Tibor Zelles

Role of sodium channel inhibition in neuroprotection: Effect of vinpocetine

Vinpocetine (ethyl apovincaminate) discovered during the late 1960s has successfully been used in the treatment of central nervous system disorders of cerebrovascular origin for decades. The increase in the regional cerebral blood flow in response to vinpocetine administration is well established and strengthened by new diagnostical techniques (transcranial Doppler, near infrared spectroscopy, positron emission tomography). The latest in vitro studies have revealed the effect of the compound on Ca(2+)/calmodulin dependent cyclic guanosine monophosphate-phosphodiesterase 1, voltage-operated Ca(2+) channels, glutamate receptors and voltage dependent Na(+)-channels; the latest being especially relevant to the neuroprotective action of vinpocetine. The good brain penetration profile and heterogenous brain distribution pattern (mainly in the thalamus, basal ganglia and visual cortex) of labelled vinpocetin were demonstrated by positron emission tomography in primates and man. Multicentric, randomized, placebo-controlled clinical studies proved the efficacy of orally administered vinpocetin in patients with organic psychosyndrome. Recently positron emission tomography studies have proved that vinpocetine is able to redistribute regional cerebral blood flow and enhance glucose supply of brain tissue in ischemic post-stroke patients.

Distance-Dependent Scaling of Calcium Transients Evoked by Backpropagating Spikes and Synaptic Activity in Dendrites of Hippocampal Interneurons

Although interactions between backpropagating action potentials and synaptic stimulations have been extensively studied in pyramidal neurons, dendritic propagation and the summation of these signals in interneurons are not nearly as well known. In this study, two-photon imaging was used to explore the basic properties of dendritic calcium signaling in CA1 stratum radiatum interneurons. In contrast to hippocampal pyramidal neurons, the backpropagating action potential-evoked calcium transients in dendrites of interneurons underwent a distance-dependent increment. Although, in proximal dendrites, an increment could be attributed to a smaller dendrite diameter, distal dendrites did not show such dependence. Calcium responses in interneurons had a smaller amplitude, slower rise time, and decay than in pyramidal neurons. To explore the factors underlying the difference, we compared the calcium-binding capacity in interneurons and in pyramidal neurons. Our finding that endogenous calcium buffers had a higher level in interneurons may primarily explain the different kinetics and amplitudes of calcium transients. Synaptic stimulation-evoked calcium transients were also larger at distant dendritic locations. The spread of these signals was restricted to 12-13 μm long dendritic compartments. Supporting the reported lack of long-term potentiation in these interneurons, we found only sublinear or linear summations of calcium responses to coincident synaptic inputs and backpropagating spikes.

Subtype-specificity of the presynaptic α2-adrenoceptors modulating hippocampal norepinephrine release in rat

In vivo brain microdialysis and high-performance liquid chromatography with electrochemical detection were used to study the effect of different selective alpha 2-antagonists on hippocampal norepinephrine (NE) release in freely moving awake rat. Systemic administration (0.5 mg/kg i.p.) of either the alpha 2AD-antagonist BRL 44408 or the alpha 2BC-antagonist ARC 239 did not significantly change the basal release of NE. At a higher dose (5 mg/kg i.p.) ARC 239 was still ineffective, whereas BRL 4408 caused a significant increase of the extracellular level of NF. Similar results were obtained from in vitro perfusion experiments. Rat hippocampal slices were loaded with [3H]NE and the electrical stimulation-evoked release of [3H]NE was determined. The alpha 2-antagonists were applied in a concentration range of 10(-8) to 10(-6) M, ARC 239 was ineffective, whereas BRL 44408 significantly increased the electrically induced release of [3H]NE. In agreement with the data of microdialysis and perfusion experiments, BRL 44408 displaced [3H]yohimbine from hippocampal and cortical membranes of rat brain with high affinity whereas ARC 239 was less effective. The pKi values of eight different alpha 2-adrenergic compounds showed a very good correlation (r = 0.98, slope = 1.11 P < 0.0001) in hippocampus and frontal cortex have the alpha 2-adrenoceptors have been characterized as alpha 2d-subtype. Our data indicate that hippocampal NE release in rat is regulated by alpha 2D-adrenoceptors, a species variation of the human alpha 2A-subtype.

Branch-Specific Ca2+ Influx from Na+-Dependent Dendritic Spikes in Olfactory Granule Cells

Two-photon laser scanning microscopy was used to correlate electrical events detected with whole-cell somatic recordings to Ca2+ transients in dendrites of olfactory bulb granule cells. A subset of spontaneous subthreshold depolarizing events recorded at the soma were shown to correspond to suprathreshold dendritic, Na-dependent action potentials [APs; dendritic spikes (D-spikes)]. These potentials were blocked by intracellular QX-314 (lidocaine N-ethyl bromide), hyperpolarizing current injection at the soma, and by partial inhibition of AMPA/kainate receptors with 0.75 μm DNQX. They were affected only slightly by 100 μm NiCl2. The majority of D-spikes recorded at the soma had a time to peak of <4 ms, comparable with somatic APs, a nonexponential decay, and amplitudes between 3 and 21 mV. Somatically recorded APs produced Ca2+ transients that were observed in spines and dendrites in all parts of the cell. Ca2+ transients from D-spikes were restricted to subsets of distal dendrites and their associated spines but were absent from the soma and dendrite within ∼50–80 μm of the soma. Ca2+ transients in different branches could be correlated with different-sized D-spikes. D-spike and backpropagating AP-induced Ca2+ transients summed in dendrites, provided the interval between them was >5–6 ms. Generation of a D-spike in a particular dendrite <5–6 ms before a somatic AP blocked backpropagation of the somatic AP into that dendrite. The temporally specific interplay between D-spikes and backpropagating APs may play a role in regulating feedback and feedforward inhibition of groups of mitral cells synapsing on different granule cell dendrites.

Effect of nicotine on dopaminergic-cholinergic interaction in the striatum

We have investigated the effect of nicotinic receptor stimulation on acetylcholine (ACh) release measured by radioassay in rat striatal slices. Since the release of ACh in the striatum is tonically inhibited by endogenous dopamine and nicotine enhances the release of dopamine, we studied the release of ACh when the dopaminergic input was impaired. We used chemical denervation (6-hydroxydopamine pretreatment) or D2-receptor-blockade by sulpiride to remove the dopaminergic control of the cholinergic neurons. In our experiments nicotine failed to increase ACh release from striatal slices taken from rats whose dopaminergic-cholinergic interaction was not impaired but it enhanced the release of ACh from slices dissected from 6-hydroxydopamine pretreated rats or in the presence of sulpiride. Our results provide neurochemical evidence for the existence of nicotinic receptors on striatal cholinergic interneurons. Since the spontaneous release of ACh enhanced by nicotine was inhibited by tetrodotoxin it seems very likely that (-)-nicotine acts on the somatodendritic part of cholinergic interneurons.

Release of acetylcholine and noradrenaline from the cholinergic and adrenergic afferents in rat hippocampal CA1, CA3 and dentate gyrus regions

An attempt was made to study the release of acetylcholine (ACh) and noradrenaline and their presynaptic modulation in isolated slice preparations dissected from different subfields of the hippocampus: CA1, CA3 and the dentate gyrus. The slices were perfused and loaded with [3H]choline or with [3H]noradrenaline. The release in response to field stimulation was determined radiochemically and the content of transmitters was assayed by a chemiluminescent method or by HPLC combined with electrochemical detection. After 30 min of loading with [3H]choline there were marked subregional differences in the specific activity of [3H]ACh content. The highest concentration was measured in the dentate gyrus and the lowest in CA3. Evidence was obtained that in all three subfields the cholinergic axon terminals are equipped with inhibitory muscarinic autoreceptors and the noradrenergic terminals with α2‐autoreceptors, as indicated by an increase in transmitter release when the tissue was exposed to selective muscarinic or α2‐adrenoceptor antagonists. In contrast, the cholinergic boutons are not equipped with α2‐adrenoceptors, and noradrenergic terminals do not possess inhibitory muscarinic receptors. It is therefore concluded that while the release of both ACh and noradrenaline is controlled by negative feedback modulation, there is no possibility of establishing a presynaptic inhibitory interaction between the two.

Layer-specific differences in reactive oxygen species levels after oxygen–glucose deprivation in acute hippocampal slices

The major role of reactive oxygen species (ROS) in the pathomechanism of ischemia have been widely recognized. Still, measurements of the precise time course and regional distribution of ischemia-induced ROS level changes in acute brain slices have been missing. By using acute hippocampal slices and the fluorescent dye CM-H2DCFDA, we showed that reoxygenation after in vitro ischemia (oxygen-glucose deprivation; OGD) increased ROS levels in the hippocampal CA1 layers vulnerable to ischemia but did not have significant effects in the resistant stratum granulosum in the dentate gyrus (DG). Production of ROS started during OGD, but, contrary to reoxygenation, it manifested as a ROS level increase exclusively in the presence of catalase and glutathione peroxidase inhibition. The mechanism of ROS production involves the activation of NMDA receptors and nitric oxide synthases. The inhibition of ROS response by either AP-5 or L-NAME together with the ROS sensitivity profile of the dye suggest that peroxynitrite, the reaction product of superoxide and nitric oxide, plays a role in the response. Direct visualization of layer-specific effects of ROS production and its scavenging, shown for the first time in acute hippocampal slices, suggests that distinct ROS homeostasis may underlie the different ischemic vulnerability of CA1 and DG.

Mechanism of the persistent sodium current activator veratridine-evoked Ca2+ elevation: Implication for epilepsy

Although the role of Na+ in several aspects of Ca2+ regulation has already been shown, the exact mechanism of intracellular Ca2+ concentration ([Ca2+]i) increase resulting from an enhancement in the persistent, non‐inactivating Na+ current (INa,P), a decisive factor in certain forms of epilepsy, has yet to be resolved. Persistent Na+ current, evoked by veratridine, induced bursts of action potentials and sustained membrane depolarization with monophasic intracellular Na+ concentration ([Na+]i) and biphasic [Ca2+]i increase in CA1 pyramidal cells in acute hippocampal slices. The Ca2+ response was tetrodotoxin‐ and extracellular Ca2+‐dependent and ionotropic glutamate receptor‐independent. The first phase of [Ca2+]i rise was the net result of Ca2+ influx through voltage‐gated Ca2+ channels and mitochondrial Ca2+ sequestration. The robust second phase in addition involved reverse operation of the Na+–Ca2+ exchanger and mitochondrial Ca2+ release. We excluded contribution of the endoplasmic reticulum. These results demonstrate a complex interaction between persistent, non‐inactivating Na+ current and [Ca2+]i regulation in CA1 pyramidal cells. The described cellular mechanisms are most likely part of the pathomechanism of certain forms of epilepsy that are associated with INa,P. Describing the magnitude, temporal pattern and sources of Ca2+ increase induced by INa,P may provide novel targets for antiepileptic drug therapy.

The Nootropic Drug Vinpocetine Inhibits Veratridine-Induced [Ca2+]i Increase in Rat Hippocampal CA1 Pyramidal Cells

The alkaloid derivative vinpocetine (14-ethoxycarbonyl-(3α,16α-ethyl)-14,15-eburnamine; Cavinton) has a well known beneficial effect on brain function in hypoxic and ischemic conditions. While it increases CNS blood flow and improves cellular metabolism, relatively little is known about vinpocetines underlying molecular mechanisms on the single cell level. Since apoptotic and necrotic cell damage is always preceded by an increase in [Ca2+]i, this study investigated the effect of vinpocetine on [Ca2+]i increases in acute brain slices. Sodium influx is an early event in the biochemical cascade that takes place during ischemia. The alkaloid veratridine can activate this Na+ influx, causing depolarization and increasing [Ca2+]i in the cells. Therefore, it can be used to simulate an ischemic attack in brain cells. Using a cooled CCD camera-based ratio imaging system and cell loading with fura 2/AM, the effect of vinpocetine on [Ca2+]i changes in single pyramidal neurons in the vulnerable CA1 region of rat hippocampal slices was investigated. Preperfusion and continuous administration of vinpocetine (10 μM) significantly inhibited the elevation in [Ca2+]i induced by veratridine (10 μM). When the drug was administered after veratridine, it could accelerate the recovery of cellular calcium levels. Piracetam, another nootropic used in clinical practice, could attenuate the elevation of [Ca2+]i only at a high, 1 mM, concentration. We have concluded that vinpocetine, at a pharmacologically relevant concentration, can decrease pathologically high [Ca2+]i levels in individual rat hippocampal CA1 pyramidal neurons; this effect might contribute to the neuroprotective property of the drug.

Chemical neuroprotection in the cochlea: The modulation of dopamine release from lateral olivocochlear efferents

The prevalence of sensorineural hearing loss is increasing worldwide, mainly due to ageing, increased noise exposure and cardiovascular risk factors. Several papers dealt with the mechanisms underlying the primary causes of impaired hearing and eventual deafness, including the damage and loss of auditory hair cells; however, very little is known about the protective mechanisms that exist for hearing. Several recent investigations have implicated dopamine (DA) in a neuroprotective circuit for the cochlea. The lateral olivocochlear (LOC) efferents provide axonal innervation of the inner hair cell afferent synapses and release DA and other substances in response to different stimuli. Under ischemic conditions or during noise exposure, DA has been proven to play a neuroprotective role against glutamate excitotoxicity. This review summarises what is currently known about the modulation of DA release in the cochlea, using primarily in vitro experimental data. Based on recent knowledge, there could be two functional subgroups within the LOC fibres, i.e., the DA- and GABA-containing projections. In this review, we attempt to show the neurochemical interactions between these two subsystems. Other aspects of cochlear neurotransmission are also discussed to provide a complete picture of cochlear dopaminergic function in physiological and pathophysiological cases with particular reference to excitotoxicity.