D. Malthe-Sørenssen

Effect of the convulsive agent 3-mercaptopropionic acid on the levels of GABA, other amino acids and glutamate decarboxylase in different regions of the rat brain

Intraperitoneal injection of 3-mercaptopropionic acid into rats caused severe convulsions which started after about 7 min. Of the amino acids examined only the level of GABA changed after 4 min and immediately before (6.5–7 min) the convulsions started. The decrease in GABA concentration detected immediately before the onset of convulsions was about 35 per cent in the cerebral cortex, corpus striatum and cerebellum, 30 per cent in pons-medulla and 20% in hippocampus. Concomitant with the fall in GABA there was a large, reversible inhibition of glutamate decarboxylase activity in the brain. The uptake of GABA into synaptosomes isolated after injection of the convulsive agent was not reduced, and the uptake of GABA into synaptosomes was not inhibited by high concentrations of 3-mercaptopropionic acid added in vitro. During convulsions levels of aspartate and taurine decreased significantly in all the brain regions investigated. A small increase in glutamine was detected in pons-medulla and in cerebellum. Major changes in the concentrations of other amino acids such as glutamate, alanine, serine and glycine were found only in corpus striatum.

Increased resting and evoked release of transmitter following repetitive electrical tetanization in hippocampus: a biochemical correlate to long-lasting synaptic potentiation

In vitro stimulation of two axonal branches from hippocampal CA3 pyramids, the CA1 afferent Schaffer collaterals and the CA3 efferents to septum through fimbria, released D-[3H]aspartate as a measure for endogenous L-glutamate. Following bursts of repetitive electrical stimuli to the Schaffer collaterals, a long-lasting and significantly increased resting efflux, as well as an increased stimulus evoked release of D-aspartate, appeared. No such persistent increase in D-aspartate efflux was recorded from the septal terminals. We propose that increased transmitter liberation may account for long-term synaptic potentiation in hippocampus.

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.

Systemic injection of kainic acid: Effect on neurotransmitter markers in piriform cortex, amygdaloid complex and hippocampus and protection by cortical lesioning and anticonvulsants

Systemic injection of kainic acid (12 mg/kg) in rats induces a well established pattern of neuronal lesions in different brain regions. These lesions are accompanied by changes in neurotransmitter markers. In the piriform cortex and amygdaloid complex, the kainic acid lesion was accompanied by a reduction in the high affinity uptake of glutamate and in the activities of glutamate decarboxylase and choline acetyltransferase, whereas in the hippocampus there was a reduction in the high affinity uptake of glutamate and in glutamate decarboxylase activity. Hemidecortication, hemitransection, a caudal knife cut in the cortex, or treatment with diazepam, all protected against the effects of kainic acid in the piriform cortex and amygdaloid complex but not in the hippocampus. Diphenylhydantoin had no effect on the neurotoxicity of kainic acid. The results indicate that the neurotoxic effects of kainic acid in the piriform cortex and amygdala are dependent on an intact cortical structure, probably due to a dependence on specific excitatory circuitry. The neurons involved may be glutamergic/aspartergic.

Molecular Properties of Choline Acetyltransferase and Their Importance for the Compartmentation of Acetylcholine Synthesis

Publisher Summary Choline acetyltransferase (ChAc) is responsible for the synthesis of the chemical transmitter acetylcholine (ACh), and is therefore an important enzyme in the nervous tissue. There is an excellent correlation between the level of ACh and the level of ChAc in different parts of the nervous system, indicating that this enzyme governs the level of ACh. The localization of ChAc is therefore synonymous with the site of synthesis of ACh and knowledge of this site gives important information as to the further processes necessary for the uptake and storage of the neurotransmitter. The compartmentation of ChAc and its consequences for the synthesis storage and release of Ach within the neurone are discussed in this chapter. This chapter shows that the binding of ChAc to membranes is a reversible process, primarily dependent upon the pH, ionic strength and the ionic properties of ChAc. The binding of ChAc to membranes resembled the ionic attraction between ChAc and a cationic exchange resin in all aspects. ChAc from different species had a different surface charge and a different isoelectric point. Isoelectric focusing of ChAc showed that only one form of ChAc was present in pigeon and guinea pig, whereas in rat and cat there were three and two (respectively) different molecular forms. Different molecular forms of ChAc had different membrane affinity. The literature on the electron microscopy histochemistry of ChAc is reviewed here.

Biochemical and pharmacological properties of acryloylcholine, an inhibitor of choline acetyltransferase

Abstract Acryloylcholine, an unsaturated choline ester was found to be a strong inhibitor of rat and pigeon brain choline acetyltransferase. Substitution of the hydrogen atoms of the C-2 and C-3 carbon atoms in the acylgroup by methylgroups resulted in less potent inhibitors Propionylcholine, the saturated choline ester analogue, was 1000-fold less potent. The rat brain enzyme was not protected against the inhibition by acryloylcholine by high concentration of acetyl-CoA or choline and the inhibition was neither affected by variation in the concentration of sodium chloride nor by variation in pH. The inhibition of rat brain choline acetyltransferase was uncompetitive with respect to choline and acetyl-CoA. The inhibition of pigeon choline acetyltransferase was completely reversible whereas the inhibition of rat choline acetyltransferase could only be reversed with difficulty. Acryloylcholine inhibited acetylcholinesterase reversibly and to the same extent as propionyllcholine. Acryloylcholine was a better substrate for buturylcholinesterase that for acethylcholinesterase. Acryloylcholine caused a neuromuscular block in the rat diaphragm, but this block was probably not due to inhibition of choline acetyltransferase. The ester contracted the esterinized frog rectus abdominus muscle to the same extent as propionylcholine.

Changes in neurotransmitter parameters in the brain induced byl-cysteine injections in the young rat

A single subcutaneous injection of L-cysteine (1.2 mg/g body wt.) to 4-day-old rats leads to atrophy of the brains examined 27-31 days later. The brains could be separated into two groups (type 1 and 2) on account of the degree of atrophy. Type-1 lesion, with a brain weight reduction of 20%, was dominated by a severe reduction in high-affinity uptake of L-glutamate in CNS regions receiving corticofugal fibers such as thalamus and striatum. Glutamate decarboxylase was only reduced in cortical structures. In type-2 lesion, with a severe brain atrophy of about 50%, high-affinity glutamate uptake was further reduced and there was a more pronounced reduction in glutamate decarboxylase activity in several brain regions. Cholinergic neurons were less affected by the lesion and the levels of choline acetyltransferase showed a relative increase in brain regions which partly compensated for their reduction in size.

Effects of organophosphates on presynaptic events in the vascularly perfused phrenic nerve-hemidiaphragm preparation from the rat.

A vascularly perfused phrenic nerve-hemidiaphragm preparation from the rat was developed to study effects of physostigmine and some organophosphate inhibitors on the synthesis and release of endogenous and deuterium-labelled (choline--D9) acetylcholine (ACh) as well as the presynaptic uptake of choline. Choline and ACh were determined by combined gas chromatography/mass spectrometry. Without stimulation the endogenous levels of ACh were 320 pmole/hemidiaphragm for unlabelled and less than 1 pmole/hemidiaphragm of deuterium-labelled ACh. After stimulation at 15 Hz for 1 hr, 460 pmole/hemidiaphragm of unlabelled and 15 pmole/hemidiaphragm of deuterium-labelled ACh were found. Without stimulation the release of unlabelled ACh was 6 pmole/min/hemidiaphragm and for deuterium-labelled 0.2 pmole/min/hemidiaphragm. Evoked release (15 Hz, 1 hr) was 22 pmole/min/hemidiaphragm for unlabelled and 1.8 pmole/min/hemidiaphragm for deuterium labelled ACh. During stimulation and treatment with high concentrations (10(-5)-10(-4) M) of soman, DFP and Vx the level of unlabelled endogenous ACh increased, but the level of deuterium labelled ACh decreased in the diaphragm. During stimulation and treatment with these inhibitors the release of both unlabelled and labelled ACh decreased. During treatment with high concentrations (10(-5)-10(-4) M) of sarin and physostigmine there were no changes in endogenous levels or release of unlabelled or deuterium labelled ACh. The different effects of cholinesterase inhibitors are probably linked to the synthesis and release mechanism of ACh rather than to the choline uptake mechanism.