Irma E. Holopainen

Early and Late Mismatch Negativity Elicited by Words and Speech-Like Stimuli in Children

In auditory perception the brains attentional and preattentional mechanisms select certain stimuli for preferential processing and filter out irrelevant input. This study investigated nonattentive auditory processing in children. Event-related potentials (ERPs) provide a means to study neural correlates related to language and speech-sound processing. Mismatch negativity (MMN) is an ERP wave that indicates attention-independent perceptual change detection. In this study cortical ERPs were elicited by complex tones, naturally spoken words, and pseudowords, with each stimulus type containing equal acoustical elements. Tones elicited a bifurcated mismatch negativity (MMN), with early MMN (peaking at 150-200 ms) being more dominant. On the other hand, words elicited a strong late MMN, peaking at about 400-450 ms after stimulus onset. The MMN wave form was significantly weaker for pseudowords than for words. The late MMN wave, especially for word differences, was found to reflect summating MMN generators and memory trace formation on gestalt bases. Results suggest that the auditory processing, even nonattended, is highly associated with the cognitive meaning of the stimuli.

Organotypic Hippocampal Slice Cultures: A Model System to Study Basic Cellular and Molecular Mechanisms of Neuronal Cell Death, Neuroprotection, and Synaptic Plasticity

The hippocampus has become one of the most extensively studied areas of the mammalian brain, and its proper function is of utmost importance, particularly for learning and memory. The hippocampus is the most susceptible brain region for damage, and its impaired function has been documented in many human brain diseases, e.g. hypoxia, ischemia, and epilepsy regardless of the age of the affected patients. In addition to experimental in vivo models of these disorders, the investigation of basic anatomical, physiological, and molecular aspects requires an adequate experimental in vitro model, which should meet the requirements for well-preserved representation of various cell types, and functional information processing properties in the hippocampus. In this review, the characteristics of organotypic hippocampal slice cultures (OHCs) together with the main differences between the in vivo and in vitro preparations are first briefly outlined. Thereafter, the use of OHCs in studies focusing on neuron cell death and synaptic plasticity is discussed.

Seizures in the developing brain : Cellular and molecular mechanisms of neuronal damage, neurogenesis and cellular reorganization

Epilepsy is a common neurological disorder that occurs more frequently in children than in adults. The extent that prolonged seizure activity, i.e. status epilepticus (SE), and repeated, brief seizures affect neuronal structure and function in both the immature and mature brain has been the subject of increasing clinical and experimental research. Earlier studies suggest that seizure-induced effects in the immature brain compared with the adult brain are different. This is manifested as differences in neuronal vulnerability, cellular and synaptic reorganization and regenerative processes. The focus of this review is first to give a short overview of currently used experimental models of epilepsy in immature rats, and then discuss more thoroughly seizure-induced acute and sub-acute cellular and molecular alterations, highlight the contribution of inflammatory-like reactions and intracellular cytoskeleton to the insult, and reveal changes in the structure and function of inhibitory GABA(A) and excitatory glutamate receptors. The role of seizure-activated reparative, plastic processes, synaptic remodelling, neurogenesis as well as the long-term consequences of seizures are briefly outlined. The main emphasis is put on studies carried out in experimental animals, and the focus of interest is the hippocampus, the brain area of great vulnerability in epilepsy. In vitro studies are discussed only to limited extent. Collectively, recent studies suggest that the deleterious effects of seizures may not solely be a consequence of neuronal damage and loss per se, but could be due to the fact that seizures interfere with the highly regulated developmental processes in the immature brain.

Glutamate receptor agonists increase intracellular Ca2+ independently of voltage-gated Ca2+ channels in rat cerebellar granule cells

Changes in membrane potential and cytosolic free Ca2+ concentrations, [Ca2+]i, in response to L-glutamate and glutamate receptor agonists were measured in rat cerebellar granule cells grown on coverslips. The membrane was depolarized by the application of L-glutamate and kainate, and by elevating the extracellular K+ concentration, as determined by using the membrane potential probe bisoxonol (DiBA-C4-(3)). The [Ca2+]i as measured with fura-2 was 220 nM on average under resting conditions and increased by raising the extracellular K+ and by applying L-glutamate, kainate, quisqualate or N-methyl-D-aspartate (NMDA). Verapamil and nifedipine reduced the high-K+ induced rise in [Ca2+]i but did not significantly affect the responses produced by NMDA, quisqualate and kainate, suggesting that the increase in intracellular Ca2+ in response to glutamate receptor agonists is primarily due to Ca2+ influx through receptor-coupled ion channels.

α-Receptor and cholinergic receptor-linked changes in cytosolic Ca2+ and membrane potential in primary rat astrocytes

Both phenylephrine and carbachol caused a sustained increase in Ca2+ influx and intracellular free Ca2+ of primary astrocytes as measured with 45Ca2+ and fura-2. The responses to phenylephrine and carbachol were additive, suggesting that they use different releasable pools of Ca2+. If extracellular Ca2+ was removed by EGTA only a transient rise in cytosolic Ca2+ was seen upon application of the agonists. Both compounds caused depolarization of the astrocyte membrane as determined with the optical probe 3,3-diethylthiadicarboxyamineiodide. Activation of protein kinase C with 12-tetradecanoylphorbol myristate acetate (TPA) or the diacylglycerol analogue dioctanoylglycerol (DiC8) also depolarized the cells. A prior activation of protein kinase C with TPA or DiC8 abolished the depolarizing effect of phenylephrine suggesting that they act through the same mediators. If the cells were made ideally permeable to K+ with the ionophore valinomycin, or the K+ channels had been blocked with Ba2+, neither TPA nor phenylephrine had any significant effect on the membrane potential. Neither TPA nor phenylephrine had any effect on the 86Rb+ equilibrium potential across the cell membrane. The results suggest that the depolarizing effect of these substances could be through a blocking of K+ channels.

Neuronal activity regulates GABAA receptor subunit expression in organotypic hippocampal slice cultures.

The postnatal expression of GABA(A) receptor subunit mRNAs in the rat brain, including the hippocampus, exhibits a unique temporal and regional developmental profile in vivo, which may be altered by external stimuli. Using the in situ hybridization technique we have now studied the in vitro expression of alpha1,alpha2, alpha 4, alpha 5, beta 1, beta 3, gamma 2, and gamma 3 subunit mRNAs of GABA(A) receptors in organotypic hippocampal slices cultured for 7 days. To find out whether neuronal activity regulates the subunit expression, a subset of cultures was chronically treated either with a GABA(A) receptor antagonist picrotoxin, or by a non-N-methyl-D-aspartate (non-NMDA)-receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX). In untreated control cultures, the expression pattern of the subunits varied regionally, the most abundantly expressed subunits being alpha 2 and alpha 5 in all subregions. All studied subunits were expressed in CA3a/b and CA1, whereas in CA3c and in granule cells of the dentate gyrus (DG) no signal of alpha 4 and gamma 3 was detected. The drug treatment differently affected the regional subunit expression. In picrotoxin-treated cultures, the expression of alpha1, alpha 5 and gamma 2 mRNAs was significantly increased in pyramidal cell layers, and in DNQX-treated cultures the expression of alpha2 mRNA in CA3c and DG, and that of beta1 in DG. Changes in the expression of GABA(A) receptor subunit mRNAs in treated cultures suggest that neuronal activity can regulate their regional expression in vitro. Since the expression profile in untreated control cultures closely resembled that observed earlier in vivo, organotypic hippocampal slice cultures could serve as a good model system to study the regulatory mechanisms of receptor expression under well-controlled experimental conditions in the developing hippocampus.

Decreased binding of [11C]flumazenil in Angelman syndrome patients with GABAA receptor β3 subunit deletions

We used positron emission tomography (PET) to study brain [11C]flumazenil (FMZ) binding in four Angelman syndrome (AS) patients. Patients 1 to 3 had a maternal deletion of 15q11‐q13 leading to the loss of β3 subunit of γ‐aminobutyric acidA/benzodiazepine (GABAA/BZ) receptor, whereas Patient 4 had a mutation in the ubiquitin protein ligase (UBE3A) saving the β3 subunit gene. [11C]FMZ binding potential in the frontal, parietal, hippocampal, and cerebellar regions was significantly lower in Patients 1 to 3 than in Patient 4. We propose that the 15q11‐q13 deletion leads to a reduced number of GABAA/BZ receptors, which could partly explain the neurological deficits of the AS patients. Ann Neurol 2001;49:110–113

Event-related desynchronization and synchronization during a memory task in children.

OBJECTIVE To examine the event-related desynchronization (ERD) and synchronization (ERS) responses of several narrow electroencephalographic (EEG) frequency bands in children during an auditory memory task. METHODS ERD/ERS responses of the 4-6, 6-8, 8-10 and 10-12 Hz EEG frequency bands were studied in 12 children (mean age 12 years) while they performed an auditory memory task. Twelve adult subjects served as a control group. RESULTS The childrens ERD/ERS responses differed from those of the adults in the 4-6, 6-8 and 8-10 Hz EEG frequency bands, especially during retrieval from memory. The childrens 4-6 Hz initial ERS responses were of lesser amplitude and of delayed latency as compared to those of the adults. In the 6-8 and 8-10 Hz frequency bands, especially during retrieval from memory, the childrens ERD responses were of lesser magnitude than those of the adults. In the 10-12 Hz frequency band, no differences were observed between the ERD/ERS responses between the children and adults. CONCLUSIONS Our results suggest that theta and alpha response systems might participate in auditory information processing already at this age, although not being fully developed. Memory systems involving retrieval may be the last to mature.

Temporal profiles of age-dependent changes in cytokine mRNA expression and glial cell activation after status epilepticus in postnatal rat hippocampus

BackgroundStatus epilepticus (SE) is proposed to lead to an age-dependent acute activation of a repertoire of inflammatory processes, which may contribute to neuronal damage in the hippocampus. The extent and temporal profiles of activation of these processes are well known in the adult brain, but less so in the developing brain. We have now further elucidated to what extent inflammation is activated by SE by investigating the acute expression of several cytokines and subacute glial reactivity in the postnatal rat hippocampus.MethodsSE was induced by an intraperitoneal (i.p.) injection of kainic acid (KA) in 9- and 21-day-old (P9 and P21) rats. The mRNA expression of interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α), interleukin-10 (IL-10), matrix metalloproteinase-9 (MMP-9), glial-derived neurotrophic factor (GDNF), interferon gamma (IFN-γ), and transforming growth factor-beta 1 (TGF-β1) were measured from 4 h up to 3 days after KA injection with real-time quantitative PCR (qPCR). IL-1β protein expression was studied with ELISA, GFAP expression with western blotting, and microglial and astrocyte morphology with immunohistochemistry 3 days after SE.ResultsSE increased mRNA expression of IL-1β, TNF-α and IL-10 mRNA in hippocampus of both P9 and P21 rats, their induction being more rapid and pronounced in P21 than in P9 rats. MMP-9 expression was augmented similarly in both age groups and GDNF expression augmented only in P21 rats, whereas neither IFN-γ nor TGF-β1 expression was induced in either age group. Microglia and astrocytes exhibited activated morphology in the hippocampus of P21 rats, but not in P9 rats 3 d after SE. Microglial activation was most pronounced in the CA1 region and also detected in the basomedial amygdala.ConclusionOur results suggest that SE provokes an age-specific cytokine expression in the acute phase, and age-specific glial cell activation in the subacute phase as verified now in the postnatal rat hippocampus. In the juvenile hippocampus, transient increases in cytokine mRNA expression after SE, in contrast to prolonged glial reactivity and region-specific microglial activity after SE, suggest that the inflammatory response is changed from a fulminant and general initial phase to a more moderate and specific subacute response.

Histaminergic Neurons Protect the Developing Hippocampus from Kainic Acid-Induced Neuronal Damage in an Organotypic Coculture System

The central histaminergic neuron system inhibits epileptic seizures, which is suggested to occur mainly through histamine 1 (H1) and histamine 3 (H3) receptors. However, the importance of histaminergic neurons in seizure-induced cell damage is poorly known. In this study, we used an organotypic coculture system and confocal microscopy to examine whether histaminergic neurons, which were verified by immunohistochemistry, have any protective effect on kainic acid (KA)-induced neuronal damage in the developing hippocampus. Fluoro-Jade B, a specific marker for degenerating neurons, indicated that, after the 12 h KA (5 μm) treatment, neuronal damage was significantly attenuated in the hippocampus cultured together with the posterior hypothalamic slice containing histaminergic neurons [HI plus HY (POST)] when compared with the hippocampus cultured alone (HI) or with the anterior hypothalamus devoid of histaminergic neurons. Moreover, α-fluoromethylhistidine, an inhibitor of histamine synthesis, eliminated the neuroprotective effect in KA-treated HI plus HY (POST), and extracellularly applied histamine (1 nm to 100 μm) significantly attenuated neuronal damage only at 1 nm concentration in HI. After the 6 h KA treatment, spontaneous electrical activity registered in the CA1 subregion contained significantly less burst activity in HI plus HY (POST) than in HI. Finally, in KA-treated slices, the H3 receptor antagonist thioperamide enhanced the neuroprotective effect of histaminergic neurons, whereas the H1 receptor antagonists triprolidine and mepyramine dose-dependently decreased the neuroprotection in HI plus HY (POST). Our results suggest that histaminergic neurons protect the developing hippocampus from KA-induced neuronal damage, with regulation of neuronal survival being at least partly mediated through H1 and H3 receptors.