Oxidative stress induced by cumene hydroperoxide produces synaptic depression and transient hyperexcitability in rat primary motor cortex neurons.

Pyramidal neurons of the motor cortex are selectively degenerated in Amyotrophic Lateral Sclerosis (ALS). The mechanisms underlying neuronal death in ALS are not well established. In the absence of useful biomarkers, the early increased neuronal excitability seems to be the unique characteristic of ALS. Lipid peroxidation caused by oxidative stress has been postulated as one of the possible mechanisms involved in degeneration motor cortex pyramidal neurons. This paper examines the effect of lipid peroxidation on layer V pyramidal neurons induced by cumene hydroperoxide (CH) in brain slices from wild type rats. CH induces a synaptic depression of pyramidal neurons in a time dependent manner, already observable on GABAergic synaptic transmission after 5min application of the drug. Altogether, our whole-cell patch-clamp recording data suggest that the functional changes induced by CH upon pyramidal neurons are due to pre- and postsynaptic mechanisms. CH did not alter mEPSCs or mIPSCs, but decreased the frequency, amplitude, and decay rate of spontaneous EPSCs and IPSCs. These effects may be explained by a presynaptic mechanism causing a decrease in action potential-dependent neurotransmitter release. Additionally, CH induced a postsynaptic inward current that underlies a membrane depolarization. Depressing the input flow from the inhibitory premotor interneurons causes a transient hyperexcitability (higher resistance and lower rheobase) in pyramidal neurons of the motor cortex by presumably altering a tonic inhibitory current. These findings, which resemble relevant cortical pathophysiology of ALS, point to oxidative stress, presumably by lipid peroxidation, as an important contributor to the causes underlying this disease.

Time and dose dependent effects of oxidative stress induced by cumene hydroperoxide in neuronal excitability of rat motor cortex neurons.

It has been claimed that oxidative stress and the production of reactive oxygen radicals can contribute to neuron degeneration and might be one factor in the development of different neurological diseases. In our study, we have attempted to clarify how oxidative damage induces dose dependent changes in functional membrane properties of neurons by means of whole cell patch clamp techniques in brain slices from young adult rats. Our research demonstrates physiological changes in membrane properties of pyramidal motor cortex neurons exposed to 3 concentrations of cumene hydroperoxide (CH; 1, 10 and 100µM) during 30min. Results show that oxidative stress induced by CH evokes important changes, in a concentration and time dependent manner, in the neuronal excitability of motor cortex neurons of the rat: (i) Low concentration of the drug (1µM) already blocks inward rectifications (sag) and decreases action potential amplitude and gain, a drug concentration which has no effects on other neuronal populations, (ii) 10µM of CH depresses the excitability of pyramidal motor cortex neurons by decreasing input resistance, amplitude of the action potential, and gain and maximum frequency of the repetitive firing discharge, and (iii) 100µM completely blocks the capability to produce repetitive discharge of action potentials in all cells. Both larger drug concentrations and/or longer times of exposure to CH narrow the current working range. This happens because of the increase in the rheobase, and the reduction of the cancelation current. The effects caused by oxidative stress, including those produced by the level of lipid peroxidation, are practically irreversible and, this, therefore, indicates that neuroprotective agents should be administered at the first symptoms of alterations to membrane properties. In fact, the pre-treatment with melatonin, acting as an antioxidant, prevented the lipid peroxidation and the physiological changes induced by CH. Larger cells (as estimated by their cell capacitance) were also more susceptible to oxidative stress. Our results provide previously unavailable observations that large size and high sensitivity to oxidative stress (even at low concentrations) make pyramidal neurons of the motor cortex, in particular corticofugal neurons, more susceptible to cell death when compared with other neuronal populations. These results could also shed some light on explaining the causes behind diseases such as Amyotrophic Lateral Sclerosis.

Oxidative stress induced by cumene hydroperoxide evokes changes in neuronal excitability of rat motor cortex neurons.

Oxidative stress and the production of reactive oxygen radicals play a key role in neuronal cell damage. This paper describes an in vitro study that explores the neuronal responses to oxidative stress focusing on changes in neuronal excitability and functional membrane properties. This study was carried out in pyramidal cells of the motor cortex by applying whole-cell patch-clamp techniques on brain slices from young adult rats. Oxygen-derived free radical formation was induced by bath application of 10µM cumene hydroperoxide (CH) for 30min. CH produced marked changes in the electrophysiological properties of neurons (n=30). Resting membrane potential became progressively depolarized, as well as depolarization voltage, with no variations in voltage threshold. Membrane resistance showed a biphasic behavior, increasing after 5min of drug exposure and then it started to decrease, even under control values, after 15 and 30min. At the same time, changes in membrane resistance produced compensatory variations in the rheobase. The amplitude of the action potentials diminished and the duration increased progressively over time. Some of the neurons under study also lost their ability to discharge action potentials in a repetitive way. Most of the neurons, however, kept their repetitive discharge even though their maximum frequency and gain decreased. Furthermore, cancelation of the repetitive firing discharge took place at intensities that decreased with time of exposure to CH, which resulted in a narrower working range. We can conclude that oxidative stress compromises both neuronal excitability and the capability of generating action potentials, and so this type of neuronal functional failure could precede the neuronal death characteristics of many neurodegenerative diseases.

Immunolocalization of human alpha-synuclein in the Thy1-aSyn ("Line 61") transgenic mouse line.

Alpha-synuclein (a-syn) is the major component of the intracytoplasmic inclusions known as Lewy bodies (LB), which constitute the hallmark of Parkinson's disease (PD). Mice overexpressing human a-syn under the Thy-1 promoter (ASO) show slow neurodegeneration and some behavioral deficits similar to those seen in human PD patients. Here, we describe a whole-brain distribution of human a-syn in adult ASO mice. We find that the human a-syn is ubiquitously distributed in the brain including the cerebellar cortex, but the intensity and sub-cellular localization of the staining differed in the various regions of the central nervous system. Among particular CNS areas with human a-syn immunoreactivity, we describe staining patterns in the olfactory bulb, cortex, hippocampus, thalamic region, brainstem nuclei and cerebellar cortex. This immunohistochemical study provides an anatomical map of the human a-syn distribution in ASO mice. Our data show that human a-syn, although not present at levels that were detectable by immunostaining in dopaminergic neurons of substantia nigra or noradrenergic neurons of locus coeruleus, was highly expressed in other PD relevant regions of the brain in different neuronal subtypes. These data will help to relate a-syn expression to the phenotypic manifestations observed in this widely used mouse line.

Postnatal development enhances the effects of cholinergic inputs on recruitment threshold and firing rate of rat oculomotor nucleus motoneurons.

Changes in the electrophysiological and morphological characteristics of motoneurons (Mns) of the oculomotor nucleus during postnatal development have been reported, however synaptic modifications that take place concurrently with postnatal development in these Mns are yet to be elucidated. We investigated whether cholinergic inputs exert different effects on the recruitment threshold and firing rate of Mns during postnatal development. Rat oculomotor nucleus Mns were intracellularly recorded in brain slice preparations and separated in neonatal (4-7 postnatal days) and adult (20-30 postnatal days) age groups. Stimulation of the medial longitudinal fasciculus evoked a monosynaptic excitatory potential in Mns that was attenuated with atropine (1.5 µM, a muscarinic antagonist). Mns were silent at their resting membrane potential, and bath application of carbachol (10 µM, a cholinergic agonist) induced depolarization of the membrane potential and a sustained firing rate that were more pronounced in adult Mns. Pharmacological and immunohistochemical assays showed that these responses were attributable to muscarinic receptors located in the membrane of Mns. In addition, compared to control Mns, carbachol-exposed Mns exhibited a higher firing rate in response to the injection of the same amount of current, and a decrease in the current threshold required to achieve sustained firing. These latter effects were more pronounced in adult than in neonatal Mns. In conclusion, our findings suggest that cholinergic synaptic inputs are already present in neonatal Mns, and that the electrophysiological effects of such inputs on recruitment threshold and firing rate are enhanced with the postnatal development in oculomotor nucleus Mns. We propose that cholinergic input maturation could provide a greater dynamic range in adult Mns to encode the output necessary for graded muscle contraction.

Temporal sequence of changes in electrophysiological properties of oculomotor motoneurons during postnatal development.

The temporal sequence of changes in electrophysiological properties during postnatal development in different neuronal populations has been the subject of previous studies. Those studies demonstrated major physiological modifications with age, and postnatal periods in which such changes are more pronounced. Until now, no similar systematic study has been performed in motoneurons of the oculomotor nucleus. This work has two main aims: first, to determine whether the physiological changes in oculomotor nucleus motoneurons follow a similar time course for different parameters; and second, to compare the temporal sequence with that in other neuronal populations. We recorded the electrophysiological properties of 134 identified oculomotor nucleus motoneurons from 1 to 40 days postnatal in brain slices of rats. The resting membrane potential did not significantly change with postnatal development, and it had a mean value of -61.8 mV. The input resistance and time constant diminished from 82.9-53.1 M omega and from 9.4-4.9 ms respectively with age. These decrements occurred drastically in a short time after birth (1-5 days postnatally). The motoneurons' rheobase gradually decayed from 0.29-0.11 nA along postnatal development. From birth until postnatal day 15 and postnatal day 20 respectively, the action potential shortened from 2.3-1.2 ms, and the medium afterhyperpolarization from 184.8-94.4 ms. The firing gain and the maximum discharge increased with age. The former rose continuously, while the increase in maximum discharge was most pronounced between postnatal day 16 and postnatal day 20. We conclude that the developmental sequence was not similar for all electrophysiological properties, and was unique for each neuronal population.

Aneurysms of the extracranial carotid arteries.

Aneurysms of the extracranial carotid arteries are unusual; they present difficulties in diagnosis and may cause death or cerebrovascular accidents in a majority of patients. This report describes three different types of aneurysm of the carotid arteries and their presentation and discusses their management.