J.E. Smith

A method for concurrent measurement of picomole quantities of acetylcholine, choline, dopamine, norepinephrine, serotonin, 5-hydroxytryptophan, 5-hydroxyindoleacetic acid, tryptophan, tyrosine, glycine, aspartate, glutamate, alanine, and gamma-aminobutyric acid in single tissue samples from different areas of rat central nervous system

A rapid and sensitive method for separation and concurrent assay of 14 compounds at the picomole level in individual rat brain parts is described. The putative amino acid neurotransmitters (aspartate, γ-aminobutyric acid, glutamate, and glycine) are extracted from a 20–30 mg portion of the tissue with 5% TCA and assayed as their respective DNP-amino acid methyl ester derivatives by glc. Four other putative neurotransmitters (acetylcholine, dopamine, norepinephrine, and serotonin) and some of their precursors and metabolites (choline, tryptophan, 5-hydroxytryptophan, 5-hydroxyindoleacetic acid, and tyrosine) are extracted in 1n formic acid-acetone (v/v:15/85) from the remaining tissue. The lipids are removed with a heptane-chloroform (v/v:8/1) wash and the aqueous phase is dried at 37°C under N2. The dried extract is dissolved in water (pH 4). With one portion of this solution, acetylcholine and choline are assayed using a radioenzymatic method whereas with the rest, dopamine, norepinephrine, serotonin, 5-hydroxyindoleacetic acid, 5-hydroxytryptophan, tryptophan, and tyrosine are separated with three ion exchange resins arranged in tandem. These compounds are assayed fluorometrically with modified microadaptations of previously reported methods.

Neurochemical changes following the administration of depleters of biogenic monoamines

Abstract A variety of drugs which were thought to selectively effect the synthesis, release or reuptake of the biogenic monoamines, and thereby deplete their stores in brain, were administered to rats. The animals were given intraperitoneal injections of reserpine, α-methyl-meta-tyrosine, α-methyl-para-tyrosine, para-cholorophenylalanine and tetrabenazine, and were killed at intervals known to cause maximum depletion of serotonin, dopamine and norepinephrine. When compared to saline controls, the drugs depleted the respective monoamines, as expected, but changes in the levels of acetylcholine, alanine, glycine, γ-aminobutyric acid, and aspartate and glutamate were also observed.

Neurochemical changes following the administration of precursors of biogenic amines

Abstract Male Wistar rats were injected intraperitoneally with either saline, L-trytophan, D,L-5hydroxytryptophan, L-tyrosine, L-DOPA or choline and killed by the near-freezing method 15 and 45 min after injection. The brains were removed, pulverized and acetylcholine, dopamine, norepinephrine, 5-hydroxytryptamine, aspartate, glutamate, glycine and γ-aminobuttyric acid wwere extracted and concurrently measured using previously reported methods. Compared to saline injected controls, some precursors not only resulted in changes in the specific neurotransmiter systems being pertubated, but also changes in the content of other neurotransmitters.

Neurochemical correlates of spasticity

Abstract Dogs were made paraplegic by complete mid-thoracic spinal cord transection. The content of glycine, glutamate, aspartate, and γ-aminobutyric acid were determined in ventral and central grey matter from the lumbar enlargement of the spinal cord at 1, 3 and 8 weeks after transection. A rapid decrease in the content of aspartate and glycine accompanied the onset of spasticity. By the eight week post-transection, aspartate and glycine had decreased to less than 50% of control levels.

Neurochemical correlates of behaviour--content of tryptophan, 5-hydroxytryptophan, serotonin, 5-hydroxyindoleacetic acid, tyrosine, dopamine and norepinephrine in four brain parts of the pigeon during behavioural depression following an injection of tryptophan.

Abstract— Pigeons working on a multiple lixed‐ratio 50, fixed interval 10 schedule of food reinforcement were injected with l‐tryptophan (300mg/kg; I.M.) and killed at various times before, during and after the period of behavioural depression following the administration of this amino acid (0, 25, 50, 90, 170 and 230 min). The levels of tryptophan, 5‐hydroxytryptophan, 5‐hydroxytryptamine, 5‐hydroxyindoleacetic acid, tyrosine, dopamine and norepinephrine were concurrently measured in 4 specific areas of the brain (telencephalon, diencephalon plus mesencephalon, pons plus medulla‐oblongata and cerebellum). The course of the increases in the level of 5‐hydroxytryptamine in the telencephalon, and subsequent return to pre‐injection levels, was temporally related to the onset of the decreased responding and gradual return to normal rates of responding. Changes in dopamine and norepinephrine were not correlated with the onset of and recovery from the decreased response rates. The data in this paper are discussed in terms of (a) the previously reported work with 5‐hydroxytryptophan and (b) the importance of the telencephalic serotonergic system in certain types of behavioural depression.

Effects of tryptophan on serotonin in nerve endings

—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.

Effects of glycine administration on canine experimental spinal spasticity and the levels of glycine, glutamate, and aspartate in the lumbar spinal cord.

Twelve female mongrel dogs were made paraplegic by midthoracic spinal cord transection. Beginning at 9 weeks posttransection, either glycine (50 mg/kg) or saline was injected intramuscularly each day and the signs of spinal spasticity were assessed clinically. After treating the dogs for 3 weeks, we removed the lumbar enlargement of each dog and microdissected it into gray and white areas which we assayed for glycine, glutamate, and aspartate content. Some of the clinical signs of spasticity improved in the animals injected with glycine compared to the saline-injected controls. The content of glycine was significantly elevated in the central gray matter and ventral medial white matter of the glycine-treated dogs. The levels of glutamate were also significantly elevated in the central, lateral ventral, and medial ventral gray matter and in the dorsal lateral and ventral medial white matter of the glycine-treated dogs. The possible role of these segmental putative neurotransmitters in spinal spasticity is discussed.

Levels of ɤ-aminobutyric acid in the dorsal grey lumbar spinal cord during the development of experimental spinal spasticity

Abstract Dogs were made paraplegic by complete mid-thoracic spinal cord transection. At one, three, eight, and twelve weeks post-transection the lumbar cord was removed and the dorsal grey matter microdissected from L 2–3 and the content of ɤ-aminobutyric acid (GABA) determined. An initial decrease in GABA levels was followed by a gradual increase in content which correlated with the progressive development of spinal spasticity. By twelve weeks post-transection, GABA was elevated 68% above controls.

Neurochemical correlates of behavior. Content of tryptophan, 5-hydroxytryptophan, serotonin, 5-hydroxyindoleacetic acid, tyrosine, dopamine and norepinephrine in a nerve ending fraction isolated from three brain areas of the pigeon during the period of behavioral depression following an injection of tryptophan.

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.

Effects ofp-chlorophenylalanine on the metabolism of serotonin from 5-hydroxytryptophan.

The effects ofD,L-ϱ-chlorophenylalanine methyl ester (PCPA-methyl ester) and two of its metabolites, 2-(ϱ-chlorophenyl)-ethylamine (PCPEA) and ϱ-chlorophenylacetic acid (PCPAA), on the metabolism of serotonin (5-HT) fromD,L-5-hydroxytryptophan (5-HTP) ware studied in vitro and in vivo using the telencephalon and brainstem of the rat. For in vivo studies and some in vitro experiments, rats were injected with either 100 mg/kg PCPA-methyl ester or saline alone on days 1, 2, and 3, and were killed on day 15. When the in vivo metabolism of 5-HT was to be studied, the saline group and the PCPA group of animals were injected with 75 μg/kg [3H]D,L-5-HTP 20 min before sacrificing. With respect to the values found for the saline-injected animals, the specific activity (S.A.; dpm/nmol) of 5-HIAA was significantly greater in the telencephanol and brainstem of the animals injected with PCPA-methyl ester. The S.A. of 5-HTP was the same in both groups; the S.A. of 5-HT was lower in the telencephalon of the PCPA group than in the saline group; in the brainstem, there was no difference. In both the saline- and PCPA-injected animals, the S.A. of 5-HIAA was greater than the S.A. of 5-HT. There was no difference between the saline- and PCPA-injected animals with regard to: (1)L-5-HTP decarboxylase activity; (2)L-5-HTP-induced release of [3H]5-HT in vitro from crude nerve ending fractions (P2); or (3) in vitro uptake of [3H]D,L-5-HTP and its conversion to [3H]5-HT using the P2 fraction. In vitro studies demonstrated that the PCPEA could directly cause a large increase in the release of [3H]5-HT from the P2 fraction, whereas PCPA and PCPAA had little or no apparent effect. The data were interpreted to suggest that in the telencephalon of the animals treated with PCPA-methyl ester, there was a higher turnover of 5-HT than was found in the saline-treated group.