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 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.

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.

Chronic ethanol treatment differentially regulates NMDA receptor subunit mRNA expression in rat brain.

Previous studies have implicated the N-methyl-D-aspartate receptor (NMDAR) complex in the physical dependence and withdrawal effects of chronic ethanol administration. In this study, we examined the effect of chronic ethanol administration and ethanol withdrawal on the NMDAR subunit R1, R2A, R2B, and R2C mRNA levels in rat hippocampus, cerebral cortex, and cerebellum. Using the RNase protection assay, we compared the levels of the NMDAR subunits mRNAs in ethanol-treated and control rats. Our results indicate that chronic ethanol administration and ethanol withdrawal do not change the NMDAR R1 subunit mRNA levels in cerebral cortex, hippocampus, or cerebellum at any time point. In contrast, 9 h after the last ethanol administration the R2A and R2B mRNA subunits were elevated by approximately 40% in cerebral cortex, and approximately 30% in hippocampus with respect to the levels in control animals. At 48 h the mRNA levels returned to the control levels. The chronic ethanol treatment did not alter R1, R2A, and R2C subunit mRNA levels in cerebellum. Our results demonstrate that chronic ethanol administration produces a differential regulation of the genes encoding the various subunits of the NMDAR.

Chronic ethanol treatment produces a selective upregulation of the NMDA receptor subunit gene expression in mammalian cultured cortical neurons.

Our previous work has shown that chronic ethanol treatment upregulated NMDA receptor function and binding in mammalian cortical neurons. However, the potential molecular mechanisms involved in these phenomenon have yet to be elucidated. In the present study, using RNase protection assay, we investigated the effect of chronic ethanol treatment on the NMDA receptor subunits R1, R2A, and R2B mRNA levels in cultured cortical neurons. We found that chronic ethanol (50 mM, 5 days) exposure did not change the NMDA receptor R1 and R2A subunits mRNA levels. In contrast, the NMDA receptor R2B subunit mRNA level was increased by approximately 40% with respect to the control values. The levels of the R2B subunit mRNA returned to the control values following the removal of ethanol for 72 h. In order to determine the involvement of the NMDA receptors in the action of chronic ethanol exposure, we further investigated the effect of the NMDA receptor antagonists on the upregulation induced by chronic ethanol exposure. The results indicate that the increased R2B subunit level was reversed by concomitant chronic exposure of the cortical neurons to the NMDA receptor competitive (10 microM; CPP), and non-competitive (1 microM; MK-801) antagonists, but not by the non-NMDA receptor antagonist, CNQX (10 microM), or the L-type calcium channel blocker, nitrendipine (10 microM). Taken together, these results suggested that chronic ethanol exposure selectively upregulated the NMDA receptor subunit R2B mRNA level in cortical neurons, and this increased NMDA receptor gene expression appears to be a NMDA receptor mediated process. The altered NMDA receptor gene expression may be responsible for the observed upregulation of the NMDA receptor binding and function in the cortical neurons following chronic ethanol exposure.

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.

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.

Increased basic fibroblast growth factor mRNA following contusive spinal cord injury.

Neurotrophic factors appear to be crucial for the survival and potential regeneration of injured neurons. Injury of the peripheral nervous system results in the induction of a number of neurotrophic molecules. Less is known about the response of central nervous tissue to injury. We have examined changes in levels of mRNA for three trophic factors, basic and acidic fibroblast growth factor (bFGF, aFGF), and nerve growth factor (NGF), after a standardized incomplete thoracic contusive spinal cord injury (SCI). RNase protection assays showed a rapid increase (3-fold) in the content of bFGF mRNA by 6 hours after SCI in tissue that included the injury site. No effect of injury was seen in segments of cervical or lumbar cord. bFGF mRNA at the injury site remained significantly increased at 1 and 7 days after SCI. Further, at 7 days, the increase was anatomically restricted to the rostral portion of the injury site suggesting the involvement of specific pathways in the maintenance of high levels of bFGF mRNA. No change in the levels of aFGF mRNA was seen after SCI. Similarly, no difference in the expression of the mRNA for NGF or its high affinity receptor (trkA), were observed at 6 h, 1 or 7 days following SCI. Our observation of a specific effect of SCI on bFGF mRNA expression supports a speculative hypothesis that bFGF may play a role in the partial recovery of function seen following incomplete contusive spinal cord injury.

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.

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.

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.

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 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.

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.

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.

Regulation of basic fibroblast growth factor and nerve growth factor mRNA by beta-adrenergic receptor activation and adrenal steroids in rat central nervous system.

Basic fibroblast growth factor (bFGF) and nerve growth factor (NGF) are two neurotrophic factors that play a role in neuronal maintenance and repair. The identification and characterization of mechanisms regulating neurotrophic factor availability in the central nervous system are vital to the development of therapeutic tools for prevention of neuronal degeneration. The lipophilic beta-adrenergic receptor (BAR) agonist clenbuterol was used to assess whether activation of central BAR changes the levels of NGF and bFGF mRNA. Within 5 hr, clenbuterol (10 mg/kg, intraperitoneally) elicited a 2-3-fold increase in bFGF and NGF mRNA content in rat cerebral cortex. The induction of bFGF and NGF mRNA expression showed anatomical specificity. Among the various brain areas examined, bFGF mRNA levels were increased in the cerebral cortex, hippocampus, and cerebellum, whereas induction of NGF mRNA was observed only in the cerebral cortex. Isoproterenol, a BAR agonist that does not cross the blood-brain barrier, also elicited a 2-3-fold increase in bFGF and NGF mRNA in the cerebral cortex. Propranolol (5 mg/kg, intraperitoneally), a lipophilic BAR antagonist, blocked the induction of NGF and bFGF mRNA mediated by either isoproterenol or clenbuterol, whereas nadolol (5 mg/kg, intraperitoneally), a BAR antagonist that does not cross the blood-brain barrier, blocked only the effect of isoproterenol. Therefore, activation of both central and peripheral BAR play a role in the regulation of bFGF and NGF mRNA expression. Moreover, in adrenalectomized rats, isoproterenol failed to increase bFGF and NGF mRNA, whereas clenbuterol elicited a 2-fold increase in bFGF mRNA in the cortex and hippocampus. Our data suggest that both adrenal steroids and noradrenaline might regulate the availability of selective neurotrophic factors in the adult central nervous system.

Regional and temporal pattern of expression of nerve growth factor and basic fibroblast growth factor mRNA in rat brain following electroconvulsive shock.

We have previously reported that focally evoked limbic motor seizures rapidly increase levels of mRNA encoding nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) in specific limbic system areas of the adult rat brain. The present studies examined the effect of both minimal and maximal electroconvulsive shock, applied via corneal electrodes, on NGF and bFGF mRNA levels in several limbic (entorhinal cortex, hippocampus, olfactory bulb) and extralimbic (striatum and cerebellum) brain regions. By 5 h following limbic motor seizures induced by low-intensity (minimal) electroshock (LES) (0.2 s, 50-70 mA; three times over a 1-h period), bFGF mRNA was significantly increased in entorhinal cortex and hippocampus, but not in the other regions examined. In contrast, tonic extensor seizures evoked by maximal electroshock (MES) (0.2 s, 150 mA; three times over a 1-h period) were associated with a significant increase in bFGF mRNA in all limbic and extralimbic regions examined. In the same animals, increases in NGF mRNA were limited to entorhinal cortex and hippocampus. Adrenal steroids were not required for the seizure-induced increase in NGF or bFGF mRNAs, based on the finding that adrenalectomized rats exhibited electroshock-induced increases in both NGF and bFGF mRNAs equivalent to the increase observed in sham-operated rats. It is suggested that the increase in mRNA levels for the neurotrophic factors occurs selectively in those regions which are especially activated by the specific seizure model, and represents an adaptive response to repeated noninjurious neuronal stimulation.

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.

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.

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.