Trigeminal nerve stimulation modulates brainstem more than cortical excitability in healthy humans.

Multiple sites in the central nervous system (CNS) have been hypothesized to explain the beneficial effects of transcutaneous trigeminal nerve stimulation (TNS) on several disorders. This work investigated the acute effects of TNS on the excitability of brainstem and intracortical circuits, as well as on sensorimotor integration processes at cortical level in physiological conditions. Brainstem excitability was evaluated in seventeen healthy subjects measuring the R1 and R2 areas of the blink reflex (BR) and its recovery cycle, with cortical excitability and sensorimotor integration assessed by probing short-interval (SICI) and long-interval (LICI) intracortical inhibition, with short-interval (SICF), intracortical facilitation (ICF), short-latency (SAI) and long-latency (LAI) inhibition measuring motor potentials evoked in the first dorsal interosseous muscle by TMS of the contralateral motor cortex. Neurophysiological parameters were assessed, in seventeen healthy subjects, before and after cyclic 20-min TNS delivered bilaterally to the infraorbital nerve. After TNS, the area of the R2 was significantly reduced (p = 0.018). By contrast, R1 area and R2 recovery cycle were unaffected. Similarly, SICI, ICF, LICI, SICF, SAI and LAI appeared unaltered after TNS. These data suggest that, in normal subjects, TNS mainly acts on brainstem polysynaptic circuits mediating the R2 component of the BR and plays a minor role in modifying the activity of higher-level structures involved in the R2 recovery cycle and in modulation of cortical excitability. A further investigation of a chronic TNS-induced effect may disclose a higher potential for TNS in producing measurable after effects on its CNS targets.

Changes in GABAA receptor gamma 2 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.

Changes in GABA(A) 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.

Role of allopregnanolone in regulation of GABA(A) receptor plasticity during long-term exposure to and withdrawal from progesterone.

Here we summarize recent data from our laboratory pertaining to the effects of fluctuations in the brain concentrations of the progesterone (PROG) metabolite allopregnanolone (3alpha,5alpha-TH PROG) on the expression and function of gamma-aminobutyric acid type A (GABA(A)) receptors. The effects of long-term exposure to progesterone and of its sudden withdrawal on the activity of GABA(A) receptors 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 3alpha,5alpha-TH PROG on GABA(A) receptor function and gene expression were examined in vivo in rats subjected to long-term administration of oral contraceptives. Our results demonstrate that long-lasting changes in the exposure of GABA(A) receptors to this PROG metabolite induce marked effects on receptor structure and function. These effects of 3alpha,5alpha-TH PROG appear to be mediated through modulation of GABA(A) receptor signaling mechanisms that control the expression of 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 3alpha,5alpha-TH PROG might thus exert differential actions on GABA(A) receptor plasticity in distinct neuronal cell populations, likely accounting for some of the physiological effects induced by these compounds.

GABAA-receptor plasticity during long-term exposure to and withdrawal from progesterone.

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.

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.

Pivagabine-induced increases in the abundance of CRF mRNA in the cerebral cortex and hypothalamus of rats.

The effect of treatment of rats with pivagabine (4-[(2,2-dimethyl-1-oxopropyl) amino] butanoic acid) for 4 days on the abundance of corticotropin-releasing factor (CRF) mRNA in the brain was investigated. Such treatment resulted in dose-dependent (100-300 mg/kg, i.p.) increases in the amount of CRF mRNA in both the hypothalamus and cerebral cortex. The maximal increases were thus apparent with the dose of 300 mg/kg in the hypothalamus (+108%) and cerebral cortex (+49%) 30 or 60 min, respectively, after the last drug injection. Foot-shock stress administered 30 min after the final drug injection had no effect on the pivagabine-induced increases in the abundance of CRF mRNA in the hypothalamus or cerebral cortex. Such stress also had no effect on the amounts of CRF mRNA in these brain regions of vehicle-treated rats. These results demonstrate that pivagabine increases the amount of CRF mRNA in both the hypothalamus and cerebral cortex of rats, effects that might be relevant to the action of this drug in preventing the stress-induced changes in CRF hypothalamic concentration.

Allopregnanolone synthesis in cerebellar granule cells: roles in regulation of GABA(A) receptor expression and function during progesterone treatment and withdrawal.

Rat cerebellar granule cells were cultured for 5 days with progesterone, resulting in the conversion of progesterone to allopregnanolone, a potent and efficacious modulator of gamma-aminobutyric acid (GABA) type-A receptors, as well as in decreases in the abundance of GABA(A) receptor alpha(1), alpha(3), alpha(5), and gamma(2) subunit mRNAs. These effects were accompanied by decreases in the efficacies of diazepam and the beta-carboline DMCM with regard to modulation of GABA-evoked Cl(-) currents. Withdrawal from such progesterone treatment resulted in a rapid and selective increase in the abundance of the GABA(A) alpha(4) subunit mRNA that was associated with a restoration of receptor sensitivity to the negative modulatory action of DMCM, a positive receptor response to flumazenil, and continued reduced responsiveness of receptors to diazepam. Prevention of allopregnanolone synthesis by the 5alpha-reductase inhibitor finasteride also prevented the changes in both GABA(A) receptor gene expression and receptor function elicited by progesterone treatment and withdrawal.

Physiological modulation of GABA(A) 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.

Changes in the gene expression of GABAA receptor subunit mRNAs in the septum of rats subjected to pentylenetetrazol-induced kindling.

Chemical kindling was induced in rats by long-term administration of pentylenetetrazol (PTZ) (30 mg/kg three times a week for 9 weeks). The effects of such kindling on the abundance of transcripts encoding subunits of the gamma-aminobutyric acid type A (GABAA) receptor in the brain were measured by RNase protection assay. Kindled rats were examined either 3 or 30 days after discontinuation of PTZ treatment. The amounts of gamma2L and gamma2S subunit mRNAs were significantly increased in the hippocampus and cerebral cortex of kindled rats 3 and 30 days after treatment discontinuation, compared with those observed in control rats, and these effects were prevented by the concomitant administration of the anticonvulsant abecarnil. In contrast, the amounts of alpha1 and beta2 subunit mRNAs in these two brain regions did not differ significantly between kindled and control rats. The abundance of alpha1, beta2, gamma2L and gamma2S subunit mRNAs was decreased in the septum of rats 3 or 30 days after discontinuation of treatment with PTZ either alone or in combination with abecarnil. The amounts of none of the four subunit mRNAs measured differed significantly between the striatum or frontal cortex of kindled rats and control rats 3 days after drug discontinuation. Immunohistochemical analysis with antibodies to choline acetyltransferase revealed a marked decrease in the number of cholinergic neurons in the septum of kindled rats 30 days after discontinuation of PTZ treatment; this effect was not prevented by the administration of abecarnil. These results suggest that long-term treatment with PTZ induces a loss of GABAA receptors in the septum.

Increased abundance of GABAA receptor subunit mRNAs in the brain of Long-Evans Cinnamon rats, an animal model of Wilson's disease.

The abundance of mRNAs encoding various subunits of the gamma-aminobutyric acid type A (GABAA) receptor was examined in different regions of the brain of Long-Evans Cinnamon (LEC) rats, an animal model of Wilson's disease (WD). The measurements were performed at two different stages of disease: at 9 weeks of age, when no symptoms are evident, and at 15 weeks of age, when 90% of the animals develop jaundice. The amounts of the gamma2L and gamma2S subunit mRNAs in the striatum, cerebellum, and cerebral cortex of LEC rats at 9 weeks of age were increased (+25 to +35%) compared with those in LE rats of the same age; these differences were no longer apparent in 15-week old animals. The amount of alpha1 subunit mRNA was also significantly increased (+30%) in the cerebellum of LEC rats at 9 weeks of age; although a smaller increase (+20%) was still evident at 15 weeks of age, this was not statistically significant. The amount of beta2 subunit mRNA was increased in the cerebellum (+32%) and hippocampus (+21%) of LEC rats at 9 weeks of age, but no differences with LE rats were apparent at 15 weeks. The onset of isoniazid-induced seizures in LEC rats at 9 weeks of age was significantly delayed compared with that in LE rats. These results demonstrate abnormal expression of GABAA receptor subunit genes in the brain of LEC rats, and they suggest that this altered expression is associated with an increase in GABAergic tone.

Role of brain allopregnanolone in the plasticity of gamma-aminobutyric acid type A receptor in rat brain during pregnancy and after delivery.

The relation between changes in brain and plasma concentrations of neurosteroids and the function and structure of gamma-aminobutyric acid type A (GABAA) receptors in the brain during pregnancy and after delivery was investigated in rats. In contrast with plasma, where all steroids increased in parallel, the kinetics of changes in the cerebrocortical concentrations of progesterone, allopregnanolone (AP), and allotetrahydrodeoxycorticosterone (THDOC) diverged during pregnancy. Progesterone was already maximally increased between days 10 and 15, whereas AP and allotetrahydrodeoxycorticosterone peaked around day 19. The stimulatory effect of muscimol on 36Cl- uptake by cerebrocortical membrane vesicles was decreased on days 15 and 19 of pregnancy and increased 2 days after delivery. Moreover, the expression in cerebral cortex and hippocampus of the mRNA encoding for gamma2L GABAA receptor subunit decreased during pregnancy and had returned to control values 2 days after delivery. Also alpha1, alpha2, alpha3, alpha4, beta1, beta2, beta3, and gamma2S mRNAs were measured and failed to change during pregnancy. Subchronic administration of finasteride, a 5alpha-reductase inhibitor, to pregnant rats reduced the concentrations of AP more in brain than in plasma as well as prevented the decreases in both the stimulatory effect of muscimol on 36Cl- uptake and the decrease of gamma2L mRNA observed during pregnancy. These results indicate that the plasticity of GABAA receptors during pregnancy and after delivery is functionally related to fluctuations in endogenous brain concentrations of AP whose rate of synthesis/metabolism appears to differ in the brain, compared with plasma, in pregnant rats.

Molecular and functional adaptation of the GABA(A) receptor complex during pregnancy and after delivery in the rat brain.

The abundance of gamma-aminobutyric acid receptor type A (GABAA receptor) subunit mRNAs and polypeptides as well as muscimol-stimulated 36Cl- uptake were measured in rat cerebral cortex or hippocampus at various times during pregnancy and after delivery. RNase protection assays revealed that the amount of the gamma2L subunit mRNA decreased progressively during pregnancy, in the cerebral cortex and hippocampus, and then returned to control values around the time of delivery. A similar pattern was observed for the alpha5 subunit mRNA in the cerebral cortex, whereas no significant changes were apparent for alpha1, alpha2, alpha3, alpha4, beta1, beta2, beta3 and gamma2S subunit mRNAs. The amounts of gamma2 and alpha1 proteins in the cerebral cortex were measured by immunoblot analysis; whereas the abundance of gamma2 protein decreased during pregnancy, no change was detected in the amount of alpha1 protein. Evaluation for functional significance of the down-regulated gamma2 and alpha5 subunit was made by determining the GABAA receptor function assessed by measurement of muscimol-stimulated 36Cl- uptake in cerebral cortical membrane vesicles. Muscimol-induced 36Cl- uptake was markedly reduced during of pregnancy compared with rats in oestrus. At this same time, the potentiating effects of diazepam and allopregnanolone on muscimol stimulation of 36Cl- uptake also were reduced. In contrast, the effects of muscimol, allopregnanolone and diazepam were significantly increased, relative to animals in oestrus, after delivery.

Brain-derived neurotrophic factor and basic fibroblast growth factor downregulate NMDA receptor function in cerebellar granule cells.

Evidence has accumulated to suggest that the NMDA glutamate receptor subtype plays an important role in neuronal degeneration evoked by hypoxia, ischemia, or trauma. Cerebellar granule cells in culture are vulnerable to NMDA-induced neuronal excitotoxicity. In these cells, brain-derived neurotrophic factor (BDNF) and basic fibroblast growth factor (FGF2) prevent the excitotoxic effect of NMDA. However, little is known about the molecular mechanisms underlying the protective properties of these trophic factors. Using cultured rat cerebellar granule cells, we investigated whether BDNF and FGF2 prevent NMDA toxicity by downregulating NMDA receptor function. Western blot and RNase protection analyses were used to determine the expression of the various NMDA receptor subunits (NR1, NR2A, NR2B, and NR2C) after BDNF or FGF2 treatment. FGF2 and BDNF elicited a time-dependent decrease in the expression of NR2A and NR2C subunits. Because NMDA receptor activation leads to increased intracellular Ca2+ concentration ([Ca2+]i), we studied the effect of the BDNF- and FGF2-induced reduction in NR2A and NR2C synthesis on the NMDA-evoked Ca2+ responses by single-cell fura-2 fluorescence ratio imaging. BDNF and FGF2 reduced the NMDA-mediated [Ca2+]i increase with a time dependency that correlates with their ability to decrease NR2A and NR2C subunit expression, suggesting that these trophic factors also induce a functional downregulation of the NMDA receptor. Because sustained [Ca2+]i is believed to be causally related to neuronal injury, we suggest that BDNF and FGF2 may protect cerebellar granule cells against excitotoxicity by altering the NMDA receptor-Ca2+ signaling via a downregulation of NMDA receptor subunit expression.

Differential induction of nerve growth factor and basic fibroblast growth factor mRNA in neonatal and aged rat brain.

Stimulation of glucocorticoid or beta-adrenergic receptors (BAR) has been shown to increase nerve growth factor (NGF) biosynthesis in adult rat brain. Little is known about the role of these receptors in the regulation of NGF expression in neonatal and aged brain. We have examined the effect of the synthetic glucocorticoid dexamethasone (DEX) and the BAR agonist clenbuterol (CLE) on the levels of NGF mRNA in neonatal (8 day old), adult (3 month old) and aged (24 month old) rats. By 3 h, DEX (0.5 mg/kg, s.c.) evoked a comparable increase in NGF mRNA in the cerebral cortex and hippocampus in both 8-day and 3-month-old rats. In contrast, CLE (10 mg/kg, i.p.) failed to change NGF mRNA levels in neonatal rats, while increasing (2-3-fold) NGF mRNA levels in the cerebral cortex of adult rats. In 24-month-old rats, both DEX and CLE elicited only a modest increase in NGF mRNA. This increase was, however, anatomically and temporally similar to that observed in adult animals. The weak effect of DEX or CLE was not related to a down-regulation of receptor function because both DEX and CLE were able to elicit a comparable increase in the mRNA levels for basic fibroblast growth factor (FGF2) in neonatal, adult and aged rat brain. Our data demonstrate that induction of NGF expression by neurotransmitter/hormone receptor activation varies throughout life and suggest that pharmacological agents might be useful tools to enhance trophic support in aging.

2,3-Dihydroxy-6-nitro-7-sulfamoyl-benzo(F)-quinoxaline (NBQX) increases fibroblast growth factor mRNA levels after contusive spinal cord injury.

We have previously demonstrated that the glutamatergic receptor (AMPA) antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)-quinoxaline (NBQX) reduces functional deficits in a standardized rat model of contusive spinal cord injury (SCI). NBQX not only acted to protect neurons from excitotoxicity but also, unexpectedly, enhanced sparing of white matter including axons of descending pathways. We have therefore investigated mechanisms through which NBQX could produce beneficial effects for white matter. We report here that NBQX elicits a rapid and selective induction of FGF2 mRNA levels in injured spinal cord. This novel effect could contribute to the therapeutic properties of NBQX in the treatment of SCI.

Postnatal phencyclidine treatment differentially regulates N-methyl-D-aspartate receptor subunit mRNA expression in developing rat cerebral cortex.

The present study was designed to determine the effects of chronic neonatal exposure to the NMDA receptor antagonist phencyclidine (PCP) on [3H]MK-801 binding and on gene expression of NMDA receptor subunits in juvenile male rats. Rat pups were injected daily with PCP from day 5 to 15 and killed on day 21. [3H]MK-801 binding was measured by quantitative autoradiography. A sensitive RNase protection assay was employed to determine simultaneously the mRNA levels of NR1 subunit (comprising all different splice variants) and three NR2 subunits (NR2A-NR2C). The relative distribution profile of NMDA receptor subunits in the cerebral cortex was NR2B > NR1 > NR2A > NR2C and in the cerebellum NR2C = NR1 > NR2A = NR2B. Chronic PCP administration in postnatal rats produced significant reduction in both [3H]MK-801 binding and mRNA level of the NR2B subunit in the cerebral cortex. Expression of the other NMDA receptor subunits in the cerebral cortex did not change following the drug treatment. In the cerebellum, neither [3H]MK-801 binding nor any of the NMDA receptor subunit expression levels showed any alteration. Together, these data provide a molecular correlate for chronic postnatal PCP-induced down-regulation of [3H]MK-801 binding in rat cerebral cortex and suggest that the NR2B subunit plays an important role in developmental plasticity.

Down-regulation of the GABA receptor subunits mRNA levels in mammalian cultured cortical neurons following chronic neurosteroid treatment.

We have recently shown that chronic neurosteroid, 5 alpha 3 alpha, treatment produced down-regulation of the GABA receptor binding and function, and heterologous uncoupling on the GABAA receptor complex in cultured mammalian cortical neurons. In order to explore the underlying mechanism of these observed down-regulation and heterologous uncoupling phenomenon, we investigated the effect of chronic 5 alpha 3 alpha (1 microM; 5 days) treatment on the GABAA receptor subunits mRNA levels, using RNase protection assay. We found that chronic neurosteroid, 5 alpha 3 alpha, treatment decreased the beta- and alpha-subunits mRNA levels while not altering the gamma 2S-subunit mRNA levels in the cortical neurons. The decrease in the beta-subunits mRNA levels suggests a decrease in the presence of the beta-subunits in the composition of GABAA receptors. This phenomenon may explain the down-regulation of the GABAA receptor binding and function. A decrease in the alpha 3-subunit mRNA level suggests a corresponding decrease in the alpha 3-subunit in the composition of GABAA receptor isoforms, relative to other isoforms. This observation may be responsible for the chronic neurosteroid-induced uncoupling and decreased efficacy. In summary, chronic 5 alpha 3 alpha treatment produced down-regulation of the GABAA receptor beta- and alpha-subunit mRNA levels, and these changes may be associated with the down-regulation, heterologous uncoupling, and decreased efficacy of GABAA receptor complex in the cultured mammalian cortical neurons.

Chronic ethanol-mediated up-regulation of the N-methyl-D-aspartate receptor polypeptide subunits in mouse cortical neurons in culture.

The goal of this study was to determine whether chronic ethanol-mediated up-regulation of the N-methyl-D-aspartate receptors (NMDAR) was associated with an augmentation of the NMDAR polypeptide subunits in the mammalian cortical neurons. The results show that chronic ethanol treatment produced an increase in the R1 and R2B polypeptide subunits. The R2A subunit was not expressed in these neurons. Chronic NMDAR antagonist ((+)-3-2-(carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP)) treatment also increased the R1 and R2B polypeptide subunits. A similar increase was observed when ethanol and CPP were used in combination. Binding studies using [3H]MK-801 ((+)-5-methyl-10, 11-dihydro-5H-dibenzo[a,d]cyclophenptan-5,10-imine maleate), a noncompetitive NMDAR antagonist, confirmed that concomitant exposure of ethanol and CPP up-regulated the NMDAR. Our results demonstrate for the first time that chronic ethanol treatment increased the NMDA receptor polypeptide subunit synthesis and that it was associated with an increase in [3H]MK-801 binding sites.

NMDA receptor upregulation: molecular studies in cultured mouse cortical neurons after chronic antagonist exposure.

We examined the possibility of changes in gene expression of the NMDA receptor subunits after chronic antagonist treatment. Exposure of neurons to the NMDA antagonist D(-)-2-amino-5-phosphonopentanoic acid (AP-5) produced an increase in the levels of the R2B mRNA subunit. Concomitant exposure of neurons to AP-5 and NMDA reversed the upregulation. Chronic AP-5 treatment increased the R1 polypeptide, whereas no change was observed in the levels of mRNA encoding the R1 subunit. A more pronounced increase was observed in the R2A/B polypeptides. These data demonstrate that chronic treatment with NMDA antagonists selectively upregulates the NMDA receptor mRNAs and polypeptides. Furthermore, antagonist treatment produced a differential regulation of the R1, R2A, and R2B subunits in cortical neurons.