Sanna Soini

[3H]Ethylketocyclazocine Binding to Brain Opioid Receptor Subtypes in Alcohol-Preferring AA and Alcohol-Avoiding ANA Rats

We measured brain regional patterns of [3H]ethylketocyclazocine binding to brain opioid receptors in ethanol-naive alcohol-preferring Alko, Alcohol (AA) and alcohol-avoiding Alko, Non-Alcohol (ANA) rats, by using quantitative autoradiography. This agonist ligand labels all opioid receptor subtypes. The proportions of mu- and delta-opioid receptor binding were evaluated by displacing the mu- and delta-opioid receptor components by the peptides Tyr-D-Ala-Gly-N(Me)Phe-Gly-ol (DAMGO, 100 nM) and Tyr-D-Pen-Gly-Phe-D-Pen (DPDPE, 100nM), respectively, the K-component being the naltrexone-sensitive binding left after removal of the above two components. The labeling patterns in the brains of the AA and ANA rats were consistent with the well-known distributions of the opioid receptor subtypes in nonselected rat strains and there was no major difference between the lines. The mu-opioid receptor binding was greater in the AA than ANA rats in several brain regions, most interestingly in the substantia nigra pars reticulata and striatal clusters with elevated shell/core ratios in the nucleus accumbens. The delta-opioid receptor binding did not differ between the lines, whereas the AA rats had more K-opioid receptors than the ANA rats in several brain regions, including limbic areas and basal ganglia. The observed results might indicate altered action of the opioidergic system on dopaminergic pathways in rats with differential alcohol preference.

Non-specific binding of [18F]FDG to calcifications in atherosclerotic plaques: experimental study of mouse and human arteries.

Purpose[18F]FDG has been used as an inflammation marker and shown to accumulate in inflammatory atherosclerotic plaques. The aim of this study was to investigate the uptake and location of [18F]FDG in atherosclerotic plaque compartments.MethodsThe biodistribution of intravenously administered [18F]FDG was analysed in atherosclerotic LDLR/ApoB48 mice (n=11) and control mice (n=9). Digital autoradiography was used to detect the ex vivo distribution in frozen aortic sections. In vitro binding of [18F]FDG in human atherosclerotic arteries was also examined.ResultsThe uptake of [18F]FDG was significantly higher in the aorta of atherosclerotic mice as compared with the control mice. Autoradiography of excised arteries showed higher [18F]FDG uptake in the plaques than in the healthy vessel wall (mean ratio ±SD 2.7±1.1). The uptake of [18F]FDG in the necrotic, calcified sites of the advanced atherosclerotic lesions was 6.2±3.2 times higher than that in the healthy vessel wall. The in vitro studies of human arterial sections showed marked binding of [18F]FDG to the calcifications but not to other structures of the artery wall.ConclusionIn agreement with previous studies, we observed [18F]FDG uptake in atherosclerotic plaques. However, prominent non-specific binding to calcified structures was found. This finding warrants further studies to clarify the significance of this non-specific binding in human plaques in vivo.

Brain Opioid Receptor Binding of [3H]CTOP and [3H]Naltrindole in Alcohol-Preferring AA and Alcohol-Avoiding ANA Rats

We compared mu- and delta-opioid receptor distributions between the brains of alcohol-preferring Alko, Alcohol (AA) and alcohol-avoiding Alko, Non-Alcohol (ANA) rat lines, using autoradiography on brain sections with mu- and delta-opioid receptor antagonist ligands [3H]CTOP and [3H]naltrindole, respectively. The labeling patterns of the ligands were consistent with the known opioid receptor distributions in both rat lines and no major genetic differences were found between the lines. However, the binding density of mu- and delta-opioid receptors differed slightly in several brain areas: in the AA brain sections, limbic areas, such as hippocampus and amygdala, showed decreased mu- and delta-opioid receptor binding, whereas the striatal patches were larger and the substantia nigra showed higher binding density of the mu-receptors compared to the ANA sections. The small differences observed between the rat lines could be due to adaptations to altered endogenous opioid peptide levels or neural circuits, and associated with the differences in alcohol drinking or other behaviors.

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.

Brain regional μ-opioid receptor function in rat lines selected for differences in alcohol preference

It has been suggested that opioid peptides play a role in the reinforcing effects of alcohol. The present study was designed to examine the function of the mu-opioid receptor system in rat lines selectively bred for alcohol preference (AA [Alko, Alcohol] rat line) and alcohol avoidance (ANA [Alko, Non-Alcohol] rat line). The functional coupling of mu-opioid receptors to G proteins was determined autoradiographically using Tyr-D-Ala-Gly-N(Me)Phe-Gly-ol-enkephalin-stimulated [35S]GTPgammaS binding in brain cryostat sections. The binding was significantly increased in the striatal patches and substantia nigra reticulata of the AA rats in comparison with that of the ANA rats. Within the AA rat line, there was a significant positive correlation between 3 mg/kg morphine-induced locomotor activity and activation of G-proteins in the substantia nigra compacta and nucleus accumbens core. These results of the selective breeding experiment suggest that brain region-specific differences in mu-opioid receptor function may correlate with innate differences in alcohol preference.

Selective changes in gamma-aminobutyric acid type A receptor subunits in the hippocampus in spontaneously seizing rats with chronic temporal lobe epilepsy.

Changes in the structure and function of inhibitory GABA(A) receptors may contribute to epileptogenesis. We have used the in situ hybridization technique to study GABA(A) receptor alpha2, alpha4, beta3 and gamma2 subunit mRNA expression in the hippocampus of spontaneously seizing rats with chronic temporal lobe epilepsy. In control rats, all four subunit mRNAs were expressed in the hippocampal subregions but the intensity of expression varied significantly between the subfields. In epileptic rats, alpha2 expression was decreased in CA3c, and alpha4 in CA1, but beta3 was increased in all subregions, in particular in the granule cell layer. Our results suggest that GABA(A) receptor undergoes region selective subunit changes during epileptogenesis in the hippocampus of rats with chronic temporal lobe epilepsy.

Maturation of cultured hippocampal slices results in increased excitability in granule cells

The preparation of hippocampal slices results in loss of input neurons to dentate granule cells, which leads to the reorganization of their axons, the mossy fibers, and alters their functional properties in long‐term cultures, but its temporal aspects in the immature hippocampus are not known. In this study, we have focused on the early phase of this plastic reorganization process by analyzing granule cell function with field potential and whole cell recordings during the in vitro maturation of hippocampal slices (from 1 to 17 days in vitro, prepared from 6 to 7‐day‐old rats), and their morphology using extracellular biocytin labelling technique. Acute slices from postnatal 14–22‐day‐old rats were analyzed to detect any differences in the functional properties of granule cells in these two preparations. In field potential recordings, small synaptically‐evoked responses were detected at 2 days in vitro, and their amplitude increased during the culture time. Whole cell voltage clamp recordings revealed intensive spontaneous excitatory postsynaptic currents, and the susceptibility to stimulus‐evoked bursting increased with culture time. In acutely prepared slices, neither synaptically‐evoked responses in field potential recordings nor any bursting in whole cell recordings were detected. The excitatory activity was under the inhibitory control of γ‐aminobutyric acid type A receptor. Extracellularily applied biocytin labelled dentate granule cells, and revealed sprouting and aberrant targeting of mossy fibers in cultured slices. Our results suggest that reorganization of granule cell axons takes place during the early in vitro maturation of hippocampal slices, and contributes to their increased excitatory activity resembling that in the epileptic hippocampus. Cultured immature hippocampal slices could thus serve as an additional in vitro model to elucidate mechanisms of synaptic plasticity and cellular reactivity in response to external damage in the developing hippocampus.

Regulation of HERG (KCNH2) potassium channel surface expression by diacylglycerol

The HERG (KCNH2) channel is a voltage-sensitive potassium channel mainly expressed in cardiac tissue, but has also been identified in other tissues like neuronal and smooth muscle tissue, and in various tumours and tumour cell lines. The function of HERG has been extensively studied, but it is still not clear what mechanisms regulate the surface expression of the channel. In the present report, using human embryonic kidney cells stably expressing HERG, we show that diacylglycerol potently inhibits the HERG current. This is mediated by a protein kinase C-evoked endocytosis of the channel protein, and is dependent on the dynein–dynamin complex. The HERG protein was found to be located only in early endosomes and not lysosomes. Thus, diacylglycerol is an important lipid participating in the regulation of HERG surface expression and function.

Stereospecific modulation of GABAA receptor function by urocanic acid isomers

A deamination product of histidine, urocanic acid, accumulates in the skin of mammals as trans-urocanic acid. Ultraviolet (UV) irradition converts it to the cis-isomer that is an important mediator in UV-induced immunosuppression. We have recently shown that urocanic acid interferes with the agonist binding to GABA(A) receptors. We now report that the effects of urocanic acid on binding of a convulsant ligand (t-butylbicyclo[35S]phosphorothionate) to GABA(A) receptors in brain membrane homogenates are dependent on pH of the incubation medium, the agonistic actions being enhanced at the normal pH of the skin (5.5). Using Xenopus laevis oocytes expressing recombinant rat alpha1beta1gamma2S GABA(A) receptors, the low pH potentiated the direct agonistic action of trans-urocanic acid under two-electrode voltage-clamp, whereas cis-urocanic acid retained its low efficacy both at pH 5.5 and 7.4. The results thus indicate clear differences between urocanic acid isomers in functional activity at one putative receptor site of immunosuppression, the GABA(A) receptor, the presence of which in the skin remains to be demonstrated.

The effects of temperature and adrenergic agonists on cardiac myocytes of perch (Perca fluviatilis) in cell culture conditions

Abstract 1. 1. Isolated cardiac myocytes of perch, Perca fluviatilis , were kept in culture conditions for 1–2 months at 12 or 22°C. In the culture most myocytes flattened, lost their spindle-shaped morphology, protruded pseudopod-like branches and many of them started visible contractions in 1–2 weeks and continued beating for several months. Myocytes did not divide in the sparse cell population used. Typical intracellular structures could be seen in electron micrographs still after 1–2 months, but the sarcoplasmic organization became gradually more irregular in the culture. 2. 2. Beat rates showed linear temperature relationship on the Arrhenius plot. Myocytes cultivated at 22°C showed higher frequencies and slightly less dependence on temperature than myocytes cultivated at 12°C (apparent activation energies ( E a ) 86 and 107 kJ/mol, respectively). 3. 3. Temperature dependence of frequencies was related to the presence of added serum or adrenergic agonists: β-adrenergic agonists increased the frequencies and rendered the cells less dependent on temperature; apparent activation energy was 43 kJ/mol for isoprenaline or adrenaline and 108 kJ/mol for noradrenaline and control group. 4. 4. Heat tolerance was greater in myocytes cultivated at 22°C than in myocytes cultivated at 12°C, and the change in tolerance appeared in 12 h after the alteration of culture temperature and the increased tolerance was persistent after that. 5. 5. It is suggested, that the processes of quick heat-hardening and of slower but persistent heat resistance acclimation developed in these cells in culture conditions but not the capacity acclimation, which seems to be dependent on adrenergic regulation of beat rate.