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
GABAA-receptor plasticity during long-term exposure to and withdrawal from progesterone
International Review of Neurobiology, 2001
The subunit composition of native gamma-aminobutyric acid type A (GABAA) receptors is an importan... more The subunit composition of native gamma-aminobutyric acid type A (GABAA) receptors is an important determinant of the role of these receptors in the physiological and pharmacological modulation of neuronal excitability and associated behavior. GABAA receptors containing the alpha 1 subunit mediate the sedative-hypnotic effects of benzodiazepines (Rudolph et al., 1999; McKernan et al., 2000), whereas the anxiolytic effects of these drugs are mediated by receptors that contain the alpha 2 subunit (Löw et al., 2000). In contrast, GABAA receptors containing the alpha 4 or alpha 6 subunits are insensitive to benzodiazepines (Barnard et al., 1998). Characterization of the functions of GABAA-receptors thus requires an understanding of the mechanisms by which the receptor subunit composition is regulated. The expression of specific GABAA-receptor subunit genes in neurons is affected by endogenous and pharmacological modulators of receptor function. The expression of GABAA-receptor subunit genes is thus regulated by neuroactive steroids both in vitro and in vivo. Such regulation occurs both during physiological conditions, such as pregnancy, and during pharmacologically induced conditions, such as pseudo-pregnancy and long-term treatment with steroid derivatives or anxiolytic-hypnotic drugs. Here, we summarize results obtained by our laboratory and by other groups pertaining to the effects of long-term exposure to, and subsequent withdrawal from, progesterone and its metabolite 3 alpha,5 alpha-tetrahydroprogesterone on both the expression of GABAA-receptor subunits and GABAA-receptor function.
... 25 Kaplan, DR, Hempstead, BL, Martin-Zanca, D., Chao, MV and Parada, LF, The trk ... 43 Riva,... more ... 25 Kaplan, DR, Hempstead, BL, Martin-Zanca, D., Chao, MV and Parada, LF, The trk ... 43 Riva, MA, Gale, K. and Mocchetti, I., Basic fibroblast growth factor mRNA increases in specific brain ... 52 Walicke, P., Cowan, WM, Ueno, N., Baird, A. and Guillemin, R. Fibroblast growth factor ...
Both benzodiazepines and ␥-hydroxybutyric acid (GHB) are used to treat alcohol withdrawal syndrom... more Both benzodiazepines and ␥-hydroxybutyric acid (GHB) are used to treat alcohol withdrawal syndrome. The molecular basis for this therapeutic efficacy was investigated with primary cultures of rat cerebellar granule cells. Long-term exposure of these cells to ethanol (100 mM, 5 days) reduced the abundance of mRNAs encoding the ␥ 2 L and ␥ 2 S subunits of the GABA type A receptor (Ϫ32 and Ϫ23%, respectively) but failed to affect that of ␣ 1 , ␣ 4 , or ␣ 6 subunit mRNAs. Subsequent ethanol withdrawal resulted in decreases in the amounts of ␣ 1 (Ϫ29%), ␣ 6 (Ϫ27%), ␥ 2 L (Ϫ64%), and ␥ 2 S (Ϫ76%),subunit mRNAs that were maximal after 6 to 12 h. In contrast, 3 h after ethanol withdrawal, the abundance of the ␣ 4 subunit mRNA was increased by 46%. Ethanol withdrawal did not affect neuronal This study was supported by Ministero dell'Istruzione dell'Universita e della Ricerca grant 2001055774.
The possible functional relation between changes in brain and plasma concentrations of neurostero... more The possible functional relation between changes in brain and plasma concentrations of neurosteroids and the plasticity of Ž. g-aminobutyric acid type A GABA receptors in the brain during pregnancy and after delivery was investigated in rats. The
Here we summarize recent data from our laboratory pertaining to the effects of fluctuations in th... more Here we summarize recent data from our laboratory pertaining to the effects of fluctuations in the brain concentrations of the progesterone (PROG) metabolite allopregnanolone (3a,5a-TH PROG) on the expression and function of g-aminobutyric acid type A (GABA) receptors. The effects of long-term exposure to progesterone and of its sudden withdrawal on the activity of GABA receptors A A and on the abundance of receptor subunit mRNAs were examined in cultured rat cerebellar granule cells and cortical neurons. The effects of a persistent reduction in the brain concentration of 3a,5a-TH PROG on GABA receptor function and gene expression were examined A in vivo in rats subjected to long-term administration of oral contraceptives. Our results demonstrate that long-lasting changes in the exposure of GABA receptors to this PROG metabolite induce marked effects on receptor structure and function. These effects of A 3a,5a-TH PROG appear to be mediated through modulation of GABA receptor signaling mechanisms that control the expression of A specific receptor subunit genes. Furthermore, the specific outcomes of such signaling appear to differ among neurons derived from different regions of the brain. Neuroactive steroids such as 3a,5a-TH PROG might thus exert differential actions on GABA receptor A plasticity in distinct neuronal cell populations, likely accounting for some of the physiological effects induced by these compounds.
The effects of ganaxolone, a synthetic analog of the endogenous neuroactive steroid allopregnanol... more The effects of ganaxolone, a synthetic analog of the endogenous neuroactive steroid allopregnanolone, on the function and expression of GABA(A) receptors were determined. Electrophysiological recordings demonstrated that ganaxolone potentiated with a potency and efficacy similar to those of allopregnanolone the Cl- currents evoked by GABA at recombinant human GABA(A) receptors (comprising alpha1beta2gamma2L or alpha2beta2gamma2L subunit assemblies) expressed in Xenopus oocytes. Exposure of cultured rat cerebellar granule cells to 1 microM ganaxolone for 5 days had no effect on the abundance of mRNAs encoding the alpha1, alpha2, alpha3, alpha4, alpha5, gamma2L, or gamma2S subunits of the GABA(A) receptor. Withdrawal of ganaxolone after such long-term treatment, however, induced an increase in the abundance of alpha2, alpha4, and alpha5 subunit mRNAs and a decrease in the amounts of alpha1, gamma2L, and gamma2S subunit mRNAs. These changes were maximal 3 to 6 h after drug withdrawal and were reversible, being no longer apparent after 24 h. These results suggest that long-term exposure of cerebellar granule cells to ganaxolone does not affect the sensitivity of the GABA(A) receptor to several positive modulators. Nevertheless, the reduction in the amounts of the alpha1 and gamma2 subunit mRNAs together with the increase in the abundance of the alpha4 subunit mRNA induced by abrupt discontinuation of long-term treatment with ganaxolone suggest that withdrawal of this drug might result in a reduced response to classic benzodiazepines.
Neuronal plasticity is achieved by regulation of the expression of genes for neurotransmitter rec... more Neuronal plasticity is achieved by regulation of the expression of genes for neurotransmitter receptors such as the type A receptor (GABA(A)R) for gamma-aminobutyric acid. We now show that two different rat neuronal populations in culture manifest distinct patterns of GABA(A)R plasticity in response to identical stimuli. Whereas prolonged exposure to ethanol had no effect on expression of the delta subunit of GABA(A)Rs at the mRNA or protein level in cerebellar granule neurons, it increased the abundance of delta subunit mRNA and protein in hippocampal neurons. Subsequent ethanol withdrawal transiently down-regulated delta 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 GABA(A)Rs. GABA(A)Rs containing the delta subunit were also distributed differentially in the cerebellar and hippocampal neurons. These findings reveal complex and distinct mechanisms of regulation of the expression of GABA(A)Rs that contain the delta subunit in different neuronal types.
Expression of specific gamma-aminobutyric acid type A (GABA(A)) receptor subunit genes in neurons... more 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.
The discovery that facilitation or inhibition of γ-aminobutyric acid (GABA)-mediated neurotransmi... more The discovery that facilitation or inhibition of γ-aminobutyric acid (GABA)-mediated neurotransmission results in anxiolytic versus anxiogenic, hypnotic versus somnolytic, and anticonvulsant versus convulsant effects, respectively, provided important insights concerning the physiology and pharmacology of central GABAergic transmission. GABAA receptors (GABAARs) are ligand-gated Cl− channels and mediate fast inhibitory transmission in the mammalian central nervous system. They are heteromeric complexes of five subunits belonging to various classes, with different subunit compositions conferring different functions as well as sensitivities to GABA, various drugs, and endogenous modulators. The subunit composition of native GABAAR plays a crucial role in defining their
functional properties in the physiological and pharmacological modulation of neuronal excitability. It has been suggested that changes in the gene expression of GABAARs represent one of the mechanisms implicated in the development of tolerance to and dependence on GABAergic drugs. Moreover, expression of specific GABAAR subunit genes is affected by endogenous modulators of receptor function such as neuroactive steroids and may contribute to the pathophysiology of anxiety disorders. This effect of steroids appears to be mediated through modulation of GABAAR signaling mechanisms that control the expression of specific receptor subunit genes. Neuroactive steroids might thus exert differential effects on GABAAR plasticity in distinct neuronal cell populations. In order to gain more information about the molecular mechanisms by which GABAergic drugs can produce plastic changes of the GABAARs, we have used a simple experimental model of neurons in culture that express most of the GABAAR subunits that are assembled in functional receptors. Here we summarize experimental data obtained with these cells in culture that show how chronic treatments with different positive modulators regulate both the expression and function of GABAARs. The understanding of these molecular mechanisms not only will provide new insights about the pathophysiology of those diseases, such as anxiety, in which GABAergic transmission plays a pivotal role, but will prove to be important to develop and design new anxiolytic, hypnotic and anticonvulsant selective drugs devoid of side effects.
Vagus nerve stimulation therapy, effective for treatment-resistant epilepsy, has recently been ap... more 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.
Vagus nerve stimulation (VNS) is used to treat pharmacotherapy-resistant epilepsy and depression.... more Vagus nerve stimulation (VNS) is used to treat pharmacotherapy-resistant epilepsy and depression. However, the mechanisms underlying the therapeutic efficacy of VNS remain unclear. We examined the effects of VNS on hippocampal neuronal plasticity and behaviour in rats. Cell proliferation in the hippocampus of rats subjected to acute (3 h) or chronic (1 month) VNS was examined by injection of bromodeoxyuridine (BrdU) and immunohistochemistry. Expression of doublecortin (DCX) and brain-derived neurotrophic factor (BDNF) was evaluated by immunofluorescence staining. The dendritic morphology of DCX+ neurons was measured by Sholl analysis. Our results show that acute VNS induced an increase in the number of BrdU+ cells in the dentate gyrus that was apparent 24 h and 3 wk after treatment. It also induced long-lasting increases in the amount of DCX immunoreactivity and in the number of DCX+ neurons. Neither the number of BrdU+ cells nor the amount of DCX immunoreactivity was increased 3 wk after the cessation of chronic VNS. Chronic VNS induced long-lasting increases in the amount of BDNF immunoreactivity and the number of BDNF+ cells as well as in the dendritic complexity of DCX+ neurons in the hippocampus. In contrast to chronic imipramine treatment, chronic VNS had no effect on the behaviour of rats in the forced swim or elevated plus-maze tests. Both chronic and acute VNS induced persistent changes in hippocampal neurons that may play a key role in the therapeutic efficacy of VNS. However, these changes were not associated with evident behavioural alterations characteristic of an antidepressant or anxiolytic action.
The actions of ethanol on gamma-aminobutyric acid type A (GABA(A)) receptors are still highly con... more The actions of ethanol on gamma-aminobutyric acid type A (GABA(A)) receptors are still highly controversial issues but it appears that some of its pharmacological effects may depend on receptor subunit composition. Prolonged ethanol exposure produces tolerance and dependence and its withdrawal alters GABA(A) receptor subunit gene expression and function. Whereas benzodiazepines are clinically effective in ameliorating ethanol withdrawal symptoms, work in our laboratory showed that benzodiazepines also prevent, in vitro, some of the ethanol withdrawal-induced molecular and functional changes of the GABA(A) receptors. In the present work, we investigated the effects, on such changes, of the benzodiazepine receptor antagonist flumazenil that can positively modulate alpha(4)-containing receptors. We here report that flumazenil prevented both the ethanol withdrawal-induced up-regulation of the alpha(4)-subunit and the increase in its own modulatory action. In contrast, flumazenil did not inhibit ethanol withdrawal-induced decrease in alpha(1)- and delta-subunit expression as well as the corresponding decrease in the modulatory action on GABA(A) receptor function of both the alpha(1)-selective ligand zaleplon and the delta-containing receptor preferentially acting steroid allopregnanolone. These observations are the first molecular and functional evidence that show a selective inhibition by flumazenil of the up-regulation of alpha(4)-subunit expression elicited by ethanol withdrawal.
Expression of specific gamma-aminobutyric acid type A (GABA(A)) receptor subunit genes in neurons... more 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. This effect of steroids appears to be mediated through modulation of GABA(A) receptor signalling mechanisms that control the expression of specific receptor subunit genes. Furthermore, the specific outcomes of such signalling appear to differ among neurons in different regions of the brain. Neuroactive steroids such as the progesterone metabolite allopregnanolone might thus exert differential effects on GABA(A) receptor plasticity in distinct neuronal cell populations, likely accounting for some of the physiological actions of these compounds. Here we summarise experimental data obtained both in vivo and in vitro that show how fluctuations in the concentration of allopregnanolone regulate both the expression and function of GABA(A) receptors and consequently affect behaviour. Such regulation is operative both during physiological conditions such as pregnancy and lactation as well as in pharmacologically induced states such as pseudopregnancy and long-term treatment with steroid derivatives or anxiolytic-hypnotic drugs. Accordingly, long-lasting exposure of GABA(A) receptors to ethanol, as well as its withdrawal, induces marked effects on receptor structure and function. These results suggest the possible synergic action between endogenous steroids and ethanol in modulating the functional activity of specific neuronal populations.
The gamma-aminobutyric acid type A (GABA(A)) receptor is an important pharmacological target of e... more The gamma-aminobutyric acid type A (GABA(A)) receptor is an important pharmacological target of ethanol. The effect of ethanol withdrawal on the expression of the alpha(2) subunit of this receptor was examined with rat cerebellar granule cells in primary culture. Long-term exposure of these cells to ethanol (100 mM, 5 days) did not affect the abundance of the mRNA for the alpha(2) subunit, as revealed by an RNase protection assay. In contrast, subsequent ethanol withdrawal for 3 h induced a marked increase in the amount of this mRNA (2.6-fold) as well as in that of the encoded polypeptide (2.2-fold), the latter revealed by immunoblot analysis. Exposure of the cells to gamma-hydroxybutyric acid (100 mM) during ethanol withdrawal prevented the increase in the amounts of both the alpha(2) mRNA and polypeptide, whereas similar treatment with diazepam (10 microM) blocked the increase in the abundance of the alpha(2) polypeptide but not that in the amount of the alpha(2) mRNA. The effect of gamma-hydroxybutyric acid was not blocked by the competitive GABA(B) receptor antagonist SCH 50911(10 microM). Given that the alpha(2) subunit of the GABA(A) receptor mediates the anxiolytic action of benzodiazepines, its up-regulation during discontinuation of long-term ethanol exposure might be relevant to the therapeutic efficacy of these drugs in the treatment of anxiety associated with ethanol withdrawal.
Changes in the expression of type A receptors for gamma-aminobutyric acid (GABA) represent one of... more Changes in the expression of type A receptors for gamma-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. We 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. Chronic treatment with and subsequent withdrawal from ethanol alter the expression of g...
Expression of specific gamma-aminobutyric acid type A (GABA(A)) receptor subunit genes in neurons... more 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. This effect of steroids appears to be mediated through modulation of GABA(A) receptor signalling mechanisms that control the expression of specific receptor subunit genes. Furthermore, the specific outcomes of such signalling appear to differ among neurons in different regions of the brain. Neuroactive steroids such as the progesterone metabolite allopregnanolone might thus exert differential effects on GABA(A) receptor plasticity in distinct neuronal cell populations, likely accounting for some of the physiological actions of these compounds. Here we summarise experimental data obtained both in vivo and in vitro that show how fluctuations in the concentration of allopregnanolone regulate both the expression and function of GABA(A) receptors and consequently affect behaviour. Such regulation is opera...
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
GABAA-receptor plasticity during long-term exposure to and withdrawal from progesterone
International Review of Neurobiology, 2001
The subunit composition of native gamma-aminobutyric acid type A (GABAA) receptors is an importan... more The subunit composition of native gamma-aminobutyric acid type A (GABAA) receptors is an important determinant of the role of these receptors in the physiological and pharmacological modulation of neuronal excitability and associated behavior. GABAA receptors containing the alpha 1 subunit mediate the sedative-hypnotic effects of benzodiazepines (Rudolph et al., 1999; McKernan et al., 2000), whereas the anxiolytic effects of these drugs are mediated by receptors that contain the alpha 2 subunit (Löw et al., 2000). In contrast, GABAA receptors containing the alpha 4 or alpha 6 subunits are insensitive to benzodiazepines (Barnard et al., 1998). Characterization of the functions of GABAA-receptors thus requires an understanding of the mechanisms by which the receptor subunit composition is regulated. The expression of specific GABAA-receptor subunit genes in neurons is affected by endogenous and pharmacological modulators of receptor function. The expression of GABAA-receptor subunit genes is thus regulated by neuroactive steroids both in vitro and in vivo. Such regulation occurs both during physiological conditions, such as pregnancy, and during pharmacologically induced conditions, such as pseudo-pregnancy and long-term treatment with steroid derivatives or anxiolytic-hypnotic drugs. Here, we summarize results obtained by our laboratory and by other groups pertaining to the effects of long-term exposure to, and subsequent withdrawal from, progesterone and its metabolite 3 alpha,5 alpha-tetrahydroprogesterone on both the expression of GABAA-receptor subunits and GABAA-receptor function.
... 25 Kaplan, DR, Hempstead, BL, Martin-Zanca, D., Chao, MV and Parada, LF, The trk ... 43 Riva,... more ... 25 Kaplan, DR, Hempstead, BL, Martin-Zanca, D., Chao, MV and Parada, LF, The trk ... 43 Riva, MA, Gale, K. and Mocchetti, I., Basic fibroblast growth factor mRNA increases in specific brain ... 52 Walicke, P., Cowan, WM, Ueno, N., Baird, A. and Guillemin, R. Fibroblast growth factor ...
Both benzodiazepines and ␥-hydroxybutyric acid (GHB) are used to treat alcohol withdrawal syndrom... more Both benzodiazepines and ␥-hydroxybutyric acid (GHB) are used to treat alcohol withdrawal syndrome. The molecular basis for this therapeutic efficacy was investigated with primary cultures of rat cerebellar granule cells. Long-term exposure of these cells to ethanol (100 mM, 5 days) reduced the abundance of mRNAs encoding the ␥ 2 L and ␥ 2 S subunits of the GABA type A receptor (Ϫ32 and Ϫ23%, respectively) but failed to affect that of ␣ 1 , ␣ 4 , or ␣ 6 subunit mRNAs. Subsequent ethanol withdrawal resulted in decreases in the amounts of ␣ 1 (Ϫ29%), ␣ 6 (Ϫ27%), ␥ 2 L (Ϫ64%), and ␥ 2 S (Ϫ76%),subunit mRNAs that were maximal after 6 to 12 h. In contrast, 3 h after ethanol withdrawal, the abundance of the ␣ 4 subunit mRNA was increased by 46%. Ethanol withdrawal did not affect neuronal This study was supported by Ministero dell'Istruzione dell'Universita e della Ricerca grant 2001055774.
The possible functional relation between changes in brain and plasma concentrations of neurostero... more The possible functional relation between changes in brain and plasma concentrations of neurosteroids and the plasticity of Ž. g-aminobutyric acid type A GABA receptors in the brain during pregnancy and after delivery was investigated in rats. The
Here we summarize recent data from our laboratory pertaining to the effects of fluctuations in th... more Here we summarize recent data from our laboratory pertaining to the effects of fluctuations in the brain concentrations of the progesterone (PROG) metabolite allopregnanolone (3a,5a-TH PROG) on the expression and function of g-aminobutyric acid type A (GABA) receptors. The effects of long-term exposure to progesterone and of its sudden withdrawal on the activity of GABA receptors A A and on the abundance of receptor subunit mRNAs were examined in cultured rat cerebellar granule cells and cortical neurons. The effects of a persistent reduction in the brain concentration of 3a,5a-TH PROG on GABA receptor function and gene expression were examined A in vivo in rats subjected to long-term administration of oral contraceptives. Our results demonstrate that long-lasting changes in the exposure of GABA receptors to this PROG metabolite induce marked effects on receptor structure and function. These effects of A 3a,5a-TH PROG appear to be mediated through modulation of GABA receptor signaling mechanisms that control the expression of A specific receptor subunit genes. Furthermore, the specific outcomes of such signaling appear to differ among neurons derived from different regions of the brain. Neuroactive steroids such as 3a,5a-TH PROG might thus exert differential actions on GABA receptor A plasticity in distinct neuronal cell populations, likely accounting for some of the physiological effects induced by these compounds.
The effects of ganaxolone, a synthetic analog of the endogenous neuroactive steroid allopregnanol... more The effects of ganaxolone, a synthetic analog of the endogenous neuroactive steroid allopregnanolone, on the function and expression of GABA(A) receptors were determined. Electrophysiological recordings demonstrated that ganaxolone potentiated with a potency and efficacy similar to those of allopregnanolone the Cl- currents evoked by GABA at recombinant human GABA(A) receptors (comprising alpha1beta2gamma2L or alpha2beta2gamma2L subunit assemblies) expressed in Xenopus oocytes. Exposure of cultured rat cerebellar granule cells to 1 microM ganaxolone for 5 days had no effect on the abundance of mRNAs encoding the alpha1, alpha2, alpha3, alpha4, alpha5, gamma2L, or gamma2S subunits of the GABA(A) receptor. Withdrawal of ganaxolone after such long-term treatment, however, induced an increase in the abundance of alpha2, alpha4, and alpha5 subunit mRNAs and a decrease in the amounts of alpha1, gamma2L, and gamma2S subunit mRNAs. These changes were maximal 3 to 6 h after drug withdrawal and were reversible, being no longer apparent after 24 h. These results suggest that long-term exposure of cerebellar granule cells to ganaxolone does not affect the sensitivity of the GABA(A) receptor to several positive modulators. Nevertheless, the reduction in the amounts of the alpha1 and gamma2 subunit mRNAs together with the increase in the abundance of the alpha4 subunit mRNA induced by abrupt discontinuation of long-term treatment with ganaxolone suggest that withdrawal of this drug might result in a reduced response to classic benzodiazepines.
Neuronal plasticity is achieved by regulation of the expression of genes for neurotransmitter rec... more Neuronal plasticity is achieved by regulation of the expression of genes for neurotransmitter receptors such as the type A receptor (GABA(A)R) for gamma-aminobutyric acid. We now show that two different rat neuronal populations in culture manifest distinct patterns of GABA(A)R plasticity in response to identical stimuli. Whereas prolonged exposure to ethanol had no effect on expression of the delta subunit of GABA(A)Rs at the mRNA or protein level in cerebellar granule neurons, it increased the abundance of delta subunit mRNA and protein in hippocampal neurons. Subsequent ethanol withdrawal transiently down-regulated delta 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 GABA(A)Rs. GABA(A)Rs containing the delta subunit were also distributed differentially in the cerebellar and hippocampal neurons. These findings reveal complex and distinct mechanisms of regulation of the expression of GABA(A)Rs that contain the delta subunit in different neuronal types.
Expression of specific gamma-aminobutyric acid type A (GABA(A)) receptor subunit genes in neurons... more 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.
The discovery that facilitation or inhibition of γ-aminobutyric acid (GABA)-mediated neurotransmi... more The discovery that facilitation or inhibition of γ-aminobutyric acid (GABA)-mediated neurotransmission results in anxiolytic versus anxiogenic, hypnotic versus somnolytic, and anticonvulsant versus convulsant effects, respectively, provided important insights concerning the physiology and pharmacology of central GABAergic transmission. GABAA receptors (GABAARs) are ligand-gated Cl− channels and mediate fast inhibitory transmission in the mammalian central nervous system. They are heteromeric complexes of five subunits belonging to various classes, with different subunit compositions conferring different functions as well as sensitivities to GABA, various drugs, and endogenous modulators. The subunit composition of native GABAAR plays a crucial role in defining their
functional properties in the physiological and pharmacological modulation of neuronal excitability. It has been suggested that changes in the gene expression of GABAARs represent one of the mechanisms implicated in the development of tolerance to and dependence on GABAergic drugs. Moreover, expression of specific GABAAR subunit genes is affected by endogenous modulators of receptor function such as neuroactive steroids and may contribute to the pathophysiology of anxiety disorders. This effect of steroids appears to be mediated through modulation of GABAAR signaling mechanisms that control the expression of specific receptor subunit genes. Neuroactive steroids might thus exert differential effects on GABAAR plasticity in distinct neuronal cell populations. In order to gain more information about the molecular mechanisms by which GABAergic drugs can produce plastic changes of the GABAARs, we have used a simple experimental model of neurons in culture that express most of the GABAAR subunits that are assembled in functional receptors. Here we summarize experimental data obtained with these cells in culture that show how chronic treatments with different positive modulators regulate both the expression and function of GABAARs. The understanding of these molecular mechanisms not only will provide new insights about the pathophysiology of those diseases, such as anxiety, in which GABAergic transmission plays a pivotal role, but will prove to be important to develop and design new anxiolytic, hypnotic and anticonvulsant selective drugs devoid of side effects.
Vagus nerve stimulation therapy, effective for treatment-resistant epilepsy, has recently been ap... more 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.
Vagus nerve stimulation (VNS) is used to treat pharmacotherapy-resistant epilepsy and depression.... more Vagus nerve stimulation (VNS) is used to treat pharmacotherapy-resistant epilepsy and depression. However, the mechanisms underlying the therapeutic efficacy of VNS remain unclear. We examined the effects of VNS on hippocampal neuronal plasticity and behaviour in rats. Cell proliferation in the hippocampus of rats subjected to acute (3 h) or chronic (1 month) VNS was examined by injection of bromodeoxyuridine (BrdU) and immunohistochemistry. Expression of doublecortin (DCX) and brain-derived neurotrophic factor (BDNF) was evaluated by immunofluorescence staining. The dendritic morphology of DCX+ neurons was measured by Sholl analysis. Our results show that acute VNS induced an increase in the number of BrdU+ cells in the dentate gyrus that was apparent 24 h and 3 wk after treatment. It also induced long-lasting increases in the amount of DCX immunoreactivity and in the number of DCX+ neurons. Neither the number of BrdU+ cells nor the amount of DCX immunoreactivity was increased 3 wk after the cessation of chronic VNS. Chronic VNS induced long-lasting increases in the amount of BDNF immunoreactivity and the number of BDNF+ cells as well as in the dendritic complexity of DCX+ neurons in the hippocampus. In contrast to chronic imipramine treatment, chronic VNS had no effect on the behaviour of rats in the forced swim or elevated plus-maze tests. Both chronic and acute VNS induced persistent changes in hippocampal neurons that may play a key role in the therapeutic efficacy of VNS. However, these changes were not associated with evident behavioural alterations characteristic of an antidepressant or anxiolytic action.
The actions of ethanol on gamma-aminobutyric acid type A (GABA(A)) receptors are still highly con... more The actions of ethanol on gamma-aminobutyric acid type A (GABA(A)) receptors are still highly controversial issues but it appears that some of its pharmacological effects may depend on receptor subunit composition. Prolonged ethanol exposure produces tolerance and dependence and its withdrawal alters GABA(A) receptor subunit gene expression and function. Whereas benzodiazepines are clinically effective in ameliorating ethanol withdrawal symptoms, work in our laboratory showed that benzodiazepines also prevent, in vitro, some of the ethanol withdrawal-induced molecular and functional changes of the GABA(A) receptors. In the present work, we investigated the effects, on such changes, of the benzodiazepine receptor antagonist flumazenil that can positively modulate alpha(4)-containing receptors. We here report that flumazenil prevented both the ethanol withdrawal-induced up-regulation of the alpha(4)-subunit and the increase in its own modulatory action. In contrast, flumazenil did not inhibit ethanol withdrawal-induced decrease in alpha(1)- and delta-subunit expression as well as the corresponding decrease in the modulatory action on GABA(A) receptor function of both the alpha(1)-selective ligand zaleplon and the delta-containing receptor preferentially acting steroid allopregnanolone. These observations are the first molecular and functional evidence that show a selective inhibition by flumazenil of the up-regulation of alpha(4)-subunit expression elicited by ethanol withdrawal.
Expression of specific gamma-aminobutyric acid type A (GABA(A)) receptor subunit genes in neurons... more 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. This effect of steroids appears to be mediated through modulation of GABA(A) receptor signalling mechanisms that control the expression of specific receptor subunit genes. Furthermore, the specific outcomes of such signalling appear to differ among neurons in different regions of the brain. Neuroactive steroids such as the progesterone metabolite allopregnanolone might thus exert differential effects on GABA(A) receptor plasticity in distinct neuronal cell populations, likely accounting for some of the physiological actions of these compounds. Here we summarise experimental data obtained both in vivo and in vitro that show how fluctuations in the concentration of allopregnanolone regulate both the expression and function of GABA(A) receptors and consequently affect behaviour. Such regulation is operative both during physiological conditions such as pregnancy and lactation as well as in pharmacologically induced states such as pseudopregnancy and long-term treatment with steroid derivatives or anxiolytic-hypnotic drugs. Accordingly, long-lasting exposure of GABA(A) receptors to ethanol, as well as its withdrawal, induces marked effects on receptor structure and function. These results suggest the possible synergic action between endogenous steroids and ethanol in modulating the functional activity of specific neuronal populations.
The gamma-aminobutyric acid type A (GABA(A)) receptor is an important pharmacological target of e... more The gamma-aminobutyric acid type A (GABA(A)) receptor is an important pharmacological target of ethanol. The effect of ethanol withdrawal on the expression of the alpha(2) subunit of this receptor was examined with rat cerebellar granule cells in primary culture. Long-term exposure of these cells to ethanol (100 mM, 5 days) did not affect the abundance of the mRNA for the alpha(2) subunit, as revealed by an RNase protection assay. In contrast, subsequent ethanol withdrawal for 3 h induced a marked increase in the amount of this mRNA (2.6-fold) as well as in that of the encoded polypeptide (2.2-fold), the latter revealed by immunoblot analysis. Exposure of the cells to gamma-hydroxybutyric acid (100 mM) during ethanol withdrawal prevented the increase in the amounts of both the alpha(2) mRNA and polypeptide, whereas similar treatment with diazepam (10 microM) blocked the increase in the abundance of the alpha(2) polypeptide but not that in the amount of the alpha(2) mRNA. The effect of gamma-hydroxybutyric acid was not blocked by the competitive GABA(B) receptor antagonist SCH 50911(10 microM). Given that the alpha(2) subunit of the GABA(A) receptor mediates the anxiolytic action of benzodiazepines, its up-regulation during discontinuation of long-term ethanol exposure might be relevant to the therapeutic efficacy of these drugs in the treatment of anxiety associated with ethanol withdrawal.
Changes in the expression of type A receptors for gamma-aminobutyric acid (GABA) represent one of... more Changes in the expression of type A receptors for gamma-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. We 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. Chronic treatment with and subsequent withdrawal from ethanol alter the expression of g...
Expression of specific gamma-aminobutyric acid type A (GABA(A)) receptor subunit genes in neurons... more 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. This effect of steroids appears to be mediated through modulation of GABA(A) receptor signalling mechanisms that control the expression of specific receptor subunit genes. Furthermore, the specific outcomes of such signalling appear to differ among neurons in different regions of the brain. Neuroactive steroids such as the progesterone metabolite allopregnanolone might thus exert differential effects on GABA(A) receptor plasticity in distinct neuronal cell populations, likely accounting for some of the physiological actions of these compounds. Here we summarise experimental data obtained both in vivo and in vitro that show how fluctuations in the concentration of allopregnanolone regulate both the expression and function of GABA(A) receptors and consequently affect behaviour. Such regulation is opera...
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Papers by Paolo Follesa
functional properties in the physiological and pharmacological modulation of neuronal excitability. It has been suggested that changes in the gene expression of GABAARs represent one of the mechanisms implicated in the development of tolerance to and dependence on GABAergic drugs. Moreover, expression of specific GABAAR subunit genes is affected by endogenous modulators of receptor function such as neuroactive steroids and may contribute to the pathophysiology of anxiety disorders. This effect of steroids appears to be mediated through modulation of GABAAR signaling mechanisms that control the expression of specific receptor subunit genes. Neuroactive steroids might thus exert differential effects on GABAAR plasticity in distinct neuronal cell populations. In order to gain more information about the molecular mechanisms by which GABAergic drugs can produce plastic changes of the GABAARs, we have used a simple experimental model of neurons in culture that express most of the GABAAR subunits that are assembled in functional receptors. Here we summarize experimental data obtained with these cells in culture that show how chronic treatments with different positive modulators regulate both the expression and function of GABAARs. The understanding of these molecular mechanisms not only will provide new insights about the pathophysiology of those diseases, such as anxiety, in which GABAergic transmission plays a pivotal role, but will prove to be important to develop and design new anxiolytic, hypnotic and anticonvulsant selective drugs devoid of side effects.
functional properties in the physiological and pharmacological modulation of neuronal excitability. It has been suggested that changes in the gene expression of GABAARs represent one of the mechanisms implicated in the development of tolerance to and dependence on GABAergic drugs. Moreover, expression of specific GABAAR subunit genes is affected by endogenous modulators of receptor function such as neuroactive steroids and may contribute to the pathophysiology of anxiety disorders. This effect of steroids appears to be mediated through modulation of GABAAR signaling mechanisms that control the expression of specific receptor subunit genes. Neuroactive steroids might thus exert differential effects on GABAAR plasticity in distinct neuronal cell populations. In order to gain more information about the molecular mechanisms by which GABAergic drugs can produce plastic changes of the GABAARs, we have used a simple experimental model of neurons in culture that express most of the GABAAR subunits that are assembled in functional receptors. Here we summarize experimental data obtained with these cells in culture that show how chronic treatments with different positive modulators regulate both the expression and function of GABAARs. The understanding of these molecular mechanisms not only will provide new insights about the pathophysiology of those diseases, such as anxiety, in which GABAergic transmission plays a pivotal role, but will prove to be important to develop and design new anxiolytic, hypnotic and anticonvulsant selective drugs devoid of side effects.