Rat Cortical Slices Research Articles

Toll-like receptor 7: A novel neuroimmune target to reduce excessive alcohol consumption

Toll-like receptors (TLRs) are a family of innate immune receptors that recognize molecular patterns in foreign pathogens and intrinsic danger/damage signals from cells. TLR7 is a nucleic acid sensing endosomal TLR that is activated by single-stranded RNAs from microbes or by small noncoding RNAs that act as endogenous ligands. TLR7 signals through the MyD88 adaptor protein and activates the transcription factor interferon regulatory factor 7 (IRF7). TLR7 is found throughout the brain and is highly expressed in microglia, the main immune cells of the brain that have also been implicated in alcohol drinking in mice. Upregulation of TLR7 mRNA and protein has been identified in postmortem hippocampus and cortex from AUD subjects that correlated positively with lifetime consumption of alcohol. Similarly, Tlr7 and downstream signaling genes were upregulated in rat hippocampal and cortical slice cultures after chronic alcohol exposure and in these regions after chronic binge-like alcohol treatment in mice. In addition, repeated administration of the synthetic TLR7 agonists imiquimod (R837) or resiquimod (R848) increased voluntary alcohol drinking in different rodent models and produced sustained upregulation of IRF7 in the brain. These findings suggest that chronic TLR7 activation may drive excessive alcohol drinking. In the brain, this could occur through increased levels of endogenous TLR7 activators, like microRNAs and Y RNAs. This review explores chronic TLR7 activation as a pathway of dysregulated neuroimmune signaling in AUD and the endogenous small RNA ligands in the brain that could perpetuate innate immune responses and escalate alcohol drinking.

Novel Activity of Oral Hypoglycemic Agents Linked with Decreased Formation of Tryptophan Metabolite, Kynurenic Acid.

Kynurenic acid is a tryptophan (Trp) metabolite formed along the kynurenine (KYN) pathway in the brain and in peripheral tissues. The disturbed formation of kynurenic acid, which targets glutamate-mediated neurotransmission, GPR35, and aryl hydrocarbon receptors of immune or redox status, was implicated in the development of neuropsychiatric and metabolic disorders among others. Kynurenic acid exerts neuroprotective and immunomodulatory effects, yet its high brain levels may negatively impact cognition. Changes in the Trp-KYN pathway are also linked with the pathogenesis of diabetes mellitus, which is an established risk factor for cardiovascular and neurological diseases or cognitive deficits. Here, the effects of metformin and glibenclamide on the brain synthesis of kynurenic acid were evaluated. Acute exposure of rat cortical slices in vitro to either of the drugs reduced kynurenic acid production de novo. Glibenclamide, but not metformin, inhibited the activity of kynurenic acid biosynthetic enzymes, kynurenine aminotransferases (KATs) I and II, in semi-purified cortical homogenates. The reduced availability of kynurenic acid may be regarded as an unwanted effect, possibly alleviating the neuroprotective action of oral hypoglycemic agents. On the other hand, considering that both compounds ameliorate the cognitive deficits in animal and human studies and that high brain kynurenic acid may hamper learning and memory, its diminished synthesis may improve cognition.

Interplay between metabotropic glutamate type 4 and adenosine type 1 receptors modulate synaptic transmission in the cerebellar cortex.

The synapses between parallel fibers and Purkinje cells play a pivotal role in cerebellar function. They are intricately governed by a variety of presynaptic receptors, notably by type 4 metabotropic glutamate (mGlu4) receptors and type 1 adenosine (A1) receptors both of which curtail glutamate release upon activation. Despite their pivotal role in regulating synaptic transmission within the cerebellar cortex, functional interactions between mGlu4 and A1 receptors have remained relatively unexplored. To bridge this gap, our study delves into how mGlu4 receptor activity influences A1 receptor-mediated alterations in excitatory transmission. Employing a combination of whole-cell patch clamp recordings of Purkinje cells and parallel fiber presynaptic fluorometric calcium measurements in acute rat and mouse cerebellar cortical slices, our results reveal functional interactions between these receptor types. These findings hold implications for understanding potential roles of these presynaptic receptors in neuroprotection during pathophysiological conditions characterized by elevated glutamate and adenosine levels.

Involvement of P2Y1, P2Y6, A1 and A2A Receptors in the Purinergic Inhibition of NMDA-Evoked Noradrenaline Release in the Rat Brain Cortex.

In the cerebral cortex, glutamate activates NMDA receptors (NMDARs), localized in noradrenergic neurons, inducing noradrenaline release that may have a permissive effect on glutamatergic transmission, and therefore, on the modulation of long-term plasticity. ATP is co-released with noradrenaline, and with its metabolites (ADP and adenosine) is involved in the purinergic modulation of electrically-evoked noradrenaline release. However, it is not known if noradrenaline release evoked by activation of NMDARs is also under purinergic modulation. The present study aimed to investigate and to characterize the purinergic modulation of noradrenaline release evoked by NMDARs. Stimulation of rat cortical slices with 30 µM NMDA increased noradrenaline release, which was inhibited by ATP upon metabolization into ADP and adenosine and by the selective agonists of A1 and A2A receptors, CPA and CGS2680, respectively. It was also inhibited by UTP and UDP, which are mainly released under pathophysiological situations. Characterization of the effects mediated by these compounds indicated the involvement of P2Y1, P2Y6, A1 and A2A receptors. It is concluded that, in the rat brain cortex, NMDA-evoked noradrenaline release is modulated by several purinergic receptors that may represent a relevant mechanism to regulate the permissive effect of noradrenaline on NMDA-induced neuroplasticity.

Bromovalerylurea modulates GABAA receptor-mediated inhibitory neurotransmission while inducing sleep

Bromovalerylurea (BU), an acyl urea derivative, was originally developed as a hypnotic/sedative. We recently reported that BU at a dose of 50 mg/kg ameliorates sepsis, Parkinson's disease, and traumatic brain injury in Wistar rat models through its anti-inflammatory actions on microglia and macrophages. However, since BU was developed more than 100 years ago, its hypnotic mechanism and characteristics are poorly understood. Herein, we conducted an electroencephalogram (EEG) study and found that BU, when administered at a dose of more than 125 mg/kg but not at a dose of 50 mg/kg in Wistar rats, significantly increased non-rapid eye movement (NREM) sleep duration and dose-dependently decreased rapid eye movement (REM) sleep duration. This characteristic of sleep induced by BU is similar to the effect of compounds such as barbiturate, benzodiazepine, and z-drugs, all of which require γ-aminobutyric acid A receptors (GABAAR) for hypnotic/sedative activity. To investigate whether BU could potentiate GABAAergic neurotransmission, we conducted a whole-cell patch-clamp recording from pyramidal neurons in rat cortical slices to detect spontaneous GABAAR-mediated inhibitory postsynaptic currents (IPSCs). We found that BU dose-dependently prolonged IPSCs. Importantly, the prolonged IPSCs were not attenuated by flumazenil, a benzodiazepine receptor antagonist, suggesting that modulation of IPSCs by BU is mediated by different mechanisms from that of benzodiazepine. Taken together, these data elucidate the basic characteristics of the hypnotic effects of BU and suggest that the enhancement of GABAAR-mediated Cl− flux may be a possible mechanism that contributes to its hypnotic/sedative activity.

Oleamide Reduces Mitochondrial Dysfunction and Toxicity in Rat Cortical Slices Through the Combined Action of Cannabinoid Receptors Activation and Induction of Antioxidant Activity.

The potential treatment of neurodegenerative disorders requires the development of novel pharmacological strategies at the experimental level, such as the endocannabinoid-based therapies. The effects of oleamide (OEA), a fatty acid primary amide with activity on cannabinoid receptors, was tested against mitochondrial toxicity induced by the electron transport chain complex II inhibitor, 3-nitropropionic acid (3-NP), in rat cortical slices. OEA prevented the 3-NP-induced loss of mitochondrial function/cell viability at a concentration range of 5nM-25µM, and this protective effect was observed only when the amide was administered as pretreatment, but not as post-treatment. The preservation of mitochondrial function/cell viability induced by OEA in the toxic model induced by 3-NP was lost when the slices were pre-incubated with the cannabinoid receptor 1 (CB1R) selective inhibitor, AM281, or the cannabinoid receptor 2 (CB2R) selective inhibitor, JTE-907. The 3-NP-induced inhibition of succinate dehydrogenase (mitochondrial Complex II) activity was recovered by 25nM OEA. The amide also prevented the increased lipid peroxidation and the changes in reduced/oxidized glutathione (GSH/GSSG) ratio induced by 3-NP. The cell damage induced by 3-NP, assessed as incorporation of cellular propidium iodide, was mitigated by OEA. Our novel findings suggest that the neuroprotective properties displayed by OEA during the early stages of damage to cortical cells involve the converging activation of CB1R and CB2R and the increase in antioxidant activity, which combined may emerge from the preservation of the functional integrity of mitochondria.

Alpha-Mangostin Alleviates the Short-term 6-Hydroxydopamine-Induced Neurotoxicity and Oxidative Damage in Rat Cortical Slices and in Caenorhabditis elegans.

The development, at the experimental level, of therapeutic strategies based on natural products to attenuate neurological alterations in degenerative disorders has gained attention. Antioxidant molecules exhibit both anti-inflammatory and neuroprotective properties. Alpha-mangostin (α-Man) is a natural xanthonoid isolated from the mangosteen tree with demonstrated antioxidant and cytoprotective properties. In this study, we investigated the antioxidant and protective properties of α-Man, both ex vivo and in vivo. We assessed the mitochondrial reductant capacity and oxidative damage to lipids in rat cortical slices, and several endpoints characteristic of physiological stress in the nematode, Caenorhabditis elegans (C. elegans), upon exposure to the parkinsonian neurotoxin, 6-hydroxydopamine (6-OHDA). In rat cortical slices, α-Man (25 and 50 µM) reduced the 6-OHDA (100 µM)-induced oxidative damage to lipid levels, but failed to reverse loss in cell viability. In wild-type (N2) C. elegans, α-Man (5-100 µM) protected against 6-OHDA (25 mM)-induced decrease in survival when administered either as pre- or post-treatment. Protective effects of α-Man were also observed on survival in the VC1772 strain (skn-1 KO-) exposed to 6-OHDA, though the extent of the protection was lesser than in the wild-type N2 strain. However, α-Man (5-50 µM) failed to attenuate the 6-OHDA-induced motor alterations in the N2 strain. The loss of lifespan induced by 6-OHDA in the N2 strain was fully reversed by high concentrations of α-Man. In addition, while 6-OHDA decreased the expression of glutathione S-transferase in the CL2166 C. elegans strain, α-Man preserved and stimulated the expression of this protein. α-Man (25 µM) also prevented 6-OHDA-induced dopaminergic neurodegeneration in the BZ555 C. elegans strain. Altogether, our novel results suggest that α-Man affords partial protection against several, but not all, short-term toxic effects induced by 6-OHDA in cortical slices and in a skn-1-dependent manner in C. elegans.

Age-dependent viscoelastic characterization of rat brain cortex

Recent efforts in biomaterial-assisted brain tissue engineering suggest that match of mechanical properties of biomaterials to those of native brain tissue may be crucial for brain regeneration. In particular, the mechanical properties of native brain tissue vary as a function of age. To date, detailed characterization of age-dependent viscoelastic properties of brain tissue throughout the postnatal development to adulthood is only available at sparse age points in animal studies. To fill this gap, we have characterized the linear viscoelastic properties of the cerebral cortex in rats at well-spaced ages from postnatal day 4 to 4 months old, the age range that is widely used in neural regeneration studies. Using an oscillatory rheometer, the viscoelastic properties of rat cortical slices were measured independently by storage moduli (G′) and loss moduli (G″). The data demonstrated increases in both the storage moduli and the loss moduli of cortex tissue over post-natal age in rats. At all ages, the damping factor (G″/G′ ratio) remained constant at low oscillatory strain frequencies (<10 rad/s) before it started to decline at medium frequency range (10-100 rad/s). Such changes were not age-dependent. The stress-relaxation response increased over post-natal age, consistent with the increasing tissue stiffness. Taken together, our study demonstrates that age is a crucial factor determining the mechanical properties of the cerebral cortex in rats during early postnatal development. This data may provide the guidelines for age-specific biomechanics study of brain tissue and help to define the mechanical properties of biomaterials for biomaterial-assisted brain tissue regeneration studies. Statement of significanceStudies about age-dependent viscoelastic properties of rat brain tissue throughout the postnatal development to adulthood is sparsely available. To fill up the gap of knowledge, in this study, we have characterized the age-dependent viscoelastic properties and the linear viscoelastic properties of the cerebral cortex throughout the postnatal development stage to adulthood in rats by measuring storage moduli (G′), loss moduli (G″), damping factor (G″/G′ ratio) and stress-relaxation response. We have found that age is a crucial factor determining the mechanical properties of the cerebral cortex in rats during early postnatal development. The findings of this study could provide guidelines for age-specific biomechanical study of brain tissue and help to define the mechanical properties of biomaterials for biomaterial-assisted brain tissue regeneration in experimental models in rats.

Astrocytes regulate ultra-slow arteriole oscillations via stretch-mediated TRPV4-COX-1 feedback.

Very-low-frequency oscillations in microvascular diameter cause fluctuations in oxygen delivery that are important for fueling the brain and for functional imaging. However, little is known about how the brain regulates ongoing oscillations in cerebral blood flow. In mouse and rat cortical brain slice arterioles, we find that selectively enhancing tone is sufficient to recruit a TRPV4-mediated Ca2+ elevation in adjacent astrocyte endfeet. This endfoot Ca2+ signal triggers COX-1-mediated "feedback vasodilators" that limit the extent of evoked vasoconstriction, as well as constrain fictive vasomotion in slices. Astrocyte-Ptgs1 knockdown invivo increases the power of arteriole oscillations across a broad range of very low frequencies (0.01-0.3Hz), including ultra-slow vasomotion (∼0.1Hz). Conversely, clamping astrocyte Ca2+invivo reduces the power of vasomotion. These data demonstrate bidirectional communication between arterioles and astrocyte endfeet to regulate oscillatory microvasculature activity.

Glutamate-induced modulation in energy metabolism contributes to protection of rat cortical slices against ischemia-induced damage.

Glutamate excitotoxicity contributes to neurodegeneration during cerebral ischemia. Recent studies in the protective effect of glutamate against ischemia and hypoxia have shown the need for questioning the role of glutamate in energy metabolism during ischemia. Current study investigates the effect of glutamate on energy substrate metabolites such as alpha-ketoglutarate, lactate, and pyruvate release during control, oxygen-glucose deprivation (OGD), and reoxygenation (REO) conditions. The effects of 0.5 and 2 mM glutamate on spontaneous alpha-ketoglutarate, lactate, and pyruvate release were tested in vitro, on acute rat cortical slices. Alpha-ketoglutarate, lactate, and pyruvate levels were determined by HPLC with UV detector. We observed that glutamate added into medium significantly increased alpha-ketogluarate release under control conditions. Although OGD and REO also had a glutamate-like effect, only REO-induced rise further enhanced by glutamate. In contrast to alpha-ketoglutarate, both OGD and REO conditions caused significant declines in pyruvate and lactate outputs. While OGD and REO-induced declines in pyruvate outputs were further potentiated, lactate output was not altered by glutamate added into the medium. Glutamate and alpha-ketoglutarate, moreover, also ameliorated OGD- and REO-induced losses in 2,3,5-triphenyltetrazolium chloride staining with a similar degree. These results indicate that glutamate probably increases alpha-ketoglutarate production as an alternative energy source for use in the TCA cycle under energy-depleted conditions. Thus, increasing the alpha-ketoglutarate production may represent a new therapeutic intervention for neurodegenerative disorders, including cerebral ischemia.

Reappraisal of anoxic spreading depolarization as a terminal event during oxygen\u2013glucose deprivation in brain slices in vitro

Anoxic spreading depolarization (aSD) has been hypothesized as a terminal event during oxygen–glucose deprivation (OGD) in submerged cortical slices in vitro. However, mechanical artifacts caused by aSD-triggered edema may introduce error in the assessment of neuronal viability. Here, using continuous patch-clamp recordings from submerged rat cortical slices, we first confirmed that vast majority of L4 neurons permanently lost their membrane potential during OGD-induced aSD. In some recordings, spontaneous transition from whole-cell to out-side out configuration occurred during or after aSD, and only a small fraction of neurons survived aSD with reperfusion started shortly after aSD. Secondly, to minimize artifacts caused by OGD-induced edema, cells were short-term patched following OGD episodes of various duration. Nearly half of L4 cells maintained membrane potential and showed the ability to spike-fire if reperfusion started less than 10 min after aSD. The probability of finding live neurons progressively decreased at longer reperfusion delays at a rate of about 2% per minute. We also found that neurons in L2/3 show nearly threefold higher resistance to OGD than neurons in L4. Our results suggest that in the OGD ischemia model, aSD is not a terminal event, and that the “commitment point” of irreversible damage occurs at variable delays, in the range of tens of minutes, after OGD-induced aSD in submerged cortical slices.

URB597 Prevents the Short-Term Excitotoxic Cell Damage in Rat Cortical Slices: Role of Cannabinoid 1 Receptors.

Endocannabinoid-based therapies constitute an emerging tool for the potential treatment of neurodegenerative disorders, requiring characterization at the experimental level. The effects of URB597, an inhibitor of the fatty acid amide hydrolase (FAAH), were tested against the quinolinic acid (QUIN)-induced early toxic effects in rat cortical slices, and compared with those effects exerted by the endocannabinoid anandamide (AEA). URB597 prevented the QUIN-induced loss of mitochondrial function/cell viability and lipid peroxidation, while reduced necrosis, and to a lesser extent, apoptosis. The protective effects of URB597 were mediated by activation of cannabinoid receptor 1 (CB1r), as evidenced by their inhibition by the selective CB1r antagonist AM281. Similar effects were observed when testing AEA against QUIN toxicity. Our findings demonstrate the neuroprotective properties of URB597 during the early stages of excitotoxic damage to cortical tissue, suggesting that these properties are mediated by FAAH inhibition, and might be linked to the protective effects of AEA, or the combination of endocannabinoids.

S-Allylcysteine Protects Against Excitotoxic Damage in Rat Cortical Slices Via Reduction of Oxidative Damage, Activation of Nrf2/ARE Binding, and BDNF Preservation.

Neuroprotective approaches comprising different mechanisms to counteract the noxious effects of excitotoxicity and oxidative stress need validation and detailed characterization. Although S-allylcysteine (SAC) is a natural compound exhibiting a broad spectrum of protective effects characterized by antioxidant, anti-inflammatory, and neuromodulatory actions, the mechanisms underlying its protective role on neuronal cell damage triggered by early excitotoxic insults remain elusive. In this study, we evaluated if the preconditioning or the post-treatment of isolated rat cortical slices with SAC (100μM) can ameliorate the toxic effects induced by the excitotoxic metabolite quinolinic acid (QUIN, 100μM), and whether this protective response involves the early display of specific antioxidant and neuroprotective signals. For this purpose, cell viability/mitochondrial reductive capacity, lipid peroxidation, levels of reduced and oxidized glutathione (GSH and GSSG, respectively), the rate of cell damage, the NF-E2-related factor 2/antioxidant response element (Nrf2/ARE) binding activity, heme oxygenase 1 (HO-1) regulation, extracellular signal-regulated kinase (ERK1/2) phosphorylation, and the levels of tumor necrosis factor-alpha (TNF-α) and the neurotrophin brain-derived neurotrophic factor (BDNF) were all estimated in tissue slices exposed to SAC and/or QUIN. The incubation of slices with QUIN augmented all toxic endpoints, whereas the addition of SAC prevented and/or recovered all toxic effects of QUIN, exhibiting better results when administered 60min before the toxin and demonstrating protective and antioxidant properties. The early stimulation of Nrf2/ARE binding activity, the upregulation of HO-1, the ERK1/2 phosphorylation and the preservation of BDNF tissue levels by SAC demonstrate that this molecule displays a wide range of early protective signals by triggering orchestrated antioxidant responses and neuroprotective strategies. The relevance of the characterization of these mechanisms lies in the confirmation that the protective potential exerted by SAC begins at the early stages of excitotoxicity and neurodegeneration and supports the design of integral prophylactic/therapeutic strategies to reduce the deleterious effects observed in neurodegenerative disorders with inherent excitotoxic events.

Experimental hypothyroidism raises brain kynurenic acid – Novel aspect of thyroid dysfunction

Hypothyroidism frequently manifests with altered mood and disturbed cognition. Kynurenic acid may influence cognition through antagonism of N-methyl-d-aspartate receptors (NMDA) and α7 nicotinic receptors. In here, thyroid hormones effects on kynurenic acid synthesis in rat cortical slices and on kynurenine aminotransferases (KATs) activity in semi-purified cortical homogenates were studied. Furthermore, brain kynurenic acid levels and KATs activities were evaluated in experimental model of hypothyroidism, induced by chronic administration of 0.05% propylthiouracil in drinking water. In vitro, L-thyroxine (T4) and 3,3,5-triiodothyronine (T3), reduced kynurenic acid synthesis and KATs activities (IC50 ~ 50–150 μM). In vivo, propylthiouracil increased cortical, hippocampal and striatal, but not cerebellar kynurenic acid content (192%, 142% and 124% of control, respectively), despite uniformly decreased KAT II activity and lower cortical and striatal KAT I activity. T4 application to hypothyroid animals restored kynurenic acid levels to control values and reversed enzymatic changes. T4 alone did not change brain kynurenic acid levels, despite increased activities of brain KATs. Hence, thyroid hormones modulate kynurenic acid levels by two opposing mechanisms, stimulation of KATs activity, most probably transcriptional, and direct, post-translational inhibition of KATs. Lack of correlation between KATs activity and kynurenic acid level may reflect the influence of T4 on organic anion transporter and result from impaired removal of kynurenic acid from the brain during hypothyroidism. Our data reveal novel mechanism linked with thyroid hormones deficiency and imply the potential involvement of increased brain kynurenic acid in the hypothyroidism-related cognitive disturbance.

Quercetin protects against cerebral ischemia/reperfusion and oxygen glucose deprivation/reoxygenation neurotoxicity

Beyond nutrition effect, quercetin is applied as a complement or an alternative for promoting human health and treating diseases. However, its complicated neuroprotective mechanisms have not yet been fully elucidated. This study provides evidence of an alternative target for quercetin, and sheds light on the mechanisms of its neuroprotection against cerebral ischemia/reperfusion (I/R) injury in Sprague–Dawley rats. Oral pretreatment using quercetin has alleviated cerebral I/R-induced neurological deficits, brain infarction, blood–brain barrier disruption, oxidative stress, TNF-α and IL-1β mRNA expression, along with apoptotic caspase 3 activity. The neuroprotective, anti-oxidative, anti-inflammatory, and anti-apoptotic effects of quercetin were replicated in rat hippocampal slice cultures and neuron/glia cultures which suffered from oxygen–glucose deprivation and reoxygenation (OGDR). Biochemical studies revealed a reduction of extracellular signal-regulated kinase (ERK) and Akt phosphorylation, along with an increase in protein tyrosine and serine/threonine phosphatase activity in cerebral I/R rat cortical tissues and OGDR hippocampal slice and neuron/glia cultures. Quercetin alleviated the changes in ERK/Akt phosphorylation and protein phosphatase activities. Inhibition of ERK or Akt alone was enough to cause apoptotic cell death and cytotoxicity in hippocampal slice cultures and neuron/glia cultures, while activators of ERK or Akt alleviated OGDR-induced cytotoxicity. Taken together, our results demonstrate that quercetin alleviated the increment of protein tyrosine and serine/threonine phosphatase activity, along with the reduction of ERK and Akt phosphorylation, which may play pivotal roles in the expansion of brain injury after cerebral I/R.

The effect of general anaesthetics on brain lactate release

The effects of anaesthetic agents on brain energy metabolism may explain their shared neurophysiological actions but remain poorly understood. The brain lactate shuttle hypothesis proposes that lactate, provided by astrocytes, is an important neuronal energy substrate. Here we tested the hypothesis that anaesthetic agents impair the brain lactate shuttle by interfering with astrocytic glycolysis. Lactate biosensors were used to record changes in lactate release by adult rat brainstem and cortical slices in response to thiopental, propofol and etomidate. Changes in cytosolic nicotinamide adenine dinucleotide reduced (NADH) and oxidized (NAD+) ratio as a measure of glycolytic rate were recorded in cultured astrocytes. It was found that in brainstem slices thiopental, propofol and etomidate reduced lactate release by 7.4 ± 3.6% (P < 0.001), 9.7 ± 6.6% (P < 0.001) and 8.0 ± 7.8% (P = 0.04), respectively. In cortical slices, thiopental reduced lactate release by 8.2 ± 5.6% (P = 0.002) and propofol by 6.0 ± 4.5% (P = 0.009). Lactate release in cortical slices measured during the light phase (period of sleep/low activity) was ~25% lower than that measured during the dark phase (period of wakefulness) (326 ± 83 μM vs 430 ± 118 μM, n = 10; P = 0.04). Thiopental and etomidate induced proportionally similar decreases in cytosolic [NADH]:[NAD+] ratio in astrocytes, indicative of a reduction in glycolytic rate. These data suggest that anaesthetic agents inhibit astrocytic glycolysis and reduce the level of extracellular lactate in the brain. Similar reductions in brain lactate release occur during natural state of sleep, suggesting that general anaesthesia may recapitulate some of the effects of sleep on brain energy metabolism.

M1 muscarinic acetylcholine receptor dysfunction in moderate Alzheimer's disease pathology.

Aggregation of amyloid beta and loss of cholinergic innervation in the brain are predominant components of Alzheimer’s disease pathology and likely underlie cognitive impairment. Acetylcholinesterase inhibitors are one of the few treatment options for Alzheimer’s disease, where levels of available acetylcholine are enhanced to counteract the cholinergic loss. However, these inhibitors show limited clinical efficacy. One potential explanation for this is a concomitant dysregulation of cholinergic receptors themselves as a consequence of the amyloid beta pathology. We tested this hypothesis by examining levels of M1 muscarinic acetylcholine receptors in the temporal cortex from seven Alzheimer’s disease and seven non-disease age-matched control brain tissue samples (control: 85 ± 2.63 years old, moderate Alzheimer’s disease: 84 ± 2.32 years old, P-value = 0.721; eight female and six male patients). The samples were categorized into two groups: ‘control’ (Consortium to Establish a Registry for Alzheimer’s Disease diagnosis of ‘No Alzheimer’s disease’, and Braak staging pathology of I–II) and ‘moderate Alzheimer’s disease’ (Consortium to Establish a Registry for Alzheimer’s Disease diagnosis of ‘possible/probable Alzheimer’s disease’, and Braak staging pathology of IV). We find that in comparison to age-matched controls, there is a loss of M1 muscarinic acetylcholine receptors in moderate Alzheimer’s disease tissue (control: 2.17 ± 0.27 arbitrary units, n = 7, Mod-AD: 0.83 ± 0.16 arbitrary units, n = 7, two-tailed t-test, t = 4.248, P = 0.00113). Using a functional rat cortical brain slice model, we find that postsynaptic muscarinic acetylcholine receptor function is dysregulated by aberrant amyloid beta-mediated activation of metabotropic glutamate receptor 5. Crucially, blocking metabotropic glutamate receptor 5 restores muscarinic acetylcholine receptor function and object recognition memory in 5XFAD transgenic mice. This indicates that the amyloid beta-mediated activation of metabotropic glutamate receptor 5 negatively regulates muscarinic acetylcholine receptor and illustrates the importance of muscarinic acetylcholine receptors as a potential disease-modifying target in the moderate pathological stages of Alzheimer’s disease.

Aging protects rat cortical slices against to oxygen-glucose deprivation induced damage

Objective: In present study, we aimed to clarify effect of aging on the susceptibility of brain tissue to neurodegeneration induced by ischemia.Methods: Damage induced by oxygen-glucose deprivation (OGD) followed by reoxygenation (REO) were compared in cortical slices prepared from young (3 months of age) and aged (22-24 months of age) male Sprague Dawley rats.Results: After incubation of the slices in an oxygen and glucose containing control condition, 2,3,5-triphenyl tetrazolium chloride (TTC) staining intensity was found significantly high in aged cortical slices. Although thirty minutes incubation of the slices in OGD medium followed by REO (OGD-REO) caused similar decline in TTC staining in young and aged cortical slices, staining intensity was still significantly higher in the slices prepared from aged animals. Thirty minutes of OGD-REO, on the other hand, also caused more increase in lactate dehydrogenase (LDH) leakage from young slices. While water contents of the slices were almost equal under control condition, it was significantly high in young cortical slices after OGD-REO incubations. In contrary to these findings, OGD and REO caused more increases in S100B output from aged rat cortical slices. S100B levels in brain regions including the cerebral cortex were also found higher in aged rats.Conclusion: All these results indicate that, cortical slices prepared from aged male rats are significantly less responsive to in vitro OGD-REO induced alterations. Since protein S100B outputs were almost doubled from aged cortical slices, a possible involvement of this enhanced S100B output seems to be likely.

Correction to: Comparing the Neuroprotective Effects of Caffeic Acid in Rat Cortical Slices and Caenorhabditis elegans: Involvement of Nrf2 and SKN-1 Signaling Pathways.

One of the authors has incorrect family name spelling in the original article. Michael Ashner should read as Michael Aschner.

PKCγ and PKCε are Differentially Activated and Modulate Neurotoxic Signaling Pathways During Oxygen Glucose Deprivation in Rat Cortical Slices.

Cerebral ischemia is known to trigger a series of intracellular events such as changes in metabolism, membrane function and intracellular transduction, which eventually leads to cell death. Many of these processes are mediated by intracellular signaling cascades that involve protein kinase activation. Among all the kinases activated, the serine/threonine kinase family, protein kinase C (PKC), particularly, has been implicated in mediating cellular response to cerebral ischemic and reperfusion injury. In this study, using oxygen-glucose deprivation (OGD) in acute cortical slices as an in vitro model of cerebral ischemia, I show that PKC family of isozymes, specifically PKCγ and PKCε are differentially activated during OGD. Detecting the expression and activation levels of these isozymes in response to different durations of OGD insult revealed an early activation of PKCε and delayed activation of PKCγ, signifying their roles in response to different durations and stages of ischemic stress. Specific inhibition of PKCγ and PKCε significantly attenuated OGD induced cytotoxicity, rise in intracellular calcium, membrane depolarization and reactive oxygen species formation, thereby enhancing neuronal viability. This study clearly suggests that PKC family of isozymes; specifically PKCγ and PKCε are involved in OGD induced intracellular responses which lead to neuronal death. Thus isozyme specific modulation of PKC activity may serve as a promising therapeutic route for the treatment of acute cerebral ischemic injury.