Rat Neocortical Slices Research Articles

Alternative strategy for driving voltage-oscillator in neocortex of rats

Information integration in the brain requires functional connectivity between local neural networks. Here, we investigated the interregional coupling mechanism from the viewpoint of oscillations using optical recording methods. Low-frequency electrical stimulation of rat neocortical slices in a caffeine-containing medium induced oscillatory activity between the primary visual cortex (Oc1) and medial secondary visual cortex (Oc2M), in which the oscillation generator was located in the Oc2M and was triggered by a feedforward signal. During to-and-fro oscillatory activity, neural excitation was marked in layer II/III. When the upper layer was disrupted between Oc1 and Oc2M, feedforward signals could propagate through the deep layer and switch on the oscillator in the Oc2M. When the lower layer was disrupted between Oc1 and Oc2M, feedforward signals could propagate through the upper layer and switch on the oscillator in the Oc2M. In the backward direction, neither the upper layer cut nor the lower layer cut disrupted the propagation of the oscillations. In all cases, the horizontal and vertical pathways were used as needed. Fluctuations in the oscillatory waveforms of the local field potential at the upper and lower layers in the Oc2M were reversed, suggesting that the oscillation originated between the two layers. Thus, the neocortex may work as a safety device for interregional communications in an alternative way to drive voltage oscillators in the neocortex.

The Virtual Electrode Recording Tool for EXtracellular Potentials (VERTEX) Version 2.0: Modelling in vitro electrical stimulation of brain tissue

Neuronal circuits can be modelled in detail allowing us to predict the effects of stimulation on individual neurons. Electrical stimulation of neuronal circuits in vitro and in vivo excites a range of neurons within the tissue and measurements of neural activity, e.g the local field potential (LFP), are again an aggregate of a large pool of cells. The previous version of our Virtual Electrode Recording Tool for EXtracellular Potentials (VERTEX) allowed for the simulation of the LFP generated by a patch of brain tissue. Here, we extend VERTEX to simulate the effect of electrical stimulation through a focal electric field. We observe both direct changes in neural activity and changes in synaptic plasticity. Testing our software in a model of a rat neocortical slice, we determine the currents contributing to the LFP, the effects of paired pulse stimulation to induce short term plasticity (STP), and the effect of theta burst stimulation (TBS) to induce long term potentiation (LTP).

The Virtual Electrode Recording Tool for EXtracellular Potentials (VERTEX) Version 2.0: Modelling in vitro electrical stimulation of brain tissue.

Neuronal circuits can be modelled in detail allowing us to predict the effects of stimulation on individual neurons. Electrical stimulation of neuronal circuits in vitro and in vivo excites a range of neurons within the tissue and measurements of neural activity, e.g the local field potential (LFP), are again an aggregate of a large pool of cells. The previous version of our Virtual Electrode Recording Tool for EXtracellular Potentials (VERTEX) allowed for the simulation of the LFP generated by a patch of brain tissue. Here, we extend VERTEX to simulate the effect of electrical stimulation through a focal electric field. We observe both direct changes in neural activity and changes in synaptic plasticity. Testing our software in a model of a rat neocortical slice, we determine the currents contributing to the LFP, the effects of paired pulse stimulation to induce short term plasticity (STP), and the effect of theta burst stimulation (TBS) to induce long term potentiation (LTP).

A comparison of anticonvulsant efficacy and action mechanism of Mannitol vs Phenytoin in adult rat neocortical slices

We show that, in adult rat neocortical slices, an anticonvulsant effect comparable to that of Phenytoin can be obtained through a Mannitol-induced increase in extracellular osmolarity of only 30mOsm/L. The anticonvulsant action of extracellular hyperosmolarity has been known for decades but has not found a feasible therapeutic application, yet. A 30mOsm/L increase in extracellular osmolarity is already utilized in neurocritical care though not as an anticonvulsant agent: the data suggest a possible effective anticonvulsant use, too, in this setting.We used multiple electrode arrays to characterize and compare the anticonvulsant mechanisms of Mannitol and Phenytoin. Phenytoin decreased the voltage, duration and spatial spread of rhythmic repetitive, ictal-like activity. In contrast, Mannitol did not significantly affect voltage, duration and spatial spread of rhythmic repetitive, ictal-like activity but rather it inhibited the rate of epileptiform discharges.

The conversion of glutamate by glutamine synthase in neocortical astrocytes from juvenile rat is important to limit glutamate spillover and peri/extrasynaptic activation of NMDA receptors.

Glutamate transporters (EAATs) are important to maintain spatial and temporal specificity of synaptic transmission. Their efficiency to uptake and transport glutamate into the intracellular space depends on several parameters including the intracellular concentrations of Na+ and glutamate, the elevations of which may slow down the cycling rate of EAATs. In astrocytes, glutamate is maintained at low concentration due to the presence of specific enzymes such as glutamine synthase (GS). GS inhibition results in cytosolic accumulation of glutamate suggesting that the conversion of glutamate by GS is important for EAATs operation. Here we recorded astrocytes from juvenile rat neocortical slices and analyzed the consequences of elevated intracellular glutamate concentrations and of GS inhibition on the time course of synaptically evoked transporter current (STC). In slices from rats treated with methionine sulfoximine (MSO), a GS inhibitor, STC evoked by short burst of high frequency stimulation (HFS; 100 Hz for 100 ms) but not by low frequency stimulation (LFS; 0.1 Hz) was twice slower than STC evoked from saline injected rats. Same results were obtained for astrocytes recorded with pipette containing 3-10 mM glutamate and compared with cells recorded with 0 or1 mM glutamate in the patch pipette. We also showed that HFS elicited significantly larger NMDAR-excitatory postsynaptic currents (EPSCs) with a stronger peri/extrasynaptic component in pyramidal cells from MSO-treated compared with saline treated rats. Taken together our data demonstrate that the conversion of glutamate by GS is fundamental to ensure an efficient clearance of glutamate by EAATs and to prevent glutamate spillover. GLIA 2017;65:401-415.

Localized adenosine signaling provides fine-tuned negative feedback over a wide dynamic range of neocortical network activities.

Although the patterns of activity produced by neocortical networks are now better understood, how these states are activated, sustained, and terminated still remains unclear. Negative feedback by the endogenous neuromodulator adenosine may potentially play an important role, as it can be released by activity and there is dense A1 receptor expression in the neocortex. Using electrophysiology, biosensors, and modeling, we have investigated the properties of adenosine signaling during physiological and pathological network activity in rat neocortical slices. Both low- and high-rate network activities were reduced by A1 receptor activation and enhanced by block of A1 receptors, consistent with activity-dependent adenosine release. Since the A1 receptors were neither saturated nor completely unoccupied during either low- or high-rate activity, adenosine signaling provides a negative-feedback mechanism with a wide dynamic range. Modeling and biosensor experiments show that during high-rate activity increases in extracellular adenosine concentration are highly localized and are uncorrelated over short distances that are certainly<500 μm. Modeling also predicts that the slow rise of the purine waveform cannot be from diffusion from distal release sites but more likely results from uptake and metabolism. The inability to directly measure adenosine release during low-rate activity, although it is present, is probably a consequence of small localized increases in adenosine concentration that are rapidly diminished by diffusion and active removal mechanisms. Saturation of such removal mechanisms when higher concentrations of adenosine are released results in the accumulation of inosine, explaining the strong purine signal during high-rate activity.

Pharmacological actions of thymol and an analogue at GABAB autoreceptors.

GABAB autoreceptors inhibit release of GABA from GABAergic nerve terminals. Agonists of these receptors (e.g. baclofen) inhibit, whereas antagonists (e.g. (+)-(S)-5,5-dimethylmorpholinyl-2-acetic acid; Sch50911) enhance release of the transmitter. The actions of thymol (2-isopropyl-5-methylphenol) and the structurally related compound 2-tert-butyl-4-methylphenol, (4MP) on the release of [(3) H]-GABA were examined in rat neocortical slices where the GABAergic nerves had been preloaded with [(3) H]-GABA and subsequently stimulated electrically on two occasions (S1 and S2 ). Test agents, baclofen and Sch50911 were added to the superfusion medium prior to the second period of stimulation (S2 ). Stimulation-induced overflow (SIO) of [(3) H]-GABA as a consequence of these stimulations (SIO1 and SIO2 ) were calculated and the effects of agents determined by comparing the SIO2 /SIO1 ratio in the presence of each agent with that in control tissue. Thymol potentiated the release of [(3) H]-GABA (EC50 170μmol/L), an action reversed by baclofen (2μmol/L). Baclofen alone had little effect on GABA release. Release of [(3) H]-GABA was inhibited by 4MP (IC50 3μmol/L) and this effect was blocked by Sch50911 (10μmol/L). Alone, Sch50911 markedly potentiated the release of GABA. These results imply that 4MP is an agonist of GABAB autoreceptors; however, further studies are needed to confirm that thymol is indeed a GABAB autoreceptor antagonist. Of interest are structural differences in these agents. Thymol has a propyl group in the ortho position relative to the phenolic hydroxyl, whereas in 4MP this is a butyl group and the methyl group moves from position 5 to 4. Whether one or both of these changes was responsible for the above actions is unknown.

Critical role of calcitonin gene-related peptide receptors in cortical spreading depression.

Cortical spreading depression (CSD) is a key pathogenetic step in migraine with aura. Dysfunctions of voltage-dependent and receptor-operated channels have been implicated in the generation of CSD and in the pathophysiology of migraine. Although a known correlation exists between migraine and release of the calcitonin gene-related peptide (CGRP), the possibility that CGRP is involved in CSD has not been examined in detail. We analyzed the pharmacological mechanisms underlying CSD and investigated the possibility that endogenous CGRP contributes to this phenomenon. CSD was analyzed in rat neocortical slices by imaging of the intrinsic optical signal. CSD was measured as the percentage of the maximal surface of a cortical slice covered by the propagation of intrinsic optical signal changes during an induction episode. Reproducible CSD episodes were induced through repetitive elevations of extracellular potassium concentration. AMPA glutamate receptor antagonism did not inhibit CSD, whereas NMDA receptor antagonism did inhibit CSD. Blockade of voltage-dependent sodium channels by TTX also reduced CSD. CSD was also decreased by the antiepileptic drug topiramate, but not by carbamazepine. Interestingly, endogenous CGRP was released in the cortical tissue in a calcium-dependent manner during CSD, and three different CGRP receptor antagonists had a dose-dependent inhibitory effect on CSD, suggesting a critical role of CGRP in this phenomenon. Our findings show that both glutamate NMDA receptors and voltage-dependent sodium channels play roles in CSD. They also demonstrate that CGRP antagonism reduces CSD, supporting the possible use of drugs targeting central CGRP receptors as antimigraine agents.

Effect of cannabinoid receptor activation on spreading depression.

The objective of this study was to evaluate the effect of cannabinoid on cortical spreading depression (CSD) in rat brain. Cannabis has been used for centuries for both symptomatic and prophylactic treatment of different types of headaches including migraine. CSD is believed to be a putative neuronal mechanism underlying migraine aura and subsequent pain. The effects of Delta9-tetrahydrocannabinol (THC), as well as, cannabinoid CB1 and CB2 receptor agonists on CSD in rat neocortical slices were investigated. Furthermore, the effect of cannabinoid CB1 agonist was tested on field excitatory postsynaptic potentials (fEPSP) and long-term potentiation (LTP). HC (1-20 microM) dose dependently suppressed CSD amplitude, duration, and propagation velocity. Cannabinoid CB1 agonist, WIN 55,212-2 mesylate (1-10 microM), also significantly suppressed all characteristic features of CSD. However, cannabinoid CB2 agonist, JWH-133 (1-20 microM), did not affect CSD. FEPSP and induction of LTP were suppressed by application of WIN55212-2. Suppression of CSD by activation of CB1 receptors points to the potential therapeutic effects of cannabinoids in migraine with aura. More research is needed before we know whether cannabinoids may be helpful in treating migraine pain.

Distinct muscarinic acetylcholine receptor subtypes mediate pre- and postsynaptic effects in rat neocortex

BackgroundCholinergic transmission has been implicated in learning, memory and cognition. However, the cellular effects induced by muscarinic acetylcholine receptors (mAChRs) activation are poorly understood in the neocortex. We investigated the effects of the cholinergic agonist carbachol (CCh) and various agonists and antagonists on neuronal activity in rat neocortical slices using intracellular (sharp microelectrode) and field potential recordings.ResultsCCh increased neuronal firing but reduced synaptic transmission. The increase of neuronal firing was antagonized by pirenzepine (M1/M4 mAChRs antagonist) but not by AF-DX 116 (M2/M4 mAChRs antagonist). Pirenzepine reversed the depressant effect of CCh on excitatory postsynaptic potential (EPSP) but had marginal effects when applied before CCh. AF-DX 116 antagonized the depression of EPSP when applied before or during CCh. CCh also decreased the paired-pulse inhibition of field potentials and the inhibitory conductances mediated by GABAA and GABAB receptors. The depression of paired-pulse inhibition was antagonized or prevented by AF-DX 116 or atropine but only marginally by pirenzepine. The inhibitory conductances were unaltered by xanomeline (M1/M4 mAChRs agonist), yet the CCh-induced depression was antagonized by AF-DX 116. Linopirdine, a selective M-current blocker, mimicked the effect of CCh on neuronal firing. However, linopirdine had no effect on the amplitude of EPSP or on the paired-pulse inhibition, indicating that M-current is involved in the increase of neuronal excitability but neither in the depression of EPSP nor paired-pulse inhibition.ConclusionsThese data indicate that the three effects are mediated by different mAChRs, the increase in firing being mediated by M1 mAChR, decrease of inhibition by M2 mAChR and depression of excitatory transmission by M4 mAChR. The depression of EPSP and increase of neuronal firing might enhance the signal-to-noise ratio, whereas the concomitant depression of inhibition would facilitate long-term potentiation. Thus, this triade of effects may represent a “neuronal correlate” of attention and learning.

Optical control of focal epilepsy in vivo with caged γ‐aminobutyric acid

There is enormous clinical potential in exploiting the spatial and temporal resolution of optical techniques to modulate pathophysiological neuronal activity, especially intractable focal epilepsy. We have recently utilized a new ruthenium-based caged compound, ruthenium-bipyridine-triphenylphosphine-γ-aminobutyric acid (RuBi-GABA), which releases GABA when exposed to blue light, to rapidly terminate paroxysmal activity in vitro and in vivo. The convulsant 4-aminopyridine was used to induce interictal activity and seizures in rat neocortical slices and anesthetized rats. We examined the effect of blue light, generated by a small, light-emitting diode (LED), on the frequency and duration of ictal activity in the presence and absence of RuBi-GABA. Neither blue light alone, nor low concentrations of RuBi-GABA, affected interictal activity or baseline electrical activity in neocortical slices. However, brief, blue illumination of RuBi-GABA, using our LED, dramatically reduced extracellular spikes and bursts. More impressively, illumination of locally applied RuBi-GABA rapidly terminated in vivo seizures induced by topical application of 4-aminopyridine. The RuBi-GABA effect was blocked by the GABA(A) antagonist picrotoxin, but not duplicated by direct application of GABA. This is the first example of optical control of in vivo epilepsy, proving that there is sufficient cortical light penetration from an LED and diffusion of caged GABA to quickly terminate intense focal seizures. We are aware that many obstacles need to be overcome before this technique can be translated to patients, but at the moment, this represents a feasible method for harnessing optical techniques to fabricate an implantable device for the therapy of neocortical epilepsy.

Dual Gamma Rhythm Generators Control Interlaminar Synchrony in Auditory Cortex

Rhythmic activity in populations of cortical neurons accompanies, and may underlie, many aspects of primary sensory processing and short-term memory. Activity in the gamma band (30 Hz up to >100 Hz) is associated with such cognitive tasks and is thought to provide a substrate for temporal coupling of spatially separate regions of the brain. However, such coupling requires close matching of frequencies in co-active areas, and because the nominal gamma band is so spectrally broad, it may not constitute a single underlying process. Here we show that, for inhibition-based gamma rhythms in vitro in rat neocortical slices, mechanistically distinct local circuit generators exist in different laminae of rat primary auditory cortex. A persistent, 30-45 Hz, gap-junction-dependent gamma rhythm dominates rhythmic activity in supragranular layers 2/3, whereas a tonic depolarization-dependent, 50-80 Hz, pyramidal/interneuron gamma rhythm is expressed in granular layer 4 with strong glutamatergic excitation. As a consequence, altering the degree of excitation of the auditory cortex causes bifurcation in the gamma frequency spectrum and can effectively switch temporal control of layer 5 from supragranular to granular layers. Computational modeling predicts the pattern of interlaminar connections may help to stabilize this bifurcation. The data suggest that different strategies are used by primary auditory cortex to represent weak and strong inputs, with principal cell firing rate becoming increasingly important as excitation strength increases.

Amperometric Measurement of Glutamate Release Modulation by Gabapentin and Pregabalin in Rat Neocortical Slices: Role of Voltage-Sensitive Ca2+ α2δ-1 Subunit

Gabapentin (GBP; Neurontin) and pregabalin (PGB; Lyrica, S-(+)-3-isobutylgaba) are used clinically to treat several disorders associated with excessive or inappropriate excitability, including epilepsy; pain from diabetic neuropathy, postherpetic neuralgia, and fibromyalgia; and generalized anxiety disorder. The molecular basis for these drugs' therapeutic effects are believed to involve the interaction with the auxiliary α(2)δ subunit of voltage-sensitive Ca(2+) channel (VSCC) translating into a modulation of pathological neurotransmitter release. Glutamate as the primary excitatory neurotransmitter in the mammalian central nervous system contributes, under conditions of excessive glutamate release, to neurological and psychiatric disorders. This study used enzyme-based microelectrode arrays to directly measure extracellular glutamate release in rat neocortical slices and determine the modulation of this release by GBP and PGB. Both drugs attenuated K(+)-evoked glutamate release without affecting basal glutamate levels. PGB (0.1-100 μM) exhibited concentration-dependent inhibition of K(+)-evoked glutamate release with an IC(50) value of 5.3 μM. R-(-)-3-Isobutylgaba, the enantiomer of PGB, did not significantly reduce K(+)-evoked glutamate release. The decrease of K(+)-evoked glutamate release by PGB was blocked by the l-amino acid l-isoleucine, a potential endogenous ligand of the α(2)δ subunit. In neocortical slices from transgenic mice having a point mutation (i.e., R217A) of the α(2)δ-1 (subtype) subunit of VSCC, PGB did not affect K(+)-evoked glutamate release yet inhibited this release in wild-type mice. The results show that GBP and PGB attenuated stimulus-evoked glutamate release in rodent neocortical slices and that the α(2)δ-1 subunit of VSCC appears to mediate this effect.

Pharmacological actions of oximino-propofol analogues at GABAB autoreceptors

1. GABA(B) autoreceptors are a subclass of GABA(B) receptors that inhibit the release of [(3) H]GABA from GABAergic nerve terminals. Baclofen is an agonist that reduces [(3)H]GABA, whilst the antagonist (+)-(S)-5,5-dimethylmorpholinyl-2-acetic acid (Sch 50911) enhances [(3)H]GABA release in electrically-stimulated rat neocortical brain slices preloaded with [(3)H]GABA. Here, the pharmacological actions of a series of compounds derived from the positive allosteric modulator, 2,6-di-tert-butyl-4-(3-hydroxy-2,2-dimethyl-propyl)-phenol (CGP7930), were examined on GABA(B) autoreceptors. 2. The compound, 3-(3,5-ditbutyl-4-hydroxyphenyl)-2,2-dimethyl-1-oximinopropane (compound 2), at 10 μmol/L had little effect on the stimulation-induced overflow of [(3)H]GABA when superfused alone, but when superfused in the presence of baclofen (2 μmol/L) inhibited the overflow of [(3)H]GABA. These effects were reversed by Sch 50911 (10 μmol/L). Although compounds 1-(4-chlorophenyl)-3-(4-hydroxy-3,5-diisopropylphenyl)-2-methyl-1-oximinopropane (compound 1), 1-[(3,5-ditbutyl-4-hydroxyphenyl)methyl]-1-oximinomethylcyclohexane (compound 3), 3-(3,5-ditbutyl-4-hydroxyphenyl)-1,2-diphenyl-1-oximinopropane (compound 4) and 4-(3,5-ditbutyl-4-hydroxyphenyl)-3-methyl-2-oximinobutane (compound 5) (each at 10 μmol/L) tended to reduce the stimulation-induced overflow in the presence of baclofen, an effect reversed by Sch 50911, their status as modulators is not confirmed in the present study. 3. Another derivative, 3-(3,5-ditbutyl-4-hydroxyphenyl)-1-(4-chlorophenyl)-2-methyl-1-oximinopropane (compound 6) (10 μmol/L), acted as an agonist as it inhibited the release of [(3)H]GABA by 32% (EC(50) of 3.3 μmol/L), an effect reversed by Sch 50911 (10 μmol/L). The other compounds, 1-[(3,5-ditbutyl-4-hydroxyphenyl)methyl]-1-methyl-2-oximinocyclohexane (compound 7), 4-(3,5-ditbutyl-4-hydroxyphenyl)-3,3-dimethyl-2-oximinobutane (compound 8) and 4-(4-hydroxy-3,5-diisopropylphenyl)-3,3-dimethyl-2-oximinobutane (compound 9) (each at 10 μmol/L), were inactive. 4. These findings indicate that this series of compounds show different modes of activity at GABA(B) autoreceptors.

VIP, CRF, and PACAP Act at Distinct Receptors to Elicit Different cAMP/PKA Dynamics in the Neocortex

The functional significance of diverse neuropeptide coexpression and convergence onto common second messenger pathways remains unclear. To address this question, we characterized responses to corticotropin-releasing factor (CRF), pituitary adenylate cyclase-activating peptide (PACAP), and vasoactive intestinal peptide (VIP) in rat neocortical slices using optical recordings of cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) sensors, patch-clamp, and single-cell reverse transcription-polymerase chain reaction. Responses of pyramidal neurons to the 3 neuropeptides markedly differed in time-course and amplitude. Effects of these neuropeptides on the PKA-sensitive slow afterhyperpolarization current were consistent with those observed with cAMP/PKA sensors. CRF-1 receptors, primarily expressed in pyramidal cells, reportedly mediate the neocortical effects of CRF. PACAP and VIP activated distinct PAC1 and VPAC1 receptors, respectively. Indeed, a selective VPAC1 antagonist prevented VIP responses but had a minor effect on PACAP responses, which were mimicked by a specific PAC1 agonist. While PAC1 and VPAC1 were coexpressed in pyramidal cells, PAC1 expression was also frequently detected in interneurons, suggesting that PACAP has widespread effects on the neuronal network. Our results suggest that VIP and CRF, originating from interneurons, and PACAP, expressed mainly by pyramidal cells, finely tune the excitability and gene expression in the neocortical network via distinct cAMP/PKA-mediated effects.

Spatiotemporal patterns of electrocorticographic very fast oscillations (&gt;80 Hz) consistent with a network model based on electrical coupling between principal neurons

We sought to characterize spatial and temporal patterns of electrocorticography (ECoG) very fast oscillations (> ∼80 Hz, VFOs) prior to seizures in human frontotemporal neocortex, and to develop a testable network model of these patterns. ECoG data were recorded with subdural grids from two preoperative patients with seizures of frontal lobe onset in an epilepsy monitoring unit. VFOs were recorded from rat neocortical slices. A "cellular automaton" model of network oscillations was developed, extending ideas of Traub et al. (Neuroscience, 92, 1999, 407) and Lewis & Rinzel (Network: Comput Neural Syst, 11, 2000, 299); this model is based on postulated electrical coupling between pyramidal cell axons. Layer 5 of rat neocortex, in vitro, can generate VFOs when chemical synapses are blocked. Human epileptic neocortex, in situ, produces preseizure VFOs characterized by the sudden appearance of "blobs" of activity that evolve into spreading wavefronts. When wavefronts meet, they coalesce and propagate perpendicularly but never pass through each other. This type of pattern has been described by Lewis & Rinzel in cellular automaton models with spatially localized connectivity, and is demonstrated here with 120,000- to 5,760,000-cell models. We provide a formula for estimating VFO period from structural parameters and estimate the spatial scale of the connectivity. These data provide further evidence, albeit indirect, that preseizure VFOs are generated by networks of pyramidal neurons coupled by gap junctions, each predominantly confined to pairs of neurons having somata separated by < ∼1-2 mm. Plausible antiepileptic targets are tissue mechanisms, such as pH regulation, that influence gap-junction conductance.

N-desmethylclozapine (NDMC) is an antagonist at the human native muscarinic M1 receptor

N-desmethylclozapine (NDMC) has been reported to display partial agonism at the human recombinant and rat native M(1) mAChR, a property suggested to contribute to the clinical efficacy of clozapine. However, the profile of action of NDMC at the human native M(1) mAChR has not been reported. The effect of NDMC on M(1) mAChR function was investigated in human native tissues by assessing its effect on (1) M(1) mAChR-mediated stimulation of [(35)S]-GTPgammaS-G(q/11)alpha binding to human post mortem cortical membranes and (2) the M(1) mAChR-mediated increase in neuronal firing in human neocortical slices. NDMC displayed intrinsic activities of 46+/-9%, compared to oxo-M, at the human recombinant M(1) receptor, in FLIPR studies and 35+/-4% at rat native M(1) receptors in [(35)S]-GTPgammaS-G(q/11)alpha binding studies. In [(35)S]-GTPgammaS-G(q/11)alpha binding studies in human cortex, oxo-M stimulated binding by 240+/-26% above basal with a pEC(50) of 6.56+/-0.05. In contrast, NDMC did not stimulate [(35)S]-GTPgammaS-G(q/11)alpha binding to human cortical membranes but antagonised the response to oxo-M (2microM) showing a pK(B) of 6.8, comparable to its human recombinant M(1) mAChR affinity (pK(i)=6.9) derived from [(3)H]-NMS binding studies. In human, contrary to the rat neocortical slices, NDMC did not elicit a significant increase in M(1) mAChR-mediated neuronal firing, and attenuated a carbachol-induced increase in neuronal firing when pre-applied. These data indicate that, whereas NDMC displays moderate to low levels of partial agonism at the human recombinant and rat native M(1) mAChR, respectively, it acts as an antagonist at the M(1) mAChR in human cortex.

Glutamine Is Required for Persistent Epileptiform Activity in the Disinhibited Neocortical Brain Slice

The neurotransmitter glutamate is recycled through an astrocytic-neuronal glutamate-glutamine cycle in which synaptic glutamate is taken up by astrocytes, metabolized to glutamine, and transferred to neurons for conversion back to glutamate and subsequent release. The extent to which neuronal glutamate release is dependent upon this pathway remains unclear. Here we provide electrophysiological and biochemical evidence that in acutely disinhibited rat neocortical slices, robust release of glutamate during sustained epileptiform activity requires that neurons be provided a continuous source of glutamine. We demonstrate that the uptake of glutamine into neurons for synthesis of glutamate destined for synaptic release is not strongly dependent on the system A transporters, but requires another unidentified glutamine transporter or transporters. Finally, we find that the attenuation of network activity through inhibition of neuronal glutamine transport is associated with reduced frequency and amplitude of spontaneous events detected at the single-cell level. These results indicate that availability of glutamine influences neuronal release of glutamate during periods of intense network activity.

Impaired function of GABAB receptors in tissues from pharmacoresistant epilepsy patients

Effects of pre- and postsynaptic γ-aminobutyric acid B (GABA(B)) receptor activation were characterized in human tissue from epilepsy surgery. Slices of human cortical tissue were investigated in a submerged-type chamber with intracellular recordings in layers II/III. Parallel experiments were performed in rat neocortical slices with identical methods. Synaptic responses were elicited with single or paired stimulations of incrementing intervals. Neurons in human epileptogenic tissue exhibited usually small inhibitory postsynaptic potentials (IPSP) mediated by GABA(B) receptor, verified by the sensitivity to the selective antagonist CGP 55845A. The IPSP(B) conductance averaged 5.8 nS in neurons from epileptogenic tissues and 15.9 nS in neurons from nonepileptogenic tissues (p < 0.0001). Application of baclofen caused small conductance increases in human neurons, which were linearly related to IPSP(B) conductances. Paired-pulse stimulation revealed constant synaptic responses in human temporal lobe epilepsy (TLE) slices at all interstimulus intervals (ISIs). Pharmacologically isolated IPSP(A) in the human tissue exhibited a small paired-pulse depression (average 10% at 500 ms ISI). Bicuculline-induced paroxysmal depolarization shifts (PDSs) were transiently depressed by 24% in human TLE tissue; and by 74% in rat neocortical slices (200 ms ISI; p = 0.015). The depressions of bicuculline-induced PDSs were antagonized by CGP 55845A in both species. Staining for GABA(B) receptors revealed significantly smaller numbers of immunopositive dots in human epileptogenic neurons versus human control neurons. The small IPSP(B), baclofen-conductances, and paired-pulse depression of PDSs and IPSPs in human TLE tissue indicate a reduced density of post- and presynaptic GABA(B) receptors. The reduced efficacy of presynaptic GABA(B) receptors facilitates the occurrence of repetitive synaptic activity.

Brivaracetam inhibits spreading depression in rat neocortical slices in vitro

Epilepsy and migraine are episodic neurological disorders with marked co-morbidity, making migraine common among epileptic patients. Conversely, several antiepileptic drugs (AEDs) are used as migraine-preventive medication. Cortical spreading depression (CSD) represents a transient suppression of bioelectric activity and is considered a key event in migraine and stroke. This study assessed the novel AED candidate brivaracetam (BRV) vs. the chemically related AED levetiracetam in a rat neocortical slice model allowing consistent quantification of drug effects on CSD. CSD episodes were regularly elicited on slices upon delivery of calibrated KCl drops and were recorded via two micropipette electrodes. After control CSDs, the drug was added to the perfusion and five subsequent CSDs were elicited during drug perfusion. Effects were assessed via CSD amplitude ( Ampl) and duration at half-amplitude ( D 1/2). BRV, 10 and 32 μM reduced the Ampl and transiently the D 1/2. Levetiracetam, 32 and 100 μM had no effect on either Ampl or D 1/2. The anti-CSD effect of BRV in this in vitro model might suggest a potential anti-migraine activity of this compound, which warrants further investigation.