Brain mitochondrial metabolic dysfunction and glutamate level reduction in the pilocarpine model of temporal lobe epilepsy in mice

Although certain metabolic characteristics such as interictal glucose hypometabolism are well established for temporal lobe epilepsy (TLE), its pathogenesis still remains unclear. Here, we performed a comprehensive study of brain metabolism in a mouse model of TLE, induced by pilocarpine–status epilepticus (SE). To investigate glucose metabolism, we injected mice 3.5–4 weeks after SE with [1,2-¹³C]glucose before microwave fixation of the head. Using ¹H and ¹³C nuclear magnetic resonance spectroscopy, gas chromatography—mass spectrometry and high-pressure liquid chromatography, we quantified metabolites and ¹³C labeling in extracts of cortex and hippocampal formation (HF). Hippocampal levels of glutamate, glutathione and alanine were decreased in pilocarpine–SE mice compared with controls. Moreover, the contents of N-acetyl aspartate, succinate and reduced nicotinamide adenine dinucleotide (phosphate) NAD(P)H were decreased in HF indicating impairment of mitochondrial function. In addition, the reduction in ¹³C enrichment of hippocampal citrate and malate suggests decreased tricarboxylic acid (TCA) cycle turnover in this region. In cortex, we found reduced ¹³C labeling of glutamate, glutamine and aspartate via the pyruvate carboxylation and pyruvate dehydrogenation pathways, suggesting slower turnover of these amino acids and/or the TCA cycle. In conclusion, mitochondrial metabolic dysfunction and altered amino-acid metabolism is found in both cortex and HF in this epilepsy model.     

Nerve activity-dependent variations in clearance of released noradrenaline: Regulatory roles for sympathetic neuromuscular transmission in rat tail artery

The aim of this study was to find out if clearance of noradrenaline released from sympathetic nerve terminals in rat isolated tail artery is a physiological variable and if so, to determine its role for the noradrenaline-mediated neurogenic contraction. The per pulse release of noradrenaline induced by electrical nerve stimulation and the fluctuations of the level of noradrenaline at the receptors driving the contractions were assessed from the electrochemically determined noradrenaline oxidation current at a carbon fibre electrode at the surface of the artery. Both were compared with the noradrenaline-mediated neurogenic contraction. The effects on these parameters of cocaine or desipramine, or of corticosterone, were used to assess the relative roles of neuronal and extraneuronal uptake, respectively. The effects of cocaine or desipramine, which enhance the noradrenaline level at the receptors by blocking neuronal reuptake, were compared with those of yohimbine, presumed to act exclusively by enhancing the per pulse release of noradrenaline.

The results seem to support the following tentative conclusions. Clearance of released noradrenaline occurs by neuronal uptake and diffusion, while extraneuronal uptake is negligible. The noradrenaline-induced neurogenic contraction is mediated via adrenoceptors on cells near the plane of the nerve plexus; the excitation spreads from these cells throughout the syncytium. The contractile response to exogenous noradrenaline may also be mediated via receptors on the innervated key cells. Reuptake of noradrenaline into the releasing varicosities, i.e. in “active junctions”, is highly efficient for single quanta but rapidly saturated by repeated release, while reuptake of noradrenaline in the “surround” of active junctions is probably rarely saturated and more independent of nerve activity. Saturation of the transporter by repeated release of quanta from the same varicosity and the consequent accumulation of “residual” noradrenaline and increased diffusion out of the junction and recruitment of noradrenaline receptors in the surround may be the cause of the rapid growth of the contraction during a high frequency train. Diffusion of released noradrenaline away from the postjunctional receptors is restricted by a local nerve activity-dependent buffering mechanism which, in spite of facling of the per pulse release, helps maintain the noradrenaline concentration at the receptors and the contraction during long high-frequency trains. Reactivation of the clearance mechanisms upon cessation of nerve activity accelerates the relaxation. This “plasticity” of noradrenaline clearance enables the vessel to virtually ignore nerve impulses at low frequency, contract briskly in response to high-frequency bursts, maintain tension during long trains at high frequency in spite of a declining per pulse release of noradrenaline, and relax rapidly upon cessation of nerve activity.     

K+ and Ca2+ channel blockers may enhance or depress sympathetic transmitter release via a Ca(2+)-dependent mechanism "upstream" of the release site.

Extracellular recording of the pre- and postjunctional electrical activity in guinea-pig or mouse vas deferens or rat tail artery was employed to study the mechanisms by which the K+ channel blockers, tetraethylammonium and 4-aminopyridine and the Ca2+ channel blockers, Cd2+, Mn2+ or nifedipine influence the nerve stimulation-induced release of adenosine 5'-triphosphate as a sympathetic co-transmitter. The K+ and Ca2+ channel blocking agents examined had no effect on the spontaneous quantal release of adenosine 5'-triphosphate. However, addition of tetraethylammonium and 4-aminopyridine inside the recording electrode broadened the nerve terminal action potential and caused it to become more resistant to local application of tetrodotoxin, and dramatically increased the magnitude and tetrodotoxin resistance of adenosine 5'-triphosphate release within the patch. Surprisingly, tetraethylammonium and 4-aminopyridine were equally effective when added outside the recording electrode; now they did not increase the duration of the nerve terminal action potential inside the patch but increased its resistance to locally applied tetrodotoxin and dramatically increased the magnitude as well as the tetrodotoxin resistance of adenosine 5'-triphosphate release from sites inside the patch. Both tetraethylammonium and 4-aminopyridine contributed to these effects, with a strong potentiating interaction. Nifedipine was without effect, but application of 1-100 microM Cd2+ or 1-5 mM Mn2+ either inside or outside the recording electrode blocked adenosine 5'-triphosphate release inside the patch. The results indicate: (i) that the nerve terminal action potential is generated by activation of voltage-gated, regenerative Na+ channels but also has a small component carried by influx of Ca2+ and that it is "normally" terminated by activation of voltage- as well as Ca(2+)-dependent K+ channels; (ii) that the release probability is tonically depressed by the resting K+ efflux, and promoted by the resting Ca2+ influx, "upstream" of the release sites; and (iii) that the upstream control of the release probability may involve both changes in properties of ionic channels in the nerve terminal membrane, and effects on the cytoskeleton leading to changes in the availability of releasable quanta in varicosities within the patch.     

"Upstream" regulation of the release probability in sympathetic nerve varicosities

The results appear to support the following tentative working hypothesis. (1) Nerve impulse-induced transmitter release from sympathetic nerve varicosities is monoquantal and highly intermittent (probability range: 0-0.03). (2) Nerve impulses invade varicosities as all-or-none, Na+ channel-dependent action potentials; invasion failure may be rare. (3) The release probability is not controlled by properties (amplitude or duration) of the invading action potential or the resulting Ca2+ current, but by the availability of an as yet unidentified permissive factor. (4) The permissive factor is actively transported intra-axonally, probably in association with organelles (LDVs?). (5) The activation and/or transport of the permissive factor are controlled "upstream" of the varicosity; they depend on Ca2+ influx through channels insensitive to nifedipine (hence, not of L-type) but blocked by Cd2+ and apparently opened by slight depolarization of the resting membrane, in this respect behaving more as T- than N-type channels. (6) A high resting K+ efflux "upstream" of the varicosity restricts the availability of the permissive factor; it is the main mechanism maintaining the (economically necessary) low release probability. (7) Prejunctional agonists do not inhibit transmitter secretion by causing a conduction block or by reducing the action potential-induced Ca2+ influx into the varicosity itself, but by depressing the Ca2(+)-dependent activation and/or transport of the permissive factor; they act at least in part via receptors "upstream" of the varicosity. (8) This hypothesis for regulation of the release probability in sympathetic nerves may apply, at least in part, to other neurons as well.