Neocortical Slice Preparation Research Articles

Investigation into the effect of the general anaesthetic etomidate on local neuronal synchrony in the mouse neocortical slice

How general anaesthetic drugs cause unconsciousness is a topic of ongoing clinical and scientific interest. It is becoming increasingly apparent that they disrupt cortical information processing, but the effects appear to depend on the spatial scale under investigation. In this study we investigated whether the intravenous anaesthetic etomidate synchronises neuronal activity on a sub-millimetre scale in mouse neocortical slices. In slices generating no-magnesium seizure-like event (SLE) field activity, we analysed the morphology of field potential activity recorded with 50µm extracellular electrodes. The analysis was based on the understanding that the amplitude and sheerness of field potential oscillations correlates with the synchrony of the underlying neural activity. When recorded from the region of the slice initiating SLE activity, etomidate consistently increased both population event amplitude (median(range) 85(24–350) to 101(30–427) µV) and slope 16.6(1.5–106.2) to 20.2(1.7–111.1) µV/ms (p=0.016 and p=0.0013, respectively). The results are consistent with an increase in neuronal synchrony within the receptive field of the recording electrode, estimated to be a circle diameter of 300µm. In conclusion, the neocortical slice preparation supports in vivo data showing that general anaesthetics increase neuronal synchrony on a local scale and provides an ideal model for investigating underlying mechanisms.

GABA Regulates the Multidirectional Tangential Migration of GABAergic Interneurons in Living Neonatal Mice

Cortical GABAergic interneurons originate from ganglionic eminences and tangentially migrate into the cortical plate at early developmental stages. To elucidate the characteristics of this migration of GABAergic interneurons in living animals, we established an experimental design specialized for in vivo time-lapse imaging of the neocortex of neonate mice with two-photon laser-scanning microscopy. In vesicular GABA/glycine transporter (VGAT)-Venus transgenic mice from birth (P0) through P3, we observed multidirectional tangential migration of genetically-defined GABAergic interneurons in the neocortical marginal zone. The properties of this migration, such as the motility rate (distance/hr), the direction moved, and the proportion of migrating neurons to stationary neurons, did not change through P0 to P3, although the density of GABAergic neurons at the marginal zone decreased with age. Thus, the characteristics of the tangential motility of individual GABAergic neurons remained constant in development. Pharmacological block of GABAA receptors and of the Na+-K+-Cl− cotransporters, and chelating intracellular Ca2+, all significantly reduced the motility rate in vivo. The motility rate and GABA content within the cortex of neonatal VGAT-Venus transgenic mice were significantly greater than those of GAD67-GFP knock-in mice, suggesting that extracellular GABA concentration could facilitate the multidirectional tangential migration. Indeed, diazepam applied to GAD67-GFP mice increased the motility rate substantially. In an in vitro neocortical slice preparation, we confirmed that GABA induced a NKCC sensitive depolarization of GABAergic interneurons in VGAT-Venus mice at P0-P3. Thus, activation of GABAAR by ambient GABA depolarizes GABAergic interneurons, leading to an acceleration of their multidirectional motility in vivo.

Connexin36 Gap Junction Blockade Is Ineffective at Reducing Seizure-Like Event Activity in Neocortical Mouse Slices

Despite much research, there remains controversy over the role of gap junctions in seizure processes. Many studies report anticonvulsant effects of gap junction blockade, but contradictory results have also been reported. The aim of this study was to clarify the role of connexin36 (Cx36) gap junctions in neocortical seizures. We used the mouse neocortical slice preparation to investigate the effect of pharmacological (mefloquine) and genetic (Cx36 knockout mice (Cx36KO)) manipulation of Cx36 gap junctions on two seizure models: low-magnesium artificial cerebrospinal fluid (ACSF) and aconitine perfusion in low-magnesium ACSF. Low-magnesium- (nominally zero) and aconitine- (230 nM) induced seizure-like event (SLE) population activity was recorded extracellularly. The results were consistent in showing that neither mefloquine (25 μM) nor genetic knockdown of Cx36 expression had anticonvulsant effects on SLE activity generated by either method. These findings call into question the widely held idea that open Cx36 gap junctions promote seizure activity.

Spatio‐temporal dynamics of oscillatory network activity in the neonatal mouse cerebral cortex

We used a 60-channel microelectrode array to study in thick (600-1000 microm) somatosensory cortical slices from postnatal day (P)0-P3 mice the spatio-temporal properties of early network oscillations. We recorded local non-propagating as well as large-scale propagating spontaneous oscillatory activity. Both types of activity patterns could never be observed in neocortical slices of conventional thickness (400 microm). Local non-propagating spontaneous oscillations with an average peak frequency of 15.6 Hz, duration of 1.7 s and maximal amplitude of 66.8 microV were highly synchronized in a network of approximately 200 microm in diameter. Spontaneous oscillations of lower frequency (10.4 Hz), longer duration (23.8 s) and larger amplitude (142.9 microV) propagated with 0.11 mm/s in the horizontal direction over at least 1 mm. These propagating oscillations were also synchronized in a columnar manner, but these waves synchronized the activity in a larger neuronal network of 300-400 microm in diameter. Both types of spontaneous network activity could be blocked by the gap junction antagonist carbenoxolone. Electrical stimulation of the subplate (SP) or bath application of the cholinergic agonist carbachol also elicited propagating network oscillations, emphasizing the role of the SP and the cholinergic system in the generation of early cortical network oscillations. Our data demonstrate that a sufficiently large network in thick neocortical slice preparations is capable of generating spontaneous and evoked network oscillations, which are highly synchronized via gap junctions in 200-400-microm-wide columns. These via synchronized oscillations coupled networks may represent a self-organized functional template for the activity-dependent formation of neocortical modules during the earliest stages of development.

Field-theoretic approach to fluctuation effects in neural networks

A well-defined stochastic theory for neural activity, which permits the calculation of arbitrary statistical moments and equations governing them, is a potentially valuable tool for theoretical neuroscience. We produce such a theory by analyzing the dynamics of neural activity using field theoretic methods for nonequilibrium statistical processes. Assuming that neural network activity is Markovian, we construct the effective spike model, which describes both neural fluctuations and response. This analysis leads to a systematic expansion of corrections to mean field theory, which for the effective spike model is a simple version of the Wilson-Cowan equation. We argue that neural activity governed by this model exhibits a dynamical phase transition which is in the universality class of directed percolation. More general models (which may incorporate refractoriness) can exhibit other universality classes, such as dynamic isotropic percolation. Because of the extremely high connectivity in typical networks, it is expected that higher-order terms in the systematic expansion are small for experimentally accessible measurements, and thus, consistent with measurements in neocortical slice preparations, we expect mean field exponents for the transition. We provide a quantitative criterion for the relative magnitude of each term in the systematic expansion, analogous to the Ginsburg criterion. Experimental identification of dynamic universality classes in vivo is an outstanding and important question for neuroscience.

How Independent Are Initiation, Propagation, and Termination of Epileptiform Activity?

Initiation, Propagation, and Termination of Epileptiform Activity in Rodent Neocortex In Vitro Involve Distinct Mechanisms Pinto DJ, Patrick SL, Huang WC, Connors BW J Neurosci 2005;25(36):8131–8140 Waves of epileptiform activity in neocortex have three phenomenological stages: initiation, propagation, and termination. We use a well studied model of epileptiform activity in vitro to investigate directly the hypothesis that each stage is governed by an independent mechanism within the underlying cortical circuit. Using the partially disinhibited neocortical slice preparation, activity is induced and modulated using neurotransmitter receptor antagonists and is measured using both intracellular recordings and a linear array of extracellular electrodes. We find that initiation depends on both synaptic excitation and inhibition and entails a slow process of recruitment at discrete spatial locations within cortical layer 5 but not layer 2 or 3. Propagation depends on synaptic excitation but not inhibition and is a fast process that involves neurons across the spatial extent of the slice and in all cortical layers. Termination is modulated by synaptic excitation and inhibition. In space, termination occurs reliably at discrete locations. In time, termination is characterized by a strong depolarizing shift (block) and recovery of neurons in all cortical layers. These results suggest that the phenomenological stages of epileptiform events correspond to distinct mechanistic stages.

Initiation, Propagation, and Termination of Epileptiform Activity in Rodent NeocortexIn VitroInvolve Distinct Mechanisms

Waves of epileptiform activity in neocortex have three phenomenological stages: initiation, propagation, and termination. We use a well studied model of epileptiform activity in vitro to investigate directly the hypothesis that each stage is governed by an independent mechanism within the underlying cortical circuit. Using the partially disinhibited neocortical slice preparation, activity is induced and modulated using neurotransmitter receptor antagonists and is measured using both intracellular recordings and a linear array of extracellular electrodes. We find that initiation depends on both synaptic excitation and inhibition and entails a slow process of recruitment at discrete spatial locations within cortical layer 5 but not layer 2/3. Propagation depends on synaptic excitation but not inhibition and is a fast process that involves neurons across the spatial extent of the slice and in all cortical layers. Termination is modulated by synaptic excitation and inhibition. In space, termination occurs reliably at discrete locations. In time, termination is characterized by a strong depolarizing shift (block) and recovery of neurons in all cortical layers. These results suggest that the phenomenological stages of epileptiform events correspond to distinct mechanistic stages.

Effect of levetiracetam on epileptiform discharges in human neocortical slices.

The anticonvulsant effects of the novel antiepileptic drug (AED) levetiracetam (LEV) were tested in neocortical slice preparations from 23 patients who underwent surgery for the treatment of refractory epilepsy. Slices were used to evaluate the effects of LEV on two different models of epilepsy: low-Mg2+-induced untriggered and bicuculline-evoked stimulus-triggered epileptiform burst discharges and spontaneously appearing rhythmic sharp waves. LEV (0.1-1 mM) did not influence spontaneously appearing rhythmic sharp waves or Mg2+-free aCSF-induced epileptiform field potentials. LEV affected neither the amplitudes or duration nor the repetition rates of burst discharges in these epilepsy models. However, LEV (100-500 microM) significantly suppressed the ictal-like discharges elicited by the gamma-aminobutyric acid subtype A (GABAA)-receptor antagonist bicuculline. A marked reduction of the amplitude and duration of bicuculline-evoked field response in the presence of LEV was observed. The results indicate the potential for LEV to inhibit epileptiform burst discharges in human neocortical tissue, which is consistent with its effects in animal models of epilepsy. These results also support the seizure reduction observed in clinical trials and support that this may, in part, be related to the ability of LEV to inhibit epileptiform discharges.

Gabapentin and vigabatrin increase GABA in the human neocortical slice

The effects of antiepileptic drugs, gabapentin and vigabatrin, on gamma-aminobutyric acid (GABA) concentrations were studied in human ( n=14) and rat ( n=6) neocortical slice preparations. In this study, neocortical slices were incubated with gabapentin, vigabatrin or no drugs for 3 h in an oxygenated environment. Proton magnetic resonance spectroscopy (MRS) of perchloric acid (PCA) extracts was used to measure GABA concentrations. Vigabatrin increased cellular GABA concentrations in both human and rat neocortical slices by 62% ( P<0.001) and 88% ( P<0.03), respectively. Gabapentin significantly increased GABA concentrations by 13% ( P<0.02) in human neocortical slices made from tissue resected during epilepsy surgery. However, in the rat neocortical slice exposed to the same conditions as the human tissue, gabapentin did not increase GABA significantly. These results confirm our MRS studies in vivo that gabapentin increases GABA levels in epileptic patients, but has minimal or no effect in a healthy rodent model. Caution must be used in extrapolating negative results obtained in rodent models to the human condition.

Effects of retigabine on rhythmic synchronous activity of human neocortical slices

The antiepileptic effects of the novel antiepileptic drug retigabine (D-23129) [ N-(2-amino-4-(4-flurobenzylamino)phenyl) carbamid acid ethyl ester] were tested in neocortical slice preparations ( n=23) from 17 patients (age, 3–42 years) who underwent surgery for the treatment of intractable epilepsy. Epileptiform events consisted of spontaneously occurring rhythmic sharp waves, as well as of epileptiform field potentials (EFP) elicited by superfusion with Mg 2+-free solution without or with addition of 10 μmol/l bicuculline. (1) Spontaneous rhythmic sharp waves ( n=6), with retigabine application, the repetition rate was decreased down to 12–47% of initial value (10 μmol/l, n=3) after 180 min or suppressed completely within 12 min (50 μmol/l, n=3). (2) Low Mg 2+ EFP ( n=9), with retigabine application, the repetition rate was decreased down to 50 and 65% of initial value (10 μmol/l; n=2) after 180 min or suppressed completely after 9–55 min (10, 50 and 100 μmol/l; n=2 in each case). In one slice only a transient reduction of the repetition rate was seen with 10 μmol/l retigabine. (3) Low Mg 2+ EFP with addition of bicuculline ( n=8), with retigabine application, the repetition rate was decreased down to 12–55% of initial value (10 μmol/l; n=4) after 180 min or suppressed completely after 6–30 min (50 and 100 μmol/l; n=2 in each case). The depressive effect of retigabine was reversible in all but one slice. The results show a clear antiepileptic effect of retigabine in human neocortical slices on spontaneously occurring rhythmic sharp waves and different types of induced seizure activity.

Effects of nifedipine on rhythmic synchronous activity of human neocortical slices

The antiepileptic effect of the dihydropyridine calcium channel blocker nifedipine was tested in neocortical slice preparations ( n=27) from patients ranging in age from four to 46 years (mean=25) who underwent surgery for the treatment of intractable epilepsy. Epileptiform events consisted of spontaneously occurring rhythmic sharp waves as well as of untriggered epileptiform field potentials induced by omission of Mg 2+ from the superfusate, or epileptiform field potentials elicited by application of bicuculline and triggered by single electrical stimuli. (1) Spontaneous rhythmic sharp waves ( n=6): with nifedipine (40 μmol/l), the repetition rate was decreased down to 30% of initial value, whereas the area under the field potential remained nearly unchanged. (2) Untriggered low Mg 2+ epileptiform field potentials ( n=6): with nifedipine (40 μmol/l) the area under the field potentials was reduced while the action on the repetition rate was ambiguous. (3) Triggered bicuculline epileptiform field potentials ( n=15): with nifedipine (40 μmol/l; n=4), no antiepileptic effect was found. There was, however, a marked increase in the area under the epileptiform field potentials. The area under the field potentials was reduced only at a dosage of 60 μmol/l ( n=11). This effect was stronger when nifedipine was applied with a K + concentration raised from 4 to 8 mmol/l. The results show that the calcium channel blocker nifedipine is able to reduce differential epileptiform discharges in human neocortical tissue. These observations are in line with previous findings, suggesting that calcium flux into neurons is involved in epileptogenesis. The present results therefore support the idea that some organic calcium antagonists may be useful in human epilepsy therapy, although the etiology of epileptic seizures seems to be a critical factor for the efficacy of the drug.

Myelination defects and neuronal hyperexcitability in the neocortex of connexin 32-deficient mice.

Morphological and electrophysiological studies were performed on neocortices of adult Connexin 32 (Cx32)-deficient mice and wild-type mice to investigate the consequences of a lack of the gap junction subunit Cx32 on neocortical structure and function. Morphometrical analysis revealed a reduced volume fraction of myelin within the neuropil and a decreased thickness of the axonal myelin sheaths in the neocortex of Cx32-deficient mice. Intracellular recordings from neurons in neocortical slice preparations provided evidence for an increased membrane input resistance in neurons of Cx32-null mutant mice as compared to neurons of wild-type mice. Consequently, neurons of Cx32-deficient mice displayed an enhanced intrinsic excitability. In addition, approximately 50% of the neurons investigated in slices of Cx32-deficient mice responded to afferent stimulation with delayed and large glutamatergic excitatory postsynaptic potentials resembling paroxysmal depolarizations. GABAergic inhibition sufficient to efficiently control synaptic excitability was virtually absent in these cells. The changes in intrinsic membrane properties observed in neurons of Cx32-null mutant mice were independent of the alterations in synaptic function, since increased membrane resistances were observed also in neurons with normal synaptic response pattern. Thus, in the neocortex, lack of Cx32 correlates with myelination defects, alterations in intrinsic membrane properties and dysfunction of inhibitory synaptic transmission.

Electrophysiological characterization of CGP6873OA a N-methyl-D-aspartate antagonist acting at the strychnine-insensitive glycine site

1. 1. Electrophysiological experiments were performed in vitro and vivo. Voltage clamp recordings were done in Xenopus oocytes. Extracellular recordings were done in vitro in the neocortical slice and in the CA1 region of the hippocampal slice and vivo in the CA1 region of the hippocampus of the anaesthetized rat. 2. 2. In oocytes expressing either the human NMDAR1 a/2 a or 1 a/2 b subunit combinations, CGP68730A [sodium (−)-9-bromo-2,3,6,7-tetrahydro-5,6-dioxo-5H-pyrazino[1,2,3-de]-1,4-benzothiazine-3-acetic acid] antagonized L-glutamate / glycine induced currents with calculated IC 50 S of 20.5 and 81.6 nM, respectively. 3. 3. In vitro, CGP68730A was tested on NMDA induced depolarizations in the neocortical slice preparation and on epileptiform activity in hippocampal slices bathed in Mg 2+-free-medium, which is known to be NMDA mediated. In both in vitro models CGP68730A exhibited antagonistic effects on the NMDA receptor mediated responses. 4. 4. In vivo CGP68730A was tested on NMDA induced excitations in the CA1 region. CGP68730A abolished NMDA induced excitations when applied microiontophoretically. However, only weak effects on NMDA induced excitation were observed after systemic administration at 100 mg/kg i.V. These results indicate that CGP68730A has poor central nervous system bioavailability. 5. 5. In oocytes, an increase in the glycine concentration from the EC 80 to the EC 95–99 shifted the inhibition curves for CGP68730A to the right. Furthermore, in neocortical slices and in anaesthetized rats CGP68730A inhibited NMDA mediated depolarizations, and this effect could be reversed by the addition of the glycine mimetic D-serine. This indicates that these effects of CGP68730A are mediated by an action on the strychnine-insensitive glycine site. 6. 6. Selectivity tests in oocytes and in the neocortical slice preparation, using NMDA, kainate and AMPA showed that CGP68730A was selective in antagonizing NMDA receptor mediated responses. In oocytes, the compound was about 1000 times less potent on the rat GluR3 and the human GluR6 receptors than on the human NMDAR1 a/2 a subunit combination. In the neocortical slice preparation CGP68730A had no effects on AMPA or kainate induced depolarizations at concentrations of 3 and 10 μM. At 30 μM CGP68730A reduced the effects of each of the three agonists tested. 7. 7. Thus, CGP68730A seems to be a selective antagonist at the strychnine-insensitive glycine coagonist site of the NMDA receptor. However, the compound showed no obvious central NMDA antagonistic effects following intravenous application.

Organotypic slice cultures for analysis of proliferation, cell death, and migration in the embryonic neocortex

Dynamic cellular interactions during neocortical neurogenesis are critical for proper cortical development, providing both trophic and tropic support. Although cell proliferation and programmed cell death have been characterized in dissociated primary cell cultures, many in vivo processes during cortical neurogenesis depend on cell–cell interactions and therefore on the three-dimensional environment of the proliferating neuroblasts and their progeny. Here we describe a murine organotypic neocortical slice preparation that retains major morphological and functional in vivo characteristics of the developing neocortex and is viable (exhibits very low levels of cell death) for up to three days. Moreover, this slice preparation is amenable to direct experimental manipulation of potential diffusible regulators of neurogenesis. Using a variety of biochemical and physiological methods including time-lapse and quantitative confocal microscopy, we demonstrate that this system can be used effectively to investigate cellular mechanisms important for brain growth and maturation, including neurogenesis, apoptosis, and neuronal migration.

Contribution of L-type calcium channels to epileptiform activity in hippocampal and neocortical slices of guinea-pigs

The aim of the present investigation was to compare the antiepileptic efficacy of the specific L-type calcium channel blocker nifedipine in hippocampal and neocortical slice preparations in the Mg 2+-free model of epilepsy. The main findings were as follows. (1) In hippocampal slices, in general, nifedipine (20–80 μmol/l) exerted a suppressive effect both on repetition rate and on area under epileptiform field potentials. This effect was clearly dose dependent. In the majority of cases, this suppression was preceded by an increase, which was transient in nature. Only in the lowest concentration (20 μmol/l) used, in normal K +, instead of a depression, a persistent increase occurred. (2) In neocortical slices, in the majority of experiments, nifedipine (20–80 μmol/l) showed a depressive action only on the area under the epileptiform field potentials. The depressive effect of nifedipine on the area was dose dependent, although to a lesser extent than in the hippocampus. In nearly half of the slices this suppression was preceded by a transient increase. By contrast, the repetition rate of epileptiform field potentials increased transiently in about 20% of the slices followed by a decrease. In the remaining 80% of the slices the repetition rate increased persistently. (3) An elevation of the K + concentration accentuated the depressive actions of nifedipine only in the hippocampus. In contrast to elevated K +, in both the hippocampus and the neocortex, epileptiform field potentials were not suppressed in all experiments in normal K +. (4) The reversibility of the depressive effects of nifedipine was differential in the two tissue types. In the hippocampus, after suppression of epileptiform field potentials they reappeared in the overwhelming majority of slices. In the neocortex, this was the case in only one experiment. These findings may indicate the existence of L-type calcium channels with a differential functional significance for epileptogenesis and/or the existence of different forms of L-type channels in hippocampal and neocortical tissue. As a whole, the differential effects of L-type calcium channel blockade in the hippocampus and neocortex point to differences in the network properties of the two tissue types.

Different Ca2+ source for slow AHP in completely adapting and repetitive firing pyramidal neurons.

Intracellular recordings in an in vitro neocortical slice preparation from immature rats were used to investigate the Ca2 source for slow afterhyperpolarization (sAHP) generation in pyramidal neurons that exhibit complete spike frequency adaptation (CA neurons). In pyramidal neurons that maintain repetitive firing for long periods of time (RF neurons), N-, P- and Q-type Ca2+ channels supply Ca2+ for sAHP generation. In CA neurons, the sAHP was reduced by only 50% by the combination of antagonists for these Ca2+ channel types and L-type channels. Ryanodine and dantrolene, blockers of Ca2(+)-induced Ca2+ release, reduced the sAHP by approximately 45% in CA neurons, but caused no reduction of the sAHP in RF neurons. Dantrolene application caused CA neurons to fire throughout a 1s suprathreshold current injection (as do RF neurons).

3H]CGP 61594, the first photoaffinity ligand for the glycine site of NMDA receptors

Activation of NMDA receptors requires the presence of glycine as a coagonist which binds to a site that is allosterically linked to the glutamate binding site. To identify the protein constituents of the glycine binding site in situ the photoaffinity label [ 3H]CGP 61594 was synthesized. In reversible binding assays using crude rat brain membranes, [ 3H]CGP 61594 labeled with high affinity ( K D=23 nM) the glycine site of the NMDA receptor. This was evident from the Scatchard analysis, the displacing potencies of various glycine site ligands and the allosteric modulation of [ 3H]CGP 61594 binding by ligands of the glutamate and polyamine sites. Electrophysiological experiments in a neocortical slice preparation identified CGP 61594 as a glycine antagonist. Upon UV-irradiation, a protein band of 115 kDa was specifically photolabeled by [ 3H]CGP 61594 in brain membrane preparations. The photolabeled protein was identified as the NR1 subunit of the NMDA receptor by NR1 subunit-specific immunoaffinity chromatography. Thus, [ 3H]CGP 61594 is the first photoaffinity label for the glycine site of NMDA receptors. It will serve as a tool for the identification of structural elements that are involved in the formation of the glycine binding domain of NMDA receptors in situ and will thereby complement the mutational analysis of recombinant receptors.

Reevaluation of ACEA 1021 as an antagonist at the strychnine-insensitive glycine site of the N-methyl- d-aspartate receptor

Electrophysiological experiments were performed in vitro and in vivo to characterize the inhibitory effects of 6,7-dichloro-5-nitro-1,4-dihydro-2,3-quinoxalinedione (ACEA 1021; licostinel) on rat brain glutamate receptors. In vitro, ACEA 1021 was tested on N-methyl- d-aspartate (NMDA)-induced depolarizations in the neocortical slice preparation and on epileptiform activity in Mg 2+-free hippocampal slices, which is known to be NMDA receptor mediated. In both in vitro models, ACEA 1021 exhibited antagonistic effects on the NMDA receptor-mediated responses. Selectivity tests in the neocortical slice preparation, using NMDA, kainate and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) showed that 10 μM ACEA 1021 reduced NMDA and kainate responses to 27.9 and 79.9% of the control value, respectively, whereas responses to AMPA were increased by 2.4% above the control value, thus showing that at this concentration ACEA 1021 acts preferentially at NMDA receptors. However, at 30 μM, all the NMDA-, AMPA- and kainate-induced responses were reduced. In vivo, ACEA 1021 was tested on NMDA-induced excitation in the CA1 region. After systemic administration of ACEA 1021, central effects were observed at 10 mg/kg i.v. in the CA1 region. These results indicate that ACEA 1021 is centrally active and inhibits NMDA receptor-mediated responses. Interestingly, selectivity tests in the CA1 region did not show clear differences in the action of ACEA 1021 on NMDA- and AMPA-induced excitations. Furthermore, ACh-induced excitations were also reduced. Thus, at low concentrations, ACEA 1021 seems to be a selective antagonist at the strychnine-insensitive glycine site of the NMDA receptor. However, at 30 μM in vitro and at 10 mg/kg in vivo, non-NMDA receptor-mediated actions of ACEA 1021 are observed. Our results suggest that these additional effects of ACEA 1021 may contribute to its anticonvulsive properties in mice as well as to its neuroprotective properties in animal models of cerebral ischemia.

Strychnine-induced epileptiform activity in hippocampal and neocortical slice preparations: suppression by the organic calcium antagonists verapamil and flunarizine

Alongside GABA, glycine is the major inhibitory transmitter in the central nervous system. Application of the glycine receptor blocker strychnine is known to evoke epileptiform phenomena. The present paper addresses the question whether postsynaptic calcium currents through L-type channels contribute to strychnine-induced epileptiform field potentials (EFP). To test for this, the antiepileptic effect of the specific L-type calcium channel blocker, verapamil, in hippocampal and neocortical slices was investigated. In parallel with this, the antiepileptic efficacy of the unspecific calcium channel modulator, flunarizine, was tested with respect to pharmacotherapy of epilepsies. In both preparations, the L-type calcium channel blocker, verapamil, was able to suppress EFP. In neocortical slices, EFP were blocked in all experiments, whereas in hippocampal slices, in 3 out of 11 experiments, no complete suppression occurred. Flunarizine acted in a similar way. It is concluded that L-type calcium channels are involved in strychnine-induced epilepsy, but to a greater extent in the neocortex than in the hippocampus.

Systemically administered cycloheximide reduces inhibition in rat neocortical slice preparation

Global cerebral ischemia leads to long lasting hyperexcitability and a reduced protein synthesis in non-infarcted tissue surrounding the lesion. In this study we investigated whether protein synthesis inhibition by pharmacological means itself changes neocortical excitability. Two hours after the last of three i.p. injections with the protein synthesis inhibitor cycloheximide (12 h interval, 1.5 mg/kg body weight) we observed a widespread reduction of neocortical inhibition. The study indicates that inhibition of protein synthesis may contribute to the altered brain excitability following ischemia.