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NEUROPHARMACOLOGY
7 Nicotinic Receptor Currents in Rat Hippocampal Neurons Is Dependent upon GABAergic Activity and Depolarization
Departmento de Farmacologia Básica e Clínica, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Centro de Ciências da Saúde, Rio de Janeiro, Brazil (H.R.S., H.S.R., P.S.-P., E.X.A., N.G.C.); and Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Baltimore, Maryland (E.X.A.)
Received April 17, 2006; accepted July 7, 2006.
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Abstract |
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7* nicotinic acetylcholine receptors modulate the release of GABA and glutamate. The control of functional receptor pools by cell firing or synaptic activity could therefore allow for a local adjustment of the sensitivity to cholinergic input upon changes in neuronal activity. We first investigated whether tonic depolarization or cell firing affected the function of 7*. The amplitude of 7*-gated whole-cell currents in cultured rat hippocampal neurons exposed to high-extracellular K+ (40 mM KCl) for 24 to 48 h increased 1.3 to 5.5 times. The proportion of 7*-responsive neurons (99%), the potency of acetylcholine, and the sensitivity to nicotinic antagonists were all unaffected. In contrast, block of spontaneous cell firing with tetrodotoxin for 24 h led to a 37% reduction in mean current amplitude. Reduced 7* responses were seen after a 24-h blockade of N-type calcium channels but not of L-type calcium channels, N-methyl-D-aspartate (NMDA), or non-NMDA receptor channels, protein kinase C, or calcium-calmodulin kinases II and IV. The N-type or L-type calcium channel antagonists 
-conotoxin GVIA and nifedipine did not prevent the current-potentiating effect of KCl. The GABAA antagonist picrotoxin led to a 44% reduction of the currents, despite increasing action potential firing, and also reversed the potentiating effect of KCl. Treatment with GABA, midazolam, or a GABA uptake blocker led to increased currents. These data indicate that 7*-gated currents in hippocampal neurons are regulated by GABAergic activity and suggest that depolarization-induced GABA release may underlie the effect of increased extracellular KCl.
; Alkondon and Albuquerque, 2001; Sharma and Vijayaraghavan, 2003) where they also mediate fast postsynaptic currents (Alkondon et al., 1998; Frazier et al., 1998; Hefft et al., 1999). The -bungarotoxin-sensitive 7*-type nAChR has been implicated in most of these cholinergic responses. This receptor channel is uniquely permeable to calcium (Castro and Albuquerque, 1995; Fucile, 2004) and is located both presynaptically and postsynaptically in hippocampal tissue (Fabian-Fine et al., 2001), which led to the suggestion that it is mostly involved in modulation of neuronal activity instead of in phasic all-or-none excitatory transmission (Jones et al., 1999; Dajas-Bailador and Wonnacott, 2004). To better serve this modulatory role, the mechanisms controlling receptor expression and function might depend upon the level of activity of the cell. Depolarization-dependent mechanisms are involved in the developmental regulation and maintenance of nAChRs in the neuromuscular junction (Fumagalli et al., 1990) and in ganglionic synapses (Fumagalli et al., 1976; De Koninck and Cooper, 1995). Unveiling such mechanisms in central neurons has become of particular interest in view of the evidence of altered hippocampal 7* nAChR number or function in major neurological diseases, including Alzheimer's and some forms of schizophrenia and epilepsy (Gotti and Clementi, 2004).
Increased extracellular K+ has often been used as an experimental paradigm emulating sustained excitation in muscle and nerve cells. Treatment with KCl for several days leads to membrane depolarization and sustained calcium influx mediated by voltage-dependent Ca2+ channels (VDCC), as demonstrated in cultured rat sympathetic neurons (Koike and Tanaka, 1991). The effect of high K+ has been studied in several cell types expressing native 7* receptors in culture (DeLorme and McGee, Jr., 1988; Betz, 1983; Smith et al., 1983; Geertsen et al., 1988, 1992; De Koninck and Cooper, 1995; Ridley et al., 2002), including hippocampal neurons (Ridley et al., 2001), and also in cells transfected with the 7 gene (Quik et al., 1996, 1997). In these diverse models, treatment with 20 to 50 mM KCl for 1 to 7 days consistently leads to increased 7 transcript levels and/or -bungarotoxin binding sites. In addition, blockade of action potentials with tetrodotoxin (TTX) or inhibition of NMDA receptors can have opposite effects to those of KCl on 7*-nAChR labeling in cultured hippocampal neurons (Kawai et al., 2002). However, TTX reduces the number of detectable 7* nAChR clusters only in a subpopulation of GABAergic neurons in the mixed hippocampal cultures, whereas total -bungarotoxin binding is in fact unchanged (Kawai et al., 2002). Furthermore, none of these previous studies evaluated the effect of depolarization on 7* receptor activity directly through ion channel current measurements, and those that used functional assays failed to demonstrate any corresponding change in responses mediated by 7* nAChRs (DeLorme and McGee, Jr., 1988; Betz, 1983; Smith et al., 1983; Geertsen et al., 1988; De Koninck and Cooper, 1995; Quik et al., 1997; Ridley et al., 2002).
The physiological significance of depolarization-induced changes in receptor expression assessed through ligand binding, protein, or transcript quantification is open to question, because even the fully mature protein inserted in the plasma membrane may not be functional, and the fraction of functional receptors may be subjected to independent regulation. Therefore, we have tested whether nAChR-mediated currents elicited by fast agonist pulses were altered in conditions that affect resting membrane potential, action potential firing, Ca2+ signaling, and postsynaptic activity in cultured rat hippocampal neurons. To our knowledge, this is the first study to address the changes in the number of functional nAChRs induced by tonic depolarization and by synaptic activity in central neurons. Our results confirm that the function of hippocampal 7* nAChR is modulated by depolarization, but we provide novel evidence implicating a GABAergic pathway in this modulation.
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Materials and Methods |
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). Usually 14 to 20 hippocampi were pooled, trypsinized, and mechanically dissociated, and then approximately 106 cells were plated per 35-mm poly-L-lysine-coated dish. Cultures were maintained in a humidified atmosphere with 10% CO2 at 35°C. Maintenance medium was minimal essential medium with Earle's salts (Invitrogen, Carlsbad, CA) supplemented with 5 g/l of d-glucose, 2 mM glutamine, 3.7 g/l NaHCO3, and 10% horse serum (Gemini Bioproducts, Woodland, CA). Plating medium further contained 10% fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA) and 20 µg/ml deoxyribonucleotidase type II and was progressively replaced by maintenance medium starting 1 or 2 days after plating. Cell proliferation was inhibited 6 to 7 days after plating, when the glial monolayer was confluent, by the addition of 14 µg/ml 5-fluoro-2'-deoxyuridine and 7 µg/ml uridine. Half of the medium was changed twice a week, and cultures were used 14 to 40 days after plating. Culture medium components were purchased from Invitrogen or Sigma (St. Louis, MO)
Electrophysiology. Whole-cell membrane currents were recorded at a membrane potential of -57 mV (corrected for measured liquid junction potentials) either with an Axopatch 200A (Axon Instruments, Forster City, CA) or an EPC-7 (List, Darmstadt, Germany) patch-clamp system. Currents were low-pass filtered at 3 kHz (8-pole Bessel) and digitized with a LabMaster interface under the control of pClamp software (Axon Instruments). The standard extracellular solution was 165 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM d-glucose, 5 mM HEPES, and 
2 mM NaOH, pH 7.3, with added TTX (0.15-0.3 µM) and atropine sulfate (1 µM). This solution replaced the culture medium approximately 20 min before the recordings and was continuously perfused at a rate of 1 ml/min throughout the experiments. A U-tube system was used to deliver fast pulses of drug solutions onto the patch-clamped neurons under computer control. Unless otherwise noted, fast nicotinic responses were evoked by 0.5-s pulses of acetylcholine chloride (1 mM) dissolved in extracellular solution. Patch micropipettes were made from borosilicate glass capillaries (WPI, Sarasota, FL) in a P-97 horizontal puller (Sutter Instruments, Novato CA). The intracellular solution was 80 mM CsCl, 80 mM CsF, 10 mM EGTA, 10 mM HEPES, and 26 mM CsOH, pH 7.3. The filled patch microelectrodes had resistances of 2 to 5 M
in the bath; the access resistance was left uncompensated. In the experiments comparing response amplitudes in treated and untreated cultures, a single response was recorded from each neuron 2 min after achieving the whole-cell configuration. Uncontrolled factors like response rundown and voltage error, if present, were expected to affect all experimental groups to the same extent. A modified intracellular ATP-regenerating solution (Castro and Albuquerque, 1995) was used in the experiments of concentration-response curves to minimize the rundown of nicotinic responses. For the calcium channel currents, a modified ("Ba-NMG") extracellular solution was used containing 133 mM N-methyl-D-glucamine (NMG), 10 mM BaCl2, 5 mM KCl, 10 mM d-glucose, 20 mM tetraethylammonium chloride, 0.15 µM TTX, and 5 mM HEPES, pH adjusted to 7.3 with HCl. The Ba-NMG solutions with or without nifedipine (5 µM) or CdCl2 (200 µM) were applied by a multibarrel delivery system positioned at approximately 100 µm from the cell. Whole-cell calcium channel currents were evoked by depolarizing square pulses to -20 mV (50 ms) from a holding potential of -80 mV. Leak currents were subtracted by a fractional method (P/N) using four scaled hyperpolarizing subpulses. In each cell, the voltage response was recorded in the following sequence: control, nifedipine, CdCl2 (200 µM), and control (wash). The small current remaining in the presence of CdCl2 was subtracted before calculation of the percentage block by nifedipine. Recordings were made at room temperature (23°C).
Drug Treatments. For some experiments, the neurons in culture were subjected to a treatment with high extracellular K+. One or 2 days before the recordings, half of the volume of medium in the culture dish was replaced by a medium containing KCl (75 mM), yielding a final concentration of 40 mM KCl. The extra KCl was added with enough water to keep the final tonicity of the medium unchanged, with minimal dilution (<10%) of all other medium components. Drugs applied to the cultures were prepared in 500 to 1000 x concentrated stock solutions in water or dimethyl sulfoxide, 20 to 40:l of which were added to the medium in the dish (2 ml) on the day before the recordings. Treatment with dimethyl sulfoxide alone (0.1-0.2%) had no significant effect in the responses. Acetylcholine chloride, GABA, atropine sulfate, nifedipine, -conotoxin GVIA, 6,7-dinitroquinoxaline-2,3(1H,4H)-dione (DNQX), d,l-2-amino-5-phosphonovalerate (APV), picrotoxin, strychnine hydrochloride, methyllycaconitine citrate (MLA), dihydro-
-erythroidine (DHE), KN-62 [1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine], GF 109203X (bisindolylmaleimide I), and NO-711 [1-[2-[[(diphenylmethylene)imino]oxy]ethyl]-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride] were from Sigma/RBI (St. Louis, MO). Tetrodotoxin was from Alomone Labs (Jerusalem, Israel). Midazolam was a generous gift from Roche Pharmaceuticals (Rio de Janeiro, Brazil). All inorganic compounds were of analytical grade.
Data Analysis. Current recordings were analyzed with pClamp software (Axon Instruments) for peak amplitude measurements. In the experiments addressing the effects of diverse culture treatments on current amplitudes, a randomized complete block design was used. The treatments were simultaneously applied to two (or three) culture dishes randomly selected from the same plating, one always being the control. The recordings were made 24 or 48 h later on seven to 12 randomly chosen neurons from each dish. Thus, experiments were performed on matched cultures from the same batch and were repeated with three to six different batches. Tests for differences between treatment groups were based on a two-way mixed model with multiple observations (replications) where the treatments, the culture batches, and the currents recorded in the same dish were the fixed factor, the random (block) factor, and the replications, respectively. Significance of the treatment effects was evaluated by the Mack-Skillings nonparametric test for unequal number of replications (Hollander and Wolfe, 1999).
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). By far the most prevalent responses were type IA currents, which reached peak within 20 ms and quickly decreased to 5% of the peak during the acetylcholine pulse, due to desensitization. Although these fast-desensitizing (type IA) responses could be unambiguously attributed to 7*-type receptors, no attempt was made to classify the few slowly desensitizing responses as being types II or III. In the 138 neurons treated with KCl, 16 responses were smaller than 10 pA (taken to be zero for statistical calculations), 121 responses were fast-desensitizing type IA currents, and one was type II or III (Alkondon and Albuquerque, 1993). In the control sample, 21 neurons did not respond, 89 responses were of type IA, two were of type IB, and one was of type II or III. The rare non-IA currents were excluded from further analysis.
KCl Did Not Affect the Pharmacological Properties of the 7* Nicotinic Responses. The most obvious difference between nicotinic responses in treated and untreated neurons was that the mean peak current amplitude was larger in the treated neurons (Fig. 1A). In four culture batches tested 24 h after KCl treatment and in five batches tested at 48 h, the ratio between the mean amplitudes in the treated and in the control neurons ranged from 1.3 to 5.5 (Table 1). The distributions of the peak amplitudes in both the KCl-treated and control cultures were markedly asymmetric and failed standard statistical tests of normality. Therefore, instead of analysis of variance, the nonparametric Mack-Skillings (M-S) procedure was used, and the data were graphed as box plots (Fig. 1, B-C). The differences in peak amplitude among all of the treated and untreated neurons were significant in both the 24- and 48 h-groups (p = 2.3 x 10-!6 and p = 2.0 x 10-!8, respectively, M-S test).
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The sensitivity of the nicotinic responses in the KCl-treated neurons to nAChR antagonists was also tested. For these experiments, acetylcholine was applied at 300 µM for 1 s. The following drugs were tested in sequence in each of the 13 KCl-treated (48 h) and seven untreated neurons: 1 nM MLA, 100 nM DHE, and 10 µM strychnine, applied both in the bath and agonist-containing solutions. At the concentrations used, MLA and DHE block the 7* and 42 receptors completely and in a selective manner, respectively (Alkondon and Albuquerque, 1993), thus allowing the dissection of the mixed nicotinic responses seen in some hippocampal neurons that coexpress both receptors. Strychnine, also known as a glycine receptor-channel antagonist, blocks competitively the 7* receptor in hippocampal neurons with an IC50 of 1.2 µM (Matsubayashi et al., 1998). The effects of the antagonists were similar in the KCl-treated neurons and untreated controls; MLA blocked the responses completely, strychnine blocked partially, and DHE had little or no effect (Fig. 2B). The effect of MLA was partially reversed upon washout, whereas that of strychnine reversed completely, in all control and treated neurons. Thus, 99% of the hippocampal neurons sampled from cultures exposed to a high concentration of KCl for 24 or 48 h showed acetylcholine-gated currents that could be identified as type IA nicotinic responses mediated by 7* nAChRs. These responses were similar to those of untreated control neurons in acetylcholine potency and sensitivity to neuronal nicotinic receptor antagonists but tended to be larger in amplitude.
Ridley et al. (2001) have shown that the increases in surface 125I--bungarotoxin binding in rat hippocampal cultures are similar after 4 or 7 days of treatment with 20 mM KCl, suggesting that the effect of KCl is stable by 4 days of exposure. In the present study, the changes in current amplitude induced by 40 mM KCl were obvious at 24 h and did not seem larger at 48 h (Fig. 1). This indicated that the mechanisms responsible for the effects of KCl on hippocampal nAChRs operate in less than 24 h, so further investigation was focused on this time frame.
Blockade of Na+ Channels Led to a Reduction of 7*-Gated Currents. If the cellular effects of the KCl treatment were akin to those of depolarization induced by intense action potential activity, suppression of action potentials would be expected to have opposite effects. Thus, neurons were treated for 24 h with the sodium channel blocker TTX at a concentration of 0.9 µM, which is several times above that required to stop all spontaneous action potential activity in the culture. A sample of 40 TTX-treated neurons from four culture batches showed smaller type IA currents than their matched controls (Fig. 3A), with an overall reduction of 37% in mean amplitude (p = 0.004, M-S test). This showed that spontaneous Na+ channel-dependent activity in the cultured neurons affected the basal level of 7* receptor function.
Blockade of VDCCs and Ca2+-Dependent Kinases Differentially Affected 7*-Gated Currents. The opposite effects of TTX and KCl seemed consistent with the previous hypothesis that 7* nAChR expression is correlated with VDCC function and Ca2+ entry, based on the inhibitory effect of L-type VDCC blockers (De Koninck and Cooper, 1995; Quik et al., 1997; Ridley et al., 2001; Dajas-Bailador et al., 2002). According to this interpretation, the effect of KCl would be mediated by depolarization and VDCC activation, whereas that of TTX would be the result of reduced VDCC activation as a consequence of action potential suppression. To test for this hypothesis, hippocampal cultures were exposed to KCl or KCl plus 5 µM nifedipine for 24 h in triple-matched experiments with an untreated control. Surprisingly, three of four cultures that were subjected to a combined treatment with nifedipine and KCl showed larger IA currents than matched cultures treated with KCl alone, which in turn showed larger currents than matched untreated cultures, as previously shown (Fig. 3B). When ignoring the group treated with KCl alone, the difference between the group receiving KCl plus nifedipine and the control was significant (p = 0.024, M-S test), with an overall increase of 62% in the mean current amplitude. However, the differences among the three treatment groups did not reach significance (p = 0.069, M-S test). In another set of experiments, cultures treated with nifedipine alone (5 µM for 24 h) showed 47% larger IA currents than matched untreated controls, but the effect was not significant (p = 0.192, M-S test) (Fig. 3C). Contrary to the results with TTX, treatment with nifedipine tended to have a potentiating effect on IA currents, and the combined effect of nifedipine and KCl was larger than that of either treatment alone.
To ascertain that the cultured hippocampal neurons expressed nifedipine-sensitive L-type VDCC currents, these were recorded with barium as the main charge carrier. VDCC currents evoked by a depolarizing pulse from -80 to -20 mV ranged from 0.3 to 2.6 nA in the Ba-NMG solution after subtracting a small cadmium-insensitive current. Nifedipine (5 µM) significantly reduced the VDCC currents (Fig. 3C, inset). The effect was reversible, but the currents failed to return to control levels, at least partially as a result of run down. The average block was of 83.3 ± 8.7%, ranging from 23.4 to 97.1% (n = 8). These results confirm that nifedipine effectively blocked dihydropyridine-sensitive (L-type) VDCCs in the cultures.
We next examined the role of N-type VDCCs by treating the cultures with -conotoxin GVIA (1 µM) with or without KCl in triple-matched experiments (Fig. 3D). The toxin alone led to a 37% reduction in the current averages (p = 0.006, M-S test) and also brought down the effect of KCl to a nonsignificant potentiation (25%) compared with untreated controls. Using the same protocol as for nifedipine, acute application of 1 µM -conotoxin GVIA blocked barium currents by more than 50%, confirming its effectiveness (data not shown). These data suggest that physiological activation of N-type (but not L-type) VDCCs contributes to maintain the basal level of IA currents in hippocampal neurons. However, the current averages in cultures treated with KCl plus toxin were 121% larger than in matched cultures treated with toxin alone (p < 0.001, M-S test), showing that blockade of N-type channels could not completely prevent the effect of KCl.
Treatment of neurons with KCl or TTX would be expected to interfere with other pathways of Ca2+ entry, independent of L-or N-type VDCCs, including other VDCCs, ligand-gated channels, and transporters. Protein kinase C and Ca2+/calmodulin-dependent kinase II are common effectors in diverse Ca2+ signaling pathways, and both enzymes have been shown to positively modulate 7* nAChR expression in different cell types (Geertsen et al., 1992; De Koninck and Cooper, 1995; Dajas-Bailador et al., 2002). The role of these Ca2+-dependent kinases in maintaining the activity of hippocampal 7* nAChRs was tested with the protein kinase C inhibitor GF 109203X and the Ca2+/calmodulin-dependent kinase II/IV inhibitor KN-62. As shown in Fig. 4A, the mean current in 29 neurons from three culture batches treated with 0.5 µM GF 109203X (Goldin and Segal, 2003) for 24 h was 74% larger than that of their matched pairs (p = 0.03, M-S test). Neurons treated with 4 µM KN-62 (De Koninck and Cooper, 1995), also applied for 24 h, exhibited a 28% larger mean current than their matched controls (Fig. 4B), but the difference did not reach statistical significance (p = 0.071, M-S test). This indicates that the major Ca2+-dependent kinases do not contribute to promote the basal level of 7* nAChR function. Altogether, the effects of nifedipine, GF 109203X, and KN-62 suggest that some Ca2+ signaling pathways known to be activated by neuronal depolarization may in fact hinder the activity of hippocampal 7* nAChRs.
Spontaneous Glutamatergic Excitation Did Not Increase 7*-Gated Currents. The hippocampal cultures used in this study presented a dense fiber network and intense synaptic activity, as judged by the frequent excitatory and inhibitory postsynaptic currents seen in nearly all recorded neurons. Treatment with TTX suppressed most of this synaptic activity, leaving only miniature postsynaptic currents. To test whether excitatory synaptic activity (and postsynaptic Ca2+ entry) contributed to the TTX-sensitive basal level of nAChR responsiveness, cultures were treated with 10 µM DNQX, an AMPA/kainate receptor antagonist (Honoré et al., 1988). As expected, in acute experiments, DNQX produced a marked suppression of synaptic events, blocking excitatory currents completely and reducing the amplitude and frequency of inhibitory currents, presumably by inhibiting excitation of GABAergic neurons in the culture. Because the culture medium contained Mg2+ (1 mM), Ca2+ entry through NMDA receptors was probably reduced as well. However, treatment with DNQX for 24 h had no significant effect on IA currents in 27 neurons from three cultures compared with matched controls (Fig. 5A) (p = 0.683, M-S test). Treatment with the NMDA receptor antagonist APV (100 µM) did not produce any significant decrease in amplitude of nicotinic responses (Fig. 5B) (p = 0.239, M-S test).
To further assess the effect of depolarization associated with synaptic excitation and cell firing, the GABAA receptor antagonist picrotoxin was used to block inhibition at 100 µM. Acute application of picrotoxin at this concentration had no effect on IA currents (data not shown) but markedly increased the frequency of action currents recorded in cell-attached mode, mimicking an epileptiform activity. This effect persisted after 24 h of continuous exposure to picrotoxin in the culture medium, because high firing rates were still observed when the treated cultures were washed and continuously perfused with solution containing picrotoxin during the recordings. The mean amplitude of IA currents recorded from 40 picrotoxin-treated neurons was 44% lower than that from matched controls (p = 0.0068, four culture batches, M-S test) (Fig. 5C). Therefore, blocking endogenous GABAergic inhibition promoted cell firing, as expected, but instead of mimicking the effect of KCl, this led to a decrease in 7* nAChR function.
GABAergic Activity Promoted an Increase in 7*-Gated Currents. Most neurons in our cultures presented spontaneous excitatory and inhibitory synaptic currents, corresponding to vesicular release of endogenous glutamate and GABA. One possibility to reconcile the inhibitory effect of TTX, which presumably led to a reduction of both glutamate and GABA release, with the inhibitory effect of picrotoxin, which presumably led to an increased release of both transmitters but selectively blocked GABAA receptors, would be that modulation of 7* currents depended on the function of GABAA receptors. To test this hypothesis, 100 µM GABA was added to the cultures for 24 h. GABA-treated cultures showed nearly 60% larger 7* currents than their matched controls (p = 0.027, M-S test) (Fig. 6A). To examine the role of endogenous GABA acting through GABAA receptors, cultures were treated for 24 h with midazolam, a benzodiazepine agonist. In our cultures, a fraction of the GABAA receptors, including those mediating tonic background currents, was sensitive to midazolam (Lopes et al., 2004). Treatment with midazolam (0.1 µM) significantly potentiated the nicotinic responses (p = 0.021, M-S test) (Fig. 6B). The percentage increase in average current ranged from 12 to 462% among the five culture batches, with an 80% increase in the grand mean.
The Na+/Cl--coupled GABA transporters (GAT) are responsible for the uptake of synaptically released GABA as well as for the maintenance of a submicromolar concentration of the transmitter in the extracellular space. The GAT-1 noncompetitive inhibitor NO-711 was then used to block uptake of endogenous GABA, prolonging GABA receptor activation (Richerson and Wu, 2003). Neurons treated with NO-711 (50 µM) for 24 h showed 51% larger 7* currents than their matched controls (p = 0.019, M-S test) (Fig. 6C).
The effect of NO-711 suggests that the transport of GABA by GAT-1 is relevant to the control of 7* nAChR responses. It is well established that increased KCl can cause reversal of the Na+-Cl--GABA symport, inducing GABA release and activating tonic GABAA receptor-gated currents in hippocampal neurons (Gaspary et al., 1998; Wu et al., 2001). Therefore, the observed potentiation of 7*-gated currents by KCl could depend on this mechanism, involving GABA and GABAA receptors. This possibility was addressed by simultaneously treating neurons with KCl and blocking GABAA receptors. In four culture batches, the neurons exposed to 40 mM KCl plus 100 µM picrotoxin showed smaller mean currents than their matched dimethyl sulfoxide-treated controls. The grand mean of the currents was reduced by 45%, ranging from 30 to 59% reduction in individual batches (p = 0.012, M-S test) (Fig. 6D). This reduction was similar to that seen with picrotoxin alone, indicating that the effect of KCl is obliterated after blockade of GABAA receptors.
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7* nAChRs, which mediate virtually all of the nicotinic currents in our cultures, are subject to strong functional regulation by activity-related mechanisms. The effects of modulating Ca2+ entry through L-and N-type VDCCs or glutamate receptor-channels were not generally consistent with a hypothesized depolarization and Ca2+-dependent mechanism for the effects of KCl and TTX. However, variations in GABA concentration and modulation of GABAA receptor function revealed that currents mediated by 7* nAChRs were specifically correlated with GABAergic activity.
Nature of the KCl-Modified Nicotinic Responses. Nearly all hippocampal neurons sampled from cultures exposed to a high concentration of KCl for 24 or 48 h showed acetylcholine-gated currents that could be identified as type IA nicotinic responses (presumed 7* nAChR). In treated neurons, the potency of acetylcholine and the sensitivity to neuronal nicotinic receptor antagonists were unchanged; however, the currents were significantly larger in amplitude. The simplest explanation of the KCl-induced increase in nicotinic responses is that more receptors have become active, these being functionally indistinguishable from those in the pre-existing active pool. In support of this view, in various cell systems expressing 7* nAChRs, the number of -bungarotoxin binding sites is increased after a depolarizing treatment (Smith et al., 1983; Geertsen et al., 1988, 1992; De Koninck and Cooper, 1995; Quik et al., 1996, 1997; Ridley et al., 2001; Kawai et al., 2002). In principle, the enhanced responses could have been due to newly inserted receptors or to activation of a pre-existing silent pool, and there is recent evidence of both types of regulation for 7* receptors. A significant intracellular pool of 7 protein is closely associated with the membrane in rat hippocampal neurons (Fabian-Fine et al., 2001), and vesicle-associated receptors can be inserted in the membrane through an exocytotic mechanism, quickly becoming functional (Cho et al., 2005). However, response potentiation by KCl (or GABA) could also occur without changes in 7* receptor number, as recently demonstrated for genistein-induced potentiation (Charpantier et al., 2005).
The relationship between depolarization-induced up-regulation of -bungarotoxin binding sites and changes in receptor-channel function remained mostly untested or unproven in previous studies (DeLorme and McGee, 1988; Smith et al., 1983; Geertsen et al., 1988). One study did show an increased receptor-mediated Ca2+ influx in GH4C1 rat pituitary cells stably expressing rat 7 receptors exposed to 50 mM K+ for 2 to 3 days, without changes in agonist potency (Quik et al., 1997). However, the calcium transients induced by nicotinic agonists in these cells are not directly coupled to nAChR activation, because they are abolished by L-type VDCC blockers. In neurons of the rat superior cervical ganglion, a selective effect of high-K+ on 7 mRNA levels and on numbers of -bungarotoxin binding sites was observed without concomitant changes in nAChR-gated whole-cell currents (De Koninck and Cooper, 1995). In most of the cell types previously studied, the small fraction of 7* among all functional nAChRs and their fast desensitization may have hindered the assessment of changes in function.
Role of Ca2+ in 7* nAChR Expression versus Function. Nifedipine did not block the potentiating effect of KCl, and nifedipine, KN-62, and GF 109203X by themselves tended to increase the magnitude of 7*-mediated currents. In contrast, previous studies have found reduced 7* receptor numbers after similar drug treatments (De Koninck and Cooper, 1995; Quik et al., 1997; Ridley et al., 2001). This discrepancy might be due to differences in cell type or to distinct Ca2+ dependencies of the mechanisms controlling the fraction of activatable receptors (or current amplitude) and the total number of receptors that bind -bungarotoxin. Indeed, alternate mechanisms unrelated to Ca2+ could lead to current potentiation without changes in the number of surface 7* receptors (Charpantier et al., 2005). On the other hand, blockade of N-type VDCCs had a marked inhibitory effect, showing that other Ca2+-dependent pathways modulate 7* responses in hippocampal neurons and may have contributed to the functional changes induced by either KCl or TTX.
Role of Glutamate-Gated Channels in 7* nAChR Expression and Function. The lack of effect of DNQX argues against a specific role of non-NMDA ionotropic glutamate receptors and postsynaptic depolarization in regulating the activity of 7* receptors in our cultures. The in vivo effect of the non-NMDA receptor antagonist CNQX on transcript levels of various nAChR subunits has been examined recently. After seven daily doses of CNQX, there are region-specific changes in 2, 4, and 2 transcripts in adult rat brains, but no changes in 7 were observed in the hippocampus or elsewhere (McCullumsmith et al., 2004). This result also suggests that hippocampal 7* receptor levels are not regulated by glutamatergic excitation in adult rats, as seen here in cultured neurons.
A major role for NMDA receptors in regulating IA currents is unlikely in view of the nonsignificant effect of a 24-h treatment with APV. However, incubation with APV for 3 days has been shown to reduce the number of fluorescence-labeled 7* nAChRs in hippocampal cultures (similarly to TTX), at least in a subpopulation of GABAergic neurons (Kawai et al., 2002).
Role of GABA in 7* nAChR Function. Both application of exogenous GABA and blockade of its uptake led to increased 7*-gated currents, whereas blockade of GABAA receptors had the opposite effect, in spite of the likely increase in GABA release associated with enhanced network activity. Although a contribution of GABAB receptors cannot be ruled out, the specific inhibition by picrotoxin and the potentiation by midazolam suggested that GABA acted mostly, if not entirely, through GABAA receptors to promote 7* nAChR function.
A 7-day treatment of hippocampal cultures with the competitive GABAA antagonist bicuculline (20 µM) has no significant effect on the number of labeled 7 protein clusters in identified GABAergic neurons and on total -bungarotoxin binding (Kawai et al., 2002). However, bicuculline is a competitive antagonist of homomeric 7 receptors, with an IC50 of 7 µM (Demuro et al., 2001). It is possible that any GABAA receptor-dependent effect of chronic bicuculline on hippocampal 7 markers has been masked by a counteracting effect mediated by its inhibition of 7* receptors. Alternatively, the lack of effect of bicuculline on surface markers and the marked effect of picrotoxin on 7* currents may reflect the independence of the mechanisms controlling receptor number and function.
Could GABA be involved in the observed effects of KCl and TTX? In this and previous studies in the same preparation (Lopes et al., 2004), we have shown that GABA is spontaneously released in culture. Changes in membrane potential or in the concentration gradients of Na+, Cl-, or GABA can change the set point of the GAT-1 transport system, which fluctuates dynamically between GABA uptake and release modes (Wu et al., 2001; Richerson and Wu, 2003). Increased extracellular KCl is known to cause release of GABA through reverse transport, leading to activation of GABAA receptors in hippocampal neurons (Gaspary et al., 1998; Wu et al., 2001). If the effects of KCl were in part independent of the GABAergic pathway, KCl treatment would be expected to partially overcome the inhibitory effect of picrotoxin; however, a similar reduction of 45% in mean current was seen when the treatments were combined (Figs. 5B and 6D). Thus, although other signaling pathways cannot be excluded, GABA release and GABAA receptor activation suffice to explain the nAChR potentiation seen with KCl.
In conclusion, the present study reveals that GABA can promote the function of 7* nicotinic receptors, which are known to enhance the release of both GABA itself and glutamate in the hippocampus. Within 24 h, 7* responses were significantly enhanced by GABAergic stimulation and reduced by blockade of GABA release (with TTX) or GABAA receptors, suggesting that the GABA-7* control mechanism has a wide dynamic range capable of affecting the system either way upon changes in the basal levels of GABA. It was also found that KCl-induced depolarization markedly increased 7*-gated currents, confirming and extending previous studies that addressed depolarization-dependent changes in 7* receptor binding sites, 7 protein labeling, or mRNA levels. The experimental results do not support a major role of Ca2+ in the effects of KCl, but an alternate mechanism is suggested through depolarization-induced GABA release. The GABA-7* control system described herein may contribute to the complex changes in hippocampal function in epilepsy and in other neurological diseases, with possible implications to therapeutic intervention.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: nAChR, nicotinic acetylcholine receptor(s); APV, d,l-2-amino-5-phosphonovalerate; DHE, dihydro--erythroidine; DNQX, 6,7-dinitroquinoxaline-2,3(1H,4H)-dione; EC50, mean effective concentration; KN-62 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine; GF 109203X, bisindolylmaleimide I; NO-711, 1-[2-[[(diphenylmethylene)imino]oxy]ethyl]-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride; MLA, methyllycaconitine; M-S, Mack-Skillings; NMG, N-methyl-D-glucamine; VDCCs, voltage-dependent calcium channels; AMPA, -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; NMDA, N-methyl-D-aspartate; GAT, GABA transporter; TTX, tetrodotoxin; CNS, central nervous system; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione.
1 Recipients of Conselho Nacional de Desenvolvimento Cientifico e Tecnológico fellowships. 
2 Recipients of Coordenação de Aperfeiçoamento de Pessoal de Nível Superior fellowships.
Address correspondence to: Dr. Edson X. Albuquerque, Department of Pharmacology and Experimental Therapeutics, UMAB, 655 West Baltimore Street, Baltimore, MD 21201-1559. E-mail: ealbuque{at}umaryland.edu
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