Nóra Szilágyi

GABAergic Neurons of the Medial Septum Lead the Hippocampal Network during Theta Activity

Information processing in the hippocampus critically relies on its reciprocal interaction with the medial septum (MS). Synchronization of the septo-hippocampal system was demonstrated during both major hippocampal activity states, the regular theta rhythm and the large amplitude irregular activity. Previous experimental and modeling data suggest that the MS provides rhythmic drive to the hippocampus, and hippocampo-septal feedback synchronizes septal pacemaker units. However, this view has recently been questioned based on the possibility of intrahippocampal theta genesis. Previously, we identified putative pacemaker neurons expressing parvalbumin (PV) and/or the pacemaker hyperpolarization-activated and cyclic nucleotide-gated nonselective cation channel (HCN) in the MS. In this study, by analyzing the temporal relationship of activity between the PV/HCN-containing medial septal neurons and hippocampal local field potential, we aimed to uncover whether the sequence of events during theta formation supports the classic view of septal drive or the challenging theory of hippocampal pacing of theta. Importantly, by implementing a circular statistical method, a temporal lead of these septal neurons over the hippocampus was observed on the course of theta synchronization. Moreover, the activity of putative hippocampal interneurons also preceded hippocampal local field theta, but by a shorter time period compared with PV/HCN-containing septal neurons. Using the concept of mutual information, the action potential series of PV/HCN-containing neurons shared higher amount of information with hippocampal field oscillation than PV/HCN-immunonegative cells. Thus, a pacemaker neuron population of the MS leads hippocampal activity, presumably via the synchronization of hippocampal interneurons.

Phase Segregation of Medial Septal GABAergic Neurons during Hippocampal Theta Activity

Septo-hippocampal GABAergic neurons immunoreactive for parvalbumin are thought to play a crucial role in the generation of hippocampal theta oscillations associated with a specific stage of memory formation. Here we use in vivo juxtacellular recording and filling in the medial septum followed by immunocytochemical identification of the recorded cells containing parvalbumin to determine their firing pattern, phase relationship with hippocampal theta, morphology, and to thereby reveal their involvement in the generation of hippocampal theta activity. We have demonstrated that GABAergic medial septal neurons form two distinct populations exhibiting highly regular bursting activity that is tightly coupled to either the trough (178°) or the peak (330°) of hippocampal theta waves. Additionally, different types of bursting as well as nonbursting activity patterns were also observed. The morphological reconstruction of theta-bursting neurons revealed extensive axon arbors of these cells with numerous local collaterals establishing symmetrical synapses; thus, synchrony among the septal pacemaker units may be brought about by their recurrent collateral interactions. Long projecting axons could also be found running dorsally toward the hippocampus and ventrally in the direction of basal forebrain regions. We conclude that GABAergic neurons in the medial septum, which are known to selectively innervate hippocampal interneurons, are in a position to induce rhythmic disinhibition in the hippocampus and other theta-related subcortical areas at two different phases of hippocampal theta.

Estrogen Induces Estrogen Receptor α-Dependent cAMP Response Element-Binding Protein Phosphorylation via Mitogen Activated Protein Kinase Pathway in Basal Forebrain Cholinergic Neurons In Vivo

In addition to classical genomic mechanisms, estrogen also exerts nonclassical effects via a signal transduction system on neurons. To study whether estrogen has a nonclassical effect on basal forebrain cholinergic system, we measured the intensity of cAMP response element-binding protein (CREB) phosphorylation (pCREB) in cholinergic neurons after administration of 17β-estradiol to ovariectomized (OVX) mice. A significant time-dependent increase in the number of pCREB-positive cholinergic cells was detected after estrogen administration in the medial septum-diagonal band (MS-DB) and the substantia innominata (SI). The increase was first observed 15 min after estrogen administration. The role of classical estrogen receptors (ERs) was evaluated using ER knock-out mice in vivo. The estrogen-induced CREB phosphorylation in cholinergic neurons was present in ERβ knock-out mice but completely absent in ERα knock-out mice in MS-DB and SI. A series of in vitro studies demonstrated that estrogen acted directly on cholinergic neurons. Selective blockade of the mitogen activated protein kinase (MAPK) pathway in vivo completely prevented estrogen-induced CREB phosphorylation in cholinergic neurons in MS-DB and SI. In contrast, blockade of protein kinase A (PKA) was effective only in SI. Finally, studies in intact female mice revealed levels of CREB phosphorylation within cholinergic neurons that were similar to those of estrogen-treated OVX mice. These observations demonstrate an ERα-mediated nonclassical effect of estrogen on the cholinergic neurons and that these actions are present under physiological conditions. They also reveal the role of MAPK and PKA–MAPK pathway activation in nonclassical estrogen signaling in the basal forebrain cholinergic neurons in vivo.

Analysis of purine and pyrimidine bases, nucleosides and deoxynucleosides in brain microsamples (microdialysates and micropunches) and cerebrospinal fluid

A new chromatographic method is reported for the synchronous analysis of endogenous purine and pyrimidine bases, ribonucleosides, and deoxyribonucleosides in brain samples. An optimized gradient chromatography system with a cooled reversed-phase column allows the detection of these compounds in very low concentrations in microsamples (microdialysates and micropunches). Chromatographic peaks were identified via the retention times of known standards, with detection at two wavelengths, and also by electrospray tandem mass spectrometry, which permits the identification of certain compounds at extremely low concentrations. The method was tested on in vivo brain microdialysis samples, micropunch tissue sample and cerebrospinal fluid of rats. Extracellular concentrations of pyrimidine metabolites in brain samples and of various purine metabolites in thalamic samples are reported here first. A comparison of the results on microdialysis and cerebrospinal fluid samples suggests that the analysis of cerebrospinal fluid provides limited information on the local extracellular concentrations of these compounds. Basic dialysis experiments revealed temporarily stable baseline levels one hour after implantation of the microdialysis probes. An elevated potassium concentration in the perfusion solution caused increases in the extracellular levels of adenosine and its metabolites, and of guanosine and the pyrimidine nucleoside uridine.

The electroretinogram and visual evoked potential of freely moving rats

The vascularised rat retina could be one of the most useful experimental objects in visual neuroscience to understand human visual physiological and pathological processes. We report here on a new method of implantation for studying the visual system of freely moving rats that provides a rat model for simultaneous recording at corneal and cortical level and is stable enough to record for months. We implanted light emitting diodes onto the skull behind the eyeball to stimulate the eye with flashes and to light adapt the retina with constant light levels. A multistrand, stainless steel, flexible fine wire electrode placed on the eyeball was used for electroretinogram recording and screw electrodes (left/right visual and parietal cortical) were used to record the visual evoked potential and the electroencephalogram. In the present report we focus on the new method of implantation for recording the corneal flash electroretinogram of normal, freely moving rats simultaneously with the visual evoked cortical potential showing examples in various visual experiments. We also introduce a program for retinogram and visual evoked potential analysis, which defines various measures (latencies, areas, amplitudes, and durations) and draw attention to the benefits of this method for those involved in visual, functional genomic, pharmacological, and human ophthalmologic research.

Post mortem degradation of nucleosides in the brain: Comparison of human and rat brains for estimation of in vivo concentration of nucleosides

There is an increasing attention paid for nucleoside metabolism and changes of nucleoside concentrations in human brain because of its pathological and physiological relevance. In order to determine the post mortem degradation of nucleosides and nucleoside metabolites, the concentrations of four nucleosides and three nucleobases were measured in rat and neurosurgical human cerebral cortical samples with 30s to 24h post mortem delay. Adenosine degradation coefficient (a multiplying factor for calculating concentrations of investigated substances for the living state) was 0.886 for human brain at 2 h post mortem time, while it was 1.976 for rats. Hypoxanthine, an adenosine degradation product had coefficients 0.564 for human brain and 0.812 for the rat brain. We provide data and degradation coefficients for the concentrations of adenosine, guanosine, inosine, uridine, uracil, hypoxanthine and xanthine with 2, 4, 6 and 24 h post mortem delay. We also report a method how to validate human neurosurgical brain samples in terms of sample preparation and statistical analysis.

B cell receptor-induced Ca2+ mobilization mediates F-actin rearrangements and is indispensable for adhesion and spreading of B lymphocytes

B cells acquire membrane‐bound cognate antigens from the surface of the APCs by forming an IS, similar to that seen in T cells. Recognition of membrane‐bound antigens on the APCs initiates adhesion of B lymphocytes to the antigen‐tethered surface, which is followed by the formation of radial lamellipodia‐like structures, a process known as B cell spreading. The spreading response requires the rearrangement of the submembrane actin cytoskeleton and is regulated mainly via signals transmitted by the BCR. Here, we show that cytoplasmic calcium is a regulator of actin cytoskeleton dynamics in B lymphocytes. We find that BCR‐induced calcium mobilization is indispensible for adhesion and spreading of B cells and that PLCγ and CRAC‐mediated calcium mobilization are critical regulators of these processes. Measuring calcium and actin dynamics in live cells, we found that a generation of actin‐based membrane protrusion is strongly linked to the dynamics of a cytoplasmic‐free calcium level. Finally, we demonstrate that PLCγ and CRAC channels regulate the activity of actin‐severing protein cofilin, linking BCR‐induced calcium signaling to the actin dynamics.

Concentration of Nucleosides and Related Compounds in Cerebral and Cerebellar Cortical Areas and White Matter of the Human Brain

1. Nucleosides potentially participate in the neuronal functions of the brain. However, their distribution and changes in their concentrations in the human brain is not known. For better understanding of nucleoside functions, changes of nucleoside concentrations by age and a complete map of nucleoside levels in the human brain are actual requirements.2. We used post mortem human brain samples in the experiments and applied a recently modified HPLC method for the measurement of nucleosides. To estimate concentrations and patterns of nucleosides in alive human brain we used a recently developed reverse extrapolation method and multivariate statistical analyses.3. We analyzed four nucleosides and three nucleobases in human cerebellar, cerebral cortices and in white matter in young and old adults. Average concentrations of the 308 samples investigated (mean±SEM) were the following (pmol/mg wet tissue weight): adenosine 10.3±0.6, inosine 69.5±1.7, guanosine 13.5±0.4, uridine 52.4±1.2, uracil 8.4±0.3, hypoxanthine 108.6±2.0 and xanthine 54.8±1.3. We also demonstrated that concentrations of inosine and adenosine in the cerebral cortex and guanosine in the cerebral white matter are age-dependent.4. Using multivariate statistical analyses and degradation coefficients, we present an uneven regional distribution of nucleosides in the human brain. The methods presented here allow to creation of a nucleoside map of the human brain by measuring the concentration of nucleosides in microdissected tissue samples. Our data support a functional role for nucleosides in the brain.

Low-[Mg2+]-induced Ca2+ fluctuations in organotypic hippocampal slice cultures.

We show here by whole field monitoring of free intracellular Ca2+ ([Ca2+]i), locally recorded field potential (fp) and external [Ca2+], that low-[Mg2+] induces seizure like events (SLEs) accompanied by simultaneous fluctuations of [Ca2+]i and [Ca2+]e in cultured hippocampal slices. Within a SLE, complex [Ca2+]e fluctuations are seen throughout phases of Ca2+ depletion (tonic) and Ca2+ recovery (clonic) of the extra-cellular space. Information theory entropy-based analyses revealed strong asymmetric associations of [Ca2+]i and [Ca2+]e kinetics. By contrast, signal-associations between SLEs were found to be weak and of symmetric nature distinguishing seizure-like and interictal events by extensive coupling and decoupling of [Ca2+]i and [Ca2+]e fluctuations, respectively.

Neurotoxicity of Lindane and Picrotoxin: Neurochemical and Electrophysiological Correlates in the Rat Hippocampus In Vivo

In the present study, we compared in vivo changes of extracellular amino acid levels and nucleotide derivatives to a single ip dose of lindane (10-60 mg/kg) and picrotoxin (5 mg/kg) in the hippocampus of halothane anaesthetized rat by microdialysis-coupled HPLC analysis. Brain activity was monitored by EEG. The effects of lindane and picrotoxin on EEG pattern of rats as well as on hippocampal amino acid and nucleotide status were studied in 0-50 min, 50-100 min and 100-150 min periods post-dosing. Significant decreases in Glu and Asp were found after picrotoxin treatment. After 50-100 min post-dosing, hippocampal hypoxanthine and inosine levels increased to both lindane (10 mg/kg) and picrotoxin whereas xanthine and uridine levels increased to picrotoxin, only. Lindane elicited a dose-dependent occurrence of negative spikes accompanied with rhythmic activity at 4-5 Hz. The picrotoxin-induced 4-5 Hz activity did not display negative sharp waves and was accompanied by 10 Hz oscillations.