Heiko J. Luhmann

Long-term changes of ionotropic glutamate and GABA receptors after unilateral permanent focal cerebral ischemia in the mouse brain

Long-term hyperexcitability was found after unilateral, permanent middle cerebral artery occlusion in exofocal neocortical areas of the adult mouse [Mittmann et al. (1998) Neuroscience 85, 15-27]. The aim of the present study was to test the hypothesis in an identical paradigm of ischemia. whether alterations in the densities of both excitatory and inhibitory amino acid receptors may underlie these pathophysiological changes. Alterations in densities of [3H]dizocilpine, [3H]D,L-amino-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, [3H]kainate and [3H]muscimol binding sites were demonstrated with quantitative in vitro receptor autoradiography. All binding sites were severely reduced in the core of the ischemic lesion. A completely different reaction was found in the exofocal, histologically inconspicuous parts of the somatosensory cortex and the more remote neocortical areas of both hemispheres. The [3H]muscimol binding sites were significantly reduced four weeks after ischemia in the motor cortex, hindlimb representation area and exofocal parts of the primary and secondary somatosensory cortices of both hemispheres. The focus of the reduction in [3H]muscimol binding sites was found in lower layer V and upper layer VI. Contrastingly, the densities of [3H]dizocilpine binding sites were found to be increased in these areas, whereas those of [3H]D,L-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and [3H]kainate binding sites did not show significant changes. The [3H]dizocilpine binding site density increased predominantly in layers III and IV. All binding sites were also reduced in the retrogradely reacting, gliotic part of the ipsilateral ventroposterior thalamic nucleus, whereas the [3H]D,L-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid binding sites were increased in the surround of the ipsilateral nucleus and no changes in binding sites were seen in the whole contralateral nucleus. We conclude that permanent local ischemia leads to a long-term and widespread impairment of the normal balance between binding sites of excitatory and inhibitory neurotransmitter receptors in neocortical areas far away from the focus of the post-ischemic tissue damage. The imbalance comprises an up-regulation of the [3H]dizocilpine binding sites in the ion channels of N-methyl-D-aspartate receptors and a down-regulation of [3H]muscimol binding sites of the GABA(A) receptors in the ipsi- and contralateral neocortex. These changes at the receptor level explain the previously observed hyperexcitability with the appearance of epileptiform field potentials and the long duration of excitatory postsynaptic potentials four weeks after ischemia.

ISCHEMIA AND LESION INDUCED IMBALANCES IN CORTICAL FUNCTION

Cortical structures are often critically affected by ischemic and traumatic lesions which may cause transient or permanent functional disturbances. These disorders consist of changes in the membrane properties of single cells and alterations in synaptic network interactions within and between cortical areas including large-scale reorganizations in the representation of the peripheral input. Prominent functional modifications consisting of massive membrane depolarizations, suppression of intracortical inhibitory synaptic mechanisms and enhancement of excitatory synaptic transmission can be observed within a few minutes following the onset of cortical hypoxia or ischemia and probably represent the trigger signals for the induction of neuronal hyperexcitability, irreversible cellular dysfunction and cell death. Pharmacological manipulation of these early events may therefore be the most effective approach to control ischemia and lesion induced disturbances and to attenuate long-term neurological deficits. The complexity of secondary structural and functional alterations in cortical and subcortical structures demands an early and powerful intervention before neuronal damage expands to intact regions. The unsatisfactory clinical experience with calcium and N-methyl-D-aspartate antagonists suggests that this result might be achieved with compounds that show a broad spectrum of actions at different ligand-activated receptors, voltage-dependent channels and that also act at the vascular system. Whether the same therapy strategies developed for the treatment of ischemic injury in the adult brain may be applied for the immature cortex is questionable, since young cortical networks with a high degree of synaptic plasticity reveal a different response pattern to hypoxic and ischemic insults. Age-dependent molecular biological, morphological and physiological parameters contribute to an enhanced susceptibility of the immature brain to these noxae during early ontogenesis and have to be investigated in more detail for the development of adequate clinical therapy.

Characterization of neuronal migration disorders in neocortical structures: quantitative receptor autoradiography of ionotropic glutamate, GABA A and GABA B receptors

Epileptiform activity was previously described [ Luhmann et al. (1998 ) Eur.J. Neurosci., 10, 3085–3094] in the neocortex of the adult rat following freeze lesioning of the newborn neocortex. After a survival time of 3 months, a small area of dysplastic cortex surrounded by histologically normal (exofocal) neocortex was observed. The dysplastic cortex is characterized by the formation of a small sulcus and a three‐ to four‐layered architecture. Two questions are addressed here: (i) is the hyperexcitability associated with changes in binding to major excitatory and inhibitory transmitter receptors in the dysplastic cortex?; and (ii) do such changes also occur in the exofocal cortex?

Long-term cellular dysfunction after focal cerebral ischemia: in vitro analyses.

The long-term (< or = six months) functional consequences of permanent middle cerebral artery occlusion were studied with in vitro extra- and intracellular recording techniques in adult mouse neocortical slices. After survival times of one to three days, 28 days and six months, intracellular recordings from layers II/III pyramidal cells in the vicinity of the infarct did not reveal any statistically significant changes in the intrinsic membrane properties when compared to age-matched control animals. However, a pronounced hyperexcitability could be observed upon orthodromic synaptic stimulation in neocortical slices obtained from mice 28 days after induction of ischemia. Low-intensity electrical stimulation of the afferents elicited particularly in this group epileptiform extracellular field potential responses and intracellular excitatory postsynaptic potentials, that were longer in duration as compared to the controls. When the N-methyl-D-aspartate receptor-mediated excitatory postsynaptic potential was pharmacologically isolated in a bathing solution containing 0.1 mM Mg2+ and 10 microM 6-cyano-7-nitroquinoxaline-2,3-dione, the synaptic responses were longer and larger in the ischemic cortex as compared to the controls. Higher stimulus intensities evoked in normal medium a biphasic inhibitory postsynaptic potential, that contained in the 28 days post-ischemia group a prominent amino-phosphonovaleric acid-sensitive component, indicating a strong concurrent activation of a N-methyl-D-aspartate receptor-mediated excitatory postsynaptic potential. This pronounced co-activation could only be observed in the 28 days ischemic group, and neither after one to three days or six months post-ischemia nor in the controls. The quantitative analysis of the efficiency of stimulus- evoked inhibitory postsynaptic potentials recorded in amino-phosphono-valeric acid revealed a reduction of GABA-mediated inhibition in ischemic cortex. Although this reduction in intracortical inhibition may already contribute to an augmentation of N-methyl-D-aspartate receptor-mediated excitation, our results do also indicate that the function of N-methyl-D-aspartate receptors is transiently enhanced in the ischemic cortex. This transient hyperexcitability does not only cause cellular dysfunction in the vicinity of the infarct, but may also contribute to neuronal damage due to excitotoxicity.

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 (∼1,600 μm2) revealed the largest input resistance (∼1.8 GΩ), and subplate cells with the largest surface area (∼6,200 μm2) showed an input resistance of ∼1 GΩ. 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 μA/cm2), 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 μA/cm2) and steady‐state (–7.6 μA/cm2) 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 μA/cm2) and steady‐state (<−3.3 μA/cm2) 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. J. Neurosci. Res. 62:574–584, 2000.

Generation and propagation of 4-AP-induced epileptiform activity in neonatal intact limbic structures in vitro.

We examined the generation, propagation and pharmacology of 4‐aminopyridine (4‐AP)‐induced epileptiform activity (EA) in the intact interconnected limbic structure of the newborn (P0–7) rat in vitro. Whole‐cell recordings of CA3 pyramidal cells and multisite field potential recordings in CA3, CA1, dentate gyrus, and lateral and medial entorhinal cortex revealed 4‐AP‐induced EA as early as P0–1. At this age, EA was initiated in the CA3 region and propagated to CA1, but not to the entorhinal cortex. Starting from P3–4, EA propagated from CA3 to the entorhinal cortex. Along the CA3 septo‐temporal axis, EA arose predominantly from the septal pole and spread towards the temporal site. Whereas the onset of 4‐AP‐induced EA decreased with age from 21.2u2003±u20031.6u2003min at P0–1 to 4.7u2003±u20030.63u2003min at P6–7, the seizure duration increased in the same age groups from 98u2003±u200314u2003s to 269.4u2003±u200385.9u2003s, respectively. The EA was blocked by 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) but not by dl‐2‐amino‐5‐phosphonovaleric acid (APV), (+)‐MK‐801 hydrogen maleate (MK‐801) or (±)‐alpha‐methyl‐4‐carboxyphenylglycine (MCPG), suggesting that they were mediated by α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA)/kainate receptor activation. We conclude that: (i) the septal pole of the hippocampal CA3 region plays a central role in the generation of EA in the neonatal limbic system; and (ii) AMPA/kainate receptor‐mediated EA can be generated in CA3 already at birth. Therefore, the recurrent collateral synapses and circuits required for the generation of EA are developed earlier than previously suggested on the basis of studies on hippocampal slices.

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.

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 136u2003mm Cl–. The sPSCs occurred at a low frequency (0.07u2003±u20030.07u2003Hz, nu2003=u200342 cells), had an average amplitude of 24.3u2003±u200312.4u2003pA (nu2003=u2003415 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 (100u2003µm), while the glutamatergic antagonists (±)‐2‐amino‐5‐phosphonopentatonic acid (APV, 30u2003µm) and 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX, 10u2003µm), as well as tetrodotoxin (1–2u2003µm), 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 GABAA receptor‐mediated spontaneous synaptic input, which may contribute to the refinement of cortical circuits.

Laminar characteristics of functional connectivity in rat barrel cortex revealed by stimulation with caged-glutamate

In rodent somatosensory (barrel) cortex input is processed by whisker-related columns before the integrated output is fed into behaviorally-relevant circuits. The layer-specific activation patterns of the rat barrel cortex were examined with a set-up for scanning functional connectivity in brain slices. Flash-induced release of caged-glutamate at a large number of stimulation sites was used in combination with simultaneous field potential recordings from layers II to VI with five electrodes. The field potentials revealed striking differences between the cortical layers. Glutamate-release in layer IV and lower layer III was most effective in evoking excitation in all other cortical layers, whereas field potentials recorded from layer IV itself were caused by stimulation of a very restricted columnar zone only. Field potentials in layers II and III were strongly driven by stimulation in layer IV and less consistently and much weaker by layer V. Layer V was the only lamina capable of responding to stimulation of all other cortical layers, thus displaying the largest input maps. Layer VI possessed functional connectivity intrinsically and with layer V. These data lead us to suggest that thalamic input may be boosted by its main target layer IV to start a sequence of excitation in layer IV, passing to the supragranular layers and finally reaching the infragranular layers. This sequence is likely to be backed-up by other simultaneous steps of transmission including a layer IV-to-V interaction. We proposed that the increasing size of the receptive fields when sampling granular, supragranular and infragranular layers in vivo, might have its functional basis in the laminar interactions described here in an in vitro preparation.

Optical recording of spreading depression in rat neocortical slices.

A spreading depression (SD) was elicited in adult rat neocortical slices by microdrop application of high potassium and the SD propagation pattern was analyzed by recording simultaneously the extracellular DC potential and the changes in the intrinsic optical signal. The electrical SD with an average peak amplitude of 13.2+/-3.4 mV showed a good spatial and temporal correlation with the optical signal. In 79% of the slices, the SD was characterized by an initial increase of light reflectance by 2.3+/-1.6%, followed by a reflectance decrease of 0.5+/-2.4% and finally a larger and long-lasting increase by 5+/-2.4%. In the remaining slices, the SD revealed an initial decrease in light reflectance by 5.8+/-1.8% followed by an increase of 1.4+/-1.2%. In all slices, the recovery in the DC recording was faster as in the optical signal. The SD preferentially propagated within layers I-IV and could be blocked in most experiments by a vertical incision through upper layers or by local glutamate receptor blockade following microdrop application of kynurenic acid in layers II-III. The SD could be also blocked by bath application of kynurenic acid, MK-801 and octanol, but not by the more specific gap junction blocker carbenoxolone. Our results indicate that the high density of dendritic processes and glutamate receptors in layers II-IV promote the horizontal spread of the SD in these cortical layers and that gap junctions are not required for the propagation of SD in neocortical slices.