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Notes: The high-affinity uptakes of [3H]serotonin, [3H]-glutamate, and γ-[3H]aminobutyric acid were studied using a myelin-free crude synaptosomal fraction prepared from the spinal cords of normal dogs and spastic dogs following sham treatment or dorsal bilateral rhizotomy surgery. Compared to sham-operated controls, rhizotomy surgery of normal dogs produced, after 1 week, a 30% reduction in the Vmax value of [3H]glutamate, but did not alter the uptake of γ-[3H]aminobutyric acid. This treatment also produced a 60% decrease in the Vmax value of [3H]serotonin. Comparison of the effect of rhizotomy surgery on normal and spastic dogs revealed that the spastic group had 60% higher Vmax values for uptakes of [3H]glutamate and γ-[3H]aminobutyric acid. Comparison of sham-operated spastic dogs and rhizotomy-treated spastic animals showed that there was a 25% decrease in the uptake of both amino acids in the rhizotomy-treated spastic group. Overall, the data (a) support the hypothesis that glutamate is the neurotransmitter from some of the primary afferents, and (b) suggest that sprouting of interneuronal amino acid transmitter systems may occur in the spinal cords of spastic dogs.

Notes: Abstract: The K+-stimulated, Ca2+-dependent release of glutamate, aspartate, -γ-aminobutyric acid (GABA), alanine, taurine, and glycine was measured in slices of cerebella obtained from control, and granule cell-, granule cell plus stellate cell-, or climbing fiber-deficient cerebella of the rat. The 55 mm-K+-stimulated release of glutamate and GABA was 10-fold greater in the presence of Ca2+ than in its absence. The stimulated release of aspartate was 4-fold higher when Ca2+ was present in the bathing media, while the value for alanine was twice as high as the amount obtained in the absence of Ca2+. There was no stimulated release of either taurine or glycine from the cerebellar slices. Increasing the Mg2+ concentration to 16 HIM inhibited the K+-stimulated, Ca2+-dependent release of glutamate, GABA, aspartate, and alanine 85% or more. The K+-stimulated, Ca2+ dependent release of glutamate, aspartate, and alanine from x-irradiated cerebella deficient in granule cells was reduced to 50–57% of control value. Additional x-irradiation treatment, which further reduced the cerebellar granule cell population and also prevented the acquisition of stellate cells, decreased the release of glutamate by 77%, aspartate by 66%, alanine by 91%, and, in addition, decreased the release of GABA by 55%. The K+-stimulated, Ca2+-dependent release of glutamate, aspartate, GABA, and alanine was not changed in climbing fiber-deficient cerebella obtained from 3-acetylpyridine-treated rats. The data support a transmitter role for GABA and glutamate in the cerebellum, but do not support a similar function for either taurine or glycine. The data also suggest that alanine and aspartate may be co-released along with glutamate from granule cells.

Notes: Abstract— The effects of i.p. injections of SO mg/kg d,l-5-hydroxytryptophan (5-HTP) and saline alone on the in uitro release of endogenous serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) were studied using preparations of axon terminals (P2 isolated from the telencephalon of rats. The level of 5-HT was 2-fold greater and the level of 5-HIAA was 5-fold greater in the P2 fraction isolated from rats given the d,l-5-HTP injection than from rats given saline injections. At 37°C the in vitro efflux of 5-HT and 5-HIAA from the P2 fractions of animals injected with 5-HTP 30min before killing was approx 3 times higher than the saline control group. The amount of 5-HT and 5-HIAA released at 37°C was 3–5 times higher than the amount released at 0°C for both the 5-HTP and saline injected rats. Increasing the concentration of potassium ions in the media to 55 mm significantly increased the release of 5-HT but not 5-HIAA in both groups of animals. The amount of 5-HT released by 55mm-K+ was about 2-fold higher from the P2 fraction isolated from rats given 5-HTP injections with respect to those given saline injections. The potassium stimulated release of 5-HT was calcium dependent. The data thus indicate that injection of 50 mg/kg d,l-5-HTP in rats can cause an increase in the level of 5-HT and 5-HIAA in a crude synaptosomal fraction and that as a result of this increase, there is a temperature dependent increased release of 5-HT and 5-HIAA under normal resting membrane conditions. There is also an increased release of 5-HT as a result of membrane depolarizing conditions induced by elevated potassium levels which is calcium dependent.

Notes: Abstract— Pigeons working on a multiple fixed ratio 50, fixed interval 10 schedule for food reinforcement were killed at 0, 50, 90, 150 and 240 min after an i.m. injection of 300mg/kg l-tryptophan. The levels of tryptophan, 5-hydroxytryptophan, 5-hydroxytryptamine, 5-hydroxyindole acetic acid, tyrosine, dopamine and norepinephrine were concurrently measured in crude nerve ending fractions (P2) isolated from the telencephalon, diencephalon plus mesencephalon and pons plus medulla-oblongata of each pigeon. Increases in 5-hydroxytryptamine levels in the nerve ending fraction from the telencephalon were correlated with the onset of the decreased response rates, whereas a return to baseline responding was correlated with a return to normal serotonin levels in this fraction. Changes in dopamine or norepinephrine were not related to the onset of or recovery from the decreased response rate. One group of pigeons were found which did not display any behavioral disruption even though each had received an injection of l-tryptophan; the content of 5-hydroxytryptophan, 5-hydroxytryptamine and 5-hydroxyindoleacetic acid in the nerve ending fraction isolated from the telencephalon of these birds did not differ from control values.

Notes: —Preparations of crude synaptosome fractions (P2) from the telencephalon and from the diencephalon plus optic lobes of the pigeon and from the telencephalon of the rat were used to study the effects of l-tryptophan on (a) the levels of serotonin (5-HT), norepinephrinc (NE) and dopamine in nerve endings and (b) the release of radioactive 5-HT, NE and dopamine from nerve endings. The level of 5-HT was significantly higher (P 〈 0–05) in the P2 fraction isolated from the telencephalon of pigeons given intramuscular injections of 300mg/kg of l-tryptophan in comparison to control values (1.11 ± 0.09 vs 0.74 ± 0.13 nmol/g original tissue wt). A smaller but not statistically significant increase in 5-HT was noted in the P2 fractions isolated from the diencephalon plus optic lobes of pigeons given injections of l-tryptophan. In vitro studies using preparations of synaptosomes (from both pigeon and rat) labelled with [3H]5–HT demonstrated that 1.0 mm-l-tryptophan caused a 30% increase (P 〈 0.05) in the release of [3H]5-HT over control values. This effect by l-tryptophan was blocked when a decarboxylase inhibitor was added to the medium. Tryptophan had no effect on the levels of NE or dopamine in these nerve endings nor did it have any effect on the release of these two amines from these preparations of synaptosomes. The results are discussed in terms of the role of serotonin in producing depression in pigeons working on a certain learned behavioural task.

Notes: —Preparations of synaptosomes (P2) from the telencephalon and from the diencephalon plus optic lobes of the pigeon and from the telencephalon of the rat were used to study the effects of 5-hydroxytryptophan (5-HTP) on (a) the levels of serotonin (5-HT) in nerve endings and (b) the release of 5-HT from nerve endings. The levels of 5-HT were significantly higher (3.21 × 0.35 nmol/g original tissue weight) in the P2 fraction isolated from the telencephalon of pigeons given intramuscular injections of 50mg/kg of d,l-5-HTP in comparison to control values (1.42 ± 0.07). A similar twofold increase was observed with the P2 fraction isolated from the diencephalon plus optic lobes. In addition, the levels of 5-HTP and 5-hydroxyindoleacetic acid also increased significantly in these P2 fractions isolated from pigeons given d,l-5-HTP injections in comparison to values obtained for pigeons given saline injections. In vitro studies using preparations of synaptosomes (from both pigeon and rat) labelled with [3H]5-HT indicated that 0.10 mil l-5-HTP increased the release of [3H]5-HT twofold over control values. A concentration as low as 0.001 mm l-5-HTP was tested on the P2 fraction from the telencephalon of the pigeon and was found to significantly increase the release of [3H]5-HT over control values. This effect by l-5-HTP was blocked if a decarboxylase inhibitor was added to the medium. l-5-HTP at a concentration of 1.5 mm had no apparent effect on the release of [3H]norepinephrine or [3H]dopamine from synaptosomes prepared from the telencephalon of the rat or pigeon. The results are discussed in terms of the role of serotonin in producing certain types of behavioral depressions exhibited by pigeons and rats given injections of 5-HTP.

Notes: Using the technique of measuring DNP-amino acid methyl esters by gas-liquid chromatography, the distribution of alanine, proline, glycine, GABA, glutamate and aspartate was determined in individual ganglia and the associated nerve bundles between these ganglia after isolation from the nervous system of the lobster, Homarus americanus. The brain or supraesophageal ganglion (27.2 mg) and the next 5 thoracic ganglia (varying from 24 to 10 mg in a rostral–caudal direction) as well as the nerve bundles connecting these ganglia were used. GABA and aspartate values varied the most among the individual ganglia; highest values were found in the second and third thoracic ganglia. The levels of alanine, proline, glycine and glutamate varied very little from ganglion to ganglion; however, the values for these amino acids did exhibit some variability among the individual connectives. The highest value for each was in the nerve bundle between the first and second thoracic ganglion. Glycine was present at the highest level of any of the amino acids whereas GABA was at the lowest level in the individual structures assayed.

Notes: Abstract— Crude synaptosomal (P2) preparations were obtained from the cerebella of rats in which the granule cell population had been selectively reduced by X-irradiation treatment and from the cerebella of control animals. In the P2 fraction from control cerebella, the level of glutamate was greater than any other of the 5 amino acids measured and was 2-fold higher than taurine, which was present at the next highest level. The content of taurine was slightly higher than that found for aspartate and was 3-fold greater than that observed for GABA. Alanine and glycine were present in the lowest amounts. The levels of glutamate and aspartate were significantly (P 〈 0.05) lower by 25 and 15%, respectively, in the P2 fraction isolated from the X-irradiated cerebella in comparison to control values. The content of taurine, GABA, glycine, and alanine were not changed by the X-irradiation treatment. The uptake of 1.0 μm-l-[3H]glutamate and l-[3H]aspartate was reduced approx 20% by X-irradiation treatment, whereas the uptake of 1.0 μm-[3H]GABA and [3H]taurine was unchanged. A more detailed kinetic analysis of l-[3H]glutamate uptake revealed there was a 20% decrease in the Vmax value with X-irradiation treatment and no change in the apparent Km value.In a second study, the uptake of l-[3H]glutamate, l-[3H]aspartate and [3H]GABA was measured using P2 fractions obtained from the cerebella of rats in which the population of granule, stellate and basket cells had been reduced by X-irradiation treatment. The uptake of 1.0μm-l-[3H]glutamate, l-[3H]aspartate and [3H]GABA was significantly (P 〈 0.05) reduced to 57, 68, and 59%, respectively, of control values. A more detailed kinetic analysis of [3H]GABA uptake revealed no significant change in the apparent Km and a 35% decrease in the Vmax value.The data are discussed in terms of glutamate being the excitatory neurotransmitter released from granule cells and GABA being the inhibitory neurotransmitter released from basket cells.

Notes: Abstract— The effects of supramaximal electrical stimulation on the metabolism of amino acids and proteins in incubated superior cervical ganglia of the rat were studied by the use of a gas-liquid chromatographic (GLC) assay procedure. Stimulation at 5 Hz for 2 h caused an apparent increase in tissue levels of free amino acids, with alanine, serine, glycine, valine, threonine, isoleucine and aspartate (+ asparagine) most noticeably affected. The amino acid composition (partial) of the TCA-insoluble proteins of resting and stimulated ganglia was approximately the same after 60 min of incubation, but there was less TCA-insoluble protein in the stimulated ganglia. The addition of amino acids (at plasma concentrations) to the standard media had no apparent affect on the amino acid composition of this protein fraction. Stimulation for 0, 5 h initially increased the efflux of alanine, valine, proline and ornithine into the incubation media but prolonged stimulation (for 4–0 h) decreased the efflux of alanine, serine, glycine and isoleucine and increased the efflux of lysine into the incubation media. The leakage of amino acids from the ganglia appeared to be a sodium-dependent process.The incorporation of 14C from [U-14C]glucose into glutamate (+ glutamine) and aspartate (+ asparagine) was greater in stimulated than in resting ganglia. However, the conversion of glutamate carbons from [U-14C]l-glutamate into aspartate was not affected by stimulation. Incorporation of 14C from [U-14C]glucose into glycine and serine was apparently not affected by stimulation during the 60 min of incubation. However, serine was the only amino acid which exhibited a higher specific radioactivity in stimulated ganglia than in resting ganglia incubated for 4 h in standard media. Lithium ions had the apparent specific effect of increasing the labelling with 14C from [U-14C]glucose into ornithine, and increasing the efflux and overall metabolism of serine in the ganglia. Incorporation of 14C from [U-14C]glucose into proteins was lower in the stimulated than in the resting ganglia if compensation was made for the higher radioactivity available in the total free amino acid pool of the stimulated ganglia.The rate of 14C incorporation from [U-14C]glutamate into the TCA-insoluble proteins of resting ganglia was greater when no other amino acids at concentrations approximating plasma levels were added to the bathing media; this rate was lower in stimulated than in resting ganglia.