Rat Neocortical Slices Research Articles

Glutamatergic Nonpyramidal Neurons From Neocortical Layer VI and Their Comparison With Pyramidal and Spiny Stellate Neurons

The deeper part of neocortical layer VI is dominated by nonpyramidal neurons, which lack a prominent vertically ascending dendrite and predominantly establish corticocortical connections. These neurons were studied in rat neocortical slices using patch-clamp, single-cell reverse transcription-polymerase chain reaction, and biocytin labeling. The majority of these neurons expressed the vesicular glutamate transporter but not glutamic acid decarboxylase, suggesting that a high proportion of layer VI nonpyramidal neurons are glutamatergic. Indeed, they exhibited numerous dendritic spines and established asymmetrical synapses. Our sample of glutamatergic nonpyramidal neurons displayed a wide variety of somatodendritic morphologies and a subset of these cells expressed the Nurr1 mRNA, a marker for ipsilateral, but not commissural corticocortical projection neurons in layer VI. Comparison with spiny stellate and pyramidal neurons from other layers showed that glutamatergic neurons consistently exhibited a low occurrence of GABAergic interneuron markers and regular spiking firing patterns. Analysis of electrophysiological diversity using unsupervised clustering disclosed three groups of cells. Layer V pyramidal neurons were segregated into a first group, whereas a second group consisted of a subpopulation of layer VI neurons exhibiting tonic firing. A third heterogeneous cluster comprised spiny stellate, layer II/III pyramidal, and layer VI neurons exhibiting adaptive firing. The segregation of layer VI neurons in two different clusters did not correlate either with their somatodendritic morphologies or with Nurr1 expression. Our results suggest that electrophysiological similarities between neocortical glutamatergic neurons extend beyond layer positioning, somatodendritic morphology, and projection specificity.

Characterizing molecular probes for diffusion measurements in the brain

Brain diffusion properties are at present most commonly evaluated by magnetic resonance (MR) diffusion imaging. MR cannot easily distinguish between the extracellular and intracellular signal components, but the older technique of real-time iontophoresis (RTI) detects exclusively extracellular diffusion. Interpretation of the MR results would therefore benefit from auxiliary RTI measurements. This requires a molecular probe detectable by both techniques. Our aim was to specify a minimum set of requirements that such a diffusion probe should fulfill and apply it to two candidate probes: the cation tetramethylammonium (TMA +), used routinely in the RTI experiments, and the anion hexafluoroantimonate ( SbF 6 − ). Desirable characteristics of a molecular diffusion probe include predictable diffusion properties, stability, minimum interaction with cellular physiology, very slow penetration into the cells, and sufficiently strong and selective MR and RTI signals. These properties were evaluated using preparations of rat neocortical slices under normal and ischemic conditions, as well as solutions and agarose gel. While both molecules can be detected by MR and RTI, neither proved an ideal candidate. TMA + was very stable but it penetrated into the cells and accumulated there within tens of minutes. SbF 6 − did not enter the cells as readily but it was not stable, particularly in ischemic tissue and at higher temperatures. Its presence also resulted in a decreased extracellular volume. These probe properties help to interpret previously published MR data on TMA + diffusion and might play a role in other diffusion experiments obtained with them.

Glutamate and dopamine receptors contribute to the lateral spread of epileptiform discharges in rat neocortical slices

The effects of AMPA-type glutamate receptor as well as dopamine D1 and D2 receptors on the lateral propagation of epileptiform field potentials (EFP) were studied across adjacent areas of rat neocortical tissues. Epileptiform burst discharges were induced by superfusion of Mg(2+)-free artificial cerebrospinal fluid. Simultaneous field potential recordings of EFP were obtained from four microelectrodes placed 2-3 mm apart across coronal slices in the third layer of the neocortex. The effects of AMPA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), dopamine D1 receptor agonist SKF 81297, and dopamine D2 receptor agonist quinpirole on lateral propagation of burst discharges were investigated. CNQX, applied focally between recording sites, blocked rapid propagation across treated areas and resulted in the emergence of spatially separate, independent pacemakers. Focal application of SKF 81297 between recording sites increased the repetition rate of EFP, but reduced the amplitude as well as the duration of epileptic discharges. However, addition of SKF 81297 to the bath medium abolished EFP. Both local and systemic applications of quinpirole irreversibly enhanced repetition rate of epileptiform burst discharges. The results indicate the prerequisite of AMPA synaptic transmission for synchronized lateral propagation of Mg(2+)-free ACSF-induced epileptic activity and the modulatory effects of dopamine D1 and D2 receptors on both EFP initiation and propagation in epileptic tissues.

Functional CB1 Receptors Are Broadly Expressed in Neocortical GABAergic and Glutamatergic Neurons

The cannabinoid receptor CB1 is found in abundance in brain neurons, whereas CB2 is essentially expressed outside the brain. In the neocortex, CB1 is observed predominantly on large cholecystokinin (CCK)-expressing interneurons. However, physiological evidence suggests that functional CB1 are present on other neocortical neuronal types. We investigated the expression of CB1 and CB2 in identified neurons of rat neocortical slices using single-cell RT-PCR. We found that 63% of somatostatin (SST)-expressing and 69% of vasoactive intestinal polypeptide (VIP)-expressing interneurons co-expressed CB1. As much as 49% of pyramidal neurons expressed CB1. In contrast, CB2 was observed in a small proportion of neocortical neurons. We performed whole cell recordings of pyramidal neurons to corroborate our molecular findings. Inhibitory postsynaptic currents (IPSCs) induced by a mixed muscarinic/nicotinic cholinergic agonist showed depolarization-induced suppression of inhibition and were decreased by the CB1 agonist WIN-55212-2 (WIN-2), suggesting that interneurons excited by cholinergic agonists (mainly SST and VIP neurons) possess CB1. IPSCs elicited by a nicotinic receptor agonist were also reduced in the presence of WIN-2, suggesting that neurons excited by nicotinic agonists (mainly VIP neurons) indeed possess CB1. WIN-2 largely decreased excitatory postsynaptic currents evoked by intracortical electrical stimulation, pointing at the presence of CB1 on glutamatergic pyramidal neurons. All WIN-2 effects were strongly reduced by the CB1 antagonist AM 251. We conclude that CB1 is expressed in various neocortical neuronal populations, including glutamatergic neurons. Our combined molecular and physiological data suggest that CB1 widely mediates endocannabinoid effects on glutamatergic and GABAergic transmission to modulate cortical networks.

Truncated TrkB‐T1 regulates the morphology of neocortical layer I astrocytes in adult rat brain slices

By altering their morphology, astrocytes, including those involved in the maintenance and plasticity of neurons and in clearance of transmitter, play important roles in synaptic transmission; however, the mechanism that regulates the morphological plasticity of astrocytes remains unclear. Recently, we reported that T1, a subtype of TrkB (a family of BDNF-specific receptors), altered astrocytic morphology through the control of Rho GTPases in primary astrocyte cultures. In this study, we extended this observation to investigate acute neocortical slices from adult rats. T1 siRNA-expression vectors were electroporated into astrocytes in neocortical layer I of living rats. In both normal slices and control vector-electroporated slices, BDNF induced the elongation of the astrocytic processes and increased the branching of processes in slices after 1 h incubation. In contrast, in T1 siRNA-electroporated slices, no such significant morphological changes were observed in the astrocytes. In addition, the number of synaptophysin+ sites in contact with GFAP+ processes increased in a BDNF-T1-dependent manner without the increase in the total synaptophysin+ sites. Therefore, the present study provides evidence of the regulation of layer I astrocytic morphology by the BDNF-T1 signal in adult rat neocortical slices.

Spontaneous and evoked synaptic rewiring in the neonatal neocortex

The local microcircuitry of the neocortex is structurally a tabula rasa, with the axon of each pyramidal neuron having numerous submicrometer appositions with the dendrites of all neighboring pyramidal neurons, but is functionally highly selective, with synapses formed onto only a small proportion of these targets. This design leaves a vast potential for the microcircuit to rewire without extensive axonal or dendritic growth. To examine whether rewiring does take place, we used multineuron patch-clamp recordings on 12- to 14-day-old rat neocortical slices and studied long-term changes in synaptic connectivity within clusters of neurons. We found pyramidal neurons spontaneously connecting and disconnecting from each other and that exciting the slice with glutamate greatly increases the number of new connections established. Evoked emergence of new synaptic connections requires action potential activity and activation of metabotropic glutamate receptor 5, but not NMDA receptor or group II or group III metabotropic glutamate receptor activation. We also found that it is the weaker connections that are selectively eliminated. These results provide direct evidence for spontaneous and evoked rewiring of the neocortical microcircuitry involving entire functional multisynaptic connections. We speculate that this form of microcircuit plasticity enables an evolution of the microcircuit connectivity by natural selection as a function of experience.

The connexin 36 blockers quinine, quinidine and mefloquine inhibit cortical spreading depression in a rat neocortical slice model in vitro

A protocol for inducing cortical spreading depression (SD) on rat neocortical slices in vitro, upon local application of calibrated ∼nl drops of KCl, 3 M was used to elicit SD events, recorded at two different points on the slice. This in vitro model was validated by the inhibition of SD episodes by the NMDA antagonist MK-801 (20 μM), the reference SD blocker. Quinine, its stereoisomer quinidine, and mefloquine consistently inhibited the SD episodes. Quinine and quinidine, 100 and 200 μM reduced the duration, while mefloquine, 100 and 200 μM reduced the amplitude of SD events, all in a concentration-dependent manner. These compounds have been reported to block gap junctions, specifically the neuronal connexin (Cx) 36, but they also exert other cellular effects. While further investigation is warranted to settle whether SD inhibition in vitro by quinine, quinidine and mefloquine reflects an involvement of neuronal Cx36 channels in SD generation/propagation, these results bear potential drug-discovery relevance for the migraine with aura.

Spatiotemporal Patterns of an Evoked Network Oscillation in Neocortical Slices: Coupled Local Oscillators

We have discovered an evoked network oscillation in rat neocortical slices and have examined its spatiotemporal patterns with voltage-sensitive dye imaging. The slices (visual and auditory cortices) were prepared in a medium of low calcium, high magnesium and with sodium replaced by choline to reduce the excito-toxicity and sodium loading. After slicing, the choline was washed out while normal calcium, magnesium, and sodium concentrations were restored. The oscillation was evoked by a single electrical shock to slices bathed in normal artificial cerebral spinal fluid (ACSF). The oscillation was organized as an all-or-none epoch containing 4-13 cycles at a central frequency approximately 25 Hz. The activity can be reversibly blocked by 6-cyano-7-nitroquinoxalene-2,3-dione (CNQX). 2-amino-5-phosphonopentanoic acid (APV), and atropine but not by bicuculline, indicating polysynaptic excitatory mechanisms. Voltage-sensitive dye imaging showed high-amplitude oscillation signals in superficial and middle cortical layers. Spatiotemporally, the oscillations were organized as waves, propagating horizontally along cortical laminar. Each oscillation cycle was associated with one wave propagating in space. The waveforms were often different at different locations (e.g., extra cycles), suggesting the co-existence of multiple local oscillators. For different cycles, the waves often initiated at different locations, suggesting that local oscillators are competing to initiate each oscillation cycle. Overall our results suggest that this cortical network oscillation is organized at two levels: locally, oscillating neurons are tightly coupled to form local oscillators, and globally the coupling between local oscillators is weak, allowing abrupt spatial phase lags and propagating waves with multiple initiation sites.

Presynaptic opioid receptors on noradrenergic and serotonergic neurons in the human as compared to the rat neocortex

1. Electrically evoked release of [3H]-noradrenaline ([3H]-NA) or [3H]-5-hydroxytryptamine ([3H]-5-HT) in slices of human and the rat neocortex was used to characterize presynaptic opioid receptors. 2. Release of [3H]-NA in rat neocortical slices was reduced only by the mu-receptor agonist DAMGO (pIC50: 7.27, CI95: [7.22, 7.32]; Imax: 77.6+/-1.6%; antagonized by naloxone: pA2: 8.88, CI95: [8.78, 8.98]). 3. Release of [3H]-NA in human neocortical slices was unaffected by DAMGO, but inhibited by the delta-receptor agonist DPDPE (Imax: 25.7+/-2.2%) and the kappa-receptor agonist U-50,488H (19.7+/-2.7% inhibition at 1 microM). Both effects were antagonized by naltrindole (1 microM). 4. Release of [3H]-5-HT in rat neocortical slices, was inhibited by DAMGO (10 microM) and U-50,488H (1 and 10 microM) only in the presence of the 5-HT receptor antagonist methiotepin (1 microM). 5. Release of [3H]-5-HT in human neocortical slices was unaffected by DPDPE, but U-50,488H (Imax: 40.8+/-8.3%; antagonized by 0.1 microM norbinaltorphimine) and DAMGO (16.4+/-3.9% inhibition at 1 microM; antagonized by 0.1 microM naloxone) acted inhibitory. 6. Release of [3H]-5-HT in human neocortical slices was reduced by nociceptin/orphanin (0.1 and 1 microM). These effects were antagonized by the ORL1 antagonist J-113397 (1-[(3R,4R)-1-cyclo-octylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one; 0.1 microM). 7. This study provides evidence for significant species differences in opioid receptor-mediated modulation of NA and 5-HT-release in human vs rat neocortex. In rats, mu-opioid receptors modulate NA release, but 5-HT release is only weakly affected by mu- and kappa-opioids. In contrast, NA release in human neocortex is modulated via delta-opioid receptors, but 5-HT release mainly via kappa-opioid receptors. In addition also the ORL1 receptor seems to be involved in 5-HT release modulation.

The H current blocker ZD7288 decreases epileptiform hyperexcitability in the rat neocortex by depressing synaptic transmission

Neurons respond to intracellular injection of hyperpolarizing current pulses by generating depolarizing sags contributed by a cation current termed I h. I h modulates neuron excitability and rhythmicity. It is, however, unclear whether the net effect of changing I h leads to facilitation or depression of cortical epileptiform activity. Here, we addressed this issue by using field and intracellular recordings to study the effects of ZD7288 (10–100 μM), a bradycardic agent known to abolish I h, on the epileptiform discharges (duration = 2.5 ± 0.3 s, mean ± SEM; interval of occurrence = 34.2 ± 3.3 s, n = 30 slices) induced in rat neocortical slices by 4-aminopyridine and GABA receptor antagonists. ZD7288 abolished the depolarizing sags seen during injection of intracellular hyperpolarizing current pulses while increasing resting membrane potential and apparent input resistance. These effects, which were fully established with 10 μM ZD7288, were associated with a dose-dependent decrease in the occurrence of spontaneous epileptiform events and a reduction in their duration (the latter change occurring at doses >20 μM). ZD7288 also caused a dose-dependent decrease of background postsynaptic potentials. Finally, ZD7288 could depress epileptiform activity during Cs + pre-treatment, a procedure known to block I h. These data indicate that ZD7288 hampers neocortical epileptiform synchronization, but also suggest that most of this action reflects the ability of ZD7288 to decrease synaptic transmission.

GABA-A Receptors Regulate Neocortical Neuronal Migration In Vitro and In Vivo

The cortical migration process depends on a number of trophic factors and on the activation of different voltage- and ligand-gated channels. We investigated the role of gamma-aminobutyric acid (GABA) type A receptors in the neuronal migration process of the newborn rat parietal cortex in vivo and in vitro. Local in vivo application of the GABA-A antagonist bicuculline methiodide (BMI) or the agonist muscimol via cortical surface Elvax implants induced prominent alterations in the cortical architecture when compared with untreated or sham-operated controls. BMI- and muscimol-treated animals revealed heterotopic cell clusters in the upper layers and a complete loss of the cortical lamination in the region underlying the Elvax implant. Immunocytochemical staining for glial fibrillary acidic protein, N-methyl-D-aspartate receptors, and GABA demonstrated that heterotopia was not provoked by glial proliferation and confirmed the presence of both glutamatergic and GABAergic neurons. In organotypic neocortical slices from embryonic day 18-19 embryos, application of BMI and to a lesser extent also muscimol induced an increase in the migration speed and an accumulation of neurons in the upper cortical layers. Spontaneous intracellular calcium ([Ca2+]i) oscillations in neocortical slices from newborn rats were abolished by BMI (5 and 20 microM) and muscimol (1 and 10 microM), indicating that both compounds interfere with [Ca2+]i signaling required for normal neuronal migration. Electrophysiological recordings from migrating neurons in newborn rat neocortical slices indicate that long-term application of muscimol causes a pronounced reduction (1 microM muscimol) or blockade (10 microM) in the responsiveness of postsynaptic GABA-A receptors due to a pronounced receptor desensitization. Our results indicate that modulation of GABA-A receptors by compounds acting as agonists or antagonists may profoundly influence the neuronal migration process in the developing cerebral cortex.

Endogenous Acetylcholine Enhances Synchronized Interneuron Activity in Rat Neocortex

Application of 4-aminopyridine (4-AP) along with EAA) receptor antagonists produces gamma-aminobutyric acid (GABAA) receptor-dependent synchronized activity in interneurons. This results in waves of activity propagating through upper cortical layers. Because interneurons in the neocortex are excited by nicotinic acetylcholine receptor (nAChR) agonists, ACh may influence synchronization of these local neocortical interneuronal networks. To study this possibility, we have used voltage-sensitive dye imaging using the fluorescent dye RH 414 (30 microM) in rat neocortical slices. Recordings were obtained in the presence of 4-AP (100 microM) and the EAA receptor antagonists D-2-amino-5-phosphonvaleric acid (20 microM) and 6-cyano-7-nitro-quinoxaline-2,3-dione (10 microM). In response to intracortical stimulation, localized or propagated activity restricted to upper cortical layers was seen. Bath application of the ACh esterase inhibitor neostigmine (10 microM) and the nAChR agonist 1,1-dimethyl-4-phenyl-piperazinium iodide (DMPP; 10 microM) increased the response amplitude, the extent of spread, and the duration of this activity. These changes were seen in 13 of 16 slices tested with neostigmine (10 microM) and 4 of 7 slices tested with DMPP (10 microM). Application of the muscarinic AChR antagonist atropine (1 microM) did not block the enhancement of activity by neostigmine (n = 7). Application of dihydro-beta-erythroidine (10 microM), known, at this concentration, to selectively antagonize alpha4beta2-like nAChRs, blocked the effect of neostigmine (n = 5). The selective alpha7-like nAChR antagonist methyllycaconitine (50 nM) was ineffective (n = 5). These results suggest that activation of alpha4beta2-like nAChRs by endogenously released ACh can enhance synchronized activity in local neocortical inhibitory networks.

Dopamine release in human neocortical slices: Characterization of inhibitory autoreceptors and of nicotinic acetylcholine receptor-evoked release

The autoinhibitory control of electrically evoked release of [ 3H]-dopamine and the properties of that induced by nicotinic receptor (nAChR) stimulation were studied in slices of the human neocortex. In both models [ 3H]-dopamine release was action potential-induced and exocytotic. The selective dopamine D 2 receptor agonist (−)-quinpirole reduced electrically evoked release of [ 3H]-dopamine, yielding IC 50 and I max values of 23 nM and 76%, respectively. Also, the effects of several other subtype-selective dopamine receptor ligands confirmed that the terminal dopamine autoreceptor belongs to the D 2 subtype. The autoinhibitory feedback control was slightly operative under stimulation conditions of 90 pulses and 3 Hz, with a biophase concentration of endogenous dopamine of 3.6 nM, and was enhanced under blockade of dopamine reuptake. [ 3H]-dopamine release evoked in an identical manner in mouse neocortical slices was not inhibited by (−)-quinpirole, suggesting the absence of dopamine autoreceptors in this tissue and underlining an important species difference. Also, nAChR stimulation-induced release of [ 3H]-dopamine revealed a species difference: [ 3H]-dopamine release was evoked in human, but not in rat neocortical slices. The nAChRs inducing [ 3H]-dopamine release most probably belong to the α 3/β 2-subtype, according to the potencies and efficacies of subtype-selective nAChR ligands. Part of these receptors may be located on glutamatergic neurons.

Volume-conducted epileptiform events between adjacent necortical slices in an interface tissue chamber

“Far-field” artifacts are presumed to contribute negligibly to the field potential activity recorded from brain slices maintained in vitro. While performing paired intracellular and field potential recordings from rat neocortical slices superfused with medium containing 4-aminopyridine + GABA receptor antagonists, we identified: (i) epileptiform discharges characterized by concomitant field oscillations (amplitude = 2.6–6.4 mV) and intracellular depolarizations as well as (ii) smaller amplitude (0.3–1.3 mV) field epileptiform events that were not associated with any intracellular activity. By placing an additional extracellular recording electrode into adjacent slices, we discovered that large amplitude, epileptiform discharges were generated concomitant to those seen in the first slice at the field potential level only. In addition, we found in these slices small amplitude, field discharges that were synchronous with those recorded intracellularly in the original slice. Analysis of the changes in field potential amplitude over space demonstrated that this parameter was reduced by approximately 60% when the recording electrode was moved from the slice generating the epileptiform activity to the bathing medium and further decreased in a quasi-linear mode when recordings were obtained from an adjacent slice. In conclusion, these observations indicate that brain slices can, under appropriate conditions, produce field potentials that are of amplitude sufficient for being recorded from other slices in the tissue chamber. These findings suggest that caution should be taken in assuming that field potential activity seen in an in vitro brain slice is generated within the recorded tissue.

Increased Extracellular K + Concentration Reduces the Efficacy of N -methyl- d -aspartate Receptor Antagonists to Block Spreading Depression-Like Depolarizations and Spreading Ischemia

Spreading depression (SD)-like depolarizations may augment neuronal damage in neurovascular disorders such as stroke and traumatic brain injury. Spreading ischemia (SI), a particularly malignant variant of SD-like depolarization, is characterized by inverse coupling between the spreading depolarization wave and cerebral blood flow. SI has been implicated in particular in the pathophysiology of subarachnoid hemorrhage. Under physiological conditions, SD is blocked by N-methyl-D-aspartate receptor (NMDAR) antagonists. However, because both SD-like depolarizations and SI occur in presence of an increased extracellular K+ concentration ([K+]o), we tested whether this increase in baseline [K+]o would reduce the efficacy of NMDAR antagonists. Cranial window preparations, laser Doppler flowmetry, and K+-sensitive/reference microelectrodes were used to record SD, SD-like depolarizations, and SI in rats in vivo; microelectrodes and intrinsic optical signal measurements were used to record SD and SD-like depolarizations in human and rat brain slices. In vivo, the noncompetitive NMDAR antagonist dizocilpine (MK-801) blocked SD propagation under physiological conditions, but did not block SD-like depolarizations or SI under high baseline [K+]o. Similar results were found in human and rat neocortical slices with both MK-801 and the competitive NMDAR antagonist D-2-amino-5-phosphonovaleric acid. Our data suggest that elevated baseline [K+]o reduces the efficacy of NMDAR antagonists on SD-like depolarizations and SI. In conditions of moderate energy depletion, as in the ischemic penumbra, or after subarachnoid hemorrhage, NMDAR inhibition may not be sufficient to block these depolarizations.

Intrinsic excitability, synaptic potentials, and short‐term plasticity in human epileptic neocortex

Although studies of epileptic human hippocampus suggest changes of synaptic and intrinsic excitability, few changes, save the appearance of spontaneous field/synaptic potentials, are known in epileptic neocortical tissue. However, invasive EEG and histological studies suggest that neocortical tissue, even in mesial temporal lobe epilepsy, can play an important role as an irritative zone or extrahippocampal focus. We hypothesized that intrinsic neuronal and synaptic excitability, as well as short-term plasticity, are altered in neocortical areas, particularly with elevated K+ levels as occur during seizures. We analyzed neuronal firing properties, synaptic responses, and paired-pulse plasticity in human neocortical slices from tissue resected during epilepsy surgery, both under normal and under pathological conditions, i.e., after elevating K+ (4/8 mM), with rat neocortical slices as controls. Neuronal firing properties were not different. We did find, however, alterations of synaptic responsiveness in epileptic tissue, i.e., an elevated network excitability with K+ elevations, and reduction of paired-pulse depression.

3-Chloro,4-methoxyfendiline is a potent GABA B receptor potentiator in rat neocortical slices

Using grease-gap recording from rat neocortical slices, the GABA B receptor agonist baclofen elicited reversible and concentration-dependent hyperpolarizing responses (EC 50=18±2.3 μM). The hyperpolarizations were antagonised by the GABA B receptor antagonist Sch 50911 [(+)-( S)-5,5-dimethylmorpholinyl-2-acetic acid). (+)- N-1-(3-chloro-4-methoxyphenyl)ethyl-3,3-diphenylpropylamine (3-chloro,4-methoxyfendiline; 3-Cl,4-MeO-fendiline) reversibly potentiated baclofen-induced hyperpolarizing responses, which were reduced by Sch 50911, producing leftward shifts of the baclofen concentration–response curves, with a marked increase in the maximal hyperpolarization (EC 50=2±0.5 μM). In slices preincubated with either [ 3H]GABA or [ 3H]glutamic acid, 3-Cl,4-MeO-fendiline (1 μM) potentiated the inhibitory effect of baclofen (2 μM) on the electrically evoked release of [ 3H]GABA and had a similar effect on the release of [ 3H]glutamic acid at a concentration of 0.5 μM, without affecting the basal release. These effects were blocked by Sch 50911 (10 μM). Our findings suggest that 3-Cl,4-MeO-fendiline is a potent potentiator of pre- and postsynaptic GABA B receptor-mediated functions.

Spiral Waves in Disinhibited Mammalian Neocortex

Spiral waves are a basic feature of excitable systems. Although such waves have been observed in a variety of biological systems, they have not been observed in the mammalian cortex during neuronal activity. Here, we report stable rotating spiral waves in rat neocortical slices visualized by voltage-sensitive dye imaging. Tissue from the occipital cortex (visual) was sectioned parallel to cortical lamina to preserve horizontal connections in layers III-V (500-mum-thick, approximately 4 x 6 mm(2)). In such tangential slices, excitation waves propagated in two dimensions during cholinergic oscillations. Spiral waves occurred spontaneously and alternated with plane, ring, and irregular waves. The rotation rate of the spirals was approximately 10 turns per second, and the rotation was linked to the oscillations in a one-cycle- one-rotation manner. A small (<128 mum) phase singularity occurred at the center of the spirals, about which were observed oscillations of widely distributed phases. The phase singularity drifted slowly across the tissue ( approximately 1 mm/10 turns). We introduced a computational model of a cortical layer that predicted and replicated many of the features of our experimental findings. We speculate that rotating spiral waves may provide a spatial framework to organize cortical oscillations.

Intracellular calcium increase in epileptiform activity: modulation by levetiracetam and lamotrigine.

Alterations in neuronal calcium (Ca2+) homeostasis are believed to play an essential role in the generation and propagation of epileptiform events. Levetiracetam (LEV) and lamotrigine (LTG), novel antiepileptic drugs (AEDs), were tested on epileptiform events and the corresponding elevations in intracellular Ca2+ concentration ([Ca2+]i) recorded from rat neocortical slices. Electrophysiological recordings were performed from single pyramidal neurons from a slice preparation. Spontaneous epileptiform events consisting of long-lasting, repetitive paroxysmal depolarization shifts (PDSs) and interictal spike activity were induced by reducing the magnesium concentration from the solution and by adding bicuculline and 4-aminopyridine. Simultaneously, microfluorimetric measurements of [Ca2+]i were performed. Optical imaging with Ca2+ indicators revealed a close correlation between Ca2+ transients and epileptiform events. Both LEV and LTG were able to reduce both amplitude and duration of PDSs, as well as the concomitant elevation in [Ca2+]i, in a dose-dependent fashion. Whole-cell patch-clamp recordings from isolated neocortical neurons revealed that LEV significantly reduced N-, and partially P/Q-type high-voltage-activated (HVA) Ca2+ currents, whereas sodium currents were unaffected. Interestingly, the inhibitory effects of LEV were mimicked and occluded by LTG or by a combination of omega-conotoxin GVIA and omega-agatoxin IVA, selective blockers of N- and P/Q-type HVA channels, respectively, suggesting a common site of action for these AEDs. These results demonstrate that large, transient elevations in neuronal [Ca2+]i correlate to epileptiform discharges. The antagonistic effects of LEV and LTG on [Ca2+]i overload might represent the basis for their anticonvulsant efficacy and could preserve neuronal viability.

Characterization of nicotinic receptors inducing noradrenaline release and absence of nicotinic autoreceptors in human neocortex

Presynaptic facilitatory nicotinic receptors (nAChRs) on noradrenergic axon terminals were studied in slices of human or rat neocortex and of rat hippocampus preincubated with [ 3 H ]noradrenaline ([ 3 H ]NA). During superfusion of the slices, stimulation by nicotinic agonists for 2 min only slightly increased [ 3 H ]NA outflow in the rat neocortex, but caused a tetrodotoxin-sensitive, Ca 2+-dependent release of [ 3 H ]NA in rat hippocampus and human neocortex. In both tissues a similar rank order of potency of nicotinic agonists was found: epibatidine⪢DMPP>nicotine∼cytisine≥acetylcholine; choline was ineffective. In human neocortex, the effects of nicotine (100 μM) were reduced by mecamylamine, methyllycaconitine, di-hydro-β-erythroidine (10 μM, each) and the α 3β 2/α 6β x -selective α-conotoxin MII (100/200 nM). The α 3β 4 selective α-conotoxin AuIB (1 μM), and the α 7 selective α-conotoxin ImI (200 nM) as well as α-bungarotoxin (125 nM) were ineffective. Glutamate receptor antagonists (300 μM AP-5, 100 μM DNQX) acted inhibitory, suggesting the participation of nAChRs on glutamatergic neurons. On the other hand, nAChR agonists were unable to evoke exocytotic release of [ 3 H ]acetylcholine from human and rat neocortical slices preincubated with [ 3 H ]choline. In conclusion: (1) α 3β 2 and/or α 6 containing nAChRs are at least partially responsible for presynaptic cholinergic facilitation of noradrenergic transmission in human neocortex; (2) nicotinic autoreceptors were not detectable in rat and human neocortex.