Methylxanthine-evoked perturbation of spontaneous and evoked activities in isolated newborn rat hippocampal networks.

Treatment of apnea of prematurity with methylxanthines like caffeine, aminophylline or theophylline can evoke hippocampal seizures. However, it is unknown at which interstitial brain concentrations methylxanthines promote such neonatal seizures or interfere with physiological 'early network oscillations' (ENOs) that are considered as pivotal for maturation of hippocampal neural networks. We studied theophylline and caffeine effects on ENOs in CA3 neurons (CA3-ENOs) and CA3 electrical stimulation-evoked monosynaptic CA1 field potentials (CA1-FPs) in sliced and intact hippocampi, respectively, from 8 to 10-days-old rats. Submillimolar doses of theophylline and caffeine, blocking adenosine receptors and phosphodiesterase-4 (PDE4), did not affect CA3-ENOs, ENO-associated cytosolic Ca(2+) transients or CA1-FPs nor did they provoke seizure-like discharges. Low millimolar doses of theophylline (⩾1mM) or caffeine (⩾5mM), blocking GABAA and glycine receptors plus sarcoplasmic-endoplasmic reticulum Ca(2+) ATPase (SERCA)-type Ca(2+) ATPases, evoked seizure-like discharges with no indication of cytosolic Ca(2+) dysregulation. Inhibiting PDE4 with rolipram or glycine receptors with strychnine had no effect on CA3-ENOs and did not occlude seizure-like events as tested with theophylline. GABAA receptor blockade induced seizure-like discharges and occluded theophylline-evoked seizure-like discharges in the slices, but not in the intact hippocampi. In summary, submillimolar methylxanthine concentrations do not acutely affect spontaneous CA3-ENOs or electrically evoked synaptic activities and low millimolar doses are needed to evoke seizure-like discharges in isolated developing hippocampal neural networks. We conclude that mechanisms of methylxanthine-related seizure-like discharges do not involve SERCA inhibition-related neuronal Ca(2+) dysregulation, PDE4 blockade or adenosine and glycine receptor inhibition, whereas GABA(A) receptor blockade may contribute partially.

Control of cortical neuronal migration by glutamate and GABA.

Neuronal migration in the cortex is controlled by the paracrine action of the classical neurotransmitters glutamate and GABA. Glutamate controls radial migration of pyramidal neurons by acting primarily on NMDA receptors and regulates tangential migration of inhibitory interneurons by activating non-NMDA and NMDA receptors. GABA, acting on ionotropic GABAA-rho and GABAA receptors, has a dichotomic action on radially migrating neurons by acting as a GO signal in lower layers and as a STOP signal in upper cortical plate (CP), respectively. Metabotropic GABAB receptors promote radial migration into the CP and tangential migration of interneurons. Besides GABA, the endogenous GABAergic agonist taurine is a relevant agonist controlling radial migration. To a smaller extent glycine receptor activation can also influence radial and tangential migration. Activation of glutamate and GABA receptors causes increases in intracellular Ca(2+) transients, which promote neuronal migration by acting on the cytoskeleton. Pharmacological or genetic manipulation of glutamate or GABA receptors during early corticogenesis induce heterotopic cell clusters in upper layers and loss of cortical lamination, i.e., neuronal migration disorders which can be associated with neurological or neuropsychiatric diseases. The pivotal role of NMDA and ionotropic GABA receptors in cortical neuronal migration is of major clinical relevance, since a number of drugs acting on these receptors (e.g., anti-epileptics, anesthetics, alcohol) may disturb the normal migration pattern when present during early corticogenesis.

Cajal-Retzius cells: update on structural and functional properties of these mystic neurons that bridged the 20th century.

Cajal-Retzius cells (CRc) represent a mostly transient neuronal cell type localized in the uppermost layer of the developing neocortex. The observation that CRc are a major source of the extracellular matrix protein reelin, which is essential for the laminar development of the cerebral cortex, attracted the interest in this unique cell type. In this review we will (i) describe the morphological and molecular properties of neocortical CRc, with a special emphasize on the question which markers can be used to identify CRc, (ii) summarize reports that identified the different developmental origins of CRc, (iii) discuss the fate of CRc, including recent evidence for apoptotic cell death and a possible persistence of some CRc, (iv) provide a detailed description of the electrical membrane properties and transmitter receptors of CRc, and (v) address the role of CRc in early neuronal circuits and cortical development. Finally, we speculate whether CRc may provide a link between early network activity and the structural maturation of neocortical circuits.

Effect of depolarizing GABA(A)-mediated membrane responses on excitability of Cajal-Retzius cells in the immature rat neocortex.

In immature neurons activation of ionotropic GABA receptors induces depolarizing membrane responses due to a high intracellular Cl(-) concentration ([Cl(-)](i)). However, it is difficult to draw conclusions about the functional consequences of subthreshold GABAergic depolarizations, since GABAergic membrane shunting and additional effects on voltage-dependent ion channels or action potential threshold must be considered. To systematically investigate factors that determine the GABAergic effect on neuronal excitability we performed whole cell patch-clamp recordings from Cajal-Retzius cells in immature rat neocortex, using [Cl(-)](i) between 10 and 50 mM. The effect of focal GABA application was quantified by measuring various parameters of GABAergic responses including the shift in minimal threshold current (rheobase). The rheobase shift was correlated with other parameters of the GABAergic responses by multiple linear regression analyses with a set of simple mathematical models. Our experiments demonstrate that focal GABA application induces heterogeneous rheobase shifts in Cajal-Retzius cells that could not be predicted reliably from [Cl(-)](i) or the GABAergic membrane depolarization. Implementation of a linear mathematical model, which takes the GABAergic membrane conductance and the difference between action potential threshold and GABA reversal potential into account, resulted in a close correlation between calculated and experimentally obtained rheobase shifts. Addition of a linear term proportional to the GABAergic membrane depolarization improved the accuracy of correlation. The main advantage of using multiple linear regression with simple models is that direction and strength of GABAergic excitability shifts can be analyzed by using only measured parameters of GABAergic responses and with minimal a priori information about cellular parameters.

Electrophysiological and morphological properties of Cajal-Retzius cells with different ontogenetic origins.

The different origins of Cajal-Retzius cells (CRc) as well as their diverse molecular profile suggest that this cell type may represent different neuronal subpopulations. In order to investigate whether CRc from different origins show distinct functional or morphological characteristics we used transgenic Dbx1(cre);ROSA26(YFP) mice in which two subpopulations of CRc, originating from the septum and ventral pallium (VP) at the pallial-subpallial border (PSB), were permanently labeled by yellow fluorescent protein (YFP) expression. Electrophysiological properties of YFP(+) and YFP(-) CRc were investigated by whole-cell patch-clamp recordings, while a thorough somatodendritic and axonal reconstruction of the biocytin labeled CRc was subsequently performed using a Neurolucida system. Our experiments revealed that no significant differences in resting membrane potential, input resistance or capacitance, hyperpolarization activated currents and most action potentials properties could be observed between YFP(+) and YFP(-) CRc. Both YFP(+) and YFP(-) CRc displayed spontaneous and carbachol-induced GABAergic postsynaptic currents with similar properties and comparable NMDA-receptor mediated glutamatergic inward currents that were equally affected by the NR2B specific antagonist ifenprodil. Morphological reconstructions revealed that dendritic and axonal parameters are similar between YFP(+) and YFP(-) CRc, while the dendritic compartment of YFP(+) CRc was slightly larger. In summary, no considerable differences in functional and morphological properties between YFP(+) and YFP(-) CRc could be observed in this study. These observations suggest that CRc of different ontogenic origins display comparable functional properties in the early postnatal cortex and therefore perform similar functions within the transient neuronal networks of the developing cortex.

GABAC receptors are functionally expressed in the intermediate zone and regulate radial migration in the embryonic mouse neocortex.

Radial neuronal migration in the cerebral cortex depends on trophic factors and the activation of different voltage- and ligand-gated channels. To examine the functional role of GABA(C) receptors in radial migration we analyzed the effects of specific GABA(A) and GABA(C) receptor antagonists on the migration of BrdU-labeled neurons in vitro using organotypic neocortical slice cultures. These experiments revealed that the GABA(A) specific inhibitor bicuculline methiodide facilitated neuronal migration, while the GABA(C) specific inhibitor (1,2,5,6-tetrahydropyridine-4-yl) methylphosphinic-acid (TPMPA) impeded migration. Co-application of TPMPA and bicuculline methiodide or the unspecific ionotropic GABA receptor antagonist picrotoxin both impeded migration, suggesting that the GABA(C) receptor mediated effects dominate. Addition of the specific GABA(C) receptor agonist cis-4-aminocrotonic acid (CACA) also hampered migration, indicating that a physiological GABAergic stimulation is required for appropriate function. RT-PCR experiments using specific probes for GABA(C) receptor mRNA and Western blot assays using an antibody directed against rho subunits revealed the expression of GABA(C) receptor mRNA and translated GABA(C) receptor protein in the immature cortex. Microfluorimetric Ca(2+) imaging in neurons of identified cortical layers using Calcium Green revealed the functional expression of GABA(A) and GABA(C) receptors in the intermediate zone, while only GABA(A) receptor mediated responses were observed in the upper cortical plate. In summary, these results demonstrate that activation of GABA(C) receptors is a prerequisite for accurate migration and that GABA(C) receptors are functionally expressed in the intermediate zone.

Glycine receptors mediate excitation of subplate neurons in neonatal rat cerebral cortex.

The development of the cerebral cortex depends on genetic factors and early electrical activity patterns that form immature neuronal networks. Subplate neurons (SPn) are involved in the construction of thalamocortical innervation, generation of oscillatory network activity, and in the proper formation of the cortical columnar architecture. Because glycine receptors play an important role during early corticogenesis, we analyzed the functional consequences of glycine receptor activation in visually identified SPn in neocortical slices from postnatal day 0 (P0) to P4 rats using whole cell and perforated patch-clamp recordings. In all SPn the glycinergic agonists glycine, beta-alanine, and taurine induced dose-dependent inward currents with the affinity for glycine being higher than that for beta-alanine and taurine. Glycine-induced responses were blocked by the glycinergic antagonist strychnine, but were unaffected by either the GABAergic antagonist gabazine, the N-methyl-d-aspartate-receptor antagonist d-2-amino-5-phosphonopentanoic acid, or picrotoxin and cyanotriphenylborate, antagonists of alpha-homomeric and alpha1-subunit-containing glycine receptors, respectively. Under perforated-patch conditions, glycine induced membrane depolarizations that were sufficient to trigger action potentials (APs) in most cells. Furthermore, glycine and taurine decreased the injection currents as well as the synaptic stimulation strength required to elicit APs, indicating that glycine receptors have a consistent excitatory effect on SPn. Inhibition of taurine transport and application of hypoosmolar solutions induced strychnine-sensitive inward currents, suggesting that taurine can act as a possible endogenous agonist on SPn. In summary, these results demonstrate that SPn express glycine receptors that mediate robust excitatory membrane responses during early postnatal development.

Model-specific effects of bumetanide on epileptiform activity in the in-vitro intact hippocampus of the newborn mouse.

The immature brain has a higher susceptibility to develop seizures, which often respond poorly to classical pharmacological treatment. It has been recently suggested that bumetanide, which blocks Na(+)-dependent K(+)-Cl(-)-cotransporter isoform 1 (NKCC1) and thus attenuates depolarizing GABAergic responses, could soothe epileptiform activity in immature nervous systems. To evaluate whether bumetanide consistently attenuates epileptiform activity, we investigated the effect of 10 microM bumetanide in five different in-vitro epilepsy models using field potential recordings in the CA3 region of intact mouse hippocampal preparations at postnatal day 4-7. Bumetanide reduced amplitude and frequency of ictal-like events (ILE) induced by 8.5 mM K(+), but it increased the frequency of ILE induced by 1 microM kainate. Inhibition of ligand-gated Cl(-) channels by 10 microM gabazine and 30 microM strychnine induced interictal activity (IA) that was only marginally affected by bumetanide. Removal of extracellular Mg(2+) induced both ILE and IA. Bumetanide had no effect on these ILE but enhanced the IA. Low-Mg(2+) solution containing 20 microM 4-AP induced late-recurrent discharges, which were slightly attenuated by bumetanide. In summary, our results demonstrate that bumetanide exerts diverse effects in different in-vitro epilepsy models.

Homogenous glycine receptor expression in cortical plate neurons and Cajal-Retzius cells of neonatal rat cerebral cortex.

Glycinergic membrane responses have been described in cortical plate neurons (CPn) and Cajal-Retzius cells (CRc) during early neocortical development. In order to elucidate the functional properties and molecular identity of glycine receptors in these two neuronal cell types, we performed whole-cell patch-clamp recordings and subsequent single-cell multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) analyses on visually identified neurons in tangential and coronal slices as well as in situ hybridizations of coronal slices from neonatal rat cerebral cortex (postnatal days 0-4). In both CPn and CRc the glycinergic agonists glycine, beta-alanine and taurine induced inward currents with larger current densities in CRc. The functional properties of these currents were similar between CPn and CRc. In both cell types the glycine receptor showed a higher affinity for glycine than for the glycinergic agonists beta-alanine and taurine. The glycinergic responses of both cells were blocked by the glycinergic antagonist strychnine and were unaffected by the GABAergic antagonist bicuculline (100 microM), the N-methyl-D-aspartic acid receptor antagonist (+/-)-2-amino-5-phosphonopentatonic acid (60 microM) and by picrotoxin (30 microM), an antagonist of alpha homomeric glycine receptors. Single-cell multiplex RT-PCR revealed the expression of glycine receptor alpha(2) and beta subunits in CPn and CRc, while no alpha(1) and alpha(3) subunits were observed. In situ hybridization histochemistry showed the expression of mRNAs for alpha(2) and beta subunits within the cortical plate and in large neurons of the marginal zone, while there were no signals for alpha(1) and alpha(3) subunits. In summary, these results suggest that CPn and CRc express glycine receptors with similar functional and pharmacological properties. The correlation of pharmacological properties and mRNA expression suggests that the glycine receptors in both cell types may consist of alpha(2)/beta heteromeric receptors.

Depolarizing glycine responses in Cajal-Retzius cells of neonatal rat cerebral cortex.

We investigated the properties of glycine-induced responses in Cajal-Retzius cells, a neuronal cell type essential for the establishment of neocortical lamination. Whole-cell and gramicidin-perforated patch-clamp recordings were performed on visually identified Cajal-Retzius cells in tangential slices from neonatal rat cortex (postnatal days 0-3). With a pipette Cl(-) concentration of 50 mM, bath application of 1 mM glycine induced a membrane depolarization of 32.8+/-7.4 mV and a massive decrease in membrane resistance by 88+/-1.4%. The membrane depolarization was abolished in the presence of the glycinergic antagonists strychnine (30 microM) and phenylbenzene-omega-phosphono-alpha-amino acid (100 microM), while the GABA(A) receptor antagonist bicuculline (100 microM) and the glutamatergic antagonist (+/-)-2-amino-5-phosphonopentatonic acid (60 microM) were without effect, suggesting that the glycine-induced membrane responses were mediated exclusively by the strychnine-sensitive glycine receptor. The EC(50) for activation of glycine receptors was 0.54 mM, 1.62 mM and 2.41 mM, for the glycinergic agonists glycine, beta-alanine and taurine, respectively. Since the reversal potential of the glycine-induced currents showed a strong dependency on the intracellular chloride concentration and was virtually unaffected under HCO(3)(-)-free conditions, the activation of glycine receptors was probably linked to Cl(-) fluxes with little contribution of HCO(3)(-) ions. Perforated patch recordings from Cajal-Retzius cells demonstrated that glycine elicited depolarizing responses mediated by Cl(-) currents which reversed at -41+/-3.7 mV. In summary, from these results we suggest that Cajal-Retzius cells of the neonatal rat cerebral cortex express functional strychnine-sensitive glycine receptors that mediate depolarizing membrane responses via Cl(-) efflux.

Modulation of Ca2+ influx in leech Retzius neurons II. Effect of extracellular Ca2+.

We investigated the cytosolic free calcium concentration ([Ca2+]i) of leech Retzius neurons in situ while varying the extracellular Ca2+ concentration via the bathing solution ([Ca2+]B). Changing [Ca2+]B had only an effect on [Ca2+]i if the cells were depolarized by raising the extracellular K+ concentration. Surprisingly, raising [Ca2+]B from 2 to 10 mm caused a decrease in [Ca2+]i, and an increase was evoked by reducing [Ca2+]B to 0.1 mm. These changes were not due to shifts in membrane potential. At low [Ca2+]B moderate membrane depolarizations were sufficient to evoke a [Ca2+]i increase, while progressively larger depolarizations were necessary at higher [Ca2+]B. The changes in the relationship between [Ca2+]i and membrane potential upon varying [Ca2+]B could be reversed by changing extracellular pH. We conclude that [Ca2+]B affects [Ca2+]i by modulating Ca2+ influx through voltage-dependent Ca2+ channels via the electrochemical Ca2+ gradient and the surface potential at the extracellular side of the plasma membrane. These two parameters are affected in a counteracting way: Raising the extracellular Ca2+ concentration enhances the electrochemical Ca2+ gradient and hence Ca2+ influx, but it attenuates Ca2+ channel activity by shifting the extracellular surface potential to the positive direction, and vice versa.

Modulation of Ca2+ influx in leech Retzius neurons. I. Effect of extracellular pH.

We investigated the cytosolic free Ca2+ concentration ([Ca2+]i) of leech Retzius neurons in situ while varying the extracellular and intracellular pH as well as the extracellular ionic strength. Changing these parameters had no significant effect on [Ca2+]i when the membrane potential of the cells was close to its resting value. However, when the cells were depolarized by raising the extracellular K+ concentration or by applying the glutamatergic agonist kainate, extracellular pH and ionic strength markedly affected [Ca2+]i, whereas intracellular pH changes appeared to have virtually no effect. An extracellular acidification decreased [Ca2+]i, while alkalinization or reduction of the ionic strength increased it. Correspondingly, [Ca2+]i also increased when the kainate-induced extracellular acidification was reduced by raising the pH-buffering capacity. At low extracellular pH, the membrane potential to which the cells must be depolarized to evoke a detectable [Ca2+]i increase was shifted to more positive values, and it moved to more negative values at high pH. We conclude that in leech Retzius neurons extracellular pH, but not intracellular pH, affects [Ca2+]i by modulating Ca2+ influx through voltage-dependent Ca2+ channels. The results suggest that this modulation is mediated primarily by shifts in the surface potential at the extracellular side of the plasma membrane.

Feedback control of intracellular pH by means of iontophoretic H+/OH- injection.

A new method for controlling intracellular pH (pH(i)) within a single cell has been developed that is based on the iontophoretic injection of the pH-affecting ions H+ and OH-. The H+/OH- injection currents are governed by an electronic feedback system, which compares the actual pH(i), monitored by pH-sensitive microelectrodes, with a command value. To avoid membrane potential (Em) alterations caused by the H+/OH- currents, the pH(i) control is performed under voltage-clamp conditions, using a two-electrode voltage-clamp design. This feedback-controlled iontophoretic injection system was capable of adjusting pH(i) between pH 6.8 and 8.0 with a precision of 0.031+/-0.026 pH units (n=51) and without affecting Em. We illustrate the application of this system by investigating the pH(i) dependence of glutamate receptors in identified leech Retzius neurons. Stimulation of these receptors with the glutamatergic agonist kainate (50 microM) under control conditions induced an intracellular acidification of 0.14+/-0.07 pH units (n=13). The feedback control system for pH(i) reduced this intracellular acidification to 0.02+/-0.01 pH units (n=15). The kainate-induced inward current was not affected by pH(i) alterations. This result demonstrates that the feedback system for pH(i) control was feasible for investigating the pH(i) dependence of physiological processes and suggests that the glutamate receptors are not modulated by pH(i).

Spontaneous synaptic activity of subplate neurons in neonatal rat somatosensory cortex.

Subplate neurons play an important role in early cortical development. To investigate whether these transient neurons receive synaptic inputs, we performed whole-cell recordings from visually identified and biocytin-labeled subplate cells in somatosensory cortical slices from postnatal day 0-3 rats. Subplate neurons had an average resting membrane potential of -55 mV and input resistance of approximately 1.1 G ohms. Suprathreshold current injection elicited in 67% of the cells repetitive action potentials at 4-13 Hz and the remaining 33% showed only one spike. Three classes of spontaneous postsynaptic currents (sPSCs) could be identified: (i) Fast sPSCs, with an average amplitude of 14 pA and a decay time of 6.3 ms, which showed a 95% decrease in their frequency during (+/-)-gamma-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA)/kainate receptor blockade. Cyclothiazide caused a 3.5-fold increase in the decay time, indicating that fast sPSCs were mediated by AMPA receptors. (ii) Slow sPSCs, with 18 pA amplitude and 51.2 ms decay time were blocked by the N-methyl-D-aspartate (NMDA) receptor antagonist CPP. (iii) Chloride-driven sPSCs, with 34.4 pA amplitude and 123 ms decay time that were blocked by the gamma-amino-butyric acid A (GABA(A)) receptor antagonist gabazine. While tetrodotoxin citrate (TTX) blocked completely NMDA-mediated slow sPSCs, the frequency of AMPA- and GABA(A)-mediated sPSCs was reduced in TTX by 55 and 90%, respectively. These results indicate that subplate neurons receive functional synaptic inputs mediated by AMPA, NMDA and GABA(A) receptors.

Spontaneous GABAergic postsynaptic currents in Cajal-Retzius cells in neonatal rat cerebral cortex.

Cajal-Retzius cells are among the first neurons appearing during corticogenesis, and play an important role in the establishment of cortical lamination. The variety of neurotransmitter receptors recently found on these cells imply that they are integrated in the neonatal cortical network. To investigate the presence and properties of spontaneous synaptic activity we performed whole-cell patch-clamp recordings from visually identified and biocytin-labelled Cajal-Retzius cells in a tangential slice preparation of neonatal rat cerebral cortex (postnatal days P0-P5). Spontaneous postsynaptic currents (sPSCs) could be observed in about 23% of the cells using a pipette solution containing 136 m M Cl-. The sPSCs occurred at a low frequency (0.07 +/- 0.07 Hz, n = 42 cells), had an average amplitude of 24.3 +/- 12.4 pA (n = 415 events) and could not be divided in subpopulations according to their amplitude distribution or kinetic properties. The sPSCs were blocked by the GABAA antagonist bicuculline (100 microM), while the glutamatergic antagonists (+/-)-2-amino-5-phosphonopentatonic acid (APV, 30 microM) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 microM), as well as tetrodotoxin (1-2 microM), a blocker of voltage-gated sodium-currents, had no significant effect on sPSCs. The incidence rate of sPSCs declined within the age of the rats and no sPSCs could be observed after P4. These results suggest that Cajal-Retzius cells transiently receive action potential-independent and GABA(A) receptor-mediated spontaneous synaptic input, which may contribute to the refinement of cortical circuits.

Cellular physiology of the neonatal rat cerebral cortex: intrinsic membrane properties, sodium and calcium currents.

The cellular physiology of the primary somatosensory cortex was studied in postnatal day (P) 0 to P5 rats using whole-cell patch-clamp recordings. Visually identified Cajal-Retzius, subplate, bifurcated pyramidal, and immature, putatively migrating neurons showed resting membrane potentials between -44 and -50 mV and TTX-sensitive action potentials. Immature pyramidal neurons with the smallest surface area ( approximately 1,600 microm(2)) revealed the largest input resistance ( approximately 1.8 GOmega), and subplate cells with the largest surface area ( approximately 6,200 microm(2)) showed an input resistance of approximately 1 GOmega. Ontogenetically older Cajal-Retzius and subplate cells revealed shorter and larger action potentials compared to bifurcated and immature pyramidal neurons. Whereas Cajal-Retzius and subplate cells responded to injection of depolarizing current pulses with a repetitive nonadapting and fast spiking firing pattern, immature pyramidal neurons showed strong adaptation. Subplate cells revealed the fastest action potentials, largest sodium current amplitude (-714 pA), and highest sodium current density (-38 microA/cm(2)), enabling these cells to transmit afferent activity faithfully to postsynaptic neurons. Whereas all cell types expressed a high-voltage-activated (HVA) calcium current, none of them showed a significant low-voltage-activated calcium current. The largest peak (-25.5 microA/cm(2)) and steady-state (-7.6 microA/cm(2)) HVA calcium current density could be observed in immature presumed migrating neurons. In contrast, Cajal-Retzius and subplate neurons showed a significantly lower peak (-4.9 microA/cm(2)) and steady-state (<-3.3 microA/cm(2)) HVA calcium current density. Whereas a large HVA calcium current may promote neuronal migration of immature neurons, low intracellular calcium levels may provoke apoptosis in Cajal-Retzius and subplate cells.

Characterization of a hyperpolarization-activated inward current in Cajal-Retzius cells in rat neonatal neocortex.

Cajal-Retzius cells are among the first neurons appearing during corticogenesis and play an important role in the establishment of cortical lamination. To characterize the hyperpolarization-activated inward current (I(h)) and to investigate whether I(h) contributes to the relatively positive resting membrane potential (RMP) of these cells, we analyzed the properties of I(h) in visually identified Cajal-Retzius cells in cortical slices from neonatal rats using the whole cell patch-clamp technique. Membrane hyperpolarization to -90 mV activated a prominent inward current that was inhibited by 1 mM Cs(+) and was insensitive to 1 mM Ba(2+). The activation time constant for I(h) was strongly voltage dependent. In Na(+)-free solution, I(h) was reduced, indicating a contribution of Na(+). An analysis of the tail currents revealed a reversal potential of -45.2 mV, corresponding to a permeability coefficient (pNa(+)/pK(+)) of 0. 13. While an increase in the extracellular K(+) concentration ([K(+)](e)) enhances I(h), it was reduced by a [K(+)](e) decrease. This [K(+)](e) dependence could not be explained by an effect on the electromotive force on K(+) but suggested an additional extracellular binding site for K(+) with an apparent dissociation constant of 7.2 mM. Complete Cl(-) substitution by Br(-), I(-), or NO(3)(-) had no significant effect on I(h), whereas a complete Cl(-) substitution by the organic compounds methylsulfate, isethionate, or gluconate reduced I(h) by approximately 40%. The I(h) reduction observed in gluconate could be abolished by the addition of Cl(-). The analysis of the [Cl(-)](e) dependence of I(h) revealed a dissociation constant of 9.8 mM and a Hill-coefficient of 2.5, while the assumption of a gluconate-dependent I(h) reduction required an unreasonably high Hill-coefficient >20. An internal perfusion with the lidocaine derivative lidocaine N-ethyl bromide blocks I(h) within 1 min after establishment of the whole cell configuration. An inhibition of I(h) by 1 mM Cs(+) was without an effect on RMP, action potential amplitude, threshold, width, or afterhyperpolarization. We conclude from these results that Cajal-Retzius cells express a prominent I(h) with characteristic properties that does not contribute to the RMP.

Mechanism of the kainate-induced intracellular acidification in leech Retzius neurons.

We examined the effect of the glutamatergic agonist kainate on the membrane potential, the intracellular Na+ concentration ([Na+]i), the intracellular-free Ca2+ concentration, and on the intracellular pH of Retzius neurons of the medicinal leech, Hirudo medicinalis, in order to investigate the mechanism responsible for the intracellular acidification caused by glutamatergic stimulation. The recordings were made with Na+- and pH-sensitive microelectrodes and iontophoretically injected Fura-2. Bath application of kainate evoked a marked membrane depolarization, a [Na+]i increase, and an intracellular acidification. The intracellular acidification was unaffected by reversal of the electromotive force for H+, suggesting that an influx of H+ from the interstitial space does not contribute to the acidification. While the Ca2+ channel blockers La3+ and Co2+ had no effect on the kainate-induced intracellular acidification, suggesting that a Ca2+ influx via voltage-dependent Ca2+ channels was not relevant, the acidification was reduced in Ca2+-free saline solution. In Na+-free saline solution the kainate-induced intracellular acidification was absent, suggesting the involvement of Na+ influx in generating the acidification. When injected iontophoretically Na+ induced an intracellular acidification but Li+, K+, Rb+ or Cs+ did not. Furthermore, a [Na+]i increase induced by blocking the Na+/K+ pump also led to an intracellular acidification. We conclude that the [Na+]i increase is the crucial event underlying the kainate-induced intracellular acidification. Possible mechanisms linking the [Na+]i increase to the intracellular acidification are discussed.