Radioenzymatic analysis of neurotransmitters.

Since the late 1960's, radioenzymatic assays have gradually come to replace the less sensitive and less specific spectrofluorometric and bioassay procedures previously used to determine many of the neurotransmitters. These assays provide the means to measure picogram quantities of most of these substances, and have enabled determinations to be made in very small volumes of body fluids, in brain perfusates and individual brain nuclei, and in large individual cells of some simple animals. This paper reviews briefly some of the radioenzymatic techniques presently available for assaying norepinephrine (NE), epinephrine (E), dopamine (DA), serotonin, and the trace amines octopamine (OA), phenylethanolamine (PEOHA), phenylethylamine (PEA), tyramine (TA) and tryptamine (T).

In vivo release of endogenous dopamine, 5-hydroxytryptamine and some of their metabolites from rat caudate nucleus by phenylethylamine.

The in vivo release of endogenous 3,4-dihydroxyphenylethylamine (DA) and its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 3-methoxytyramine (3-MT), and of 5-hydroxytryptamine (5-HT) and its metabolite, 5-hydroxyindoleacetic acid (5-HIAA), has been measured in the caudate nucleus of the anesthetized rat. A push-pull cannula was implanted into the brain, and the tissue perfused with artificial CSF or artificial CSF containing 5 X 10(-4) M phenylethylamine. The perfusate was collected and analyzed for DA, 5-HT and their metabolites by high performance liquid chromatography with electrochemical detection (HPLC-ECD). DA was released by phenylethylamine at rates significantly greater than its basal rate. 3-MT and 5-HT were undetectable in perfusates collected under basal conditions, but could be detected readily during phenylethylamine stimulation. DOPAC, HVA and 5-HIAA concentrations were not significantly affected by phenylethylamine. The results suggest (1) that phenylethylamine may exert its behavioural effects through increased release of both DA and 5-HT, and (2) that in vivo measurements of the acid metabolites alone may not be indicative of the release of the amines.

In vivo release of endogenous dopamine from rat caudate nucleus by beta-phenylethylamine and alpha,alpha,-dideutero-beta-phenylethylamine.

The release of endogenous dopamine (DA) has been measured in the rat striatum following the intracardial administration of various doses of beta-phenylethylamine (PEA) or alpha,alpha-dideutero-beta-phenylethylamine (deuterated PEA). The release was significantly increased for a period of approximately 15 minutes by a dose of 25 mg/kg PEA. Both the dose required to stimulate DA release and the duration of the effect were in good agreement with previously reported behavioral and locomotor effects of administered PEA. When the animals were given 25 mg/kg of deuterated PEA, the increase in DA release was both longer lasting and significantly greater in magnitude than that observed in response to the non-deuterated amine. The results of these experiments provide direct evidence that DA release is stimulated by amounts of PEA known to cause behavioral effects and locomotor activity in rats, and suggest that these effects are likely to be mediated, at least in part, by DA.

Yawning elicited by systemic and intrastriatal injection of piribedil and apomorphine in the rat.

The behavioural effects of systemic and intrastriatal injections of the dopamine agonists piribedil and apomorphine in male rats were examined. Bilateral application of piribedil (50 and 100 micrograms) or apomorphine (5, 10 and 20 micrograms) to the striatum produced yawning and chewing mouth movements accompanied by intermittent stretching and sexual arousal. Low doses of piribedil (1.25 and 2.5 mg/kg) and apomorphine (0.1 and 0.2 mg/kg) injected SC produced an identical yawning syndrome. Previous work has suggested that yawning elicited by systemic dopamine agonist treatment is a consequence of dopamine autoreceptor stimulation. Similarly, the most likely explanation of the present data is that yawning elicited by systemic and central dopamine agonist treatment was due to activation of dopamine autoreceptors. Systemic injection of haloperidol and scopolamine abolished yawning induced by intrastriatal piribedil and these data provide tentative support for the proposal that a dopamine-acetylcholine link may be involved in the expression of yawning.

Modification of nocturnal spontaneous and adrenergic-induced feeding in the rat following either A5 or A7 lesions.

The ingestive profiles of intact, A5 and A7 damaged animals were examined during the 2-hr nocturnal period following onset of the dark cycle. A5, A7 and intact rats consumed comparable amounts of food following initial access to food nocturnally. Sebsequent feeding declined in A5 animals below control values and failed to return to baseline at the end of the nocturnal period examined. A7 damaged rats appeared more resistant to the appetite suppressing effects of initial meal taking and consumed more food than control animals. Only A5 damaged rats were noted to be hyperdipsic during the immediate 10 postoperative days. Intracranial injection of 1-norepinephrine bitartrate (10 ug/ul) into the paraventricular nucleus (PVN) of the hypothalmus produced a reliable facilitation of feeding in A5, A7 and intact rats during the first hour of the dark cycle. A5 rats exhibited the largest increase in feeding elicited by NE administration into the PVN. This feeding response was observed in rats with A5 lesions regardless of whether testing was carried out during the initial hours of the dark cycle or during a predetermined "satiation" test. A5 lesions also effected a marked hyperglycemia while A7 lesions were ineffective in this respect. Taken together these data suggest the A5 and A7 cell groupings regulate spontaneous feeding within a rostrally coursing feeding circuitry and appear to interact with the PVN in the elicitation of noradrenergic feeding.

In vivo release of endogenous dopamine from rat caudate nucleus by phenylethylamine.

The in vivo release of endogenous dopamine (DA) from the rat caudate nucleus has been measured in the presence and absence of beta-phenylethylamine. A push-pull cannula was implanted into the brain and the tissue was perfused with artificial cerebrospinal fluid (CSF) containing phenylethylamine in concentrations ranging from 5 X 10(-3) to 5 X 10(-7) M. The DA released into the perfusate was determined radioenzymatically. Dopamine was released at rates significantly greater than its resting rate by concentrations of phenylethylamine of 5 X 10(-3) to 5 X 10(-5)M; 5 X 10(-6)M phenylethylamine caused a slight increase in release, but the difference from the resting rate was not significant. The absence of calcium in the perfusing medium did not significantly alter either the unstimulated release rate of DA or the release rate stimulated by 5 X 10(-5)M phenylethylamine. The concentrations of phenylethylamine required to increase release of DA in vivo are discussed briefly in relation to the doses required to elicit behavioural effects.

Amphetamine-stimulated release of endogenous dopamine from the rat caudate nucleus in vivo.

The in vivo release of endogenous dopamine (DA) has been measured from the rat caudate nucleus. A push-pull cannula was implanted into the brain and the tissue was perfused with artificial cerebrospinal fluid (CSF) containing amphetamine in concentrations ranging from 5 X 10(-3) to 5 X 10(-7) M. The DA released into the perfusate was determined by a radioenzymatic procedure. DA release was increased to levels significantly above its resting rate by amphetamine concentrations of 5 X 10(-6) M or greater. Release stimulated by 5 X 10(-5) M amphetamine was significantly reduced by removing calcium from the perfusing fluid; the unstimulated release rate was not significantly affected. The concentrations of amphetamine required to increase DA release in vivo would appear to be similar to those found in the brain following intraperitoneal doses which produce increases in locomotor activity and stereotyped behavior.

Sensitive and specific radioenzymatic assay for norepinephrine, epinephrine and dopamine based on the thin-layer chromatographic separation of their Dns-O-methyl derivatives.

A radioenzymatic assay is described in which norepinephrine, epinephrine and dopamine are converted to their tritiated 3-O-methyl derivatives by reaction with S-[methyl-3H]adenosyl-L-methionine in the presence of catechol-O-methyltransferase. The methylated compounds are then reacted with Dns chloride, and the Dns derivatives are extracted into ethyl acetate, isolated by thin-layer chromatography and quantified by liquid scintillation spectrometry. The assay displays a high degree of specificity for each compound, due in large part to the chromatographic properties of the Dns derivatives. It is capable of measuring 2 pg of each catecholamine, and is linear to at least 5 ng. Approximately 50 samples can be assayed in 1.5 days.

In vivo release of endogenous dopamine from rat caudate putamen and nucleus accumbens by 40 mM potassium chloride.

The resting and K+-stimulated release rates of endogenous dopamine (DA) have been measured in vivo at four different sites in the rat caudate putamen and nucleus accumbens. A push-pull cannula was inserted into the brain sites chosen, and the tissue was perfused with artificial cerebrospinal fluid (CSF) containing 2.6 or 40 mM KC1. The DA content of the perfusates was determined by a radioenzymatic procedure. DA release was significantly increased above unstimulated levels by 40 mM KC1 in all areas tested. Neither unstimulated nor K+-stimulated release rates varied significantly among the regions examined. K+-stimulated DA release was not significantly diminished by perfusing the tissue with calcium-free medium, suggesting that release was probably supported by residual amounts of calcium in the tissue.

Unstimulated and amphetamine-stimulated release of endogenous noradrenaline and dopamine from rat brain in vivo.

The in vivo release rates of endogenous noradrenaline from the hypothalamus and dopamine from the caudate nucleus of the rat have been determined. Artificial CSF perfusates collected from a push-pull cannula inserted into specific areas of the brain were assayed for the amines by a sensitive radioenzymatic procedure. The release rates of noradrenaline and dopamine into artificial CSF perfusates were 38 +/- 6 and 46 +/- 6 pg/h (225 +/- 36 and 301 +/- 39 fmol/h), respectively; when 0.5 mM amphetamine was added to the CSF, the release rates of noradrenaline and dopamine increased to 176 +/- 50 and 1183 +/- 453 ph/h (1041 +/- 296 and 7732 +/- 2961 fmol/h), respectively.

Effects of tranylcypromine on the concentration of some trace amines in the diencephalon and hippocampus of the rat.

Concentrations of 4 trace amines in diencephalon and hippocampus of the rat were measured by integrated-ion-current mass spectrometry after administration of the antidepressant drug, tranylcypromine. Much larger increases were observed for 2-phenylethylamine and tryptamine than for m- and p-tyramine.

Arylalkylamines in the adrenal medulla.

The trace amines p-tyramine, m-tyramine, tryptamine, and phenylethylamine have been determined in the bovine adrenal medulla and the rat adrenal gland by the mass spectrometric integrated ion current technique, using the corresponding deuterated amines as internal standards. In the bovine adrenal medulla, these amines were present at levels ranging from 21.7 ng/g for tryptamine to 37.3 ng/g for phenylethylamine, while in the rat adrenal, they occurred in amounts ranging from 5.2 ng/g for m-tyramine to 11.4 ng/g for tryptamine. In the rat, intraperitoneal administration of pargyline (100 mg/kg) increased the adrenal levels of each of the trace amines. Adrenaline, noradrenaline, and p-tyramine were significantly depleted by reserpine (3 or 10 mg/kg), but the level of m-tyramine was unaffected. It is suggested that the mode of uptake of the tyramine isomers into the adrenal medullary granules may depend on the position of the bydroxyl group relative to the side chain on the aromatic nucleus.

Evidence for the presence of m-tyramine, p-tyramine, tryptamine, and phenylethylamine in the rat brain and several areas of the human brain.

Postmortem human brains have been obtained from four nonpsychiatric patients, aged 59-70 years. Regional analysis of the trace amines phenylethylamine, p-tyramine, m-tyramine, and tryptamine has indicated that the amines are distributed heterogeneously throughout the brain, but are most concentrated in the basal ganglia. Although the levels are very low, evidence obtained from animal studies has indicated that the trace amines have a very rapid turnover rate. Their presence in a brain synaptosomal fraction suggests a possible involvement in the process of neurotransmission. Postmortem changes in human brain amines are discussed in relation to those occurring postmortem in the rat brain, in which phenylethylamine, p-tyramine, and tryptamine have been shown to increase to levels greater than those prevailing in vivo.

Effects of tranylcypromine and pargyline on brain tryptamine.

Tranylcypromine produces behavioral excitation while pargyline produces depression. Tranylcypromine increased brain tryptophan which led to an accumulation of tryptamine. The levels of tryptamine after tranylcypromine were found to be 3 times those found after pargyline.

The effects of reserpine and 6-hydroxydopamine on the concentrations of some arylakylamines in rat brain.

1 The concentrations of p- and m-tyramine were measured in the caudate nucleus of the rat brain following subcutaneous injection of reserpine or intraventricular injection of 6-hydroxydopamine, beta-Phenylethylamine was analysed in the hypothalamus after reserpine. 2 Endogenous levels of p-tyramine and m-tyramine in the caudate nucleus, and beta-phenylethylamine in the hypothalamus were 8.02, 2.25 and 2.52 ng/g respectively. 3 Tyramine concentrations were reduced to less than 20% of control values one day after a reserpine injection of 1 or 10 mg/kg. A single dose of reserpine (0.4 mg/kg) significantly decreased the content of both tyramines in the caudate nucleus. The effects became apparent as early as 45 min after drug case of m-tyramine. 4 The hypothalamic content of beta-phenylethylamine was unaffected by reserpine. 5 Ten days after an intraventricular injection of 6-hydroxydopamine (250 mug), p- and m-tyramine concentrations in the caudate nucleus were significantly below control levels. 6 The results suggest that p- and m-tyramine may be stored by an intraneuronal reserpine-sensitive storage mechanism. Alternatively, the tyramines may replace some of the catecholamines from their storage granules and then be released as false transmitters by the nervous impulse. The observed changes in tyramine levels might also the fact that these amines may be metabolically related to another amine which is stored in reserpine-sensitive granules.

Arylalkylamines inOctopus tissues.

A number of phenylethylamines and indoleamines have been analyzed in the circumoesophageal ganglia and posterior salivary gland of the normal and pargyline-treated maleOctopus dofleini martini. ß-Phenylethylamine,m-tyramine, and tryptamine are present in the optic lobes in amounts of 3, 0.6, and 0.6 ng/g, and in the posterior salivary gland at levels of 1, 64, and 52 ng/g, respectively, in contrast to the much higher levels observed forp-tyramine, octopamine, dopamine, noradrenaline, and 5-hydroxytryptamine. Although pargyline causes a substantial increase in the content of ß-phenylethylamine,m-tyramine,p-tyramine, and tryptamine in the optic lobes, no significant changes are observed in the posterior salivary gland. Their relatively rapid metabolism suggests an active role for these amines in the function of nervous tissue in theOctopus.