Cortical Brain Slices Research Articles

Protection of Respiratory Chain Enzymes from Ischaemic Damage in Adult Rat Brain Slices

The effect of aglycaemic hypoxia (AH) on the activity of the mitochondrial respiratory chain complexes was measured in superfused adult cortical brain slices. After 15 min of AH the activity of complex II-III was significantly reduced (by 45%) with no change in complex I or IV. Following 30 min of reperfusion the activities of complex II-III and IV were significantly reduced (by 45% and 20% respectively). These reductions in enzyme activity were abolished by removing the external calcium or by the addition of N omega-nitro-L-arginine (LNNA) or an analogue of superoxide dismutase (SOD) manganese [III] tetrakis 4-benzoic acid porphyrin (Mn-TBAP). These data suggest that a reactive oxygen species (ROS) such as peroxynitrite is involved in the reduction of mitochondrial complex activities following AH.

Spatiotemporally Graded NMDA Spike/Plateau Potentials in Basal Dendrites of Neocortical Pyramidal Neurons

Glutamatergic inputs clustered over approximately 20-40 microm can elicit local N-methyl-D-aspartate (NMDA) spike/plateau potentials in terminal dendrites of cortical pyramidal neurons, inspiring the notion that a single terminal dendrite can function as a decision-making computational subunit. A typical terminal basal dendrite is approximately 100-200 microm long: could it function as multiple decision-making subunits? We test this by sequential focal stimulation of multiple sites along terminal basal dendrites of layer 5 pyramidal neurons in rat somatosensory cortical brain slices, using iontophoresis or uncaging of brief glutamate pulses. There was an approximately sevenfold spatial gradient in average spike/plateau amplitude measured at the soma, from approximately 3 mV for distal inputs to approximately 23 mV for proximal inputs. Spike/plateaus were NMDA receptor (NMDAR) conductance-dominated at all locations. Large Ca(2+) transients accompanied spike/plateaus over a approximately 10- to 40-microm zone around the input site; smaller Ca(2+) transients extended approximately uniformly to the dendritic tip. Spike/plateau duration grew with increasing glutamate and depolarization; high Ca(2+) zone size grew with spike/plateau duration. The minimum high Ca(2+) zone half-width (just above NMDA spike threshold) increased from distal (approximately 10 microm) to proximal locations (approximately 25 microm), as did the NMDA spike glutamate threshold. Depolarization reduced glutamate thresholds. Simulations exploring multi-site interactions based on this demonstrate that if appropriately timed and localized inputs occur in vivo, a single basal dendrite could correspond to a cascade of multiple co-operating dynamic decision-making subunits able to retain information for hundreds of milliseconds, with increasing influence on neural output from distal to proximal. Dendritic NMDA spike/plateaus are thus well-suited to support graded persistent firing.

Memantine increases brain production of kynurenic acid via protein kinase A-dependent mechanism

We describe a novel aspect of action of memantine ex vivo, in the brain cortical slices and in vitro, in mixed glial cultures. The drug potently increased the production of kynurenic acid, an endogenous tryptophan metabolite blocking N-methyl-D-aspartate (NMDA) and nicotinic alpha7 receptors. In cortical slices memantine, an open-channel NMDA blocker (100-150 microM), but not the competitive NMDA receptor antagonist, LY235959 increased the production of kynurenic acid. Neither SCH23390, D1 receptor antagonist (50 microM) nor raclopride, D2 receptor antagonist (10 microM) changed the memantine-induced effects. Propranolol (100 microM) has partially reduced its action. Selective cAMP-dependent protein kinase (PKA) inhibitor, KT5720 (1 microM), but not selective protein kinase C (PKC) inhibitor, NPC15437 (30 microM) totally reversed the action of memantine. In mixed glial cultures, 2-24 h incubation with memantine (2-50 microM) enhanced the production of kynurenic acid. Memantine (up to 0.5 mM) has not affected the activity of kynurenic acid biosynthetic enzymes. The obtained data suggest that memantine enhances the production of kynurenic acid in PKA-mediated way. This effect may partially contribute to the therapeutic actions of memantine and be of a potential clinical importance.

Halothane induces vesicular and carrier-mediated release of [ 3H]serotonin from rat brain cortical slices

The serotonergic system may play a role during general anesthesia but the effect of the volatile anesthetic halothane on the release of serotonin (5-HT) is not fully understood. Rat brain cortical slices were labeled with [ 3H]5-HT to investigate the effects of halothane on the release of this neurotransmitter from the central nervous system. Halothane induced an increase on the release of [ 3H]5-HT that was dependent on incubation time and anesthetic concentration (0.006, 0.012, 0.024, 0.036, 0.048 and 0.072 mM). This effect was independent of extracellular calcium and was not affected by tetrodotoxin (blocker of voltage dependent Na + channels). In contrast, the halothane-evoked [ 3H]5-HT release was reduced by BAPTA-AM, a membrane-permeable BAPTA analog that chelates intracellular Ca 2+. The anesthetic-induced [ 3H]5-HT release depends on the ryanodine-sensitive intracellular calcium store since it was blocked by dantrolene and azumolene (inhibitors of the calcium-release through ryanodine receptors) but was not affected by aminoethoxydiphenylborate (2-APB), an inhibitor of inositol 1,4,5-triphosphate receptor. The [ 3H]5-HT release induced by halothane comes mainly from the vesicular pool since it was reduced in about 70% by reserpine, a blocker of vesicular monoamine transporter. The halothane-evoked release of [ 3H]5-HT release is reduced by fluoxetine, an inhibitor of 5-HT uptake, and the volatile agent also decreased the uptake of [ 3H]5-HT into rat brain cortical slices. Moreover, a decrease on halothane-induced release of [ 3H]5-HT was also observed when the brain cortical slices were incubated at low temperature, which is known to interfere with the carrier-mediated release of the neurotransmitter. Ouabain, a Na +/K + ATPase pump inhibitor, which induces 5-HT release through reverse transport, also decreased [ 3H]5-HT release induced by halothane, confirming the involvement of a carrier-mediated release of the neurotransmitter in the presence of halothane. In conclusion, these data suggest that halothane induces vesicular and carrier-mediated release of [ 3H]5-HT in rat brain cortical slices.

Neuroprotection Afforded by GABA Against Oxygen-Glucose Deprivation-Induced Injury in Rat Cortical Brain Slices: An Hormetic Dose-Response Effect

The aim of the present investigation was to assess neuroprotection exerted by GABA (100 nM-100 mM) in rat cortical brain slices subjected to oxygen-glucose deprivation and reoxygenation. Neuronal injury and neuroprotection were assessed by measuring the release of glutamate and lactate dehydrogenase and tissue water content. Results demonstrate that GABA exerted neuroprotective effects according to a U-shaped, hormetic-like, concentration-response curve, with an efficacy window of 10-100 I¼M concentration. In order to verify whether GABA-neuroprotective effects were GABAA- or GABAB-mediated, Muscimol (GABAA agonist) and Baclofen (GABAB agonist) were tested. Results demonstrated that only Muscimol exerted neuroprotection according to a U-shaped, hormetic-like, concentration-response curve, while baclofen was ineffective. In conclusion, the hormetic response of GABA or GABAA agonists should be taken into consideration when designing experiments aimed at assessing neuroprotection by these agents against ischemia/reoxygenation injury.

Modulation of brain metabolism by very low concentrations of the commonly used drug delivery vehicle dimethyl sulfoxide (DMSO)

Dimethyl sulfoxide (DMSO) has long been used in studies as a vehicle to enhance the solubility and transport of ligands in biological systems. The effects of this drug on the outcomes of such studies are still unclear, with concentrations of DMSO reported as "safe" varying considerably. In the present work, we investigated the effects of very low concentrations of DMSO on the brain metabolism of [3-(13)C]pyruvate and D-[1-(13)C]glucose using (1)H/(13)C NMR spectroscopy and a guinea pig cortical brain slice model. Our results show that DMSO is accumulated by brain slices. DMSO at all concentrations [0.000025%-0.25% (v/v)] increased the metabolic rate when [3-(13)C]pyruvate was used as a substrate and also in the presence of D-[1-(13)C]glucose (0.00025%-0.1% DMSO). These results are consistent with DMSO stimulating respiration, which it may do through altering the kinetics of ATP-requiring reactions. Our results also emphasize that there is no practical concentration of DMSO that can be used in metabolic experiments without effect. Therefore, care should be taken when evaluating the actions of drugs administered in combination with DMSO.

Halothane increases non-vesicular [(3)H]dopamine release from brain cortical slices.

Experimental data suggest that halothane anesthesia is associated with significant changes in dopamine (DA) concentration in some brain regions but the mechanism of this effect is not well known. Rat brain cortical slices were labeled with [(3)H]DA to further characterize the effects of halothane on the release of this neurotransmitter from the central nervous system. Halothane induced an increase on the release of [(3)H]DA that was dependent on incubation time and anesthetic concentration (0.012, 0.024, 0.048, 0.072 and 0.096 mM). This effect was independent of extracellular or intracellular calcium. In addition, [(3)H]DA release evoked by halothane was not affected by TTX (blocker of voltage-dependent Na(+) channels) or reserpine (a blocker of vesicular monoamine transporter). These data suggest that [(3)H]DA release induced by halothane is non-vesicular and would be mediated by the dopamine transporter (DAT) and norepinephrine transporter (NET). GBR 12909 and nomifensine, inhibitors of DAT, decreased the release of [(3)H]DA evoked by halothane. Nisoxetine, a blocker of NET, reduced the release of [(3)H]DA induced by halothane. In addition, GBR 12909, nisoxetine and, halothane decrease the uptake of [(3)H]DA into rat brain cortical slices. A decrease on halothane-induced release of [(3)H]DA was also observed when the brain cortical slices were incubated at low temperature and low extracellular sodium, which are known to interfere with the carrier-mediated release of the neurotransmitter. Ouabain, a Na(+)/K(+) ATPase pump inhibitor, which induces DA release through reverse transport, decreased [(3)H]DA release induced by halothane. It is suggested that halothane increases [(3)H]DA release in brain cortical slices that is mediated by DAT and NET present in the plasma membrane.

Functional mapping of cortical areas with optical imaging

Sensory areas in mammalian cortex compute sensory inputs of different modalities in order to perceive the environment. Much is known about the anatomical pattern of inter-laminar connections, which form the basis of the computational process. Nevertheless, less is known about the functional relevance of these wiring patterns. We used intrinsic optical signals (IOSs) in vitro to investigate functional properties of inter-laminar connections in cortical brain slices of rat sensory cortex. By electrical stimulation in layer VI, a columnar-shaped IOS in all cortical areas was found. We detected different laminar patterns of activation in different cortical areas. In primary sensory areas, like primary visual cortex and primary somatosensory cortex, the peak intensity of IOSs occurred in layer IV, which receives the main thalamic input. In secondary sensory areas, like the secondary visual cortex or the secondary somatosensory cortex, the maximum of IOSs amplitude was shifted to layer II/III. In motor areas, IOS peak amplitude is located in layer II/III. In the hind limb area, considered as amalgam between sensory and motor function, a mixture of the activity patterns observed in primary sensory and a motor area occurred with a peak amplitude in layers II and IV. At different stimulation sites within one cortical area, the shape of columnar IOSs remained very similar, reflecting a canonical architecture of functional micro-circuitry. We conclude that both primary and secondary sensory cortical areas display their characteristic functional activation pattern, regardless of their sensory modalities.

Alanine metabolism, transport, and cycling in the brain

Brain glutamate/glutamine cycling is incomplete without return of ammonia to glial cells. Previous studies suggest that alanine is an important carrier for ammonia transfer. In this study, we investigated alanine transport and metabolism in Guinea pig brain cortical tissue slices and prisms, in primary cultures of neurons and astrocytes, and in synaptosomes. Alanine uptake into astrocytes was largely mediated by system L isoform LAT2, whereas alanine uptake into neurons was mediated by Na(+)-dependent transporters with properties similar to system B(0) isoform B(0)AT2. To investigate the role of alanine transport in metabolism, its uptake was inhibited in cortical tissue slices under depolarizing conditions using the system L transport inhibitors 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid and cycloleucine (1-aminocyclopentanecarboxylic acid; cLeu). The results indicated that alanine cycling occurs subsequent to glutamate/glutamine cycling and that a significant proportion of cycling occurs via amino acid transport system L. Our results show that system L isoform LAT2 is critical for alanine uptake into astrocytes. However, alanine does not provide any significant carbon for energy or neurotransmitter metabolism under the conditions studied.

Hypoosmotic swelling and ammonia increase oxidative stress by NADPH oxidase in cultured astrocytes and vital brain slices

The role of NADPH oxidase (NOX) and the regulatory subunit p47(phox) for hypoosmotic ROS generation was studied in cultured rat astrocytes and brain slices of wilde type and p47(phox) knock-out mice. Cultured rat astrocytes express mRNAs encoding for the regulatory subunit p47(phox), NOX1, 2, and 4, and the dual oxidases (DUOX)1 and 2, but not NOX3. Hypoosmotic (205 mosmol/L) swelling of cultured astrocytes induced a rapid generation of ROS that was accompanied by serine phosphorylation of p47(phox) and prevented by the NADPH oxidase inhibitor apocynin. Apocynin also impaired the hypoosmotic tyrosine phosphorylation of Src. Both, hypoosmotic ROS generation and p47(phox) serine phosphorylation were sensitive to the acidic sphingomyelinase inhibitors AY9944 and desipramine, the protein kinase C (PKC)zeta-inhibitory pseudosubstrate peptide, the NMDA receptor antagonist MK-801 and the intracellular Ca(2+) chelator BAPTA-AM. Also hypoosmotic exposure of wilde type mouse cortical brain slices increased ROS generation, which was allocated in part to the astrocytes and which was absent in presence of apocynin and in cortical brain slices from p47(phox) knock-out mice. Also ammonia induced a rapid ROS production in cultured astrocytes and brain slices, which was sensitive to apocynin. The data suggest that astrocyte swelling triggers a p47(phox)-dependent NADPH oxidase-catalyzed ROS production. The findings further support a close interrelation between osmotic and oxidative stress in astrocytes, which may be relevant to different brain pathologies including hepatic encephalopathy.

The effect of sevoflurane on the release of [ 3H]dopamine from rat brain cortical slices

Dopamine is a neurotransmitter that exerts major control on important brain functions and some lines of studies suggest that dopaminergic neurotransmission may be a potential target for volatile anesthetics. In the present study, rat brain cortical slices were labeled with [ 3H]dopamine to investigate the effects of sevoflurane on the release of this neurotransmitter. [ 3H]dopamine release was significantly increased in the presence of sevoflurane (0.46 mM) and this effect was independent of extracellular or intracellular calcium. In addition, [ 3H]dopamine release evoked by sevoflurane was not affected by TTX (blocker of voltage-dependent sodium channels) or reserpine (a blocker of the vesicular monoamine transporter). These data suggest that the dopamine release induced by sevoflurane is non-vesicular, independent of exocytosis and, would be mediated by the dopamine transporter (DAT). GBR12909 and nomifensine, inhibitors of DAT, decreased the release of [ 3H]dopamine evoked by sevoflurane. The same effect was also observed when the brain cortical slices were incubated at low temperature and low extracellular sodium. Ouabain, a Na +/K + ATPase pump inhibitor, which is known to induce dopamine release through reverse transport, decreased [ 3H]dopamine release induced by sevoflurane. In conclusion, the present study suggests that sevoflurane increases [ 3H]dopamine release in brain cortical slices that is mediated by DAT located at the plasma membrane.

Neuroprotection afforded by diazepam against oxygen/glucose deprivation-induced injury in rat cortical brain slices

The aim of the present investigation was to assess neuroprotection exerted by diazepam (0.1–25 μM) in rat cortical brain slices subjected to oxygen–glucose deprivation and reoxygenation. Neuronal injury and neuroprotection were assessed by measuring the release of glutamate and lactate dehydrogenase and tissue water content. Results demonstrate that diazepam exerted neuroprotective effects according to a “U-shaped”, hormetic-like, concentration-response curve, with an efficacy window of 0.5–5 μM concentration. Flumazenil (20 μM) fully antagonised neuroprotection afforded by 5 μM diazepam. In conclusion, the hormetic response of diazepam should be taken into consideration when designing experiments aimed at assessing diazepam neuroprotection against ischemia/reoxygenation injury.

Role of Ca2+‐activated K+ channels in 11,12‐epoxyeicosatrienoic acid‐induced vasodilation of parenchymal arterioles: possible role of astrocytes

A current hypothesis suggests that glutamate-induced Ca2+ changes in astrocytes leads to arachidonic acid mobilization and production and release of epoxyeicosatrienoic acids (EETs), which diffuse to smooth muscle cells and mediate vasodilation through their action on Ca2+-activated K+ channels (BK channels). Here, we present an alternative hypothesis where EETs’ action in the neurovascular response is initiated at the level of the astrocytes, in particular the endfeet, resulting in BK channel activation contributing to K+-induced vasodilation. Using confocal Ca2+ imaging techniques, electrophysiology and video microscopy from cortical brain slices, we observed a robust increase in astrocytic Ca2+in response to 11,12-EET. Interestingly, while this response occurred rapidly (~2–15 sec), EET-induced arteriolar dilation (~46% from baseline) was significantly delayed (~2 min), suggesting that the locus of EETs’ primary neurovascular action might be at the level of the astrocytes. In addition, we also observed a significant increase in whole-cell outward currents from astrocytes in response to 11,12-EET, and suppression of EET-induced arteriolar dilation in the presence of the BK channel blocker TEA (1mM). Our data support the novel hypothesis that EETs, by increasing intracellular Ca2+ in astrocytes, may modulate BK channel activity and thus K+-induced dilation of parenchymal arterioles.

Dynamic Inositol Trisphosphate-mediated Calcium Signals within Astrocytic Endfeet Underlie Vasodilation of Cerebral Arterioles

Active neurons communicate to intracerebral arterioles in part through an elevation of cytosolic Ca2+ concentration ([Ca2+]i) in astrocytes, leading to the generation of vasoactive signals involved in neurovascular coupling. In particular, [Ca2+]i increases in astrocytic processes (“endfeet”), which encase cerebral arterioles, have been shown to result in vasodilation of arterioles in vivo. However, the spatial and temporal properties of endfoot [Ca2+]i signals have not been characterized, and information regarding the mechanism by which these signals arise is lacking. [Ca2+]i signaling in astrocytic endfeet was measured with high spatiotemporal resolution in cortical brain slices, using a fluorescent Ca2+ indicator and confocal microscopy. Increases in endfoot [Ca2+]i preceded vasodilation of arterioles within cortical slices, as detected by simultaneous measurement of endfoot [Ca2+]i and vascular diameter. Neuronal activity–evoked elevation of endfoot [Ca2+]i was reduced by inhibition of inositol 1,4,5-trisphosphate (InsP3) receptor Ca2+ release channels and almost completely abolished by inhibition of endoplasmic reticulum Ca2+ uptake. To probe the Ca2+ release mechanisms present within endfeet, spatially restricted flash photolysis of caged InsP3 was utilized to liberate InsP3 directly within endfeet. This maneuver generated large amplitude [Ca2+]i increases within endfeet that were spatially restricted to this region of the astrocyte. These InsP3-induced [Ca2+]i increases were sensitive to depletion of the intracellular Ca2+ store, but not to ryanodine, suggesting that Ca2+-induced Ca2+ release from ryanodine receptors does not contribute to the generation of endfoot [Ca2+]i signals. Neuronally evoked increases in astrocytic [Ca2+]i propagated through perivascular astrocytic processes and endfeet as multiple, distinct [Ca2+]i waves and exhibited a high degree of spatial heterogeneity. Regenerative Ca2+ release processes within the endfeet were evident, as were localized regions of Ca2+ release, and treatment of slices with the vasoactive neuropeptides somatostatin and vasoactive intestinal peptide was capable of inducing endfoot [Ca2+]i increases, suggesting the potential for signaling between local interneurons and astrocytic endfeet in the cortex. Furthermore, photorelease of InsP3 within individual endfeet resulted in a local vasodilation of adjacent arterioles, supporting the concept that astrocytic endfeet function as local “vasoregulatory units” by translating information from active neurons into complex InsP3-mediated Ca2+ release signals that modulate arteriolar diameter.

Local potassium signaling couples neuronal activity to vasodilation in the brain

The mechanisms by which active neurons, via astrocytes, rapidly signal intracerebral arterioles to dilate remain obscure. Here we show that modest elevation of extracellular potassium (K+) activated inward rectifier K+ (Kir) channels and caused membrane potential hyperpolarization in smooth muscle cells (SMCs) of intracerebral arterioles and, in cortical brain slices, induced Kir-dependent vasodilation and suppression of SMC intracellular calcium (Ca2+) oscillations. Neuronal activation induced a rapid (<2 s latency) vasodilation that was greatly reduced by Kir channel blockade and completely abrogated by concurrent cyclooxygenase inhibition. Astrocytic endfeet exhibited large-conductance, Ca2+-sensitive K+ (BK) channel currents that could be activated by neuronal stimulation. Blocking BK channels or ablating the gene encoding these channels prevented neuronally induced vasodilation and suppression of arteriolar SMC Ca2+, without affecting the astrocytic Ca2+ elevation. These results support the concept of intercellular K+ channel-to-K+ channel signaling, through which neuronal activity in the form of an astrocytic Ca2+ signal is decoded by astrocytic BK channels, which locally release K+ into the perivascular space to activate SMC Kir channels and cause vasodilation.

Anaesthesia in patients suffering from organophosphorus intoxication—interactions between general anaesthetics and acetylcholine in cortical networks in vitro

In scenarios of mass destruction it is likely that victims are intoxicated by organophosphates and, at the same time, physically injured. Organophosphate compounds produce excessive cholinergic overstimulation in the CNS via blocking acetylcholinesterase activity. The specifics of acute care and anaesthesia in physically traumatized and intoxicated patients are largely unknown. Recent studies in animals and human subjects demonstrated that acetylcholinesterase inhibitors reverse anaesthesia. Two distinct mechanisms are potentially involved. First, acetylcholine produces an excitatory drive onto neurons, thereby counterbalancing the inhibitory actions of anaesthetics. Anaesthesia is reversed because it critically depends on a distinctive depression of several central nervous functions. Second, cholinergic stimulation may affect the mechanisms by which anaesthetics mediate their depressant actions on central neurons. In this case acetylcholine reverses anaesthesia by decreasing the potency of anaesthetic agents. In order to identify potential mechanisms involved in cholinergic reversal of anaesthesia we have investigated interactions between acetylcholine and the volatile anaesthetic sevoflurane in isolated cortical brain slices. Our results provide evidence that cholinergic stimulation counterbalances the effects of general anaesthetics by increasing neuronal excitability, and, in addition, by decreasing anaesthetic potency. These findings imply that in patients suffering from organophosphorus intoxication dose requirements for general anaesthetics are considerably increased.

A Metabolomic Approach to Ionotropic Glutamate Receptor Subtype Function: A Nuclear Magnetic Resonance in vitro Investigation

A range of behaviours are elucidated via ionotropic glutamate receptors (iGluR). In this work, we examined the acute activation of iGluRs by a range of receptor ligands and effectors to see whether distinguishable metabolic sequelae were elucidated by the activity. We used a guinea-pig brain cortical tissue slice model using targeted receptor ligands ((RS)-(tetrazol-5-yl)glycine (TZG), (5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801, dizocilpine), cis-4-[phosphomethyl]-piperidine-2-carboxylic acid (CGS 19755), (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, (2S, 3S, 4S)-2-carboxy-4-(1-methylethenyl)-3-pyrrolidineacetic acid (kainate) and D-serine (D-Ser), as well as compounds (quinolinic acid and kynurenic acid (KynA)) involved in some neuroinflammatory responses. The data were derived using 13C and 1H NMR spectroscopy, and analysed by metabolomic approaches and multivariate statistics. The metabolic effects of agonists at the three major classes of iGluR were easily separated from each other using this method. The classical N-methyl-D-aspartate receptor agonist TZG and the antagonist CGS 19755 produced excitatory and inhibitory metabolic responses, respectively, while the blocker MK-801 resulted in a significant decrease in net metabolism and produced the largest decrease in all metabolite pool sizes seen by any glutamatergic ligand we have studied. Quinolinic acid and KynA produced similar acute metabolic responses, which were unlike those to TZG or CGS 19755, but similar to that of D-Ser. D-Ser was highly stimulatory of net flux into the Krebs cycle. These data show that the metabolic response to iGluR perturbation in vitro is a sensitive discriminator of function.

Constitutive activity of H 3 autoreceptors modulates histamine synthesis in rat brain through the cAMP/PKA pathway

We previously described that agonist-activated histamine H 3 autoreceptors inhibit the stimulation of histamine synthesis mediated by calcium/calmodulin- and cAMP-dependent protein kinases (CaMKII and PKA respectively) in histaminergic nerve endings. In the absence of an agonist H 3 receptors show partial constitutive activity, so we hypothesized that suppression of constitutive activity by an inverse agonist could stimulate these transduction pathways. We show here that the H 3 inverse agonist thioperamide increases histamine synthesis in rat brain cortical slices independently from the amounts of extracellular histamine. Thioperamide effects were mimicked by the inverse agonists clobenpropit and A-331440, but not by the neutral antagonist VUF-5681. In contrast, coincubation with VUF-5681 suppressed thioperamide effects. The effects of thioperamide were completely blocked by the PKA inhibitor peptide myristoyl-PKI 14–22, a peptide that did not block depolarization stimulation of histamine synthesis. In addition, thioperamide effects required depolarization and were impaired by blockade of N-type calcium channels (mediating depolarization), but not by CaMKII inhibition. These results indicate that constitutive activity of H 3 receptors in rat brain cortex inhibits the adenylate cyclase/PKA pathway, and perhaps also the opening of N-type voltage sensitive calcium channels, but apparently not CaMKII.

MeasuringD-serine efflux from mouse cortical brain slices using online microdialysis-capillary electrophoresis

Efflux of a number of important neurochemicals, including D-serine, L-serine, taurine, glutamate, and gamma-aminobutyric acid (GABA), from mouse cortical brain slices housed in a 7 microL perfusion chamber was monitored using online microdialysis-CE (MD-CE). Analyte concentrations could be measured every 20-27 s using the MD-CE instrument. Stimulation with high potassium induced increased release of D-serine. Kainic acid (KA) induced D-serine release, but this release was not blocked by 6-cyano-7-nitroquinoxaline-2,3-dione, suggesting that alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid/KA receptors do not mediate D-serine release. Application of L-serine, the precursor of D-serine, resulted in increased extracellular D-serine concentrations. L-Cysteine also increased extracellular D-serine levels in a partially Na+-dependent manner. The observed effects upon application of L-serine and L-cysteine support the involvement of ASC neutral amino acid transporters in regulating the extracellular concentration of D-serine concentration through competitive inhibition of uptake or increased release through heteroexchange.

Inhibition of rabbit brain 4-aminobutyrate transaminase by some taurine analogues: A kinetic analysis

The use of the antiepileptic drug, 4-aminobutyrate transaminase (GABA-T) inhibitor vigabatrin (VIGA), has been recently cautioned because it is associated to irreversible field defects from damage of the retina. Since novel GABA-T inhibitors might prove useful in epilepsy or other CNS pathologies as VIGA substitutes, the aim of the present investigation was to characterize the biochemical properties of some taurine analogues (TA) previously shown to act as GABA-T inhibitors. These include (±)piperidine-3-sulfonic acid (PSA), 2-aminoethylphosphonic acid (AEP), (±)2-acetylaminocyclohexane sulfonic acid (ATAHS) and 2-aminobenzenesulfonate (ANSA). Kinetic analysis of the activity of partially purified rabbit brain GABA-T in the presence of VIGA and TA showed that PSA and AEP caused a linear, mixed-type inhibition ( K i values 364 and 1010 μM, respectively), whereas VIGA, ANSA and ATAHS behaved like competitive inhibitors ( K i values 320, 434 and 598 μM, respectively). Among the compounds studied, only VIGA exerted a time-dependent, irreversible inhibition of the enzyme, with K i and k inact values of 773 μM and 0.14 min −1, respectively. Furthermore, the ability of VIGA and TA to enhance GABA-ergic transmission was assessed in rabbit brain cortical slices by NMR quantitative analysis. The results demonstrate that VIGA as well as all TA promoted a significant increase of GABA content. In conclusion, PSA, ANSA and ATAHS, reversible GABA-T inhibitors with K i values close to that of VIGA, represent a new class of compounds, susceptible of therapeutic exploitation in many disorders associated with low levels of GABA in brain tissues.