Marina Samoilova

Epileptiform activity in hippocampal slice cultures exposed chronically to bicuculline: increased gap junctional function and expression

Chronic (18 h) exposure of cultured hippocampal slices to the type‐A GABA receptor blocker, bicuculline methiodide (BMI) 10 μm increased the levels of connexin 43 (Cx43) and connexin 32 (Cx32) mRNAs, but not connexin 26 and connexin 36, as demonstrated by RNase protection assays. The levels of Cx43 and Cx32 proteins in membrane fractions detected by western blotting were also significantly increased. Immunoblotting indicated that BMI also promoted a significant expression of the transcription protein c‐fos. The rate of fluorescence recovery after photobleaching, an index of gap junctional coupling, was also significantly increased, whereas it was blocked by the gap junctional blocker, carbenoxolone (100 μm). Extracellular recordings in CA1 stratum pyramidale, performed in BMI‐free solution, demonstrated that BMI‐exposed cultures possessed synaptic responses characteristic of epileptiform discharges: (i) significantly greater frequency of spontaneous epileptiform discharges, (ii) post‐synaptic potentials with multiple population spikes, and (iii) significantly longer duration of primary afterdischarges. Carbenoxolone (100 μm), but not its inactive analog, oleanolic acid (100 μm), reversibly inhibited spontaneous and evoked epileptiform discharges. The findings of BMI‐induced parallel increases in levels of gap junction expression and function, and the increase in epileptiform discharges, which were sensitive to gap junctional blockers, are consistent with the hypothesis that increased gap junctional communication plays an intrinsic role in the epileptogenic process.

Connexin 43 mimetic peptides inhibit spontaneous epileptiform activity in organotypic hippocampal slice cultures.

Gap junctions are cytoplasmic channels connecting adjacent cells and mediating their electrical and metabolic coupling. Different cell types in the CNS express various gap junction forming proteins, the connexins, in a cell-specific manner. Using the general gap junctional blocker, carbenoxolone, and two synthetic connexin mimetic peptides, corresponding to amino acid sequences of segments within the second extracellular loop of connexin 43, we studied the role of gap junctions in the generation of epileptiform activity in rat organotypic hippocampal slice cultures. While carbenoxolone inhibited both spontaneous and evoked seizure-like events, connexin mimetic peptides selectively attenuated spontaneous recurrent epileptiform activity, and only after prolonged (>10 h) treatment. The effects were mediated through reduced gap junctional coupling as indicated by suppressed fluorescent dye transfer between the cells. Assuming a selective inhibition of a connexin 43-dependent process by the mimetic peptides and preferential localization of this connexin isoform in astrocytes, the data suggest that, in developing hippocampal networks, the generation and/or initiation of spontaneous recurrent seizure-like activity may depend in large part upon the opening of glial gap junctions. Furthermore, this study shows that the use of a synthetic peptide that mimics a short sequence of a specific connexin isoform and, hence, blocks gap junctional communication in targeted cell types in the CNS, is a viable strategy for the modulation of cerebral activity.

Hippocampal seizures alter the expression of the pannexin and connexin transcriptome.

J. Neurochem. (2010) 112, 92–102.

General anesthetics inhibit gap junction communication in cultured organotypic hippocampal slices.

Gap junctions are protein channels that directly connect the cytosol of neighboring cells, thus forming electrical synapses and promoting synchronous neuronal activities. Such activities lead to the initiation and propagation of electroencephalogram oscillations implicated in cognition and consciousness. In this study, we investigated the effects of propofol, thiopental, and halothane on gap junction communication in cultured organotypic hippocampal slices by recovery of fluorescence after photo bleaching (FRAP) technique and electrophysiological recordings. Propofol 15 &mgr;M and thiopental 10 &mgr;M attenuated gap junction communication in slice cultures by 46.7% ± 4.5% and 48.8% ± 5.5%, respectively, as measured by FRAP. Smaller concentrations of propofol 5 &mgr;M and thiopental 2 &mgr;M did not change gap junction coupling. Accompanying the decreased gap junction communication, hippocampus slice cultures exposed to propofol 15 &mgr;M and thiopental 10 &mgr;M were found to have reduced electrophysiologic spontaneous discharges and primary after discharges evoked by a tetanic train of 50 Hz for 2 s. On the other hand, halothane 0.64 mM, a concentration slightly larger than twice its minimum alveolar concentration had no effect on gap junction coupling while halothane 2.8 mM blocked FRAP by 70%. The current study illustrates that anesthetic concentrations of propofol and thiopental, but not halothane, attenuate gap junction communication in cultured hippocampal slices. Suppression of gap junction function could compound the mechanisms of anesthetic actions.

Calcium chelation improves spatial learning and synaptic plasticity in aged rats

Impaired regulation of intracellular calcium is thought to adversely affect synaptic plasticity and cognition in the aged brain. Comparing young (2-3 months) and aged (23-26 months) Fisher 344 rats, stratum radiatum-evoked CA1 field EPSPs were smaller and long-term potentiation (LTP) was diminished in aged hippocampal slices. Resting calcium, in presynaptic axonal terminals in the CA1 stratum radiatum area, was elevated in aged slices. Loading the slice with the calcium chelator, BAPTA-AM, depressed LTP in young slices, but enhanced this plasticity in old slices. Forty-five minutes following LTP-inducing high frequency stimulation, resting calcium levels were significantly increased in both young and old presynaptic terminals, and significantly reduced by pretreatment with BAPTA-AM. In vivo, intraperitoneal administration of BAPTA-AM prior to training in the reference memory version of the Morris water maze test, significantly improved the acquisition of spatial learning in aged animals, without a significant effect in young rats. These results support the hypothesis that increasing intracellular neuronal buffering power for calcium in aged rats ameliorates age-related impaired synaptic plasticity and learning.

Chronic in vitro ketosis is neuroprotective but not anti-convulsant.

J. Neurochem. (2010) 113, 826–835.

Modular double sucrose gap apparatus for improved recording of compound action potentials from rat and mouse spinal cord white matter preparations.

Compound action potential (CAP) recording is a powerful tool for studying the conduction properties and pharmacology of axons in multi-axonal preparations. The sucrose gap technique improves CAP recording by replacing the extracellular solution between the recording electrodes with a non-conductive sucrose solution to minimize extracellular shunting. The double sucrose gap (DSG), conferring similar advantages at the stimulation site, has been extensively used on guinea pig spinal cord white matter (WM) in vitro. Establishing the DSG methodology for WM preparations from smaller animals such as rats and mice is appealing due to their extensive use in basic and translationally oriented research. Here we describe a versatile modular DSG apparatus with rubber membrane separation of the compartments, suitable for WM strips from rat and mouse spinal cord. The small volumes of compartments (<0.1 ml) and the air-tight design allow perfusion rates of 0.5-1 ml/min with faster refreshment rates compared to commonly used 2-3 ml/min and larger compartments, providing economical usage of expensive pharmacological drugs. Our improved DSG design is particularly efficient for uncovering slower conducting, higher threshold CAP components, as demonstrated by recordings of C-wave (non-myelinated axons) in rat dorsal WM. In myelin-deficient Shiverer mice with genetically dysmyelinated axons, our DSG apparatus recordings revealed a multi-peak C-wave without preceding faster components. The improved stimulation and recording with our DSG apparatus, lowering the range of required stimulus intensities and reducing the artifact interference with recorded CAPs provide for critical technical advantages that allow for more detailed analysis of CAPs in relatively short preparations.

Contribution of Fast and Slow Conducting Myelinated Axons to Single-Peak Compound Action Potentials in Rat Spinal Cord White Matter Preparations

Unlike recordings derived from optic nerve or corpus callosum, compound action potentials (CAPs) recorded from rodent spinal cord white matter (WM) have a characteristic single-peak shape despite the heterogeneity of axonal populations. Using a double sucrose gap technique, we analyzed the CAPs recorded from dorsal, lateral, and ventral WM from mature rat spinal cord. The CAP decay was significantly prolonged with increasing stimulus intensities suggesting a recruitment of higher threshold, slower conducting axons. At 3.5 mm conduction distance, a hidden higher threshold, slower conducting component responsible for prolongation of CAP decay was uncovered in 22 of 25 of dorsal WM strips by analyzing the stimulus-response relationships and a normalization-subtraction procedure. This component had a peak conduction velocity (CV) of 5.0 ± 0.2 (SE) m/s as compared with 9.3 ± 0.5 m/s for the lower threshold peak (P < 0.0001). Oxygen-glucose deprivation (OGD), along with its known effects on CAP amplitude, significantly (P < 0.015) shortened the CAP decay. The hidden higher threshold, slower conducting component showed greater sensitivity to OGD compared with the lower threshold, faster conducting component, suggesting a differential sensitivity of axonal populations of spinal cord WM. At longer conduction distances and lower temperatures (9.8 mm, 22-24°C), the slower peak could be directly visualized in CAPs at higher stimulation intensities. A detailed analysis of single-peak CAPs to identify their fast and slow conducting components may be of particular importance for studies of axonal physiology and pathophysiology in small animals where the conduction distance is not sufficiently long to separate the CAP peaks.

Factors which abolish hypoglycemic seizures do not increase cerebral glycogen content in vitro

The brain is heavily dependant on glucose for its function and survival. Hypoglycemia can have severe, irreversible consequences, including seizures, coma and death. However, the in vivo content of brain glycogen, the storage form of glucose, is meager and is a function of both neuronal activity and glucose concentration. In the intact in vitro hippocampus isolated from mice aged postnatal days 8-13, we have recently characterized a novel model of hypoglycemic seizures, wherein seizures were abolished by various neuroprotective strategies. We had hypothesized that these strategies might act, in part, by increasing cerebral glycogen content. In the present experiments, it was found that neither decreasing temperature nor increasing glucose concentrations (above 2 mM) significantly increased hippocampal glycogen content. Preparations of isolated frontal neocortex in vitro do not produce hypoglycemic seizures yet it was found they contained significantly lower glycogen content as compared to the isolated intact hippocampus. Further, the application of either TTX, or a cocktail containing APV, CNQX and gabazine, to block synaptic activity, did not increase, but paradoxically decreased, hippocampal glycogen content in the isolated intact hippocampus. Significant decreases in glycogen were noted when neuronal activity was increased via incubation with l-aspartate (500 muM) or low Mg(2+). Lastly, we examined the incidence of hypoglycemic seizures in hippocampi isolated from mice aged 15-19 and 22-24 days, and compared it to the incidence of hypoglycemic seizures of hippocampi isolated from mice aged 8-13 days described previously (Abdelmalik et al., 2007 Neurobiol Dis 26(3):646-660). It was noted that hypoglycemic seizures were generated less frequently, and had less impact on synaptic transmission in hippocmpi from PD 22-24 as compared to hippocampi from mice PD 15-19 or PD 8-13. However, hippocampi from 8- to 13-day-old mice had significantly more glycogen than the other two age groups. The present data suggest that none of the interventions which abolish hypoglycemic seizures increases glycogen content, and that low glycogen content, per se, may not predispose to the generation of hypoglycemic seizures.

White Matter: Basic Principles of Axonal Organization and Function

White matter occupies nearly half of the human brain and accommodates a variety of pathways interconnecting different areas of the CNS via predominantly myelinated axons. Being topographically segregated from gray matter, the white matter can be viewed, in a functional sense, as the “nerves within the brain”. However, unlike peripheral nerves, the white matter does not have protective layers of connective tissue around it or its constituent axonal bundles, which makes them potentially susceptible to diffusional influences from neighboring tissues. White matter receives less blood supply compared to gray matter, and its axons are well adapted to minimal energy supply while maintaining high fidelity delivery signals from one gray matter area to another. The CNS myelinated axons are well designed space-savers, having smaller diameters compared to PNS and more compact myelin sheaths. Myelin covers nearly 99 % of the length of myelinated axons, the rest being nodes of Ranvier that serve as “relay stations” for saltatory propagation of action potentials. The axonal conduction velocities in white matter are well tuned to specific physiological needs and provide timely delivery of signals to target neurons for summation with other synaptic inputs. A minor decrease in conduction velocity or in the number of conducting axons due to injury or stroke may have catastrophic consequences due to disturbed coordination of signal arrivals to target neurons and their summation with other synaptic inputs, potentially halting further transfer of signals to other neurons. The myelin-forming cells of the CNS, the oligodendrocytes, myelinate different numbers of axons depending on their calibers, ranging from one in case of large (10 μm) axons to 50–60 in case of smallest (<0.5 μm) axons. Myelinating multiple axons by single oligodendrocyte has its drawback, as an injury to one oligodendrocyte may have a “multiplication effect,” shutting down a number of axons at once. The chapter discusses in detail the organization of myelin sheaths and their relationships with axons and periaxonal glia.