Giovanni Biggio

Time-Dependent Changes in Rat Brain Neuroactive Steroid Concentrations and GABAA Receptor Function after Acute Stress

The time courses of changes in rat brain neuroactive steroid concentrations and gamma-aminobutyric acid type A (GABAA) receptor function elicited by acute stress were investigated in animals exposed to CO2 for 1 min, a treatment known to induce stress in rats and panic attacks in humans. Inhalation of CO2 induced increases in cerebral cortical steroid concentrations, the time dependence of which varied with the steroid examined. Thus, progesterone and deoxycorticosterone showed maximal increases (10- and 4-fold, respectively) 10 min after CO2 inhalation and had returned to basal values by 30 and 60 min, respectively. In contrast, pregnenolone and 3alpha-hydroxy-5alpha-pregnan-20-one (allopregnanolone) concentrations showed maximal increases (+174 and + 200%, respectively) at 30 min, were still higher than control at 60 min and returned to control values 120 min after stress. Inhalation of CO2 also resulted in increases in plasma steroid concentrations, most of which peaked at 30 min and had returned to control values by 60 min. A parallel analysis of the stress-induced changes in GABAA receptor function, assessed either biochemically by t-[35S]butylbicyclophosphorothionate ([35S]TBPS) binding to cerebral cortical membranes or behaviorally by the punished responding score in Vogels test, showed that the effects of CO2 inhalation on both parameters were maximal (+51 and -40%, respectively) after 10 min; the behavioral reaction returned to normal after 60 min, whereas [35S]TBPS binding had returned to control values 120 min after stress. The results show that: (a) the maximal increase in the brain concentrations of allopregnanolone, a potent and efficacious positive modulator of GABAA receptors, occurred at a time (30 min) when both conflict behavior and [35S]TBPS binding begun to decrease, and (b) both allopregnanolone concentrations and [35S]TBPS binding had returned to control values 120 min after CO2 inhalation. The data are thus consistent with a physiological role of neuroactive steroids in restoring GABAergic tone after stress.

Social Isolation‐Induced Decreases in Both the Abundance of Neuroactive Steroids and GABAA Receptor Function in Rat Brain

The effects of social isolation on behavior, neuroactive steroid concentrations, and GABAA receptor function were investigated in rats. Animals isolated for 30 days immediately after weaning exhibited an anxiety‐like behavioral profile in the elevated plus‐maze and Vogel conflict tests. This behavior was associated with marked decreases in the cerebrocortical, hippocampal, and plasma concentrations of pregnenolone, progesterone, allopregnanolone, and allotetrahydrodeoxycorticosterone compared with those apparent for group‐housed rats ; in contrast, the plasma concentration of corticosterone was increased in the isolated animals. Acute footshock stress induced greater percentage increases in the cortical concentrations of neuroactive steroids in isolated rats than in group‐housed rats. Social isolation also reduced brain GABAA receptor function, as evaluated by measuring both GABA‐evoked Cl‐ currents in Xenopus oocytes expressing the rat receptors and tert‐[35S]butylbicyclophosphorothionate ([35S]TBPS) binding to rat brain membranes. Whereas the amplitude of GABA‐induced Cl‐ currents did not differ significantly between group‐housed and isolated animals, the potentiation of these currents by diazepam was reduced at cortical or hippocampal GABAA receptors from isolated rats compared with that apparent at receptors from group‐housed animals. Moreover, the inhibitory effect of ethyl‐β‐carboline‐3‐carboxylate, a negative allosteric modulator of GABAA receptors, on these currents was greater at cortical GABAA receptors from socially isolated animals than at those from group‐housed rats. Finally, social isolation increased the extent of [35S]TBPS binding to both cortical and hippocampal membranes. The results further suggest a psychological role for neurosteroids and GABAA receptors in the modulation of emotional behavior and mood.

Free tryptophan in serum controls brain tryptophan level and serotonin synthesis

Abstract Tryptophan (Try) is the only aminoacid present in plasma bound to serum proteins. The administration of Try to rats increased free and total Try in serum. Changes in free Try were much more pronounced and longer lasting than in total Try. Brain Try and 5-hydroxyindoleacetic acid (5-HIAA) underwent changes, parallel and proportional to the changes in free Try in serum. Rats fasted for 24 hours had higher concentrations of free serum tryptophan, brain tryptophan and 5-HIAA than rats fed for 2 hours. In contrast, total serum tryptophan was lower in fasted than in fed rats. These results indicate that brain tryptophan and serotonin turnover are controlled by free serum tryptophan and that free serum tryptophan levels are independent from total serum tryptophan concentrations.

Rapid depletion of serum tryptophan, brain tryptophan, serotonin and 5-hydroxyindoleacetic acid by a tryptophan-free diet.

Abstract Rats were trained for 20 days to eat their normal daily meal in a period of 2 hours. On the twentyfirst day they received a diet in which tryptophan was omitted instead of the usual balanced diet. The ingestion of the tryptophan-free diet produced a marked depletion of free serum tryptophan (90%), brain tryptophan (85%), brain 5-HT (58%) and brain 5-HIAA (76%). These changes were almost maximal within 2 hours after food presentation and persisted for more than 24 hours. The mechanism of these changes is discussed.

Propofol in Anesthesia. Mechanism of Action, Structure-Activity Relationships, and Drug Delivery

Propofol (2,6-diisopropylphenol) is becoming the intravenous anesthetic of choice for ambulatory surgery in outpatients. It is extensively metabolized, with most of the administered dose appearing in the urine as glucuronide conjugates. Favorable operating conditions and rapid recovery are claimed as the main advantages in using propofol, whereas disadvantages include a relatively high incidence of apnea, and blood pressure reductions. Besides a literature summary of the pharmacodynamics, pharmacokinetics, toxicology, and clinical use, this review provides a deeper discussion on the current understanding of mechanism of action and structure-activity relationships, and recent findings on drug delivery technologies as applied to the improvement of propofol formulations. The action of propofol involves a positive modulation of the inhibitory function of the neurotransmitter gama-aminobutyric acid (GABA) through GABAA receptors. Recent results from recombinant human GABAA receptor experiments and findings from the work exploring the effects at other receptors (e.g., glycine, nicotinic, and M1 muscarinic receptors) are reviewed. Studies showing its antiepileptic and anxiolytic properties are also discussed. The structure-activity relationships (SAR) of series of alkylphenols and p-X-substituted congeners have been reinvestigated. Interestingly, unlike the other congeners tested sofar, p-iodo-2,6-diisopropylphenol displayed anticonvulsant and anticonflict effects, but not sedative-hypnotic and anesthetic properties. Due to its high lipid-solubility, propofol was initially formulated as a solution with the surfactant Cremophor EL, but the occurrence of pain on injection and anaphylactoid reactions prompted to search for alternative formulations. Results from using cyclodextrins, water-soluble prodrugs, and adopting Bodors approach to the site-specific chemical delivery system (CDS), as well as the advantages provided by computer-controlled infusion systems, are examined in some detail.

Brain steroidogenesis mediates ethanol modulation of GABAA receptor activity in rat hippocampus.

An interaction with the GABA type A (GABAA) receptor has long been recognized as one of the main neurochemical mechanisms underlying many of the pharmacological actions of ethanol. However, more recent data have suggested that certain behavioral and electrophysiological actions of ethanol are mediated by an increase in brain concentration of neuroactive steroids that results from stimulation of the hypothalamic-pituitary-adrenal (HPA) axis. Neuroactive steroids such as 3α-hydroxy-5α-pregnan-20-one (3α,5α-THP) are, in fact, potent and efficacious endogenous positive modulators of GABAA receptor function. Because neurosteroids can be synthesized de novo in the brain, we have investigated whether ethanol might affect both neurosteroid synthesis and GABAA receptor function in isolated rat hippocampal tissue. Here, we show that ethanol increases the concentration of 3α,5α-THP as well as the amplitude of GABAA receptor-mediated IPSCs recorded from CA1 pyramidal neurons in isolated hippocampal slices. These effects are shared by the neurosteroid precursor progesterone, the peripheral benzodiazepine receptor-selective agonist CB34, and γ-hydroxybutyrate, all of which are known to increase the formation of neuroactive steroids in plasma and in the brain. The action of ethanol on GABAA receptor-mediated IPSC amplitude is biphasic, consisting of a rapid, direct effect on GABAA receptor activity and an indirect effect that appears to be mediated by neurosteroid synthesis. Furthermore, ethanol affects GABAA receptor activity through a presynaptic action, an effect that is not dependent on neurosteroid formation. These observations suggest that ethanol may modulate GABAA receptor function through an increase in de novo neurosteroid synthesis in the brain that is independent of the HPA axis. This novel mechanism may have a crucial role in mediating specific central effects of ethanol.

Stress and neuroactive steroids.

The discovery that the endogenous steroid derivatives 3 alpha-hydroxy-5 alpha-pregnan-20-one (allopregnanolone, or 3 alpha,5 alpha-TH PROG) and 3 alpha,21-dihydroxy-5 alpha-pregnan-20-one (allotetrahydrodeoxycorticosterone, or 3 alpha,5 alpha-TH DOC) elicit marked anxiolytic and anti-stress effects and selectively facilitate gamma-aminobutyric acid (GABA)-mediated neurotransmission in the central nervous system (see Chapter 3) has provided new perspectives for our understanding of the physiology and neurobiology of stress and anxiety. Evidence indicating that various stressful conditions that downregulate GABAergic transmission and induce anxiety-like states (Biggio et al., 1990) also induce marked increases in the plasma and brain concentrations of these neuroactive steroids (Biggio et al., 1996, 2000) has led to the view that stress, neurosteroids, and the function of GABAA receptors are intimately related. Changes in the brain concentrations of neurosteroids may play an important role in the modulation of emotional state as well as in the homeostatic mechanisms that counteract the neuronal overexcitation elicited by acute stress. Indeed, neurosteroids not only interact directly with GABAA receptors but also regulate the expression of genes that encode subunits of this receptor complex. This chapter summarizes observations from our laboratories and others, suggesting that neurosteroids and GABAergic transmission are important contributors to the changes in emotional state induced by environmental stress.

Effect of the oral administration of tryptophan-free amino acid mixtures on serum tryptophan, brain tryptophan and serotonin metabolism

TRYPTOPHAN (Try) is the only amino acid circulating in plasma which is bound to serum proteins (MCMENAMY & ONCLEY, 1958). Previous studies from our (TAGLIAMONTE et al., 1971a; GESSA et al., 1972; TAGLIAMONTE et al., 1973a; 1973b) and Curzons laboratories (KNOTT & CURZON, 1972) have shown that the concentration of free Try in serum parallels in many instances the concentration of Try in brain which, in turn, parallels the synthesis rate of brain serotonin (TAGLIAMONTE et al., 1971~; TAGLIAMONTE, et al., 1971b). On the other hand, studies on transport of amino acids from blood to brain on uptake of amino acids by brain slices GUROFF & UDENFRIEND, 1962; OLDENWRF, 1971; &HEN & LAJTHA, 1972) or synaptosomes (GRAHAME-SMITH & PARFITT, 1970) have shown that many of the aromatic acids share the same transport mechanism from blood to brain and that a high concentration of one can lower the uptake or transport of others. These considerations suggest that Try concentration in brain may depend not only on the concentration of free serumTry but also, as suggested by FERNSTROM & WURTMAN (1971), on the plasma concentration of the other amino acids competing for the same transport mechanism. In order to test this hypothesis, we studied the effect of the intragastric administration of different amino acid mixtures not containing Try, on brain Try level and serotonin metabolism. Results show that certain amino acid combinations produce a dramatic fall in Try, serotonin and 5-h ydroxyindoleacetic acid in brain and in free and total Try in serum. MATERIALS AND METHODS

The effects of inhibitors of GABAergic transmission and stress on brain and plasma allopregnanolone concentrations

This study was undertaken to investigate the relationship between a reduction in brain GABAA receptor function and the cerebro‐cortical content of 3α‐hydroxy‐5α‐pregnan‐20 one (allopregnanolone, AP), a potent endogenous positive modulator of γ‐aminobutyric acid (GABA) action at GABAA receptors, with anticonflict and anticonvulsant effects in rodents. An acute depletion of the cerebral content of GABA or an attenuation of GABAA receptor‐mediated transmission by systemic injections of isoniazid (375 mg kg−1, s.c.) or FG 7142 (15 mg kg−1, i.p.) induced a transient increase in the cerebro‐cortical and plasma concentrations of AP in handling‐habituated (not stressed) rats. Two stress paradigms, handling in naive rats and mild foot shock in handling‐habituated rats, that reduce central GABAergic tone mimicked the effects of isoniazid and FG 7142 on cortical AP content; foot shock in handling‐habituated rats, but not handling in naive animals, also increased plasma AP. Isoniazid, FG 7142, and foot shock also each increased the concentrations of the AP precursors, pregnenolone and progesterone, in both brain and plasma of handling‐habituated rats, whereas handling in naive rats increased the concentrations of these steroids only in brain. Pretreatment of handling‐habituated rats with the anxiolytic β‐carboline derivative abecarnil, a positive allosteric modulator of GABAA receptors, which per se failed to affect the AP concentration in brain or plasma, prevented the increase in brain and plasma AP induced by foot shock or isoniazid. In adrenalectomized and castrated rats foot shock or isoniazid failed to increase AP both in brain cortex and plasma. These observations indicate that inhibition of GABAergic transmission, induced by foot shock or pharmacological manipulations, results in an increase in the concentrations of AP in brain and plasma, possibly via a modulation of hypothalamic‐pituitary‐adrenal (HPA) axis. Given that AP enhances GABAA receptor function with high efficacy and potency, an increase in brain AP concentration may be important in the fine tuning of the GABA‐mediated inhibitory transmission in the central nervous system.

Developmental and age-related changes in D1-dopamine receptors and dopamine content in the rat striatum

The relationship between the postnatal development of dopaminergic (DAergic) nerve endings and the maturation of D1 DA receptors in the rat striatum was analyzed by measuring the content of DA and dihydroxyphenylacetic acid (DOPAC), two biochemical markers of DAergic nerve terminal proliferation, and the ontogenetic changes in [3H]SCH 23390 binding sites. DA-stimulated adenylate cyclase (AC) activity was also measured in order to characterize the coupling of [3H]SCH 23390 binding sites to the responses mediated by the activation of D1 DA receptors. Striatal levels of DA and DOPAC, as well as the density and affinity of [3H]SCH 23390 binding sites and DA-stimulated AC activity were also measured in senescent rats. The striatal content of DA increased slowly after birth, reaching adult levels by postnatal day 60 and remaining constant through adulthood and senescence (up to 20 months of age). The density of [3H]SCH 23390 binding sites increased 14-fold from birth to postnatal day 35, when a peak value was reached, whereas a significant decrease was observed in the striatum of aged rats. In contrast, the affinity of D1 DA receptors for [3H]SCH 23390 remained unchanged from birth through senescence. The stimulation of cyclic AMP formation induced by 100 microM DA increased 4-fold from birth to postnatal day 14, when the maximal responsiveness to DA was observed and then returned to adult levels. No significant alterations were observed in the Km values during development, whereas the stimulatory effect of 100 microM DA on AC activity was significantly decreased in senescent rats.(ABSTRACT TRUNCATED AT 250 WORDS)