Locus Coeruleus Neurons' Firing Pattern Is Regulated by ERG Voltage-Gated K+ Channels.

Locus coeruleus (LC) neurons, with their extensive innervations throughout the brain, control a broad range of physiological processes. Several ion channels have been characterized in LC neurons that control intrinsic membrane properties and excitability. However, ERG (ether-à-go-go-related gene) K+ channels that are particularly important in setting neuronal firing rhythms and automaticity have not as yet been discovered in the LC. Moreover, the neurophysiological and pathophysiological roles of ERG channels in the brain remain unclear despite their expression in several structures. By performing immunohistochemical investigations, we found that ERG-1A, ERG-1B, ERG-2 and ERG-3 are highly expressed in the LC neurons of mice. To examine the functional role of ERG channels, current-clamp recordings were performed on mouse LC neurons in brain slices under visual control. ERG channel blockade by WAY-123,398, a class III anti-arrhythmic agent, increased the spontaneous firing activity and discharge irregularity of LC neurons. Here, we have shown the presence of distinct ERG channel subunits in the LC which play an imperative role in modulating neuronal discharge patterns. Thus, we propose that ERG channels are important players behind the changes in, and/or maintenance of, LC firing patterns that are implicated in the generation of different behaviors and in several disorders.

Aminonaphthalimide hybrids of mitoxantrone and amonafide as anticancer and fluorescent cellular imaging agents.

Novel water-soluble 4-aminonaphthalimides were synthesised and their cellular fluorescent imaging, cytotoxicity and ability to induced apoptosis evaluated. The lead compound 1 was designed from the cross-fertilisation of the basic hydrophilic amino pharmacophore of mitoxantrone, and an aminonaphthalimide scaffold of the drug candidate, amonafide. The compounds are also fluorescent pH probes based on photoinduced electron transfer (PET) and internal charge transfer (ICT). The compounds are sensitive to solvent polarity with large Stoke shifts (>90 nm) and provide emissive-coloured solutions (blue to yellow). Excited state pKas of 9.0-9.3 and fluorescence quantum yields of 0.47-0.58 were determined in water. The cytotoxicity and cellular fluorescent imaging properties of the compounds were tested on human cancer cell lines K562 and MCF-7 by the MTT assay, phase contrast and fluorescence microscopy. Compounds 1 and 3 with flexible aminoalkyl chains exhibited GI50 comparable to amonafide, while 2 and 4 with a rigid piperazine moiety and butyl chain are less cytotoxic. Fluorescence microscopy with 1 allowed for the visualization of the intracellular microenvironment exemplifying the potential utility of such hybrid molecules as anticancer and fluorescent cellular imaging agents.

Dexamethasone in Glioblastoma Multiforme Therapy: Mechanisms and Controversies.

Glioblastoma multiforme (GBM) is the most common and malignant of the glial tumors. The world-wide estimates of new cases and deaths annually are remarkable, making GBM a crucial public health issue. Despite the combination of radical surgery, radio and chemotherapy prognosis is extremely poor (median survival is approximately 1 year). Thus, current therapeutic interventions are highly unsatisfactory. For many years, GBM-induced brain oedema and inflammation have been widely treated with dexamethasone (DEX), a synthetic glucocorticoid (GC). A number of studies have reported that DEX also inhibits GBM cell proliferation and migration. Nevertheless, recent controversial results provided by different laboratories have challenged the widely accepted dogma concerning DEX therapy for GBM. Here, we have reviewed the main clinical features and genetic and epigenetic abnormalities underlying GBM. Finally, we analyzed current notions and concerns related to DEX effects on cerebral oedema, cancer cell proliferation and migration and clinical outcome.

Vesicular glutamate release from central axons contributes to myelin damage.

The axon myelin sheath is prone to injury associated with N-methyl-D-aspartate (NMDA)-type glutamate receptor activation but the source of glutamate in this context is unknown. Myelin damage results in permanent action potential loss and severe functional deficit in the white matter of the CNS, for example in ischemic stroke. Here, we show that in rats and mice, ischemic conditions trigger activation of myelinic NMDA receptors incorporating GluN2C/D subunits following release of axonal vesicular glutamate into the peri-axonal space under the myelin sheath. Glial sources of glutamate such as reverse transport did not contribute significantly to this phenomenon. We demonstrate selective myelin uptake and retention of a GluN2C/D NMDA receptor negative allosteric modulator that shields myelin from ischemic injury. The findings potentially support a rational approach toward a low-impact prophylactic therapy to protect patients at risk of stroke and other forms of excitotoxic injury.

Early Loss of Blood-Brain Barrier Integrity Precedes NOX2 Elevation in the Prefrontal Cortex of an Animal Model of Psychosis.

The social isolation rearing of young adult rats is a model of psychosocial stress and provides a nonpharmacological tool to study alterations reminiscent of symptoms seen in psychosis. We have previously demonstrated that social isolation in rats leads to increased oxidative stress and to cerebral NOX2 elevations. Here, we investigated early alterations in mRNA expression leading to increased NOX2 in the brain. Rats were exposed to a short period of social isolation (1 week) and real-time polymerase chain reaction (PCR) for mRNA expression of genes involved in blood-brain barrier (BBB) formation and integrity (ORLs, Vof 21 and Vof 16, Leng8, Vnr1, and Trank 1 genes) was performed. Real-time PCR experiments, immunohistochemistry, and Western blotting analysis showed an increased expression of these genes and related proteins in isolated rats with respect to control animals. The expression of specific markers of BBB integrity, such as matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 9 (MMP9), occludin 1, and plasmalemmal vesicle associated protein-1 (PV-1), was also significantly altered after 1 week of social isolation. BBB permeability, evaluated by quantification of Evans blue dye extravasation, as well as interstitial fluid, was significantly increased in rats isolated for 1 week with respect to controls. Isolation-induced BBB disruption was also accompanied by a significant increase of Interleukin 6 (IL-6) expression. Conversely, no differences in NOX2 levels were detected at this time point. Our study demonstrates that BBB disruption precedes NOX2 elevations in the brain. These results provide new insights in the interplay of mechanisms linking psychosocial stress to early oxidative stress in the brain, disruption of the BBB, and the development of mental disorders.

Acute nicotine induces anxiety and disrupts temporal pattern organization of rat exploratory behavior in hole-board: a potential role for the lateral habenula.

Nicotine is one of the most addictive drugs of abuse. Tobacco smoking is a major cause of many health problems, and is the first preventable cause of death worldwide. Several findings show that nicotine exerts significant aversive as well as the well-known rewarding motivational effects. Less certain is the anatomical substrate that mediates or enables nicotine aversion. Here, we show that acute nicotine induces anxiogenic-like effects in rats at the doses investigated (0.1, 0.5, and 1.0 mg/kg, i.p.), as measured by the hole-board apparatus and manifested in behaviors such as decreased rearing and head-dipping and increased grooming. No changes in locomotor behavior were observed at any of the nicotine doses given. T-pattern analysis of the behavioral outcomes revealed a drastic reduction and disruption of complex behavioral patterns induced by all three nicotine doses, with the maximum effect for 1 mg/kg. Lesion of the lateral habenula (LHb) induced hyperlocomotion and, strikingly, reversed the nicotine-induced anxiety obtained at 1 mg/kg to an anxiolytic-like effect, as shown by T-pattern analysis. We suggest that the LHb is critically involved in emotional behavior states and in nicotine-induced anxiety, most likely through modulation of monoaminergic nuclei.

The central role of aquaporins in the pathophysiology of ischemic stroke.

Stroke is a complex and devastating neurological condition with limited treatment options. Brain edema is a serious complication of stroke. Early edema formation can significantly contribute to infarct formation and thus represents a promising target. Aquaporin (AQP) water channels contribute to water homeostasis by regulating water transport and are implicated in several disease pathways. At least 7 AQP subtypes have been identified in the rodent brain and the use of transgenic mice has greatly aided our understanding of their functions. AQP4, the most abundant channel in the brain, is up-regulated around the peri-infarct border in transient cerebral ischemia and AQP4 knockout mice demonstrate significantly reduced cerebral edema and improved neurological outcome. In models of vasogenic edema, brain swelling is more pronounced in AQP4-null mice than wild-type providing strong evidence of the dual role of AQP4 in the formation and resolution of both vasogenic and cytotoxic edema. AQP4 is co-localized with inwardly rectifying K(+)-channels (Kir4.1) and glial K(+) uptake is attenuated in AQP4 knockout mice compared to wild-type, indicating some form of functional interaction. AQP4-null mice also exhibit a reduction in calcium signaling, suggesting that this channel may also be involved in triggering pathological downstream signaling events. Associations with the gap junction protein Cx43 possibly recapitulate its role in edema dissipation within the astroglial syncytium. Other roles ascribed to AQP4 include facilitation of astrocyte migration, glial scar formation, modulation of inflammation and signaling functions. Treatment of ischemic cerebral edema is based on the various mechanisms in which fluid content in different brain compartments can be modified. The identification of modulators and inhibitors of AQP4 offer new therapeutic avenues in the hope of reducing the extent of morbidity and mortality in stroke.

Hsp60 response in experimental and human temporal lobe epilepsy.

The mitochondrial chaperonin Hsp60 is a ubiquitous molecule with multiple roles, constitutively expressed and inducible by oxidative stress. In the brain, Hsp60 is widely distributed and has been implicated in neurological disorders, including epilepsy. A role for mitochondria and oxidative stress has been proposed in epileptogenesis of temporal lobe epilepsy (TLE). Here, we investigated the involvement of Hsp60 in TLE using animal and human samples. Hsp60 immunoreactivity in the hippocampus, measured by Western blotting and immunohistochemistry, was increased in a rat model of TLE. Hsp60 was also increased in the hippocampal dentate gyrus neurons somata and neuropil and hippocampus proper (CA3, CA1) of the epileptic rats. We also determined the circulating levels of Hsp60 in epileptic animals and TLE patients using ELISA. The epileptic rats showed circulating levels of Hsp60 higher than controls. Likewise, plasma post-seizure Hsp60 levels in patients were higher than before the seizure and those of controls. These results demonstrate that Hsp60 is increased in both animals and patients with TLE in affected tissues, and in plasma in response to epileptic seizures, and point to it as biomarker of hippocampal stress potentially useful for diagnosis and patient management.

Cerebral white matter injuries following a hypoxic/ischemic insult during the perinatal period: pathophysiology, prognostic factors, and future strategy of treatment approach. A minireview.

Recent advances in medical care have significantly improved the survival rate of neonates who suffer a hypoxic/ ischemic event, before, during, or after birth. These infants are extremely vulnerable to brain injury and are at high risk of developing motor and cognitive abnormalities later on in life. The regional distribution of perinatal brain injury varies, and depends primarily on; the severity, pattern and type of insult, the metabolic status, and on the gestational age. The principal neuropathological substrate that is affected in the premature infant is cerebral white matter. The aim of this article is to re-examine the current knowledge on the ischemic pathophysiology of all cellular components that comprise the white matter, pred ict the consequences of the long-term neurological outcome, and analyze possible therapeutic strategies. Although oligodendrocytes have long been regarded as the hallmark of perinatal white matter injury, axons, astrocytes and microglia, all contribute to the complex pattern of brain injury that occurs in this cohort of individuals. It is hoped that a better understanding of the pathophysiology of white matter injury and its underlying prognostic factors, may lead to the development of new therapeutic strategies for such a complex and debilitating condition.

Oligodendrocyte pathophysiology and treatment strategies in cerebral ischemia.

Oligodendrocytes (OLs), the myelin-forming cells of the central nervous system, form a functional unit with axons and play a crucial role in axonal integrity. An episode of hypoxia-ischemia causes rapid and severe damage to these particularly vulnerable cells via multiple pathways such as overactivation of glutamate and ATP receptors, oxidative stress, and disruption of mitochondrial function. The cardinal effect of OL pathology is demyelination and dysmyelination, and this has profound effects on axonal function, transport, structure, metabolism, and survival. The OL is a primary target of ischemia in adult-onset stroke and especially in periventricular leukomalacia and should be considered as a primary therapeutic target in these conditions. More emphasis is needed on therapeutic strategies that target OLs, myelin, and their receptors, as these have the potential to significantly attenuate white matter injury and to establish functional recovery of white matter after stroke. In this review, we will summarize recent progress on the role of OLs in white matter ischemic injury and the current and emerging principles that form the basis for protective strategies against OL death.

High dose of 8-OH-DPAT decreases maximal dentate gyrus activation and facilitates granular cell plasticity in vivo.

Although several studies have emphasized a crucial role for the serotonergic system in the control of hippocampal excitability, the role of serotonin (5-HT) and its receptors in normal and pathologic conditions, such as temporal lobe epilepsy (TLE), is still unclear. The present study was therefore designed firstly to investigate the acute effect of 8-OH-DPAT, a mixed 5-HT1A/7 receptor agonist, at a high dose (1 mg/kg, i.p.) known to have antiepileptic properties, in a model of acute partial epilepsy in rats. For this purpose, a maximal dentate activation (MDA) protocol was used to measure electrographic seizure onset and duration. In addition, the effect of 8-OH-DPAT on in vivo dentate gyrus cell reactivity and short- and long-term plasticity was studied. Rats injected with 8-OH-DPAT exhibited a significant reduction in MDA and epileptic discharges, a decrease in paired-pulse facilitation and an increase in long-term potentiation. This study suggests that 8-OH-DPAT or in general 5-HT1A/7 agonists might be useful for the treatment of TLE and also have some beneficial effects on the comorbid cognitive disorders seen in epileptic patients.

Kv1.1 knock-in ataxic mice exhibit spontaneous myokymic activity exacerbated by fatigue, ischemia and low temperature.

Episodic ataxia type 1 (EA1) is an autosomal dominant neurological disorder characterized by myokymia and attacks of ataxic gait often precipitated by stress. Several genetic mutations have been identified in the Shaker-like K(+) channel Kv1.1 (KCNA1) of EA1 individuals, including V408A, which result in remarkable channel dysfunction. By inserting the heterozygous V408A, mutation in one Kv1.1 allele, a mouse model of EA1 has been generated (Kv1.1(V408A/+)). Here, we investigated the neuromuscular transmission of Kv1.1(V408A/+) ataxic mice and their susceptibility to physiologically relevant stressors. By using in vivo preparations of lateral gastrocnemius (LG) nerve-muscle from Kv1.1(+/+) and Kv1.1(V408A/+) mice, we show that the mutant animals exhibit spontaneous myokymic discharges consisting of repeated singlets, duplets or multiplets, despite motor nerve axotomy. Two-photon laser scanning microscopy from the motor nerve, ex vivo, revealed spontaneous Ca(2+) signals that occurred abnormally only in preparations dissected from Kv1.1(V408A/+) mice. Spontaneous bursting activity, as well as that evoked by sciatic nerve stimulation, was exacerbated by muscle fatigue, ischemia and low temperatures. These stressors also increased the amplitude of compound muscle action potential. Such abnormal neuromuscular transmission did not alter fiber type composition, neuromuscular junction and vascularization of LG muscle, analyzed by light and electron microscopy. Taken together these findings provide direct evidence that identifies the motor nerve as an important generator of myokymic activity, that dysfunction of Kv1.1 channels alters Ca(2+) homeostasis in motor axons, and also strongly suggest that muscle fatigue contributes more than PNS fatigue to exacerbate the myokymia/neuromyotonia phenotype. More broadly, this study points out that juxtaparanodal K(+) channels composed of Kv1.1 subunits exert an important role in dampening the excitability of motor nerve axons during fatigue or ischemic insult.

Central axons preparing to myelinate are highly sensitive [corrected] to ischemic injury.

OBJECTIVE: Developing central white matter is subject to ischemic-type injury during the period that precedes myelination. At this stage in maturation, central axons initiate a program of radial expansion and ion channel redistribution. Here we test the hypothesis that during radial expansion axons display heightened ischemic sensitivity, when clusters of Ca(2+) channels decorate future node of Ranvier sites. METHODS: Functionality and morphology of central axons and glia were examined during and after a period of modeled ischemia. Pathological changes in axons undergoing radial expansion were probed using electrophysiological, quantitative ultrastructural, and morphometric analysis in neonatal rodent optic nerve and periventricular white matter axons studied under modeled ischemia in vitro or after hypoxia-ischemia in vivo. RESULTS: Acute ischemic injury of central axons undergoing initial radial expansion was mediated by Ca(2+) influx through Ca(2+) channels expressed in axolemma clusters. This form of injury operated only in this axon population, which was more sensitive to injury than neighboring myelinated axons, smaller axons yet to initiate radial expansion, astrocytes, or oligodendroglia. A pharmacological strategy designed to protect both small and large diameter premyelinated axons proved 100% protective against acute ischemia studied under modeled ischemia in vitro or after hypoxia-ischemia in vivo. INTERPRETATION: Recent clinical data highlight the importance of axon pathology in developing white matter injury. The elevated susceptibility of early maturing axons to ischemic injury described here may significantly contribute to selective white matter pathology and places these axons alongside preoligodendrocytes as a potential primary target of both injury and therapeutics.

Nitric oxide modulation of the basal ganglia circuitry: therapeutic implication for Parkinson's disease and other motor disorders.

Several recent studies have emphasized a crucial role for the nitrergic system in movement control and the pathophysiology of the basal ganglia (BG). These observations are supported by anatomical evidence demonstrating the presence of nitric oxide synthase (NOS) in all the basal ganglia nuclei. In fact, nitrergic terminals have been reported to make synaptic contacts with both substantia nigra dopamine-containing neurons and their terminal areas such as the striatum, the globus pallidus and the subthalamus. These brain areas contain a high expression of nitric oxide (NO)-producing neurons, with the striatum having the greatest number, together with important NO afferent input. In this paper, the distribution of NO in the BG nuclei will be described. Furthermore, evidence demonstrating the nitrergic control of BG activity will be reviewed. The new avenues that the increasing knowledge of NO in motor control has opened for exploring the pathophysiology and pharmacology of Parkinson's disease and other movement disorders will be discussed. For example, inhibition of striatal NO/guanosine monophosphate signal pathway by phosphodiesterases seems to be effective in levodopa-induced dyskinesia. However, the results of experimental studies have to be interpreted with caution given the complexities of nitrergic signalling and the limitations of animal models. Nevertheless, the NO system represents a promising pharmacological intervention for treating Parkinson's disease and related disorders.

Identification of oligodendrocytes in experimental disease models.

The ability to identify oligodendrocytes in culture, in fixed tissue, and in vivo using unique markers is a requisite step to understanding their responses in any damage, recovery, or developmental process. Their nuclei are readily seen in histological preparations of healthy white and gray matter, and their cell bodies can be reliably identified with a variety of immunocytochemical markers. However, there is little consensus regarding optimal methods to assess oligodendrocyte survival or morphology under experimental injury conditions. We review common approaches for histological and immunocytochemical identification of these cells. Transgenic and viral methods for cell type-selective transfer of genes encoding fluorescent proteins offer promising new approaches for manipulating and visualizing oligodendrocytes in models of health and disease.

Malignant infarction in cats after prolonged middle cerebral artery occlusion: glutamate elevation related to decrease of cerebral perfusion pressure.

BACKGROUND AND PURPOSE: To study the putative role and predictive significance of glutamate elevation in space-occupying ischemic stroke, we investigated the correlation between perfusional disturbances and glutamate alterations in a transient ischemia model in cats that is susceptible to secondary deterioration after reperfusion. METHODS: In 10 halothane-anesthetized cats, the left middle cerebral artery was occluded for 3 hours, followed by 6 hours of reperfusion. Laser-Doppler flowmetry (LDF) probes, microdialysis/high-performance liquid chromatography, and pressure sensors measured simultaneously regional cerebral blood flow (CBF), extracellular amino acids, mean arterial blood pressure, and intracranial pressure, respectively. Cerebral perfusion pressure (CPP) was calculated. In complementary experiments (n=2), regional CBF was assessed by sequential positron emission tomography. RESULTS: Middle cerebral artery occlusion reduced LDF-measured CBF in all animals to <25% of control. In 5 of 10 cats, glutamate rose approximately 30-fold during ischemia. LDF-measured CBF and glutamate primarily recovered after reperfusion. Glutamate rose again in the late reperfusion phase, when CPP decreased to <60 mm Hg, and symptoms of transtentorial herniation were recognized. Positron emission tomography revealed ischemic thresholds of 15 to 20 mL/100 g per minute for secondary deterioration. In the other 5 cats, ischemic elevation of glutamate was significantly smaller, and signs of secondary deterioration were not recognized. CONCLUSIONS: Glutamate determinations during ischemia predict fatal outcome, as do intracranial pressure and CPP measurements during early reperfusion. Secondary amino acid elevation during reperfusion is presumably caused by a drastic decrease of CPP to <50 mm Hg in the final stage of space-occupying, malignant focal ischemia. At this stage, a further progression of injury due to increased glutamate may be irrelevant with respect to fatal outcome.