K. K. Skrede

Localization of Neurotransmitters, Particularly Glutamate, in Hippocampus, Septum, Nucleus Accumbens and Superior Colliculus

Publisher Summary This chapter presents a survey to identify the neurotransmitter in certain well-defined fibers in the hippocampal–septal system including the nucleus accumbens septi. The transmitters present in the neurons are identified by correlating the distribution of the transmitter marker to the distribution of axon terminals as defined by previous anatomical investigation; by studying the changes in the marker after interruption of specific afferent fibers and after destruction of intrinsic neurons by local injection of kainic acid; and also by studying the release of transmitter marker after stimulating specific nerve fiber bundles. The chapter discusses the structure as a ventral part of striatum, and its efferent projection as a ventral striato-pallidal pathway. Superior colliculus is, like the hippocampus, a laminar structure where many of the afferent fibers are well known. Hippocampus is a laminated structure where the pyramidal and granular cells are distributed in single layers that extend throughout the hippocampus proper and area dentata, respectively. The hippocampal neurons are excited by a series of known fibers such as commissural, perforant path, mossy fibers, and Schaffer collaterals, and inhibited by for example basket cells. The fibers are organized in a layered manner parallel to the cell bodies. In addition, all major neuronal excitatory members as well as the arteries to the hippocampal formation are arranged in lamellae parallel to each other in a plane nearly transverse to the longitudinal axis of the hippocampus.

Calcium-dependent release of D-[3H]aspartate evoked by selective electrical stimulation of excitatory afferent fibres to hippocampal pyramidal cells in vitro.

Abstract Selective electrical stimulation of CA1 excitatory afferent fibres in the stratum radiatum of a transverse slice of the hippocampus released d -[ 3 H]aspartate in a stimulus dependent manner, d -aspartate being used to mimic endogenous l -glutamate. The release fulfilled several of the requirements for neurotransmitter identification in as much as it was Ca 2+ -dependent and specific. Neither [ 3 H]γ-aminobutyrate nor l -[ 3 H]leucine were released in response to stimulation. The stimulus dependent release of d -[ 3 H]aspartate could also be demonstrated in an isolated preparation of the hippocampal slice containing the CA3/CA1 region, but devoid of the area dentata and subiculum. A cut carefully placed in the isolated CA3/CA1 slice dividing all CA1 afferents coursing through the stratum radiatum, without sectioning the pyramidal layer nor the stratum oriens/alveus, abolished the stimulus-evoked release of d -[ 3 H]aspartate. The results, together with previous work, favour l -glutamate as the neurotransmitter of the Schaffer collaterals of the CA3 pyramidal cells.

Release ofd-[3H]aspartate from the dorsolateral septum after electrical stimulation of the fimbria in vitro

A new in vitro preparation from guinea-pig brain is described. The preparation consists of a slice of dorsolateral septum with adhering fimbria. The viability of the preparation was demonstrated. Fibre volleys, single and multiple unit discharges and evoked postsynaptic field potentials could be detected essentially as in vivo. Electrical stimulation of the CA3 hippocampal-septal excitatory connections through the fimbria releasedd-[3H]aspartate, as a marker forl-glutamate, from the dorsolateral septum. The release was specific. The presence of presynaptic fibre volleys at both high (1.5 mm) and low (0.1 mm) concentrations of Ca2+, but efflux ofd-[3H]aspartate only at high Ca2+ concentrations strongly favour a transmitter-related release. The results strongly suggest that two axon collaterals from the same hippocampal CA3 pyramidal cells usel-glutamate as neurotransmitter.

Glutamate transport, glutamine synthetase and phosphate-activated glutaminase in rat CNS white matter. A quantitative study.

Glutamatergic signal transduction occurs in CNS white matter, but quantitative data on glutamate uptake and metabolism are lacking. We report that the level of the astrocytic glutamate transporter GLT in rat fimbria and corpus callosum was ∼ 35% of that in parietal cortex; uptake of [3H]glutamate was 24 and 43%, respectively, of the cortical value. In fimbria and corpus callosum levels of synaptic proteins, synapsin I and synaptophysin were 15–20% of those in cortex; the activities of glutamine synthetase and phosphate‐activated glutaminase, enzymes involved in metabolism of transmitter glutamate, were 11–25% of cortical values, and activities of aspartate and alanine aminotransferases were 50–70% of cortical values. The glutamate level in fimbria and corpus callosum was 5–6 nmol/mg tissue, half the cortical value. These data suggest a certain capacity for glutamatergic neurotransmission. In optic and trigeminal nerves, [3H]glutamate uptake was < 10% of the cortical uptake. Formation of [14C]glutamate from [U‐14C]glucose in fimbria and corpus callosum of awake rats was 30% of cortical values, in optic nerve it was 13%, illustrating extensive glutamate metabolism in white matter in vivo. Glutamate transporters in brain white matter may be important both physiologically and during energy failure when reversal of glutamate uptake may contribute to excitotoxicity.

Evidence for a higher glycolytic than oxidative metabolic activity in white matter of rat brain

Different values exist for glucose metabolism in white matter; it appears higher when measured as accumulation of 2-deoxyglucose than when measured as formation of glutamate from isotopically labeled glucose, possibly because the two methods reflect glycolytic and tricarboxylic acid (TCA) cycle activities, respectively. We compared glycolytic and TCA cycle activity in rat white structures (corpus callosum, fimbria, and optic nerve) to activities in parietal cortex, which has a tight glycolytic-oxidative coupling. White structures had an uptake of [(3)H]2-deoxyglucose in vivo and activities of hexokinase, glucose-6-phosphate isomerase, and lactate dehydrogenase that were 40-50% of values in parietal cortex. In contrast, formation of aspartate from [U-(14)C]glucose in awake rats (which reflects the passage of (14)C through the whole TCA cycle) and activities of pyruvate dehydrogenase, citrate synthase, alpha-ketoglutarate dehydrogenase, and fumarase in white structures were 10-23% of cortical values, optic nerve showing the lowest values. The data suggest a higher glycolytic than oxidative metabolism in white matter, possibly leading to surplus formation of pyruvate or lactate. Phosphoglucomutase activity, which interconverts glucose-6-phosphate and glucose-1-phosphate, was similar in white structures and parietal cortex ( approximately 3 nmol/mg tissue/min), in spite of the lower glucose uptake in the former, suggesting that a larger fraction of glucose is converted into glucose-1-phosphate in white than in gray matter. However, the white matter glycogen synthase level was only 20-40% of that in cortex, suggesting that not all glucose-1-phosphate is destined for glycogen formation.

Adrenaline stimulated cyclic adenosine monophosphate response in leucocytes is reduced after prolonged physical activity combined with sleep and energy deprivation

SummaryThe mechanism for adrenergic desensitisation during physical stress was studied by measuring [125I] cyanopindolol ([125I]CYP) binding sites and the adrenaline stimulated cyclic adenosine monophosphate (cAMP) responses in peripheral blood leucocytes from ten male cadets during a 5-day military training course. The cadets had physical activities around the clock corresponding to a daily energy consumption of about 40,000 kJ but with an intake of only 2,000 kJ, and only 1–3 h of sleep in the 5 days. During the course, the maximal cAMP response to adrenaline stimulation was reduced to about 45% in granulocytes and to 52% in mononuclear cells, and the half maximal response was obtained only at 5–10 times higher adrenaline concentrations than in the control experiment. The binding sites for [125I]-CYP in mononuclear cells increased during the course. However, [125I]-CYP measured not only surface receptors but also intracellular receptors and might even have represented other binding sites. In conclusion, this study showed that decreased cAMP response to adrenergic stimulation would seem to be one of the mechanisms behind adrenergic desensitisation during stress.

Calprotectin (S100A8/S100A9) and myeloperoxidase: co-regulators of formation of reactive oxygen species.

Inflammatory mediators trigger polymorphonuclear neutrophils (PMN) to produce reactive oxygen species (ROS: O2-, H2O2, ∙OH). Mediated by myeloperoxidase in PMN, HOCl is formed, detectable in a chemiluminescence (CL) assay. We have shown that the abundant cytosolic PMN protein calprotectin (S100A8/A9) similarly elicits CL in response to H2O2 in a cell-free system. Myeloperoxidase and calprotectin worked synergistically. Calprotectin-induced CL increased, whereas myeloperoxidase-triggered CL decreased with pH > 7.5. Myeloperoxidase needed NaCl for CL, calprotectin did not. 4-hydroxybenzoic acid, binding ∙OH, almost abrogated calprotectin CL, but moderately increased myeloperoxidase activity. The combination of native calprotectin, or recombinant S100A8/A9 proteins, with NaOCl markedly enhanced CL. NaOCl may be the synergistic link between myeloperoxidase and calprotectin. Surprisingly- and unexplained- at higher concentration of S100A9 the stimulation vanished, suggesting a switch from pro-oxidant to anti-oxidant function. We propose that the ∙OH is predominant in ROS production by calprotectin, a function not described before.

Atrial natriuretic peptide in plasma after prolonged physical strain, energy deficiency and sleep deprivation

SummaryPlasma concentrations of atrial natriuretic peptide (ANP) were investigated daily in 16 male cadets during a 6-day military training course with continuous heavy physical activities, sleep and energy deficiency (course 1). At the end of another similar course (course 11) 15 cadets were studied during 30-min cycle exercise at 50% maximal oxygen uptake with and without glucose infusion. A small, but not significant increase was found in the plasma concentrations of ANP during course I from 9.6 (SEM 1.1) pmol·l−1 in the control experiment to 11.1 (SEM 0.5) pmol·l−1 on day 5. During course II a small but significant increase was found from 7.8 (SEM 0.5) pmol·l−1 in the control experiment to 9.1 (SEM 0.5) pmol·l−1 at the end of the course. Plasma osmolality and chloride concentration decreased during the course. During the exercise test a significant increase was seen in ANP concentration from 8.2 (SEM 0.8) to 13.1 (SEM 2.0) pmol·l−1 in the control experiment and from 9.4 (SEM 0.7) to 13.5 (SEM 1.2) pmol·l−1 during the course. This response was attenuated by glucose infusion, an effect which may have been due to an exercise induced increase in plasma chloride concentration being abolished. In contrast, the potassium concentration response to exercise was increased during the course but unaffected by glucose infusion. In conclusion, the large increases in endogenous plasma catecholamine concentration shown to take place during previous courses were not reflected in the plasma concentrations of ANP, indicating only a moderate cardiac stress or no cardiac work overload during such courses.

Glutamergic Neurons: Localization and Release of the Transmitter

Studies have accumulated showing that amino acids may constitute the group of neurotransmitters which dominate quantitatively in the mammalian brain (5,6,10,26,27). Glutamate is the most important candidate as an excitatory neurotransmitter. But it has several other important functions to fulfill and the problem is therefore to locate the transmitter pool of glutamate. By analogy to other amino acids and amines that function as neurotransmitters, this pool can be identified by three methods: 1) High affinity uptake process. 2) Ca++ dependent release on depolarization of brain slices or synaptosomes. 3) A high intraterminal localization.