Frode Fonnum

A rapid radiochemical method for the determination of choline acetyltransferase.

RADIOCHEMICAL assays of choline acetyltransferase (ChAT) must be based on reproducible, rapid and specific procedures for isolating ACh from the incubation mixture. In our laboratory this has been achieved by isolating labelled ACh by liquid cation exchange using sodium tetraphenylboron (Kalignost) in ethyl butyl ketone (FONNUM, 1969a. b). In the present report I will show that this method can be simplified considerably by using the scintillation mixture as extraction solvent and carrying out the extraction directly in the scintillation vial. Labelled ACh can subsequently be determined by liquid scintillation counting at high efficiency in the biphasic aqueous: toluene scintillation solution mixture. The principle involved would be applicable to other radiochemical enzyme assays based on different forms of organic solvent extraction such as in methods for AChE (POTTER, 1967) and aromatic amino acid decarboxylase (BROCH & FONNUM, 1972). Particular attention has been paid to the possible interference of acetylcarnitine in the assay of ChAT and the effect of acetyl-CoA concentration on the ChAT activity.

Glutamate: A Neurotransmitter in Mammalian Brain

Glutamate is ubiquitously distributed in brain tissue, where it is present in a higher concentration than any other amino acid. During the last 50 years glutamate in brain has been the subject of numerous studies, and several different functions have been ascribed to it. Early studies by Krebs (1935) suggested that glutamate played a central metabolic role in brain. The complex compartmentation of glutamate metabolism in brain was first noted by Waelsch and coworkers (Berl et al., 1961). These studies were precipitated by the claim that glutamate improved mental behaviour and was beneficial in several neurological disorders including epilepsy and mental retardation. Other scientists pointed out its function in the detoxification of ammonia in brain (Weil-Malherbe, 1950). Glutamate is also an important building block in the synthesis of proteins and peptides, including glutathione (Meister, 1979). The toxic effect of administered glutamate and its analogues kainic acid, ibotenic acid, and N-methyl aspartic acid on CNS neurones has become a large and independent line of research (Lucas and Newhouse, 1957; Olney et al., 1974; Lund-Karlsen and Fonnum, 1976; Coyle, 1983). Attention has also been focused on the role of glutamate as a precursor for the inhibitory neurotransmitter y-aminobutyric acid (GABA) (Roberts and Frankel, 1950). Electrophysiological studies (Curtis and Watkins, 1961) focused early on the powerful and excitatory action of glutamate on spinal cord neurones. Since the action was widespread and effected by both the Dand Lforms, it was at first difficult to believe that glutamate could be a neurotransmitter. During the last 15 years, however, several studies have provided support for the concept that glutamate is a transmitter in brain (for review see Curtis and Johnston, 1974; Fonnum, 1978; 1981; Roberts et al., 1981; DiChiara and Gessa, 1981). Glutamate satisfies today to a large extent the four main criteria for classification as a neurotransmitter: (1) it is presynaptically localized in specific neurones; (2) it is specifically released by physiological stimuli in concentrations high enough to elicit postsynaptic response; (3) it demonstrates identity of action with the naturally occurring transmitter, including response to antagonists; and (4) mechanisms exist that will terminate transmitter action rapidly. The evidence for glutamate as a transmitter at the locust neuromuscular junction has recently been carefully evaluated by Usherwood (1981). In that case the identity of action of glutamate with the naturally occurring transmitter on the neuromuscular receptor, the release from nerve terminals, and its similarity to acetylcholine at the mammalian neuromuscular junction with regard to presynaptic pharmacology and denervation supersensitivity are compelling evidence for glutamate as a neurotransmitter. The main methods used to identify glutamergic pathways in brain will be critically reviewed and discussed. The effect of lesions on high-affinity uptake and release are particularly important, but immunohistochemical methods to study enzymes and glutamate itself are becoming more interesting. The release of glutamate has been demonstrated by several different procedures using both in vivo and in vitro preparations. The synthesis of large groups of specific agonists and antagonists has been important both for identification and characterization of the glutamate receptor by electrophysiological techniques and for the isolation of glutamate receptors. High and perhaps low-affinity uptake into nerve terminals and glial cells is important for the termination of transmitter action. Particular attention is given in this review to the complex compartmentation of glutamate synthesis and the possibility of identifying the transmitter pool of glutamate.

Biochemical evidence for glutamate as neurotransmitter in corticostriatal and corticothalamic fibres in rat brain

Abstract The effects of ablation of frontal, occipital or entire hemicortex on several neurotransmitter parameters in the rostral and caudal neostriatum, thalamus and the contralateral anterior medial cortex were investigated. In particular the effects on the high affinity uptake of d -aspartate and on the endogenous level of amino acids, especially glutamate and aspartate, were studied in order to identify glutamate- or aspartate-containing nerve terminals in these regions. The results show a specific decrease in high affinity uptake of d -aspartate in both rostral and caudal neostriatum ipsilateral to the lesion after frontal or entire hemidecortication. There was also a small but significant decrease in d -aspartate uptake on the contralateral side. Only the level of endogenous glutamate decreased in the neostriatum after hemidecortication. There was a specific decrease in d -aspartate uptake in the thalamus only ipsilateral to the cortical lesions. In thalamus there was a significant decrease both in the level of glutamate and to a smaller extent in that of aspartate after hemidecortication. Anterior medial cortex showed a very active high affinity uptake of d -aspartate, which was slightly reduced after removal of the contralateral hemicortex. The high affinity uptake of d -aspartate was in all cases mainly due to uptake in synaptosomes. The results show that the neostriatum receives glutamate-containing fibres from the neocortex, particularly the frontal part. This projection is mainly ipsilateral with a small element derived from the contralateral side. The thalamus, both the rostral and caudal parts, receives glutamate-containing fibres from the whole extent of the ipsilateral neocortex. Some of the corticothalamic fibres may also contain aspartate. The anterior medial cortex probably contains a high proportion of glutamate- and/or aspartate-containing nerve terminals but only a low proportion of these are derived from the contralateral cortex.

Origin and distribution of glutamate decar☐ylase in substantia nigra of the cat

The topographical distribution of glutamate decar☐ylase (GAD) in substantia nigra in unoperated and operated cats was studied in samples microdissected from freeze-dried tissue sections. The concentration of GAD, the enzyme synthesizing γ-aminobutyric acid (GABA), was highest in the medial part of pars reticulata, and decreased in the mediolateral direction. In pars compacta, on the other hand, the highest enzyme activity was found in the lateral part which merges with pars reticulata, and it decreased gradually in the latero-medial direction. The activity of GAD was always lower in the medial part of pars compacta, which contains the highest concentration of cell bodies. GAD in substantia nigra decreased after lesions in putamen, nucleus caudatus, globus pallidus and nucleus entopeduncularis. The loss of enzyme activity was strictly localized and was related to the site of termination of the degenerating striato-nigral fibers. The reduction of GAD in substantia nigra following lesions of globus pallidus or nucleus entopeduncularis may be ascribed to the interruption of striato-nigral fibers passing through these regions. The results thus indicate that the fibers of the GAD-containing axon terminals in substantia nigra of the cat originate in putamen and nucleus caudatus. Subcellular fractionation showed that about 85% of GAD and about 25% of lactate dehydrogenase were present in particles (probably synaptosomes) from substantia nigra in unoperated animals. Electron microscopic examination revealed that 11.5% of the tissue volume of pars reticulata was occupied by boutons compared to 5.9% for pars compacta. The concentration of GABA in pars reticulata was found to be 9 mM. From these data the intraterminal concentration of GABA was estimated to be at least 60 mM, probably over 100 mM. DOPA decar☐ylase was mainly found in pars compacta. Acetylcholin-esterase showed a very high activity in substantia nigra, the highest concentration being found in the medial part of pars reticulata. In contrast, the concentration of choline acetyltransferase was very low. The ratio of acetylcholinesterase activity to choline acetyltransferase activity was 1000. DOPA decar☐ylase and the cholinergic enzymes were little affected by the above described lesions.

Distribution of glutamate decarboxylase, choline acetyl-transferase and aromatic amino acid decarboxylase in the basal ganglia of normal and operated rats. Evidence for striatopallidal, striatoentopeduncular and striatonigral GABAergic fibres.

Abstract The topographical distribution of glutamate decarboxylase (GAD), aromatic amino acid decarboxylase (AAD) and choline acetyltransferase (ChAT) were studied in striatum (i.e. caudate-putamen), globus pallidus, nucleus entopeduncularis and substantia nigra. There were only small differences in the rostrocaudal distribution of enzymes in striatum. The highest concentration of GAD was found in the ventrocaudal part, whereas AAD was highest in the rostral part. ChAT did not show any distinct distribution pattern. Globus pallidus and nucleus entopeduncularis were similar in their content of GAD, AAD and ChAT. They were rich in GAD but poor in AAD and ChAT. The highest concentration of GAD was found in the rostralmost part of globus pallidus. In substantia nigra AAD was concentrated in the rostral part; contents of GAD and ChAT did not differ distinctly in the rostral and caudal nigra. GAD and ChAT were highly localized in a particulate fraction, probably in all 4 regions, whereas AAD was localized in particulate fraction in the striatum and globus pallidus and soluble in the nucleus entopeduncularis and substantia nigra. Transverse and/or oblique hemitransections which passed through the striatum but not through the globus pallidus resulted in substantial loss of GAD in the globus pallidus, nucleus entopeduncularis and substantia nigra. There was a good correlation between the anteroposterior level of hemitransection and the decline in GAD activities, which was generally highest following posterior lesions. The reduction of GAD was largest in the globus pallidus and smallest in the substantia nigra, in which a significant loss of GAD occurred only following transections of the postcommissural part of caudate-putamen. A very high decrease of GAD in the nucleus entopeduncularis and substantia nigra was obtained following posterior oblique hemitransections which passed through posterior part of striatum and rostral globus pallidus. The results indicate that the majority of GABAergic terminals in the globus pallidus belong to striatopallidal fibers. They suggest, furthermore, that a large number of striatoentopeduncular and striatonigral fibers are GABAergic, the latter arising preferentially from the posterior part of caudate-putamen.

Biochemical evidence for γ-aminobutyrate containing fibres from the nucleus accumbens to the substantia nigra and ventral tegmental area in the rat

Abstract Glutamate decarboxylase activity, a specific marker for γ-aminobutyrate-containing neurons, has been analysed in microdissected samples from rat mesencephalon following unilateral electrocoagulations of the nucleus accumbens. This lesion resulted in a consistent decrease of 50% in the enzyme activity in the rostromedial substantia nigra, and a slight, but insignificant decrease (−15%) in the medial parts of the caudal pars compacta of the substantia nigra. No change was found in the lateral pars compacta or the central pars reticulata. In the ventral tegmental area, the highest activity was found in the rostromedial part, adjacent to the mammillary body. At this level, a significant decrease of 20% was found in the ventral tegmental area on the lesioned side. In contrast, the activities in the medial accessory optic nucleus and the caudal ventral tegmental area adjacent to the interpenduncular nucleus were unchanged. The results indicate that the nucleus accumbens sends γ-aminobutyrate-containing fibres to the rostromedial substantia nigra and to the rostral ventral tegmental area. The caudal ventral tegmental area, the lateral pars compacta and the central pars reticulata do not receive measurable amounts of such fibres.

An in vivo model for studying function of brain tissue temporarily devoid of glial cell metabolism: the use of fluorocitrate.

Abstract: The effect of intrastriatal injection of fluorocitrate on amino acid pattern, cell enzyme markers, and ultrastruc‐tural appearance was investigated. A dose of 1 nmol of fluorocitrate resulted in temporarily decreased levels of glutamine, glutamate, and aspartate, whereas the level of alanine was increased. The glutamine level was severely reduced after 4 h but was reversed after 24 h. The activity of different cellular enzyme markers did not change markedly after this dose. Ultrastructural changes in glial cells were observed, concomitant with the biochemical changes. A dose of ≥2 nmol of fluorocitrate resulted in more marked and irreversible changes in amino acid levels. By 24–72 h after the injection of this dose, several marker enzyme activities decreased markedly. The ultrastructural changes affected the neurons as well as the glial cells and were not reversible. The use of microinjection of 1 nmol of fluorocitrate into the neostria‐tum of the rat to provide a model for studying transmitter amino acid metabolism in brain devoid of glial cell activity is discussed.

The effect of brominated flame retardants on neurotransmitter uptake into rat brain synaptosomes and vesicles.

The environmental levels of brominated flame retardants (BFRs) are increasing, but little is known about their toxic effects. In this paper, we show that some of the most important BFRs in commercial use today, have a neurotoxicological potential. Hexabromocyclododecane (HBCD) and tetrabromobisphenol-A (TBBPA) inhibit plasma membrane uptake of the neurotransmitters dopamine, glutamate and gamma-amino-n-butyric acid (GABA) at a concentration level similar to what previously found for polychlorinated biphenyls (PCBs) and even for ecstasy. The IC(50) value for HBCD on dopamine uptake was 4 microM, and the IC(50) values for TBBPA were 9, 6 and 16 microM for dopamine, glutamate and GABA, respectively. HBCD also inhibited glutamate uptake at low concentrations, but never achieved more than 50% inhibition. The inhibition was primarily due to their effect on the membrane potential, measured by the membrane potential marker tetraphenylphosphonium bromide (TPP(+)). Other brominated flame retardants such as octaBDE and decaBDE did not have any effects on uptake. TBBPA, HBCD and even the pentabrominated diphenylether mixture (pentaBDE, DE-71, Great Lakes) also inhibited the vesicular uptake of dopamine with an IC(50) value of 3, 3 and 8 microM, respectively. The neurotoxicological consequences of these findings for environmental contaminants such as BFRs and PCBs are discussed.

Use of fluorocitrate and fluoroacetate in the study of brain metabolism.

Fluoroacetate and its toxic metabolite fluorocitrate cause inhibition of aconitase. In brain tissue, both substances are preferentially taken up by glial cells and leads to inhibition of the glial TCA cycle. It is important to realise, however, that the glia‐specificity of these compounds depends both on the dosage and on the model used. The glia‐inhibitory effect of fluorocitrate as obtained by intracerebral microinjection in vivo is reversible within 24 h. A substantial inhibition of the glial TCA cycle by systemic administration of fluoroacetate requires a lethal dose.  Inhibition of the glial aconitase leads to accumulation of citrate and to a reduction in the formation of glutamine. Whereas the former is likely to be responsible for the main toxic effect of these compounds possibly by chelation of free calcium ions, it is the latter that has received most attention in the study of glial‐neuronal interactions, since glutamine is an important precursor for transmitter glutamate and GABA. GLIA 21: 106–113, 1997.

Biochemical evidence for glutamate as a transmitter in hippocampal efferents to the basal forebrain and hypothalamus in the rat brain.

Abstract The effects of bilateral transection of the fornix bundle on the high affinity uptake of glutamate and on the amino acid content in several nuclei of rat forebrain and hypothalamus were studied in order to investigate the possible role of glutamate as a transmitter of these fibres. This lesion decreased the high affinity uptake of l -glutamate by 60–70% in the mammillary body and lateral septum, and by 40–50% in the anterior diagonal band nucleus, the bed nucleus of the stria terminalis, the mediobasal hypothalamus and the nucleus accumbens. The content of endogenous glutamate in samples dissected from freeze-dried tissue also decreased significantly in these regions. Endogenous aspartate was slightly decreased in the anterior diagonal band nucleus and the mammillary body, but unchanged in the other regions. No significant changes were seen in the levels of serine, γ-aminobutyric acid, glutamine and taurine, except for an increase in glutamine and taurine in the bed nucleus of the stria terminalis. The high affinity uptake of γ-aminobutyric acid, tested in the bed nucleus of the stria terminalis, the mediobasal hypothalamus and the mammillary body, was unchanged after the lesion. The results indicate that allocortical efferents innervating subcortical nuclei through the fornix might use glutamate as a transmitter. The study further supports the concept that glutamate plays an important role as transmitter of several different corticofugal fibre systems in mammalian brain.