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A method is reported for the quantitative analysis of free amino acids and related substances by the dansyl reaction, and the technique has been used for the analysis of tissues of the dorsal sensory neuron of the rat. A comparison of dorsal and ventral roots revealed no major qualitative differences, but glutamate and the very much less abundant amino acids, threonine and arginine occurred in significantly higher concentrations in dorsal roots as compared with ventral roots. After an 8 h period of dorsal root ligation in vivo, an apparently selective accumulation of alanine, glutamate, glycine and tyrosine occurred. These findings are compatible with the postulated transmitter role of glutamate at the terminals of primary afferent fibres and may indicate its subsequent transport towards the cord after synthesis in the ganglion. After the injection of [14C]glucose into the dorsal root ganglion, no rapid transport of any radiolabelled material along the axon could be detected. This finding is discussed in relation to current knowledge of the metabolic pathways involved in the synthesis of glutamate and related amino acids.

— Brain slices were incubated with [3H]GABA in a medium containing aminooxyacetic acid to prevent metabolism of [3H]GABA by GABA-glutamate transaminase. The slices, which rapidly accumulated radioactivity, were then continuously perfused and the efflux of [3H]GABA from the tissue was measured. The spontaneous efflux of [3H]GABA consisted of an initial rapid phase followed by a much slower release of [3[H]GABA. After 40 min perfusion 90 per cent of the radioactivity remained in the tissue. The slices were depolarized by electrical stimulation or by perfusion with a medium containing a high potassium concentration (40 mM). These procedures caused a striking increase in the efflux of [3H]GABA. The increased efflux produced by potassium, but not that produced by electrical stimulation, was dependent on calcium ions in the medium. The effect of electrical stimulation on [3H]GABA release was considerably reduced by a raised concentration (10 mM) of magnesium in the medium. High potassium concentrations and electrical stimulation did not cause an increase in the efflux of [14C]urea, L-[3H]leucine or [14C]α-amino-isobutyric acid from brain slices. These results are consistent with the suggestion that GABA may be an inhibitory transmitter in the cerebral cortex.

IT has been suggested that enkephalin might act as a neuro-transmitter or neuromodulator in the mammalian brain1, and recent experiments2,3 have shown that microiontophoretic application of enkephalin will depress brainstem neurones in both rat and cat. We have confirmed these observations on neurones in other regions of the rat brain and have extended the study to an investigation of the action of iontophoretic met-enkephalin on neurones activated by painful stimuli.

1 The release of endogenous acetylcholine (ACh) from cerebral cortical slices stimulated at 0.25, 1, 4, 16 and 64 Hz was measured in the presence either of physostigmine or of physostigmine and atropine. 2 Atropine potentiated the evoked release of endogenous ACh especially at low frequencies resulting in an output per stimulus which sharply declined with increasing frequency of stimulation, while in the absence of atropine the output of ACh per stimulus was low and fairly constant. 3 The evoked release of [3H]-ACh per stimulus following the incubation of the slices with [3H]-choline, as estimated by means of rate constants of the evoked release of total radioactivity, showed a frequency dependence similar to endogenous ACh when the two were tested under identical conditions. 4 In the absence of an anticholinesterase the evoked release of [3H]-ACh per stimulus was dependent on frequency of stimulation in a similar way to that in the presence of physostigmine and atropine. 5 Results suggest that under physiological conditions, i.e. in the absence of an anti-cholinesterase, the release of ACh per stimulus decreases with increasing frequency of stimulation and that this decrease is due to a lag in the mobilization of stored ACh rather than in the synthesis of new ACh.

The efflux of [ 3 H]GABA from the dorsal surface of adult rat medulla overlying the dorsal column nuclei (DCN) was found not be influenced by increasing the concentration of potassium in the superfusing solution to 40 mequiv. Similarly, raised potassium was found not to influence the efflux from slices of the dorsal region of the caudal medulla containing the dorsal column nuclei. This lack of effect of raised potassium is not thought to be due to lack of GABAergic terminals in this region because there is good pharmacological evidence for their presence and because both electrical stimulation and 100 μM veratridine increased the efflux of [ 3 ]GABA from such slices. Also, 40 mequiv potassium was found to increase the efflux of both endogenous and [ 3 H]GABA from crude synaptosome preparations of this region without influencing the efflux of [ 14 C]sucrose of [ 3 H]leucine. This release of [ 3 H]GABA was calcium-dependent, and was similar whether produced by 20, 40 or 60 mequiv potassium and occurred whether eos or AOAA was used to inhibit GABA metabolism. Release from synaptosomes could also be induced with 100 μM veratridine. Raised potassium was additionally found to prevent the increased efflux from slices produced by electrical stimulation and to increase the efflux from slices prepared from the brains of rats 14 days old. It is suggested that the astrocytic swelling produced by raised potassium concentration restricts the diffusion of GABA away from depolarized terminals.

— Each of the four convulsants used significantly influenced the release of [3H]-GABA from brain slices, without affecting [3H]GABA uptake. Bicuculline (10−5M, but not 10-fold higher or lower concentrations) potentiated the electrically evoked release of [3H]GABA but not the resting release, whereas metrazol (10−4 to 10−6 M) was found to inhibit resting but not electrically evoked release. Strychnine (10−4 and 10−5 M) and picro-toxin (10−4 M) inhibited electrically evoked release.

1. Ergothioneine has been detected and quantitatively estimated in the brains of mice, rats, guinea-pigs, rabbits, cats and sheep. It is present in the cerebellum in amounts ranging from about 3·0 μg/g in the cat to 10 μg/g in the guinea-pig. Amounts in the cerebral hemispheres are much smaller, ranging from < 0·1 μg/g in the cat to 1·6 μg/g in the guinea-pig. 2. Large amounts of ergothioneine (about 30 μg/g) were found in the optic nerves of the rabbit. None was detected in the dorsal columns of the spinal cord of the same species. 3. Ergothioneine has an excitatory action on the electrical activity of the cerebellum of the decerebrate rabbit and the excitatory actions of cerebellar extracts on this preparation are completely accounted for by their contained ergothioneine. 4. It is concluded that the cerebellar factor of Crossland & Mitchell (1956) is identical with ergothioneine.

SEVERAL attempts have been made in the past to correlate the amount of acetylcholine released during activity at nerve endings with the amount needed to produce activation of the effector cell1–3. It is clearly of crucial importance for the acetylcholine hypothesis that there should be quantitative agreement between these amounts. Until recently, a very substantial discrepancy has remained, supporting the suggestion that acetylcholine is merely a byproduct of transmission. One source of difficulty in making such correlations has been the paucity of results concerning the release of acetylcholine and the excitation produced by its application in the same preparation.