Graziano Pinna

Brain allopregnanolone regulates the potency of the GABAA receptor agonist muscimol

Allopregnanolone (ALLO), a potent positive-allosteric modulator of the action of GABA at GABA(A) receptors, is synthesized in the brain from progesterone by the sequential action of two enzymes: 5alpha-reductase and 3alpha-hydroxysteroidoxidoreductase. The concentration of ALLO in various parts of the mouse brain varies substantially, from 15 pmol/g in the olfactory bulb, to approximately 6 pmol/g in the frontoparietal cortex, and 2.7 pmol/g in the cerebellum. The systemic administration of 48 micromol/kg of the Type I and Type II 5alpha-reductase inhibitor, (17beta)-17-[bis (1-methylethyl) amino carbonyl)] androsta-3, 5-diene-3-carboxylic acid (SKF 105,111), reduced brain ALLO content by 80-90% in 30 min; the rate constant (k) of ALLO decrease in each brain area can be utilized to establish the rate of ALLO biosynthesis, which is higher in the olfactory bulb (62 pmol/g/h) than in the frontoparietal cortex (24 pmol/g/h) or cerebellum (11 pmol/g/h). The duration of the righting reflex loss elicited by the potent GABA(A) receptor agonist muscimol was reduced in SKF 105,111-treated ALLO-depleted mice. SKF 105,111 treatment had no effect on muscimol metabolism or on brain levels of pregnenolone and progesterone; however, the brain levels of 5alpha-DHP, the precursor of ALLO, were also decreased. Administration of ALLO at a dose of 15 micromol/kg i.p. by itself did not alter the muscimol-induced loss of the righting reflex; but it completely blocked the effect of SKF 105,111. To elucidate the possible molecular mechanism by which a decrease of brain ALLO content can shorten the duration of the righting reflex loss elicited by muscimol, we patch-clamped neocortical pyramidal neurons of mice pretreated with SKF 105,111 or vehicle, and studied the efficiency of muscimol in eliciting Cl- currents. The current amplitude was significantly smaller in neurons from SKF 105,111-treated mice, especially at lower doses (0.1-1 microM) of muscimol, and the muscimol dose-response (0.1-10 microM) relationship displayed cooperativity (nH=1.4). These data suggest that ALLO synthesized in brain plays an important physiological permissive role in the modulation of GABA-gated Cl- channel function.

Rat Brain Type II 5′-Iodothyronine Deiodinase Activity Is Extremely Sensitive to Stress

Abstract: The effects of different kinds of acute stressor on thyroid hormone concentrations and deiodinase activities were investigated in four brain regions (frontal cortex, amygdala, hypothalamus, and cerebellum) and in the pituitaries and livers of adult male rats. Five groups of rats were killed after each of the following stressors: (a) an intraperitoneal injection of saline, (b) intragastric intubation, (c) and (d) two different forms of handling, being grasped as for intraperitoneal injection and being moved from one cage to another, and (e) a 2‐h period spent in a slowly rotating drum. Two other groups were placed in the rotating drums for 10 and 19 h (sleep deprivation experiment), respectively. All stressors induced significant (in some cases up to 200%) increases in the activity of type II 5′‐iodothyronine deiodinase, which catalyzes the deiodination of the prohormone l‐thyroxine (T4) to the active metabolite 3,3′,5‐triiodo‐l‐thyronine (T3). As a consequence, the tissue concentrations of T4 fell, and those of T3 rose (sometimes by up to 300%). However, these changes were limited to selected areas of the brain that were specific for each stressor and were not seen in all brain regions investigated in any group. No clear‐cut effects of stress were seen on the activities of the type III 5‐iodothyronine deiodinase isoenzyme, which catalyzes the inactivation of T3, on liver or serum thyroid hormone concentrations or on liver of brain type I 5′‐iodothyronine deiodinase activities. In summary, our results show that even mild and very brief stress can induce marked increases in T3 concentrations specifically in brain but not in liver or blood. Thus, contrary to common opinion, thyroid hormones may play an important physiological role in stress reactions, at least in tissues that contain type II 5′‐iodothyronine deiodinase, such as brain and pituitary.

Concentrations of Seven Iodothyronine Metabolites in Brain Regions and the Liver of the Adult Rat

The concentrations of the iodothyronine metabolites T4 ,T 3, 3,5-diiodothyronine (3,5-T2), 3,3-diiodothyronine (3,3-T2), reverse T3 (rT3), 3,3-T2 sulfate (3,3T2S), and T3 sulfate (T3S) were measured in 12 regions of the brain, the pituitary gland, and liver in adult male rats. Quantification of iodothyronine was performed by RIA following a newly developed method of purification and separation by HPLC. 3,5-T2, 3,3-T2 ,r T 3 and T2S were detectable in the low femtomolar range (20 –200 fmol/g) in most areas of the rat brain. T3S was detectable only in the hypothalamus. The concentrations of T3 and T4 were approximately 20- to 60-fold higher, ranging between 1 and 6 pmol/g. There was a significant negative correlation between the activities of inner-ring deiodinase and T3 concentrations across brain areas. In the liver, 3,5-T2 ,r T3, and T3S were measurable in the low femtomolar range, whereas 3,3-T2 and 3,3T2S were not detectable. 3,5-T2 and 3,3-T2 were not detectable in mitochondrial fractions of the brain regions. Tissue concentrations of 3,5-T2 exhibited a circadian variation closely parallel to those of T3 in the brain regions and liver. T3 was not a substrate for outer-ring deiodination under different experimental conditions; thus, it remains unclear which substrate(s) and enzyme(s) are involved in the production of 3,5-T2. These results indicate that five iodothyronine metabolites other than T3 and T4 are detectable in the low femtomolar range in the rat brain and/or liver. The physiological implications of this finding are discussed. (Endocrinology 143: 1789 –1800, 2002)

Extraction and quantification of thyroid hormones in selected regions and subcellular fractions of the rat brain.

There is increasing evidence of an involvement of thyroid hormones in numerous physiological processes of the adult vertebrate brain. However, the only valid method available for measuring triiodothyronine (T3) in brain tissue is time-consuming and not sufficiently sensitive to determine hormone concentrations in small, but physiologically important areas such as the amygdala and septum. We therefore developed a protocol for reliable measurement of the concentrations of thyroxine (T4) and T3 in brain tissue. This was achieved by combining a new method of extracting iodothyronines with highly sensitive, accurate and reproducible radioimmunoassays (RIAs) in order to be able to detect T4 and T3 in homogenates and even subcellular fractions (nuclear, synaptosomal and mitochondrial) in up to 11 regions of the rat brain. The iodothyronines were extracted from tissue samples by adding 100% methanol containing 1 mM PTU. Recoveries of 72.8 +/- 5.5% and 83.2 +/- 3.3% for T4 and T3, respectively, were obtained. The RIA detection thresholds were 10 fmol/g for T4 and 18 fmol/g for T3. Only 0.2% of the antibody for T4 cross-reacted with T3 and 0.95% reverse T3. T3 antibody (0.05%) reacted with T4 and 0.01% with 3,5-T2. The T4 concentrations in the homogenates of selected areas of the brain ranged between 1 and 4 pmol/g, whereas those of T3 ranged between 0.5 and 4 pmol/g. The T3 levels ranged between 190 and 470 fmol/mg protein, 38 and 110 fmol/g protein and 25 and 180 fmol/mg protein in the nuclei, synaptosomes and mitochondriae, respectively. In conclusion, the newly developed method enabled us to determine both T4 and T3 concentrations in homogenates and T3 in subcellular fractions of regions of the brain as small as the septum and amygdala.

Gene expression of receptors and enzymes involved in GABAergic and glutamatergic neurotransmission in the CNS of rats behaviourally dependent on ethanol

The steady state levels of the messenger RNA (mRNA) of eight GABAA receptor subunits, five glutamate receptor subunits and seven enzymes involved in the synthesis of glutamate and GABA were measured in eight regions of rat brain in a recently developed animal model of ‘behavioural dependence’ on ethanol. ‘Behavioural dependence’ including loss of control was induced by offering the rats the choice between ethanol and water over a 9‐month period (Group A). This group was compared with a group given the choice between ethanol and water for only 2 months (not yet ‘behaviourally dependent’, Group B), a group forced to consume ethanol as sole fluid over a 9‐month period (also not ‘behaviourally dependent’, Group C) and ethanol‐naive control rats (Group D). All groups were sacrificed 1 month after the ethanol was withdrawn. The mRNA concentrations of all eight GABA receptor subunits, four out of the five subunits of different glutamate receptors and those of seven enzymes involved in GABA and glutamate production were reduced almost exclusively in the parieto‐occipital cortex in Groups A and B, but not Group C. These data suggest that the synthesis of glutamate and GABA and the activities of their respective neurons are selectively impaired in the parieto‐occipital cortex in the groups having consumed ethanol in a free‐choice design, in which its rewarding properties can better take effect than after forced administration. As the parieto‐occipital cortex is believed to contain emotional memory structures, it may be hypothesized that the glutamatergic and GABAergic neuronal systems in this area are involved in the development of memory for reward from ethanol. However, they are not specifically associated with ‘behavioural dependence’.

Effects of acute administration of ethanol and the μ-opiate agonist etonitazene on thyroid hormone metabolism in rat brain

Abstract The effects of acute, low-dose administration of ethanol (1 g/kg bodyweight) and the μ-opioid receptor agonist etonitazene (30 μg/kg bodyweight) on the activities of the iodothyronine deiodinase isoenzymes were investigated in nine regions of the rat brain. The experiments were performed at three different times of the 24-h cycle (1300, 2100 and 0500 hours) and the rats were decapitated 30 and 120 min after administration of the respective drugs. Interest was focused on changes in the two enzymes that catalyze 1) 5′-deiodination of thyroxine (T4) to the biologically active triiodothyronine (T3), i.e. type II 5′-deiodinase (5′D-II) and 2) 5 (or inner-ring) deiodination of T3 to the biologically inactive 3′3-T2, i.e. type III deiodinase (5D-III). 120 min after administration of ethanol and etonitazene 5D-III activity was selectively inhibited in the frontal cortex (at 1300 and 1700 hours) and the amygdala (at all three measuring times). The 5′D-II activity was significantly enhanced 30 min after administration of etonitazene in the frontal cortex, amygdala and limbic forebrain, and after administration of ethanol in the amygdala alone. These effects on 5′D-II activity were seen at 2100 hours only. In conclusion, the two different addictive drugs both reduced the inactivation of the physiologically active thyroid hormone T3 and enhanced its production. These effects occurred almost exclusively in the brain regions which were most likely to be involved in the rewarding properties of addictive drugs. As thyroid hormones have stimulating and mood-elevating properties, an involvement of these hormones in the reinforcing effects of addictive drugs seems conceivable.