 36 stained cells showed the typical morphological features of Cajal-Retzius cells: an ovoid soma, a thick tapered dendrite, and poorly ramified dendritic processes (Fig. 1B).

The mean RMP of the Cajal-Retzius cells was −45.8 ± 7.7 mV (n = 109), and the mean input resistance was 1,367 ± 510 MΩ (n = 109). The cells elicited action potentials at a threshold of −28.4 ± 10.8 mV (n= 94) and with an amplitude of 35.7 ± 12.6 mV if depolarizing currents were applied (Fig. 1C). The first action potential had a width at half-maximal spike amplitude of 10.1 ± 5.3 ms, while a distinct action potential broadening during repetitive spiking could be observed.

Hyperpolarization activates a Cs⁺-sensitive voltage sag

Figure 1C also demonstrates the characteristic response of a Cajal-Retzius cell to the injection of hyperpolarizing currents (Zhou and Hablitz 1996a). After the membrane was hyperpolarized to below −90 mV, an obvious sag in the membrane potential (E_m) was observed. The amplitude of this voltage sag depended on the maximal amplitude of the hyperpolarization (Fig. 1, C and D). The hyperpolarization-activated voltage sag was reversibly blocked by extracellular application of 1 mM Cs⁺(n = 8, Fig.2A) while extracellular application of 1 mM Ba²⁺ had no effect on the voltage sag (n = 8, Fig. 2B), although Ba²⁺ led to a membrane depolarization of 13.6 ± 7.4 mV (n = 8) and increased the input resistance by 121 ± 22% (n = 8). The depolarizing effect of Ba²⁺ in combination with the observed increase in the input resistance may suggest an inhibitory effect of Ba²⁺ on a tonically active K⁺ conductance (Sutor and Hablitz 1993). The decline time of the voltage sag appeared to be voltage dependent (see Fig. 1C), which made the analysis of the underlying inward currents complex ifE_m was not constant. Thus for further analysis voltage-clamp experiments were performed.

The voltage sag is due to a slowly activating Cs⁺-sensitive inward current

Figure 3A shows a typical voltage-clamp recording of the hyperpolarization-activated inward current. A membrane hyperpolarization below a threshold potential of about −90 mV induced an additional component of the inward current that could be distinguished by its slow activation. Corresponding to the observations in the current-clamp experiments, the application of 1 mM Ba²⁺ had no significant effect on the slowly activating inward current (n = 8, Fig. 3B), while the application of 1 mM Cs⁺(n = 8) caused a complete block of the slowly activating component of the inward current, without affecting the capacitive and the “leakage” (= Cs⁺insensitive) components (Fig. 3C).

For a first analysis, we investigated the Cs⁺-sensitive component of the inward current (Fig. 3D), which was obtained by subtracting the current traces recorded in the presence of 1 mM Cs⁺ from the control traces. If the amplitude of the Cs⁺-sensitive component was plotted againstE_m (Fig. 3E), the data could be fitted with the Boltzmann equation

with a maximal current amplitude (I_max) of −63 pA, a voltage at half-maximal current activation (E_1/2) of −117 mV, and a slope factor (s) of 13.2 mV. From a linear regression of the data between −100 and −130 mV, where the current-voltage relation was nearly linear, it was calculated that the Cs⁺-sensitive component of the inward current activated at −90.3 mV (Fig. 3E). The time course for activation of the Cs⁺-sensitive component could be fitted with a single-exponential function [I_t =I₀+s*exp(t/τ)]. The time constant (τ) of the current activation showed a strong dependence from E_m in the examined voltage range with a slope of 14 ms/mV (Fig. 3F).

To facilitate the analysis of the current, the slowly activating component of the inward current (I_s) was isolated from the leakage current (or instantaneous current =I_i) by fitting the current trace with an exponential function (see Fig.4A), a method also used byPerkins and Wong (1995). The current traces of all experiments could be fitted with a single exponential function. The isolated I_s component displayed a nearly linear current-voltage relation between −100 and −130 mV, activated at −89.0 mV (Fig. 4B) and disappeared in the presence of 1 mM Cs⁺ (n = 19, data not shown). The plotted data could be fitted by a Boltzmann equation using an I_max of −68 pA, anE_1/2 of −118 mV, and a sof 14.8 mV. The activation time constant showed a strong voltage dependence with a slope of 18.4 ms/mV (Fig. 4C). Because these results were similar to the results obtained from the Cs⁺-sensitive current component, the method ofPerkins and Wong (1995) was used for the further analysis of the hyperpolarization-activated inward current.

To examine whether the hyperpolarization-activated inward current depends on the basal E_m, a 30-s lasting depolarization to 0 mV was applied prior to the hyperpolarizing steps (Fig. 4D). The solution used for these experiments contained 1 μM TTX, 20 mM TEA, 6 mM 4AP, 300 μM Cd²⁺, and 100 μM Ni²⁺ to prevent the activation of voltage gated Na⁺, K⁺, and Ca²⁺ currents during the depolarization. The predepolarization to 0 mV (n = 4) shifted the threshold potential of theI_s component (−75.8 mV) andE_1/2 (−109.0 mV) to more positive potentials (Fig. 4E). In addition, we analyzed very slow activation kinetics of the I_scomponent at supra- and subthreshold potentials by investigating the time course of current activation for ≤30 s. However, we could not find an additional slowly activating component of the current (n = 7; data not shown). Furthermore, it had been shown that E_1/2 critically depends on complete current activation, which requires longer hyperpolarized pulses at more positive potentials due to the strong time dependence of the activation kinetic (Seifert et al. 1999). Thus we examined the effect of an increased length of the hyperpolarizing pulse on E_1/2. Increasing the pulse length from 2 to 5 s shifted E_1/2 to −105 mV.

Ionic nature of the slowly activating inward current

For the analysis of an influence of [Na⁺]_e on theI_s component of the inward current, Na⁺ was completely removed from the bathing solution. Figure 5A shows that under these conditions, the I_scomponent was reduced by 70.4 ± 12.6% (n = 6; analyzed at −120 mV), while the I_icomponent was not significantly affected (112.8 ± 17.6%).

To determine the reversal potential of theI_s component, an analysis of the tail currents was necessary because it activates at potentials negative to the expected reversal potential of a mixed cation current (Pape 1996). To enable the examination of the reversal potential, voltage-gated Na⁺, K⁺, and Ca²⁺ currents were blocked by 1 μM TTX, 20 mM TEA, 6 mM 4-AP, 300 μM Cd²⁺, and 100 μM Ni²⁺ during the experiments. A characteristic experiment is shown in Fig. 5B. The membrane was hyperpolarized to −110 mV for 2 s to activate the slowly activating inward current and subsequently depolarized to potentials ranging from −60 to 0 mV. For the analysis of the tail currents, an exponential function was fitted to the current traces, using a time frame from 0.2 until 2 s after the depolarizing step, to avoid contaminations by capacitive currents. A single-exponential function was sufficient to fit the tail currents. The exponential curve was extrapolated to the beginning of the depolarizing step and this value was taken as the expected maximal amplitude of the tail current (I_t0) (see Fig. 5B, inset). In Fig. 5C, this I_t0 was plotted against the E_m. The current-voltage relation of the tail currents was linear in the examined voltage range and reversed at anE_m of −45.2 mV. The time constants for the inactivation of the tail currents showed no significant dependence from E_m.

The slowly activating inward current is affected by extracellular K⁺

It has been reported that [K⁺]_e affects the hyperpolarization-activated inward current (Edman and Grampp 1989; Hestrin 1987; Maccaferri et al. 1993; McCormick and Pape 1990a; Solomon and Nerbonne 1993a). Thus we tested the effect of lowered and raised [K⁺]_e on theI_i andI_s components of the hyperpolarization-activated inward current. A typical experiment in K⁺-free saline solution is shown in Fig.6A. TheI_s component of the inward current was nearly completely abolished under these conditions (9.1 ± 7.0%;n = 6; analyzed at −120 mV), while theI_i component was not significantly affected. A [K⁺]_eincrease to 6, 8, 16, and 20 mM enhanced theI_s component, while it was decreased by a [K⁺]_e reduction to 2 mM (Fig. 6B). Boltzmann fits of the data revealed that [K⁺]_e affected onlyI_max and had no significant effect onE_1/2 and s (Table1). Neither a [K⁺]_e increase nor a [K⁺]_e decrease had a significant effect on the time constant of theI_s component or on theI_i component. For a quantitative analysis of the relation between I_sand [K⁺]_e, the cord conductance (g_h =I_s/E_m; analyzed at −120 mV) was plotted against [K⁺]_e in a double-reciprocal diagram (Fig. 6C) and fitted by linear regression to obtain the maximal g_h(g_max) and the apparent dissociation constant for K⁺(K_app). From this plot, ag_max of 1.1 fS and aK_app of 7.2 mM were calculated. IfI_s was plotted versus [K⁺]_e (Fig.6D) the data could be fitted using a Michaelis-Menten-like function

with the determined values forg_max andK_app. On the other hand, the plotted data could not be fitted by a function derived from the Goldman equation(with s = Nernst slope of 60.4 mV at 32°C) if for all parameters the values determined in this examination were used (see Fig. 6D). This finding suggests thatI_s depends on [K⁺]_e in a way that could not be explained by the effect of [K⁺]_e on the electromotive force.

The slowly activating inward current is affected by extracellular Cl⁻

Previous investigations demonstrated that the hyperpolarization-activated inward current declined in Cl⁻-reduced solutions (Frace et al. 1992; Mayer and Westbrook 1983; McCormick and Pape 1990a; Yanagihara and Irisawa 1980). While it was proposed for neuronal cells that this decline in [Cl⁻]_e-reduced solutions was caused by a blocking action of the Cl⁻substitutes (Mayer and Westbrook 1983; McCormick and Pape 1990a), Frace et al. (1992) suggested for sino-atrial node cells that the hyperpolarization-activated inward current depends directly on small anions. Thus we tested the effect of various organic and inorganic substances as Cl⁻substitutes on the I_i andI_s component of the hyperpolarization-activated inward current. A characteristic experiment is shown in Fig. 7A. The inorganic Cl⁻ substitutes Br⁻ (122 ± 7%, n = 7), I⁻ (130 ± 11%, n = 8), and NO₃⁻ (112 ± 5%,n = 5) had no significant effect on the amplitude of the hyperpolarization-activated inward current. The organic substitutes isethionate (n = 11), methylsulfate (n = 9), and gluconate (n = 5) caused a significant decrease in the I_scomponent by 38 ± 5, 41 ± 10, and 42 ± 8%, respectively, while the time constant and theI_i component were not significantly altered. The Boltzmann fits of these data (Fig. 7B) revealed that the I_s reduction was caused by a reduction in I_max, whileE_1/2 or s were not affected.

To investigate whether the effect of Cl⁻substitution was caused by a blocking action of the organic anions, we examined the effect of different [Cl⁻]_e on theI_s component. In these experiments, Cl⁻ was substituted for gluconate. Figure7C shows that a [Cl⁻]_e increase led to a recovery of the I_s component. The [Cl⁻]_e dependence of theI_s component could be fitted using a Hill-like function (Fig. 7C)

This fit revealed a value of 9.8 mM forK_app and a Hill-coefficient (h) of 2.5. On the other hand, if a gluconate dependence of the I_s component was assumed and analyzed using a Hill plot (i.e., [I_max/(1 − I)] plotted versus log[gluconate]_e), a Hill-coefficient of 20.3 was obtained.

The slowly activating inward current is blocked by intracellular QX-314

To test whether the slowly activating inward current was blocked by intracellular administration of the lidocaine derivative QX-314 (Perkins and Wong 1995), we used an electrode solution containing 5 mM QX-314. A depolarizing step from −60 to −110 mV was applied immediately (8 s) after the establishment of the whole cell configuration and was repeated every 10 s for up to 4 min. While the I_i component was not affected during this experiment, the amplitude of theI_s component declined within 40 s to <10% (n = 6) compared to the first hyperpolarizing pulse (Fig. 8). Because the analysis of the QX-314 effect required an immediate start of hyperpolarizing pulses, these six cells could not be tested for action potential properties and were thus identified only by their morphological and passive electrophysiological characteristics.

The slowly activating inward current has no effect on RMP and action potential properties

To determine whether the hyperpolarization-activated inward current contributes to the RMP of Cajal-Retzius cells, we tested the effect of 1 mM Cs⁺ on the RMP of these cells. After a 4-min incubation with Cs⁺, no significant difference in the RMP was observed (−47.0 ± 2.6 mV vs. −46.5 ± 2.0 mV, n = 10). In addition we examined the effect of Cs⁺ on action potential properties. The application of 1 mM Cs⁺ had no effect on action potential amplitude, threshold, width, and frequency or on the amplitude of the afterhyperpolarization (n = 7, data not shown).

DISCUSSION

Identification of Cajal-Retzius cells

Cajal-Retzius cells possess an unique morphological appearance, which makes it easy to identify them visually. In addition, all cells appearing in this investigation displayed electrophysiological properties typical for Cajal-Retzius cells. They had a relatively positive RMP, had a high input resistance, and showed action potentials of small amplitude and slow time course that were elicited at a low threshold and expressed a pronounced broadening (Hestrin and Armstrong 1996; Mienville and Pesold 1999;Zhou and Hablitz 1996a). All 36 histologically recovered cells showed the morphological features typical for Cajal-Retzius cells, such as an ovoid soma, one thick, tapered dendrite, and sparsely branched dendritic processes (Hestrin and Armstrong 1996; Zhou and Hablitz 1996b). This finding demonstrates that the identification of Cajal-Retzius cells by their appearance in the infrared videomicroscope in combination with their electrophysiological properties is sufficient for an unambiguous identification.

Properties of the slowly activating inward current

For a characterization of the slowly activating inward current, it is important to demonstrate that it is not caused by a shift in driving forces evoked by ion accumulation or depletion but that it relates to the direct activation of a membrane conductance (Pape 1996). The findings that the inward current was well described with the Boltzmann equation, expressed a strong [K⁺]_e dependence, which cannot be explained by an effect on the driving force of K⁺ ions, and could be selectively blocked strongly suggest a conventional membrane conductance as the physical correlate of this current. Our results clearly confirm that the slowly activating inward current examined in this investigation resembles the characteristic properties for I_h(Pape 1996). It showed a slow activation after a hyperpolarizing step, depended on extracellular K⁺ and Na⁺, and was blocked by intracellular QX-314 and extracellular Cs⁺ but was not affected by 1 mM Ba²⁺. Thus we can refer to the slowly activating inward current of Cajal-Retzius cells asI_h.

The activation of I_h could be well described by a single-exponential function, resembling previous reports (but see Solomon and Nerbonne 1993b). The activation time constant showed a strong voltage dependence and was in the range found in other examinations (McCormick and Pape 1990a;Santoro et al. 1998; Solomon and Nerbonne 1993b). On the other hand, I_hactivated at more negative E_m than reported for other preparations (Ludwig et al. 1998;Maccaferri et al. 1993; Mayer and Westbrook 1983; Pape and McCormick 1989; Santoro et al. 1998; Solomon and Nerbonne 1993a;Wollmuth 1995). The negative activation threshold ofI_h was puzzling because the RMP of Cajal-Retzius cells is more positive as compared with the cells in these reports. This discrepancy between the RMP and the activation threshold was not effected by errors in the determination of these potentials due to liquid-junction potentials because the measured liquid junction potential would shift both values by ∼10 mV toward positive direction. It had been shown by Seifert et al. (1999) that the I_h current amplitude at more positive potentials might be underestimated if the hyperpolarizing pulse was too short becauseI_h may not reach steady-state values at positive potentials due to the strong voltage dependence of theI_h activation kinetic. Thus the estimation of E_1/2 critically depends on the length of the hyperpolarizing pulse. In our investigation, an attenuation of the hyperpolarizing pulse to 5 s indeed shiftedE_1/2 by 13 mV in positive direction. However, even under these conditions, the activation threshold ofI_h was far more negative than the RMP of Cajal-Retzius cells. These findings suggests thatI_h was not activated under resting conditions and thus did not contribute to the maintenance of the RMP. This suggestion was confirmed by the finding that the RMP was unaffected by a block of I_h with 1 mM Cs⁺.

To test whether I_h was partially inactivated at a holding potential of −60 mV, theI_h activation threshold was determined after a 30-s lasting depolarization to 0 mV. This massive depolarization prior to the hyperpolarizing step had only a minor effect on I_h activation threshold, which was still far more negative than the RMP. ThusI_h could not be activated near RMP even under these conditions. If the observed shift in activation threshold was due to the release of inactivation, a mechanism never reported for I_h (Pape 1996), or due to shifts in driving forces evoked by ion accumulation or depletion could not be answered unequivocally from our observation.

Ionic nature of the inward current

Due to the equilibration of the cell interior with the pipette solution occurring in whole cell mode the intra- as well as the extracellular concentrations of Na⁺ and K⁺ are known. Thus it is possible to estimate the ion permeabilities of I_h without analyzing shifts in the reversal potential caused by alterations in extracellular ion concentrations. From the determined reversal potential of −45.2 mV a permeability coefficient (α = pNa⁺/pK⁺) of 0.13 was calculated using the Goldman equation (Hille 1984). This value for α was in the range reported for other neuronal preparations (α = 0.24, Ludwig et al. 1998; 0.25,Hestrin 1987; 0.40, Solomon and Nerbonne 1993a) and demonstrates thatI_h is a mixed Na⁺/K⁺ current in Cajal-Retzius neurons. We observed that complete substitution of Na⁺ with choline caused an incomplete block ofI_h. A fraction of ∼30% (analyzed at −120 mV) preserved under this conditions. This remaining fraction cannot be explained sufficiently by a K⁺ influx due to the inwardly directed electromotive force on K⁺ under these conditions because we estimate from the Goldman equation that only 3% ofI_h should maintain in Na⁺-free solution. This finding may be an indication that the Na⁺ influx interferes with K⁺ fluxes by the same mechanisms as described byWollmuth (1995) for the inhibiting action of submillimolar [Na⁺]_e.

The complete omission of K⁺ from the extracellular solution blocked I_hnearly completely, a finding that could not be explained by the effect of [K⁺]_e on driving forces. Thus an alternative explanation for this effect must be considered. Alterations in [K⁺]_e led to massive shifts in I_h amplitude that also could not sufficiently be explained by the effect of [K⁺]_e on the driving force. However, the observed relation betweenI_h and [K⁺]_e could be modeled with a Michaelis-Menten-like equation, suggesting an additional regulatory binding site for K⁺. The calculatedK_app for K⁺ of 7.2 mM is lower than reported for other preparations (20 mM,Edman and Grampp 1989; 25.7 mM, Solomon and Nerbonne 1993a). The observed relation between [K⁺]_e andI_h predicts relatively great shifts in I_h evoked by small [K⁺]_e alterations in the physiological [K⁺]_erange. Thus in Cajal-Retzius cells, I_hmay act as a sensor for [K⁺]_e, which may be influenced by various physiological and pathophysiological events (Coles and Poulain 1991; Perez-Pinzon et al. 1995).

The complete Cl⁻ substitution with the organic anions isethionate, methylsulfate, or gluconate evoked a significant reduction in I_h that could be abolished by the readdition of Cl⁻. Our results argue against the suggestion that this reduction was caused by a blocking action of the organic substitute itself (Mayer and Westbrook 1983; McCormick and Pape 1990a). We observed an almost identical blocking effect for all organic compounds used in this investigation despite their different molecular structure. We also observed that the analysis of the [Cl⁻]_e dependence ofI_h reduction demonstrates that an unreasonable high Hill coefficient >20 is required, if a blocking action of gluconate is assumed. Thus we suggest that the Cl⁻ anions itself modulateI_h. Corresponding to investigations on cardiac cells (Frace et al. 1992), we observed that substitution of Cl⁻ with the small inorganic anions Br⁻, I⁻, or NO₃⁻ had no significant effect on I_h. This result suggests that at least some small anions can replace Cl⁻ inI_h modulation. Although no complete block of I_h could be observed in Cl⁻-free solution, small anions may be essential for I_h activation because the HCO₃⁻ anions in the Cl⁻-free solutions may probably contribute to the I_h activation by small anions.

Physiological relevance of the hyperpolarization-activated inward current

Although a Na⁺ influx is involved in the determination of the relatively positive RMP of Cajal-Retzius cells, the RMP was not influenced by inhibitors of excitatory neurotransmitter receptors or by the Na⁺ channel blocker TTX (Mienville and Pesold 1999). These observations may indicate the contribution of an inward current throughI_h channels to the RMP. However, our observations that I_h activated at potentials negative to −90 mV and that a block ofI_h by Cs⁺ was without effect on the RMP strongly suggest thatI_h does not contribute to the relatively positive RMP of Cajal-Retzius cells.

It was already shown that I_hcontributes to membrane oscillations by intervening with the afterhyperpolarization (McCormick and Pape 1990a). Because Cajal-Retzius cells express a strong afterhyperpolarization, we also tested if a block of I_h by Cs⁺ had an effect on the properties of action potentials. However, we could not find an effect of Cs⁺ on the afterhyperpolarization nor on other action potential properties. This observation suggests thatI_h does probably not contribute to the active membrane properties. Because we could not find an effect of blocking I_h on any electrophysiological properties, the physiological relevance ofI_h for Cajal-Retzius cells is currently unknown.

One possible hypothesis for the role ofI_h may be that during development either the activation threshold of I_his shifted to more positive potentials or that RMP is shifted to more negative potentials. However, no developmental alteration in the hyperpolarization-activated voltage sag had been found between P0 and P6 (Zhou and Hablitz 1996a) and the RMP of Cajal-Retzius cells remained also relatively positive during their development (Mienville and Pesold 1999). Another possibility is thatI_h could be activated by hyperpolarizations, which occur during hypoxic events (Higashi et al. 1988; Luhmann 1996; Luhmann and Heinemann 1992). And finally, it had been reported for a variety of preparations that neurotransmitters can affect theI_h by a shift in voltage dependence (Colino and Halliwell 1993; Li et al. 1993; McCormick and Pape 1990a; Pape and McCormick 1989). Because monoaminergic and serotonergic fibers are among the first projections reaching the neocortex during early development (Bayer and Altman 1991), the neurotransmitters released from these fibers may shiftI_h activation threshold to more physiological E_m. A functional alteration of I_h due to noradrenaline or other monoamines may thus be involved in the regulation of cortical development because it had been shown that noradrenaline determines the fate of Cajal-Retzius cells (Naqui et al. 1999).

FOOTNOTES

• Address for reprint requests: W. Kilb, Institut für Neurophysiologie, Heinrich-Heine-Universität Düsseldorf, Postfach 101007, D-40001 Düsseldorf, Germany (E-mail:[email protected]de).

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