Francesca Biggio

Vagus nerve stimulation increases norepinephrine concentration and the gene expression of BDNF and bFGF in the rat brain

Vagus nerve stimulation therapy, effective for treatment-resistant epilepsy, has recently been approved also for treatment-resistant depression; nevertheless, the molecular mechanism(s) underlying its therapeutic action remains unclear. Given that neurotrophic factors and monoamines could play a crucial role in the pathophysiology of depression, we tested whether vagus nerve stimulation increases the expression of brain-derived neurotrophic factor, fibroblast growth factor, and nerve growth factor as well as the concentration of norepinephrine in the rat brain. Rats were implanted with a vagus nerve stimulator device and the effects of acute stimulation were evaluated on the growth factors mRNA levels and norepinephrine concentration by ribonuclease protection assay and microdialysis, respectively. We found that acute vagus nerve stimulation increased the expression of brain-derived neurotrophic factor and fibroblast growth factor in the hippocampus and cerebral cortex, decreased the abundance of nerve growth factor mRNA in the hippocampus, and, similar to the antidepressant drug venlafaxine, increased the norepinephrine concentration in the prefrontal cortex. This study demonstrates that acute vagus nerve stimulation triggers neurochemical and molecular changes in the rat brain involving neurotransmitters and growth factors known to play a crucial role in neuronal trophism. These new findings contribute to the elucidation of the molecular mechanisms underlying the therapeutic actions of vagus nerve stimulation in both treatment-resistant depression and epilepsy.

Neurosteroids, GABAA receptors, and ethanol dependence

RationaleChanges in the expression of type A receptors for γ-aminobutyric acid (GABA) represent one of the mechanisms implicated in the development of tolerance to and dependence on ethanol. The impact of such changes on the function and pharmacological sensitivity of GABAA receptors (GABAARs) has remained unclear, however. Certain behavioral and electrophysiological actions of ethanol are mediated by an increase in the concentration of neuroactive steroids in the brain that results from stimulation of the hypothalamic–pituitary–adrenal (HPA) axis. Such steroids include potent modulators of GABAAR function.ObjectivesWe have investigated the effect of ethanol exposure and withdrawal on subunit expression and receptor function evaluated by subunit selective compounds, as well as the effects of short-term exposure to ethanol on both neurosteroid synthesis and GABAAR function, in isolated neurons and brain tissue.ResultsChronic treatment with and subsequent withdrawal from ethanol alter the expression of genes for specific GABAAR subunits in cultured rat neurons, and these changes are associated with alterations in receptor function and pharmacological sensitivity to neurosteroids, zaleplon, and flumazenil. Acute ethanol exposure increases the amount of 3α-hydroxy-5α-pregnan-20-one (allopregnanolone) in hippocampal slices by a local action independent of the activity of the HPA axis. This effect of ethanol was associated with an increased amplitude of GABAAR-mediated miniature inhibitory postsynaptic currents recorded from CA1 pyramidal neurons in such slices.ConclusionsChronic ethanol exposure elicits changes in the subunit composition of GABAARs, which, in turn, likely contribute to changes in receptor function associated with the altered pharmacological and behavioral sensitivity characteristic of ethanol tolerance and dependence. Ethanol may also modulate GABAAR function by increasing the de novo synthesis of neurosteroids in the brain in a manner independent of the HPA axis. This latter mechanism may play an important role in the central effects of ethanol.

Changes in GABAA receptor γ2 subunit gene expression induced by long-term administration of oral contraceptives in rats

The effects of oral contraceptives (OCs) on neurosteroid concentrations were evaluated in female rats and women. In rats, ethynylestradiol and levonorgestrel (0.030 and 0.125 mg, respectively, subcutaneously) administered daily for 6 weeks reduced the concentrations of pregnenolone (-41%) progesterone (-74%) and allopregnanolone (-79%) in the cerebral cortex; the plasma concentrations of these steroids were also reduced but by smaller extents. OC administration for 3 months also reduced the serum concentrations of pregnenolone, progesterone and allopregnanolone in women. Chronic administration of OCs in rats increased the abundance of gamma-aminobutyric acid type A (GABA(A)) receptor gamma 2L and gamma 2S subunit mRNAs and the relative protein in the cerebral cortex, while the amounts of various alpha and beta subunit mRNAs were unaffected. Ovariectomy did not modify the effect of OCs administration on the concentrations of neurosteroids in the rat cerebral cortex (but not in the plasma) as well as on the GABA(A) receptor gene expression, suggesting a direct effect of OCs in brain. Finally, rats treated with OCs exhibited an anxiety-like behavior in the elevated plus-maze test. These results indicate that long-term treatment with OCs induced a persistent reduction in the concentrations of pregnenolone, progesterone and its GABA(A) receptor-active metabolite, allopregnanolone, both in rats and women. In rats this effect was associated with a plastic adaptation of GABA(A) receptor gene expression in the rat cerebral cortex.

Increase in expression of the GABA(A) receptor alpha(4) subunit gene induced by withdrawal of, but not by long-term treatment with, benzodiazepine full or partial agonists.

The effects of long-term exposure to, and subsequent withdrawal of, diazepam or imidazenil (full and partial agonists of the benzodiazepine receptor, respectively) on the abundance of GABA(A) receptor subunit mRNAs and peptides were investigated in rat cerebellar granule cells in culture. Exposure of cells to 10 microM diazepam for 5 days significantly reduced the amounts of alpha(1) and gamma(2) subunit mRNAs, and had no effect on the amount of alpha(4) mRNA. These effects were accompanied by a decrease in the levels of alpha(1) and gamma(2) protein and by a reduction in the efficacy of diazepam with regard to potentiation of GABA-evoked Cl- current. Similar long-term treatment with 10 microM imidazenil significantly reduced the abundance of only the gamma(2)S subunit mRNA and had no effect on GABA(A) receptor function. Withdrawal of diazepam or imidazenil induced a marked increase in the amount of alpha(4) mRNA; withdrawal of imidazenil also reduced the amounts of alpha(1) and gamma(2) mRNAs. In addition, withdrawal of diazepam or imidazenil was associated with a reduced ability of diazepam to potentiate GABA action. These data give new insights into the different molecular events related to GABA(A) receptor gene expression and function produced by chronic treatment and withdrawal of benzodiazepines with full or partial agonist properties.

Changes in GABAA receptor gene expression induced by withdrawal of, but not by long-term exposure to, zaleplon or zolpidem

The effects of long-term treatment with and subsequent withdrawal of the two hypnotic drugs zaleplon and zolpidem on the abundance of gamma-aminobutyric acid type A (GABA(A)) receptor subunit mRNAs in cultured rat cerebellar granule cells were investigated. Incubation of neurons with either drug at a concentration of 10 microM for 5 days did not significantly affect the amounts of mRNAs encoding the alpha(1), alpha(4), beta(1), beta(2), beta(3), gamma(2)L, or gamma(2)S subunits. As expected, similar treatment with the nonselective benzodiazepine diazepam resulted in a decrease in the abundance of alpha(1), beta(2), gamma(2)L, and gamma(2)S subunit mRNAs as well as an increase in that of the beta(1) subunit mRNA. Withdrawal of zaleplon or zolpidem, like that of diazepam, induced a marked increase in the amount of the alpha(4) subunit mRNA. In addition, discontinuation of treatment with either hypnotic drug resulted in a decrease in the amounts of alpha(1), beta(2), gamma(2)L, and gamma(2)S subunit mRNAs as well as an increase in that of the beta(1) subunit mRNA. These effects of zaleplon and zolpidem on GABA(A) receptor gene expression are consistent with the reduced tolerance liability of these drugs, compared with that of diazepam, as well as with their ability to induce both physical dependence and withdrawal syndrome.

Molecular mechanisms of tolerance to and withdrawal of GABAA receptor modulators

Here, we summarize recent data pertaining to the effects of GABA(A) receptor modulators on the receptor gene expression in order to elucidate the molecular mechanisms behind tolerance and dependence induced by these drugs. Drug selectivity and intrinsic activity seems to be important to evidence at the molecular level the GABA(A) receptor tolerance. On the contrary, we suggested that all drug tested are equally potentially prone to induce dependence. Our results demonstrate that long-lasting exposure of GABA(A) receptors to endogenous steroids, benzodiazepines and ethanol, as well as their withdrawal, induce marked effects on receptor structure and function. These results suggest the possible synergic action between endogenous steroids and these drugs in modulating the functional activity of specific neuronal populations. We report here that endogenous steroids may play a crucial role in the action of ethanol on dopaminergic neurons.

Distinct patterns of expression and regulation of GABAA receptors containing the δ subunit in cerebellar granule and hippocampal neurons

Neuronal plasticity is achieved by regulation of the expression of genes for neurotransmitter receptors such as the type A receptor (GABAAR) for γ‐aminobutyric acid. We now show that two different rat neuronal populations in culture manifest distinct patterns of GABAAR plasticity in response to identical stimuli. Whereas prolonged exposure to ethanol had no effect on expression of the δ subunit of GABAARs at the mRNA or protein level in cerebellar granule neurons, it increased the abundance of δ subunit mRNA and protein in hippocampal neurons. Subsequent ethanol withdrawal transiently down‐regulated δ subunit expression in cerebellar granule neurons and gradually normalized that in hippocampal neurons. These effects of ethanol exposure and withdrawal were accompanied by corresponding functional changes in GABAARs. GABAARs containing the δ subunit were also distributed differentially in the cerebellar and hippocampal neurons. These findings reveal complex and distinct mechanisms of regulation of the expression of GABAARs that contain the δ subunit in different neuronal types.

Voluntary ethanol consumption induced by social isolation reverses the increase of α 4 /δ GABA A receptor gene expression and function in the hippocampus of C57BL/6J mice

Post-weaning social isolation (SI) is a model of prolonged mild stress characterized by behavioral and neurochemical alterations. We used SI in C57BL/6J mice to investigate the effects of ethanol (EtOH) in the free-choice drinking paradigm on gene expression and function of γ-aminobutyric acid type A receptors (GABAARs) and the role of neuroactive steroids in the actions of EtOH in the hippocampus. SI stress induced a marked reduction in hippocampal 3α-hydroxy-5α-pregnan-20-one (3α,5α-TH PROG) and was associated with molecular and functional changes of the GABAAR. The gene expression of the α4 and δ subunits was increased in the hippocampus of SI C57BL/6J mice; the expression of the γ2 subunit was decreased whereas that of the α1 did not change. Patch-clamp recordings in dentate gyrus (DG) granule cells obtained from SI C57BL/6J mice revealed a greater enhancement of tonic currents induced by α-(4,5,6,7-tetrahydroisoxazolo[5,4-c] pyridin-3-ol (THIP) compared to that in control C57BL/6J mice. These neurochemical, molecular and functional changes observed in SI C57BL/6J mice were associated with an increased EtOH intake and EtOH preference. Nevertheless, the increase in EtOH consumption did not restore the reduction in hippocampal 3α,5α-TH PROG induced by SI. EtOH self-administration blocked the changes in gene expression of the α4 subunit but not those of the δ and γ2 subunits induced by SI. In addition, EtOH self-administration did not block the SI-induced changes in GABAAR-mediated tonic inhibition in hippocampal granule cells but increased the frequency of basal GABAergic sIPSCs in DG granule cells. We conclude that self-administration of EtOH selectively abolishes the increase of α4 subunit but not other neurochemical, molecular, and functional modifications induced by SI prolonged mild stress.

Maternal separation attenuates the effect of adolescent social isolation on HPA axis responsiveness in adult rats

Adverse early life experiences that occur during childhood and adolescence can have negative impacts on behavior later in life. The main goal of our work was to assess how the association between stressful experiences during neonatal and adolescent periods may influence stress responsiveness and brain plasticity in adult rats. Stressful experiences included maternal separation and social isolation at weaning. Three hours of separation from the pups (3-14 PND) significantly increased frequencies of maternal arched-back nursing and licking-grooming across the first two weeks postpartum. Separation also induced a long-lasting increase in dams blood levels of corticosterone. Maternal separation did not modify brain and plasma allopregnanolone and corticosterone levels in adult offspring, but they demonstrate partial recovery from the reduction induced by social isolation during adolescence. Moreover, the enhancement of corticosterone and allopregnanolone levels induced by foot shock stress in socially isolated animals that were subjected to maternal separation was markedly reduced with respect to that observed in animals that were just socially isolated. All experimental groups showed a significant reduction of BDNF and Arc protein expression in the hippocampus. However, the reduction of BDNF observed in animals that were maternally separated and subjected to social isolation was less significantly pronounced than in animals that were just socially isolated. The results sustained the mismatch hypothesis stating that aversive experiences early in life trigger adaptive processes, thereby rendering an individual to be better adapted to aversive challenges later in life.

Plastic neuronal changes in GABAA receptor gene expression induced by progesterone metabolites: In vitro molecular and functional studies

Expression of specific gamma-aminobutyric acid type A (GABA(A)) receptor subunit genes in neurons is affected by endogenous modulators of receptor function such as neuroactive steroids. Neuroactive steroids such as the progesterone metabolite allopregnanolone might thus exert differential effects on GABA(A) receptor plasticity in neurons, likely accounting for some of the physiological actions of these compounds. Here we summarise experimental data obtained in vitro that show how fluctuations in the concentration of progesterone regulate both the expression and function of GABA(A) receptors. The data described in this manuscript are in agreement with the notion that fluctuations in the concentrations of progesterone and its metabolite allopregnanolone play a major role in the temporal pattern of expression of various subunits of the GABA(A) receptor. Thus, rapid and long-lasting increases or decreases in the concentrations of these steroid derivatives observed in physiological and patho-physiological conditions, or induced by pharmacological treatments, might elicit selective changes in GABA(A) receptor gene expression and function in specific neuronal populations. Given both the importance of GABA(A) receptors in the regulation of neuronal excitability and the large fluctuations in the plasma and brain concentrations of neuroactive steroids associated with physiological conditions and the response to environmental stimuli, these compounds are likely among the most relevant endogenous modulators that could affect emotional and affective behaviors.