Albert J. Azzaro

Prenatal stress increases corticotropin-releasing factor (CRF) content and release in rat amygdala minces

Corticotropin-releasing factor (CRF) is a neuropeptide found throughout the central nervous system that has a proposed role in modulating emotional and behavioral states, including stress and anxiety. The amygdala, which is important in the control of emotional and autonomic responses to stress, contains CRF nerve terminals, CRF cell bodies, and CRF receptors. In rats, exposure to prenatal stress results in offspring that display a hyperemotional state and increased anxiety. In this study the effects of prenatal stress on CRF release was measured in amygdala minces (1 mm3) obtained from adult (8-16 weeks of age) male offspring of dams subjected to daily saline injection (0.1 ml, s.c.) from gestational day 14 to 21. CRF release from amygdala was time- and calcium-dependent, and stimulated by KCl-induced depolarization. Depolarization-induced CRF release was significantly increased by 42% from the amygdala of prenatally stressed offspring versus controls. Prenatally stressed offspring also showed a 49% increase in CRF levels in the amygdala. The increased amounts of CRF released in response to depolarization were likely the consequence of increased tissue content of CRF, as fractional release under basal or KCl-stimulated conditions was not different in the prenatal stress group versus control. This suggests that a long-lasting up-regulation of the CRFergic neurotransmission may occur in the amygdala, which may be important in the generation of hyperemotional offspring after exposure to prenatal stress.

Specificity of Endogenous Substrates for Types A and B Monoamine Oxidase in Rat Striatum

Abstract: Studies were designed to evaluate specificity of the transmitter amines serotonin (5‐hydroxytryptamine, 5‐HT) and dopamine (DA), as well as the trace amines p‐tyramine (p‐TA) and β‐phenylethylamine (PEA) for types A and B monoamine oxidase (MAO) in rat striatum. 5‐HT was found to be a specific substrate for the type A enzyme. However, the specificity of PEA for the type B enzyme was found to be concentration‐dependent. When low concentrations of PEA and 5‐HT were used to measure type B and type A activities, respectively, both clorgyline and deprenyl were highly selective for the sensitive form of MAO in vivo. However, as the concentration of PEA was increased, the type B inhibitor deprenyl became less effective in preventing deamination of PEA. Conversely, the type A inhibitor clorgyline became more effective in this regard. Kinetic analysis following selective in vivo inhibition showed PEA deamination by both forms of MAO with a 13‐fold greater affinity for the type B enzyme. In vivo dose‐response curves obtained with the common substrates DA and p‐TA showed approximately 20% deamination by the B enzyme. Kinetic values for DA and p‐TA deamination in in vivo‐treated tissue possessing only type A or type B MAO activity, revealed a 2.5‐fold greater affinity for the type A enzyme. These studies show the importance of concentration on substrate specificity in striatal tissue. The results obtained characterize the common substrate properties of DA and p‐TA as well as of PEA in rat striatum. In addition, the presence of regional specificity for 5‐HT deamination by only type A MAO is demonstrated.

Guinea Pig Striatum as a Model of Human Dopamine Deamination: The Role of Monoamine Oxidase Isozyme Ratio, Localization, and Affinity for Substrate in Synaptic Dopamine Metabolism

Abstract: The kinetic properties of type A and type B monoamine oxidase (MAO) were examined in guinea pig striatum, rat striatum, and autopsied human caudate nucleus using 3,4‐dihydroxyphenylethylamine (dopamine, DA) as the substrate. MAO isozyme ratio in guinea pig striatum (28% type A/72% type B) was similar to that in human caudate nucleus (25% type A/75% type B) but different from that in rat striatum (76% type A/24% type B). Additional similarities between guinea pig striatum and human caudate nucleus were demonstrated for the affinity constants (Km) of each MAO isozyme toward DA. Endogenous concentrations of DA, 3‐methoxytyramine, 3,4‐dihydroxyphenylacetic acid, and homovanillic acid were also measured in guinea pig and rat striatum following selective type A (clorgyline‐treated) and type B (deprenyl‐treated) MAO inhibition. In guinea pig, DA metabolism was equally but only partially affected by clorgyline or deprenyl alone. Combined treatment with clorgyline and deprenyl was required for maximal alterations in DA metabolism. By contrast, DA metabolism in rat striatum was extensively altered by clorgyline but unaffected by deprenyl alone. Finally, the deamination of DA in synaptosomes from guinea pig striatum was examined following selective MAO isozyme inhibition. Neither clorgyline nor deprenyl alone reduced synaptosomal DA deamination. However, clorgyline and deprenyl together reduced DA deamination by 94%. These results suggest that the isozyme localization and/or isozyme affinity for DA, rather than the absolute isozyme content, determines the relative importance of type A and type B MAO in synaptic DA deamination. Moreover, based on the enzyme kinetic properties of each MAO isozyme, guinea pig striatum may serve as a suitable model of human DA deamination.

Stimulation of the Amygdala by Glutamate Facilitates Corticotropin-Releasing Factor Release from the Median Eminence and Activation of the Hypothalamic-Pituitary-Adrenal Axis in Stressed Rats

The role of the amygdala in the regulation of hypothalamic release of corticotropin-releasing factor (CRF) was investigated. Microinjection of glutamate (50 nmol) into the amygdala resulted in increased plasma corticosterone in male rats previously subjected to a 14-day unpredictable stressor paradigm (p < or = 0.05 vs. saline-injected controls). A long-lived increase in corticosterone levels was also observed in rats which were urethane-anesthetized (1.35 g/kg) 3 h prior to glutamate microinjection (p < or = 0.01 vs. saline-injected controls). These effects on plasma corticosterone were observed despite the presence of high basal levels of corticosterone. Furthermore, microperfusion of glutamate (3-300 microM) into the amygdala of urethane-anesthetized rats resulted in a dose-dependent increase in CRF release from the median eminence, as assessed by in vivo microdialysis (p < or = 0.025 vs. basal). These results indicate a facilitating role for the amygdala in stress-induced increases in CRF release and subsequent adrenocortical activation.

Effects of Intrastriatal Kainic Acid Injection on [3H]Dopamine Metabolism in Rat Striatal Slices: Evidence for Postsynaptic Glial Cell Metabolism by Both the Type A and B Forms of Monoamine Oxidase

Abstract: Intrastriatal injections of kainic acid (KA) were utilized to investigate the cellular localization of postsynaptic dopamine (DA) metabolism by type A and B monoamine oxidase (MAO) in rat striatum. At 2 days postinjection, maximal degeneration of cholinergic and γ‐aminobutyric acid (GABA)ergic neurons was observed and found to be associated with a significant decrease in both type A and B MAO activity. However, over the next 8‐day period, when only the process of gliosis appeared to be occurring, a selective return to control of type B MAO activity was seen. When the metabolism of [3H]DA (10−7 M) was examined in 8‐day KA‐lesioned rat striatal slices, an increase in [3H]dihydroxyphenylacetic acid (DOPAC) and [3H]homovanillic acid (HVA) formation was observed. The KA‐induced elevation of [3H]DOPAC formation (but not [3H]HVA) was abolished by the DA neuronal uptake inhibitor nomifensine. This is consistent with earlier findings suggesting that HVA is formed exclusively within sites external to DA neurons. Experiments with clorgyline and/or deprenyl revealed that the relative roles of type A and B MAO in striatal DA deamination remained unchanged following KA (90% deamination by type A MAO) even though total deamination was substantially enhanced. At high concentrations of [3H]DA (10−5 M), deamination by type B MAO could be increased to 30% of the total MAO activity; however, this was observed in both control and KA‐lesioned striata. These results suggest that KA‐sensitive neurons contain type A and/or type B MAO. Moreover, whereas these neurons may metabolize DA, a major portion of postsynaptic DA deamination appears to occur within glial sites of rat striatal tissue. Furthermore, glial cells would appear to contain functionally important quantities of both type A and B MAO.

Daily transdermal administration of selegiline to guinea-pigs preferentially inhibits monoamine oxidase activity in brain when compared with intestinal and hepatic tissues

Selegiline has been formulated in an acrylic polymer adhesive mixture to be employed as a constant release topical patch for daily transdermal administration. Application of this selegiline transdermal system (STS) to guinea‐pigs resulted in an average delivery of 1.185 mg selegiline/cm2 patch/24 h. STS dose‐response curves were generated by altering patch size (cm2). A transdermal dose range was identified which inhibited guinea‐pig brain monoamine oxidase‐B (MAO‐B) by greater than 95 % yet provided for a dose‐dependent inhibition of monoamine oxidase‐A (MAO‐A) activity. The ID50 for inhibition of MAO‐A activity in response to a 21‐day daily regimen with transdermal selegiline was approximately 7.5‐fold lower for cortical and striatal brain regions compared with that obtained for duodenum; hepatic MAO‐A was unaffected following the same dosing regimen. By contrast, orally administered selegiline inhibited brain and duodenal MAO‐A to the same extent, and generated a shallower dose–inhibition curve for brain MAO‐A inhibition. In addition, transdermal delivery was approximately 6–8‐times more potent than oral selegiline for the inhibition of brain MAO‐A activity. It is concluded that daily transdermal selegiline administration may provide therapeutic advantages over oral treatment, based on its preferential, dose‐dependent inhibition of brain vs peripheral MAO‐A activity.

Role of type A and type B monoamine oxidase in the metabolism of released [3H]dopamine from rat striatal slices☆

The effects of selective inhibition of multiple forms of monoamine oxidase (MAO) on the in vitro release and metabolism of newly-synthesized [3H]dopamine (DA) were examined using rat brain slices. Striatal slices were preincubated in the presence of [3H]l-tyrosine (20 μM) followed by a short incubation period in the presence of the selective irreversible MAO-inhibitor agents clorgyline (type A) and deprenyl (type B). Tissue pretreated in this manner was then subjected to a release incubation, and DA release and metabolism were determined under spontaneous and depolarizing conditions. Pretreatment with clorgyline (10−7 M) significantly reduced the spontaneous, as well as K+-evoked, formation of both 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). Deprenyl (10−7 M) pretreatment did not significantly affect these variables, but clorgyline + deprenyl pretreatment resulted in a reduction of both DOPAC and HVA that was greater than that produced by clorgyline alone. By contrast, deprenyl pretreatment significantly decreased both DOPAC and HVA under depolarizing conditions, but only in the presence of the DA uptake inhibitor nomifensine (10−5 M). In the absence of MAO inhibition, nomifensine increased K+-evoked formation of DOPAC and HVA, while spontaneous formation was not affected. The results suggest that released DA is deaminated primarily by the type A form of MAO; however, in the absence of the type A MAO, or under conditions that promote exclusive postsynaptic deamination, minor but significant metabolism occurs via the type B enzyme. Data obtained are further discussed in relation to the mechanism of MAO-inhibitor drug action and pre- versus postsynaptic formation of DOPAC and HVA.

Oral Versus Transdermal Selegiline: Antidepressant-Like Activity in Rats

These studies compared the dose-response effects of oral vs. transdermal selegiline on antidepressant-like activity and brain monoamine oxidase (MAO) activities in rats. Rats received selegiline by gavage (0-100 mg/kg) or via transdermal patches (0-4.8 cm2, 0-8.7 mg/kg) daily for 7 days; antidepressant-like activity was determined using the forced-swim test. Following behavioral testing, cerebral cortices were assayed for MAO-A and MAO-B activities. Doses of selegiline that selectively inhibited MAO-B (3 and 10 mg/kg/day by gavage and 0.4 mg/kg/day via patch) did not alter either immobility or latency time. However, the oral administration of 30 or 100 mg/kg/day or the transdermal administration of 8.7 mg/kg/day, doses that led to greater than 70% inhibition of MAO-A, decreased immobility time significantly. The IC50s for inhibition of MAO-A following oral and transdermal administration for 7 days were 19.8 and 1.1 mg/kg, respectively. Results indicate that both oral and transdermal selegiline have antidepressant-like activity as assessed by the forced-swim test, and that transdermal administration, which bypasses first-pass metabolism, allows for using lower doses than oral administration.

Effect of repeated amiflamine administration on serotonergic and noradrenergic neurotransmission: electrophysiological studies in the rat CNS

SummaryAmiflamine is a selective and reversible inhibitor of monoamine oxidase (MAO) type A which exerts a preferential effect on serotonin (5-HT) catabolism. The present studies were undertaken to compare the effects of repeated administration of amiflamine (2 mg/kg, twice daily) on several aspects of the functioning of the 5-HT and norepinephrine (NE) systems in the rat CNS. The activity of MAO-A and B was assessed in forebrain slices and the whole brain contents of the neurotransmitters and their metabolites were determined by HPLC after 2-, 7- and 21-day treatments. MAO-A was inhibited by about 50% 2 h after the last dose and its activity was back to normal in rats sacrificed 12 h after the last dose. The activity of MAO-B was unaffected two or 12 h after the last dose. Whole brain concentration of 5-HT was increased to a greater degree than that of NE following repeated administration of amiflamine. These increases in 5-HT and NE were accompanied by decreased levels of their respective metabolites 5-hydroxy-indoleacetic acid and 3-methoxy-4-hydroxyphenylethyleneglycol. The firing activity of dorsal raphe 5-HT neurons, but not that of NE neurons, was markedl decreased 2–6 h after the last dose of a 2-day treatment. However, 2–6 h after a 21-day treatment, the firing activity of 5-HT neurons was back to normal, whereas that of NE neurons was decreased by 30%. The recovery of firing activity of 5-HT neurons following long-term amiflamine is attributable to a desensitization of the somatic 5-HT autoreceptor as indicated by the decreased responsiveness of 5-HT neurons to intravenous LSD. In contrast, the sensitivity of the somatic NE autoreceptor was not modified, as verified by the unchanged responsiveness of NE neurons to clonidine, an α2 agonist. The duration of the suppression of firing activity of hippocampus pyramidal neurons produced by the electrical activation of the ascending 5-HT pathway was prolonged in amiflamine-treated rats as compared to controls. This increased synaptic efficacy was attributable to a presynaptic modification since the responsiveness of the same neurons to microiontophoretically-applied 5-HT was not changed. The efficacy of the stimulation of the dorsal NE bundle, as well as that of the microiontophoretic applied of NE, were unchanged in the same rats. Since the efficacy of the stimulation of the 5-HT pathway is increased at the time 5-HT neurons have regained their normal firing rate, it is concluded that 5-HT neurotransmission is enhanced following the long-term treatment with amiflamine.

Dopaminergic nature of feeding-induced behavioral stereotypies in stressed pigeons☆

Dopamine dependence of feeding-induced behavioral stereotypies (FIBS) was demonstrated by FIBS facilitation following chronic treatment with a dopamine (DA) agonist, apomorphine, and by FIBS inhibition following the administration of haloperidol, a DA antagonist. However, individuals that emitted FIBS were differentiated from those not emitting FIBS not by assayed telencephalic DA concentrations alone but by a higher stereotyping index (SI), a score positively related to the ratio of telencephalic DA-norepinephrine (NE) concentrations. These latter findings support the hypothesis of Antelman and Caggiula [3] which indicates that a catecholamine interaction in the brain serves to facilitate or inhibit some behavioral actions associated with stress.