Maria Luisa Barbaccia

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.

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.

Down-regulation of β-adrenergic receptors following repeated injections of desmethylimipramine: Permissive role of serotonergic axons

The injection of desmethylimipramine (DMI) twice daily for 3 weeks reduced the density of beta-adrenergic receptor recognition sites located in crude synaptic membranes prepared from the cortex and hippocampus and attenuated the stimulation of the membrane-bound adenylate cyclase by isoproterenol. Both actions were abolished if prior to treatment with desmethylimipramine the serotonergic axons were destroyed by an intraventricular injection of 5,7-dihydroxytryptamine. These results show that the down-regulation of beta-adrenergic receptors elicited by repeated injections of desmethylimipramine occurs only if the serotonergic axons are intact.

Ethanol markedly increases “GABAergic” neurosteroids in alcohol-preferring rats

Alcohol administration (1 g/kg, i.p.) increased the levels of the neurosteroids 3alpha-hydroxy-5alpha-pregnan-20-one (allopregnanolone) and 3alpha,21-dihydroxy-5alpha-pregnan-20-one (allotetrahydrodeoxycorticosterone, THDOC) in the cerebral cortex and hippocampus both in alcohol-naive Sardinian alcohol-preferring (sP) and -non-preferring (sNP) rats (two rat lines selectively bred for alcohol preference and non-preference, respectively). However, the increase reached several fold higher levels in sP than in sNP rats (6-24 vs. 2-11 fold the basal levels, respectively). Since the two neurosteroids are the most potent endogenous positive modulators of GABA(A) receptors and elicit anxiolytic and rewarding effects, while voluntary alcohol consumption produces anxiolytic and rewarding effects in sP but not in sNP rats, the results suggest that the neurosteroids may play a role in the anxiolytic and rewarding effects of alcohol in sP rats.

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.

Physiological modulation of GABAA receptor plasticity by progesterone metabolites

The possible functional relation between changes in brain and plasma concentrations of neurosteroids and the plasticity of gamma-aminobutyric acid type A (GABA(A)) receptors in the brain during pregnancy and after delivery was investigated in rats. The concentrations in the cerebral cortex and plasma of pregnenolone as well as of progesterone and its neuroactive derivatives allopregnanolone (3alpha-hydroxy-5alpha-pregnan-20-one) and allotetrahydrodeoxycorticosterone (5alpha-hydroxy-3alpha,21-diol-20-one) increased during pregnancy, peaking around day 19, before returning to control (estrus) values immediately before delivery (day 21). In the postpartum period, steroid concentrations in plasma and brain did not differ from control values. The densities of [3H]GABA, [3H]flunitrazepam, and t-[35S]butylbicyclophosphorotionate (TBPS) binding sites in the cerebral cortex also increased during pregnancy, again peaking on day 19 and returning to control values on day 21; receptor density was decreased further 2 days after delivery and again returned to control values within 7 days. These changes were accompanied by a decrease in the apparent affinity of the binding sites for the corresponding ligand on day 19 of pregnancy. The amount of the gamma2L subunit mRNA decreased progressively during pregnancy, in the cerebral cortex and hippocampus, returned to control value around the time of delivery and did not change in the postpartum period. On the contrary, the amount of alpha4 subunit mRNA was not modified during pregnancy both in the cerebral cortex and hippocampus whereas significantly increased 7 days after delivery only in the hippocampus. No significant changes were apparent for alpha1, alpha2, alpha3, beta1, beta2, beta3 and gamma2S subunit mRNAs. Administration of finasteride, a specific 5alpha-reductase inhibitor, to pregnant rats from days 12 to 18 markedly reduced the increases in the plasma and brain concentrations of allopregnanolone and allotetrahydrodeoxycorticosterone as well as prevented both the increase in the densities of [3H]flunitrazepam and [35S]TBPS binding sites and the decrease of gamma2L mRNA normally observed during pregnancy. The results demonstrate that the changes in the plasticity of GABA(A) receptors that occur in rat brain during pregnancy and after delivery are related to the physiological changes in plasma and brain concentrations of neurosteroids.

Age‐related changes of anandamide metabolism in CB1 cannabinoid receptor knockout mice: correlation with behaviour

Anandamide (N‐arachidonoylethanolamine, AEA) and 2‐arachidonoylglycerol (2‐AG) are the most active endocannabinoids at brain (CB1) cannabinoid receptors. CD1 mice lacking the CB1 receptors (‘knockout’[KO] mutants) were compared with wildtype (WT) littermates for their ability to degrade AEA through an AEA membrane transporter (AMT) and an AEA hydrolase (fatty acid amide hydrolase, FAAH). The age dependence of AMT and FAAH activity were investigated in 1‐ or 4‐month‐old WT and KO animals, and found to increase with age in KO, but not WT, mice and to be higher in the hippocampus than in the cortex of all animals. AEA and 2‐AG were detected in nmol/mg protein (µm) concentrations in both regions, though the hippocampus showed approximately twice the amount found in the cortex. In the same regions, 2‐AG failed to change across groups, while AEA was significantly decreased (≈ 30%) in hippocampus, but not in cortex, of old KO mice, when compared with young KO or age‐matched WT animals. In the open‐field test under bright light and in the lit‐dark exploration model of anxiety, young KO mice, compared with old KO, exhibited a mild anxiety‐related behaviour. In contrast, neither the increase in memory performance assessed by the object recognition test, nor the reduction of morphine withdrawal symptoms, showed age dependence in CB1 KO mice. These results suggest that invalidation of the CB1 receptor gene is associated with age‐dependent adaptive changes of endocannabinoid metabolism which appear to correlate with the waning of the anxiety‐like behaviour exhibited by young CB1 KO mice.

Stress-induced increase in brain neuroactive steroids: Antagonism by abecarnil.

Acute foot shock stress elicits a selective and time-dependent increase of neuroactive steroid (pregnenolone, progesterone, allotetrahydrodeoxycorticosterone) concentrations in rat brain cortex, accompanied by a marked increase of plasma corticosterone. The brain cortical neuroactive steroid levels peaked between 10 and 30 min poststress and returned to control values by 2 h. Abecarnil (0.3 mg/kg), i.p.), a beta-carboline derivative with anxiolytic properties, completely antagonized the effect of foot shock on brain cortical neuroactive steroids. A single administration of the anxiogenic beta-carboline FG 7142 (15 mg/kg, i.p.), in contrast, mimicked the effect of foot shock. These data support the hypothesis for the existence of a functional relationship between brain neuroactive steroid concentrations and GABAA receptor function/emotional state of the animal.

Clozapine, but not Haloperidol, Increases Brain Concentrations of Neuroactive Steroids in the Rat

The extrapyramidal side effects of typical antipsychotics, which are induced to a markedly reduced extent by clozapine, have been linked to a dysfunction of central γ-aminobutyric acid (GABA)-mediated neurotransmission. The effects of clozapine on the brain concentrations of 3α-hydroxy-5α-pregnan-20-one (allopregnanolone, AP) and 3α,21-dihydroxy-5α-pregnan-20-one (allotetrahydrodeoxycorticosterone, THDOC), two potent and endogenous positive allosteric modulators of GABA-mediated chloride current intensities at GABAA receptors, were compared with those of the typical antipsychotic haloperidol. A single administration of clozapine (1.25–20 mg/kg, IP), but not of haloperidol (0.1 or 0.5 mg/kg, IP), induced dose- and time-dependent increases in the concentrations of progesterone, AP, and THDOC in the cerebral cortex and striatum of rats. Clozapine (at 10 mg/kg, but not at lower doses) also increased the concentrations of these steroids as well as that of corticosterone in plasma in intact rats, but failed to increase the cortical concentrations of AP and THDOC in adrenalectomized-orchidectomized rats. An acute challenge with clozapine (10 mg/kg), administered 48 h after the termination of daily treatment with the same dose for 19 days, still increased the cortical concentrations of progesterone, AP, and THDOC. These results suggest that the clozapine-induced increases in neuroactive steroid concentrations in the brain may contribute to the atypical pharmacological profile of this antipsychotic drug.

2-Phenyl-imidazo[1,2-a]pyridine derivatives as ligands for peripheral benzodiazepine receptors: stimulation of neurosteroid synthesis and anticonflict action in rats

Selective activation of peripheral benzodiazepine receptors (PBRs) in adrenal cells and brain oligodendrocytes promotes steroidogenesis. Three 2‐phenyl‐imidazo[1,2‐a]pyridine derivatives (CB 34, CB 50 and CB 54) have now been investigated with regard to their selectivity for PBRs and their ability to stimulate central and peripheral steroidogenesis in rats. The three CB compounds (10−10–10−4 M) potently inhibited the binding of the PBR ligand [3H]‐PK 11195 to brain and ovary membranes in vitro, without substantially affecting [3H]‐flunitrazepam binding to central benzodiazepine receptors. These compounds (10−7–10−4 M) also had little or no marked effects on GABA‐evoked Cl− currents in voltage‐clamped Xenopus oocytes expressing human α1β2γ2S GABAA receptors. In addition, they failed to affect ligands binding to GABAB, D1/D2 dopamine, muscarinic acetylcholine, N‐methyl‐D‐aspartic acid and opiate receptors. Intraperitoneal administration of CB compounds (3–50 mg kg−1) induced a dose‐dependent increase in the concentrations of neuroactive steroids in plasma and brain. The brain concentrations of pregnenolone, progesterone, allopregnanolone and allotetrahydrodeoxycorticosterone (THDOC) showed maximal increases in 96±3, 126±14, 110±12 and 70±13% above control, respectively, 30 to 60 min after injection of CB 34 (25 mg kg−1). CB 34 also increased the brain concentrations of neuroactive steroids in adrenalectomized‐orchiectomized rats, although to a lesser extent than in sham‐operated animals, suggesting that CB compounds stimulate brain steroidogenesis independently of their effects on peripheral tissues. The increase in brain and plasma neurosteroid content induced by CB 34 was associated with a marked anticonflict effect in the Vogel test. Our results indicate that the three CB compounds tested are specific and potent agonists at peripheral benzodiazepine receptors, and that they stimulate steroidogenesis in both the brain and periphery.