Electron microscopic cytochemical correlates of histofluorescence

The biogenic amines, norepinephrine and dopamine, have been localized electron microscopically in the substantia nigra, pineal body, adrenal medulla and stellate ganglion of squirrel monkeys. The method of localization has been by the use of a specific cytochemical technique, utilizing a chromium (Cr) complex with an isoquiniline derivative formed by the reaction of the unsubstituted biogenic amine with glutaraldehyde. The final reaction product, i.e., amine-glutaraldehyde-Cr, has been identified using energy dispersive X-ray analysis and the positive Cr reactive sites have been correlated with light microscopy conducted by using glyoxylic acid histofluorence. All tissues were taken from squirrel monkeys which had either been perfused with the fixative or had been perfused with an F-12 buffer prior to fixation and/or histochemical treatment. The best results were obtained from the F-12 perfusion followed by tissue being taken through the various histochemical-cytochemical procedures. The electron microscopic localization of deposits shows significantly large structures containing the Cr positive amine material. These large deposits have not heretofore been described and correlate well in size and location with what is seen with histofluorescence. This electron microscopic technique should lead to further advances in the study of biogenic amines in nervous tissue when fine structural localization, plus well preserved morphology, are essential.     

A role for biogenic amines in developmental language disorders

The authors discuss the current uncertainty regarding etiology, treatment, and classification of developmental language disorders (DLD). Referring to previous reports in the literature, they propose a specific subclass of DLD associated with a hypothesized dysfunction of the dopaminergic system of the basal ganglia. Mechanisms of dysfunction and treatment implications are discussed.     

Characterization of the adrenergic activity of arbutamine, a novel agent for pharmacological stress testing

In this study, we characterized the interactions of arbutamine, a novel catecholamine developed for use as a cardiac stress testing agent, with different adrenergic receptor subtypes in vitro. These effects were compared with those of isoproterenol. In the electrically stimulated left atria of rats, arbutamine increased contractile force. The pD₂ values (-log of the dose that produces 50% of the maximal responses) for arbutamine and isoproterenol were 8.45±0.15 and 8.55±0.02, respectively. Metoprolol shifted the concentration-effect curves for both isoproterenol and arbutamine to the right with a pA₂ value (-log of the dose of the antagonist that reduces the maximal responses of an agonist to 50%) of 7.22–7.5. Both arbutamine and isoproterenol increased the rate of spontaneously beating rat right atria with pD₂ values of 9.0±0.19 and 8.82±0.18, respectively. The affinity constants (K_A) of arbutamine and isoproterenol for cardiac beta₁-adrenergic receptors, as determined by competition binding assays, were found to be 7.32 and 6.04, respectively. In guinea pig trachea, arbutamine and isoproterenol produced a concentration-dependent relaxation that was blocked by propranolol. Their pD₂ values were 7.9±0.1 and 8.2±0.1, respectively. Arbutamine contracted isolated rat aortic rings with a maximal increase of 38.1±6.7% that of 10 μM of norepinephrine. In rat white adipocytes, arbutamine, isoproterenol, and BRL-37344 stimulated glycerol release, with the order of potency being BRL-37344 > arbutamine > isoproterenol. In hamster brown adipocytes, the order was arbutamine > isoproterenol > BRL-37344. Moreover, arbutamine stimulated beta₃-adrenergic receptors in guinea pig ileum. In conclusion, arbutamine is a novel catecholamine with similar potency and efficacy to that of isoproterenol. It stimulates cardiac beta₁-, tracheal beta₂-, and adiopocyte beta₃-adrenergic receptors. Arbutamine does not stimulate alpha-adrenergic receptors at concentrations that wer high enough to maximally activate the beta-adrenergic receptors.     

Deficits in inhibitory avoidance after neurotoxic lesions of the ventral striatum are neurochemically and behaviorally selective

Inhibitory avoidance (step-in type) was investigated in rats subjected to neurochemical lesions of the ventral striatum. The neurotoxins 6-hydroxydopamine or 5,7-dihydroxytryptamine were used to produce selective depletions of either dopamine, norepinephrine or serotonin. Only lesions which decreased the dopamine content of the ventral striatum impaired post-shock step-in behavior. Measurement of footshock reactivity by the Flinch-Jump technique indicated that only serotonin depletion altered reactivity to footshocks. Assessment of open-field locomotor behavior showed that the dopamine-denervated rats were hypoactive (fewer rearings) compared to controls, whereas serotonin-depleted rats were hyperactive. It is concluded that the deficit in inhibitory avoidance behavior following ventral striatal dopamine loss was dissociated from its effect on locomotor activity.     

Characterization of the urinary metabolites of 5-amino-1-naphthol in the rat

The metabolic fate of [¹⁴C]5-amino-1-naphthol (5A1N) was investigated in Sprague-Dawley rats. [¹⁴C]5A1N was administered by gastric intubation to male rats at doses 1, 37 and 135 mg/kg body weight. In a separate experiment the rats were also dosed with 150 mg/kg of unlabeled 5A1N daily for 4 consecutive days. Between 74% and 85% of the administered dose was excreted in the urine. Over 98% of the urinary radioactivity was characterized as unchanged 5A1N, 5-acetamido-1-naphthol (5AA1N) and glucuronic and sulfuric acid conjugates of both 5A1N and 5AA1N. Unchanged 5A1N and 5AA1N accounted for less than 3% of the dose. The amount of 5A1N converted to 5AA1N and its conjugates varied inversely with the dose. Two minor metabolites were not identified. Rats dosed repeatedly with 150 mg/kg of 5A1N showed no significant change in metabolite excretion patterns compared to rats dosed singly. These findings indicate that in the rodent model the metabolism of 5A1N was dose dependent, and occurred predominantly by phase II reaction involving N-acetylation and conjugation with glucuronic and sulfuric acids. N-Acetylation predominated at lower doses and O-sulfate conjugation at higher doses. There was no evidence for the formation of N-hydroxylated metabolites over the dose range studied.     

Attempted antagonism of adenosine analogue induced depression of respiration

Intracerebroventricular (ICV) administration of the stable adenosine analogue 2-chloroadenosine (2CA) to hyperoxic halothane-anesthetized rats produced a dose-dependent depression of respiration largely as a result of a decrease in tidal volume. Similar changes were noted after another adenosine analogue, phenylisopropyladenosine (PIA). Higher doses shifted the minute ventilation-PaCO2 curve to the right and decreased its slope. Bradycardia and hypotension were produced at doses which altered respiration. Neonatal destruction of brain serotonin or dopamine-containing nerve terminals did not alter the 2CA-induced respiratory depression. Naloxone significantly antagonized the respiratory and circulatory changes produced by 2CA though the changes produced by PIA were not significantly antagonized. Peripherally and intracerebroventricularly administered theophylline were largely ineffective in reversing the 2CA-induced respiratory depression. Thus, these data suggest that a major part of the respiratory depression produced by 2CA is due to indirect activation of opioid receptors. In contrast, very little of the respiratory depression after PIA is via mechanisms antagonized by naloxone. Thus, putative adenosine agonists appear to vary in the extent to which respiratory depression is provoked by interactions with opioid systems.     

Neurotransmitter functions in depression

The understanding and management of depression has now progressed beyond the limitations imposed by clinical examination. Biochemical and pharmacological studies based on the biogenic amine hypothesis have investigated neurotransmitter mechanisms to varying degrees.

1. Subgroups of depressions may be identified and treated based on MHPG execution.

2. HVA correlates more with activity than with mood.

3. CSF-5HIAA may be helpful in categorising some depressions.

4. Acetylcholine has some effect on mood most probably through indirect action on other neurotransmitters.

5. GABA is still not adequately investigated.

6. Desensitization of presynaptic adrenergic autoreceptors may explain some of the mechanisms of antidepressant action of drugs.

7. Decreased post-synaptic adrenergic activity is a common effect of most antidepressants and of ECT.

Racemization studies in peptide synthesis through the separation of protected epimeric peptides by reversed-phase high performance liquid chromatography

Separation of protected epimeric peptides, Z-Gly-Xaa-Xbb-OMe (where Xaa and Xbb = chiral amino acid residues), by reversed-phase HPLC was utilized for studying racemization in peptide synthesis. Thus, the following factors which might affect the extent of racemization during the coupling by the carbodiimide method were investigated: the combination of amino acid residues to be coupled, coexisting tertiary amine salts, and the relative configuration of the amino acid residues. The following points were revealed: the combination of bulky residues at the coupling site results in extensive racemization in a polar solvent such as DMF, the amine hydrochlorides cause less racemization than the p-toluenesulfonates in DMF, and the influence of relative configuration differs depending on the solvent and the individuality of the amino components. Furthermore, the racemization-suppressing effect of some additives in the carbodiimide method was reevaluated by employing the same procedure.     

Abnormalities in norepinephrine turnover rate in the central nervous system of the genetically epilepsy-prone rat

Norepinephrine turnover rates were estimated in the hypothalamus-thalamus, midbrain, pons-medulla and telencephalon of genetically epilepsy-prone rats (GEPR). In each of these 4 brain areas the endogenous norepinephrine levels were significantly lower in the GEPR than in control animals. In the hypothalamus-thalamus, midbrain and telencephalon the calculated norepinephrine turnover rates were also significantly lower in GEPRs than in control. These studies confirm and extend earlier observations relating seizures in the GEPR to decrements in central nervous system noradrenergic function.     

A correlational analysis of the effects of surgical transections of three components of the MFB on ingestive behavior and hypothalamic, striatal, and telencephalic amine concentrations.

A retractable wire knife was used to transect medial or lateral components of the MFB or its lateral projections to the striatum and amygdaloid complex. All cuts produced significant depletions of NE, DA, and 5-HT from telencephalon and striatum but little or no effect on hypothalamic NE or 5-HT. Two of our cuts resulted in aphagia and adipsia, the third in hyperphagia and obesity. A detailed correlational analysis of the magnitude and direction of the behavioral and biochemical consequences of our cuts indicated that the ingestive behavior of all of our experimental animals (including animals which had been aphagic and adipsic after surgery as well as animals which were hyperphagic and obese) was positively correlated with the concentration of DA in striatum and telencephalon and negatively correlated with telencephalic 5-HT. Less consistent evidence for facilitatory noradrenergic influences on food intake was also obtained. Our results suggest that the regulation of food intake may be the result of an interaction between telencephalic serotonergic mechanisms and dopaminergic pathways which exert opposite effects on ingestive behavior.