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Nicotinic receptors mediate increased GABA release in brain through a tetrodotoxin-insensitive mechanism during prolonged exposure to nicotine
PII: S 0 3 0 6 - 4 5 2 2 ( 0 2 ) 0 0 3 7 1 - 8
Neuroscience Vol. 115, No. 1, pp. 137^144, 2002 Published by Elsevier Science Ltd on behalf of IBRO Printed in Great Britain 0306-4522 / 02 $22.00+0.00
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NICOTINIC RECEPTORS MEDIATE INCREASED GABA RELEASE IN BRAIN THROUGH A TETRODOTOXIN-INSENSITIVE MECHANISM DURING PROLONGED EXPOSURE TO NICOTINE P. J. ZHU and V. A. CHIAPPINELLI Department of Pharmacology, The George Washington University Medical Center, Washington, DC 20037, USA
Abstract0The e¡ects of nicotine on the spontaneous release of GABA from nerve terminals in the chick lateral spiriform nucleus were examined using whole cell patch-clamp recording in brain slices. Exposure to 1 WM nicotine produced an early immediate increase in the frequency of spontaneous postsynaptic GABAergic currents. This e¡ect was blocked in the presence of 0.5 WM tetrodotoxin. However, a prolonged application of 0.1^1 WM nicotine ( s 3 min) caused a tetrodotoxin-insensitive increase in the frequency of spontaneous GABAergic currents. This late tetrodotoxin-insensitive e¡ect was blocked by the nicotinic antagonists dihydro-L-erythroidine (30 WM) and mecamylamine (10 WM), but not by methyllycaconitine (50^100 nM), indicating that activation of high a⁄nity nicotine receptors was mainly responsible for this e¡ect. This enhancement was inhibited by the high threshold Ca2þ channel blocker Cd2þ (100 WM), but not by dantrolene or ryanodine. The tetrodotoxin-insensitive enhancement of the frequency of GABA currents by nicotine was reduced by inhibition of cAMP-dependent protein kinase with HA1004 (30 WM), but not by inhibition of protein kinase C with staurosporine (1 WM), and was facilitated by forskolin (10 WM) or bromo-cAMP (50 WM). The results indicate that nicotine-enhanced GABA release can operate through both tetrodotoxin-sensitive and -insensitive mechanisms in a single brain region and that a second messenger cascade may be involved in the tetrodotoxin-insensitive enhancement by nicotine. Published by Elsevier Science Ltd on behalf of IBRO. Key words: chick lateral spiriform nucleus, cadmium, second messenger cascade.
TTX-sensitive responses are thought to be mediated primarily by receptors located at some distance from the release sites, and hence activation of sodium channels is required to transmit the nAChR-initiated depolarization to the transmitter release sites (Le¤na et al., 1993; McMahon et al., 1994b). The nAChRs mediating these TTX-sensitive responses have been called ‘preterminal’ receptors, in contrast to the ‘presynaptic’ nAChRs involved in TTX-insensitive enhancement of transmitter release. Since this terminology is based on functional studies, it does not provide a precise cellular location for the nAChRs involved in the responses (see Wonnacott, 1997). Nicotinic agonists can increase the frequency of spontaneous postsynaptic GABAergic currents by activating preterminal nAChRs (Le¤na et al., 1993; McMahon et al., 1994b) or presynaptic nAChRs (McMahon et al., 1994a; Le¤na and Changeux, 1997). In the chick SPL, a TTXsensitive nicotinic enhancement of GABA release is mediated by nAChRs with a high a⁄nity for nicotine (McMahon et al., 1994b; Yum et al., 1996). Immunohistochemical studies (Nong et al., 1999) reveal numerous nAChR-positive ¢bers in the chick SPL nucleus, many of which could be located on presynaptic terminals. The aim of this study was to further investigate synaptic mechanisms of nicotine enhancement of transmitter release in chick SPL neurons. Our results indicate that nicotine enhancement of GABA release involves both TTX-sensitive and TTX-insensitive mechanisms. The TTX-insensitive enhancement is only observed after pro-
Numerous subtypes of nicotinic acetylcholine receptors (nAChRs) are present in the CNS and can be distinguished both pharmacologically and functionally (Role and Berg, 1996). Some somatodendritic nAChRs have a postsynaptic functional role in fast synaptic transmission in the rat hippocampus and chick lateral spiriform nucleus (SPL) (Alkondon et al., 1998; Nong et al., 1999). Many other central nAChRs have a presynaptic functional role and mediate enhanced release of various neurotransmitters from nerve terminals. This enhancement of transmitter release has been further divided into two groups, based on sensitivity of the nAChRmediated responses to the sodium channel blocker tetrodotoxin (TTX). Responses that are insensitive to blockade by TTX are generally assumed to be mediated by nAChRs located nearby transmitter release sites (McMahon et al., 1994a; Gray et al., 1996). In contrast,
*Corresponding author. Present address: Laboratory of Molecular and Cellular Neurobiology, NIAAA/NIH, 12420 Parklawn Drive, Park Building/Room 118, Rockville, MD 20852, USA. Tel.: +1-301443-8164; fax: +1-301-480-6882. E-mail address: [email protected] (P. J. Zhu). Abbreviations : K-BgTx, K-bungarotoxin; BIC, bicuculline ; DHLE, dihydro-L-erythroidine; EGTA, ethylene glycol-bis(2-aminoethylether)-N,N,NP,NP-tetraacetic acid; HEPES, N-(2-hydroxyethyl)piperazine-NP-(2-ethanesulfonic acid); MLA, methyllycaconitine ; nAChR, nicotinic acetylcholine receptor ; SPL, lateral spiriform nucleus; TTX, tetrodotoxin; VDCC, voltage-dependent calcium channel. 137
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longed exposure to nicotine and it appears that a second messenger cascade is involved in this process.
EXPERIMENTAL PROCEDURES
Brain slices (400 Wm) containing the SPL were prepared from White Leghorn chick embryos (18 days of incubation). Slices were cut with a Vibroslicer in cold bu¡er bubbled with 95% O2 /5% CO2 . The composition of the external recording bu¡er was (in mM): 126 NaCl, 2.5 KCl, 2.5 CaCl2 , 1.3 MgCl2 , 1.2 Na2 HPO4 , 25 NaHCO3 and 10 glucose. The slice was continuously perfused (4 ml/min) at room temperature (23‡C) in a recording chamber as previously described (Zhu and Chiappinelli, 1999). The SPL neurons were visualized with a
Zeiss Axioskop ¢xed stage upright microscope at 400U for voltage clamp recording. Patch pipettes were made by a two-stage microelectrode puller and had resistances of 4^7 M6 after ¢lling with internal solution containing (in mM): 140 KCl, 10 HEPES, 5.5 EGTA, 2.0 MgCl2 , 2.0 Mg-ATP, and 5 QX314 (added to block fast sodium channels). Conventional patch-clamp techniques were used with an Axoclamp-2A or Axopatch 200B ampli¢er. Holding potential was 360 to 370 mV. The spontaneous postsynaptic currents were recorded with a video cassette recorder. The data were sampled by pClamp7 software through a Digidata 1200 interface. Drugs were applied by bath perfusion using manually controlled valves. TTX was pre-applied for 2^ 3 min and then co-applied with nicotine. Likewise, other antagonists were also pre-applied (15 min) and then co-applied with nicotine when examining their e¡ects on the responses to the agonist. A nicotine-free period of at least 15 min separated consecutive nicotine applications. This was su⁄cient time for
Fig. 1. Identi¢cation of TTX-sensitive and TTX-insensitive enhancements of spontaneous GABAergic events by nicotine. (Aa) Nicotine 1 WM rapidly increased the frequency of spontaneous GABAergic events and produced an inward postsynaptic current. (Ab) In the presence of 0.5 WM TTX, nicotine still produced a sustained inward current, but synaptic event frequency was only increased after several minutes of exposure to nicotine. (Ac) The e¡ect of TTX on nicotinic enhancement was reversible after 90 min of washing out TTX. (B) TTX-sensitive and TTX-insensitive enhancements of synaptic events are shown in detail. (C) The e¡ects of TTX on cumulative distributions of GABAergic event interval. (a) Before TTX, nicotine rapidly increased the frequency of spontaneous GABA events; (b) in the presence of 0.5 WM TTX, nicotine increased GABA event frequency after 4 min exposure, but not after 0.5 min. (c) Recovery of the rapid response to nicotine was observed after washing out TTX. Vh was 370 mV.
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Fig. 2. DHLE (30 WM), a competitive nicotinic receptor antagonist, blocked TTX-insensitive enhancement of spontaneous GABA events induced by nicotine. (A) All traces were obtained in the presence of 0.5 WM TTX. Nicotine (1 WM) produced a delayed enhancement of spontaneous GABAergic currents (left); this response was blocked by DHLE (middle). Recovery of the response to nicotine appears after washing out DHLE (right). (B) Plots of the cumulative distributions of GABA event interval. The reduced spontaneous event interval produced by 4 min exposure to nicotine (a) was prevented by DHLE (b), and recovered partially after 1.5 h washing out DHLE (c). All recordings were from the same neuron and Vh was 370 mV.
full recovery of TTX-sensitive responses. Spontaneous events were analyzed as described previously by McMahon et al. (1994a), using MINI Ver.1.2 software package. Our detection threshold was set at a di/dt of 5 pA/ms, with minimum and maximum rise times set at 0.1 and 10 ms, respectively. The minimal acceptable amplitude for a spontaneous event was 7 pA. The ¢gures were plotted with SigmaPlot. Drugs were obtained as follows: (3)-nicotine bitartrate and bicuculline methiodide (BIC) from Sigma (St. Louis, MO, USA); TTX from Calbiochem (San Diego, CA, USA) and other drugs were from RBI/Sigma (Natick, MA, USA). Data are expressed as mean S S.E.M.; di¡erences between means were examined by the paired t-test unless otherwise indicated, with P 6 0.05 indicating signi¢cance.
RESULTS
Perfusion of slices with arti¢cial cerebrospinal £uid containing the nicotinic agonist nicotine (1 WM) caused an immediate enhancement in the frequency of spontaneous currents and a steady-state inward current in SPL neurons (Fig. 1Aa). These spontaneous GABAergic inward currents were completely blocked by the GABAA receptor antagonist, BIC (McMahon et al., 1994b). In 31 cells tested, nicotine (1 WM) increased the frequency of spontaneous currents by 410 S 43% (mean S S.E.M.) and increased the holding current by 3116 S 6 pA from the control level of 377 S 9 pA. TTX-insensitive enhancement of spontaneous synaptic currents The immediate enhancement of spontaneous GABAer-
gic currents produced by nicotine (Fig. 1Aa) was blocked in the presence of 0.5 WM TTX (Fig. 1Ab). However, nicotine caused a delayed, TTX-insensitive enhancement of spontaneous currents after it was perfused for a prolonged time (usually s 3 min, Fig. 1Ab). To determine the magnitude of the TTX-sensitive and TTX-insensitive nicotinic responses, 60 s sampling at 0.5 min exposure to nicotine was used to assess early immediate responses (which were TTX-sensitive) and unless otherwise indicated, sampling at 4 min was done to assess the e¡ects of prolonged exposure to nicotine (Fig. 1B, C). In 18 cells tested in the presence of TTX (0.5 WM), brief exposure to nicotine (sampling started at 0.5 min exposure) had no signi¢cant e¡ect on the frequency of spontaneous GABAergic current events (di¡ered from control by 10 S 12%; P s 0.05), whereas prolonged exposure to nicotine (4 min) produced a 168 S 21% (P 6 0.001) increase in the frequency of spontaneous GABAergic events. TTX did not prevent nicotine-induced sustained inward current in SPL neurons (inward shift by 148 S 13 pA from the control value of 380 S 9 pA) due to direct activation of nAChRs located on the SPL neuron’s soma/dendrites. Nicotine increased baseline current noise at both 0.5 min and 4 min after nicotine application, consistent with increased nAChR channel activities. A lower concentration of nicotine was also found to increase the frequency of GABAergic spontaneous currents in a TTX-insensitive manner. After 5 min exposure to nicotine (100 nM), the frequency of spontaneous GABAergic events recorded in SPL neurons was increased by 41 S 10% (P 6 0.01, n = 10) with an inward shift of the holding current by 11 S 2 pA.
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Fig. 3. Mecamylamine, but not MLA, blocked the TTX-insensitive increase in the frequency of GABAergic currents. (A) Traces (top) were recorded in the presence of 0.5 WM TTX and 10 WM mecamylamine, an open channel blocker of nAChRs. Nicotine (1 WM) did not produce detectable changes in the intervals of spontaneous GABAergic currents under these conditions. Plots (bottom) show the cumulative distributions of event intervals with and without nicotine for the traces above. GABAergic event numbers were 169 in control, and 164 and 160 in the presence of nicotine at 0.5 min and 4 min, respectively. (B) In the presence of 0.5 WM TTX and 50 nM MLA, a blocker of K7-containing nAChRs, nicotine (1 WM) still produced a delayed enhancement of the frequency of spontaneous GABAergic currents. Plots (bottom) of cumulative distributions show that nicotine decreases the spontaneous event interval only at 4 min of exposure. GABAergic event numbers were 81 in control, and 90 and 188 in the presence of nicotine at 0.5 min and 4 min, respectively.
Pharmacology of the TTX-insensitive component Dihydro-L-erythroidine (DHLE, 30 WM), a competitive nAChR antagonist, blocked the TTX-insensitive enhancement of spontaneous responses induced by nicotine (Fig. 2). In seven cells tested, nicotine (1 WM) for 4 min had no signi¢cant e¡ect on spontaneous GABA events in the presence of DHLE (di¡ered from control by 8 S 4%, P s 0.05). An inward shift in membrane current of 34 S 7 pA (n = 7) was produced by nicotine in the presence of DHLE, which was signi¢cantly smaller (P 6 0.01, group t-test) than the inward shift produced by nicotine alone in the presence of TTX (di¡erence of 3148 S 13 pA, n = 18). The non-competitive nAChR blocker mecamylamine (10 WM), an open channel blocker, also abolished this TTX-insensitive enhancement by nicotine (di¡ered from control by 310 S 7%, n = 6, P s 0.1, Fig. 3A). Mecamylamine fully blocked the nicotine-induced inward shift of holding current (difference of 7 S 4 pA, P s 0.05, n = 6). The SPL expresses a diversity of nAChRs. About 90% of SPL neurons exhibit immunoreactivity to the K2 and/ or K4 subunits, components of high a⁄nity nicotine receptors, and 20% of neurons are immunoreactive to the K7 subunit (Ullian and Sargent, 1995). Presynaptic
K7-containing nAChRs sensitive to K-bungarotoxin (K-BgTx) have been reported to enhance transmitter release in brain (McGehee et al., 1995; Gray et al., 1996; Guo et al., 1998). We used methyllycaconitine (MLA), a blocker of K7-containing nAChRs (Alkondon et al., 1992), to examine if this type of receptor mediates the TTX-insensitive enhancement of GABAergic events by nicotine. The nicotine-induced enhancement of spontaneous GABAergic events persisted in the presence of 50^100 nM MLA (Fig. 3B), which is su⁄cient to block typical K7-containing nAChRs (Alkondon et al., 1992). In seven cells tested in the presence of TTX (0.5 WM) and MLA (50^100 nM), 4 min perfusion with 1 WM nicotine increased spontaneous events by 66 S 22% (P 6 0.01) and caused a 3122 S 18 pA increase in holding current (P 6 0.01). These results indicate that the presynaptic, TTX-insensitive nicotine enhancement of spontaneous GABAergic events in the SPL is mediated primarily by high a⁄nity nicotine receptors. Role of high threshold voltage-dependent calcium channels We next examined whether the TTX-insensitive enhancement of GABAergic currents by nicotine was due to (1) release of Ca2þ from intracellular Ca2þ stores,
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Fig. 4. Blockade of high threshold calcium channels by Cd2þ , but not inhibition of Ca2þ release by ryanodine and dantrolene, prevented TTX-insensitive enhancement of spontaneous GABAergic events by nicotine. (A) (Top) Ryanodine/dantrolene (10/10 WM) did not prevent TTX-insensitive enhancement of spontaneous GABAergic currents by nicotine (1 WM). Plots of data show a decreased GABAergic event interval at 4 min (bottom). GABAergic event numbers were 80 for control, and 89 and 175 at 0.5 or 4 min nicotine application, respectively. (B) Traces (top) were recorded in the presence of 0.5 WM TTX and 100 WM Cd2þ . Nicotine (1 WM) did not have a signi¢cant e¡ect on spontaneous GABAergic event interval (bottom). GABAergic event numbers were 48 for control, and 58 and 60 at 0.5 or 4 min nicotine application, respectively.
(2) depolarization of neurons and subsequent opening of high threshold calcium channels or (3) Ca2þ £ux directly through nAChRs. In the presence of ryanodine (10 WM) and dantrolene (10 WM), 1 WM nicotine (4 min exposure) still produced a 103 S 33% increase (P 6 0.05, n = 6) in the frequency of spontaneous GABAergic events (Fig. 4A) which was not signi¢cantly di¡erent from prolonged nicotine plus TTX (P s 0.05, group t-test). In contrast, Cd2þ (100 WM), a blocker of high threshold calcium channels, prevented the enhancement of spontaneous events following prolonged application of nicotine (Fig. 4B), but did not suppress the inward shift of holding current in SPL neurons. In eight cells tested, 4 min exposure to nicotine produced a 3227 S 25 pA increase in holding current (P 6 0.001), but had no signi¢cant e¡ect (di¡ered from control by 8 S 10%, P s 0.4) on the frequency of GABAergic spontaneous events.
presence of 1 WM staurosporine (P 6 0.01, Fig. 5B). However, 30 WM HA1004, a blocker of cAMP-dependent protein kinase (Yang et al., 1996; Lee et al., 1995), prevented the nicotine-induced enhancement (differed from control by 34.4 S 5.6% after 4 min exposure to nicotine, P s 0.3, n = 7, Fig. 5A). Furthermore, treatment with forskolin (10 WM) or bromo-cAMP (50 WM), both of which should increase intracellular cAMP, facilitated nicotine-induced TTX-insensitive enhancement of GABAergic currents (Fig. 6). In the presence of TTX and either forskolin or bromo-cAMP, a 0.5 min exposure to nicotine produced an increase in the frequency of spontaneous GABAergic events of 66 S 20% (P 6 0.03, n = 5) and 32 S 2.2% (P 6 0.001, n = 5), respectively.
DISCUSSION
The role of second messenger cascades in the TTX-insensitive enhancement of spontaneous currents When SPL neurons were exposed to nicotine in the presence of TTX, the induced sustained inward current preceded the TTX-insensitive enhancement of GABAergic events (Fig. 1). This delay suggests that a second messenger cascade might be involved in the TTX-insensitive increase in GABA events. Staurosporine, an inhibitor of protein kinase C (Sauve et al., 1999; Freyssenet et al., 1999; Gao et al., 1999), did not inhibit the TTXinsensitive enhancement. In three cells tested, 4 min exposure to nicotine produced a 210 S 6% increase in the frequency of spontaneous GABAergic events in the
The present study demonstrates that both TTX-sensitive and TTX-insensitive enhancements of spontaneous GABAergic currents are produced by nicotine in SPL neurons. The TTX-insensitive enhancement of spontaneous GABAergic events was produced only after prolonged exposure to nicotine. The data indicate that in the SPL, nAChRs are di¡erentially distributed on neurons and may have distinct roles in modulating synaptic function. The blockade of high threshold calcium channels abolished the TTX-insensitive enhancement. Furthermore, this delayed enhancement was suppressed following the inhibition of cAMP-dependent protein kinase and was facilitated by agents that increase the intra-
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(McMahon et al., 1994b) and as presynaptic receptors. The preterminal nAChRs mediate an early immediate enhancement of GABA release (McMahon et al., 1994b, and present study), while activation of presynaptic nAChRs is responsible for a delayed and more persistent enhancement of transmitter release. High threshold voltage-dependent calcium channels (VDCCs)
Fig. 5. HA1004 (30 WM), but not staurosporine (1 WM), suppressed TTX-insensitive enhancement of spontaneous GABAergic current by nicotine. All recordings were obtained in the presence of 0.5 WM TTX. (A) (Top) The normal delayed enhancement of GABAergic currents by 1 WM nicotine was suppressed by the cAMP/cGMP-dependent protein kinase inhibitor, HA1004. (Bottom) Cumulative distributions of GABAergic event intervals. (B) Staurosporine (1 WM) did not signi¢cantly alter the TTX-insensitive enhancement produced by 1 WM nicotine. Similar arrangement to A: (top) chart recording trace ; (bottom) cumulative distribution of GABAergic event intervals. A and B are from di¡erent neurons.
Two distinct mechanisms have been proposed for nicotine-induced enhancement of transmitter release. In the ¢rst, enhanced release is due to an increased Ca2þ in£ux directly through open nAChR channels, such as K7-containing nAChRs having a high permeability for Ca2þ (McGehee et al., 1995; Gray et al., 1996). In the second mechanism, enhanced release is due to Ca2þ in£ux through VDCCs that open following the depolarization initiated by activated nAChRs. In a previous study (Tredway et al., 1999), functional high threshold N-type VDCCs were required for the immediate, TTXsensitive enhancement of GABA release produced by nicotine in the SPL. In the present study the delayed, TTX-insensitive enhancement of GABA release by nicotine was also dependent on functional calcium channels. Furthermore, Cd2þ (100 WM) blocked this enhancement without reducing the nicotine-mediated postsynaptic inward current in SPL neurons, suggesting that Cd2þ did not directly alter nAChR function. Thus, the nicotine-induced TTX-insensitive enhancement of GABA release in the present study appears to be due to secondary activation of VDCCs following depolarization of GABAergic terminals by activated nAChRs. Release of Ca2þ from intracellular stores has been shown to modulate transmitter release (Cochilla and Alford, 1998; Wang and Kelly, 1997; Peng, 1996). However, in the SPL it appears that the release of Ca2þ from intracellular stores does not play a major role in the TTX-insensitive enhancement of GABA release by nicotine, since neither ryanodine nor dantrolene prevented the enhancement of GABA release. Second messenger cascade
cellular level of cAMP, indicating a second messenger cascade was likely involved. Presynaptic nAChRs Nicotinic receptors at presynaptic sites can modulate synaptic transmission by regulating transmitter release. A well-established example is nicotine-induced enhancement of excitatory transmission in the rat hippocampus, which is TTX-insensitive. The enhancement is blocked by K-BgTx or 10 nM MLA, indicating that the nAChRs responsible are K7-containing (McGehee et al., 1995; Gray et al., 1996). In contrast, the presynaptic enhancement of GABA release by nicotine in the SPL was mediated primarily through activation of non-K7-containing nAChRs, since they were blocked by DHLE, but not by 100 nM MLA. Therefore in the SPL, high a⁄nity nicotine receptors function both as preterminal receptors
A striking feature of the TTX-insensitive enhancement of GABA release is that it is delayed by several minutes after nicotine exposure. One explanation for this delay could be that the number of nAChRs on the presynaptic terminals is low, and activation of these receptors may not immediately depolarize the terminal su⁄ciently to open voltage-dependent Ca2þ channels. A problem with this explanation is that the nicotine-induced postsynaptic current reaches steady-state before the enhancement of spontaneous GABA currents begins. Further, prolonged application of nicotine may actually reduce, not enhance, nicotinic channel function due to desensitization. A second and more likely explanation for the delay would be that second messenger pathways are involved. In the present study, TTX-insensitive enhancement of GABA release by nicotine was reduced by an inhibitor of cAMP-dependent protein kinase and facilitated by
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Fig. 6. E¡ects of forskolin and Br-cAMP on TTX-insensitive enhancement of GABAergic events produced by nicotine. Similar arrangement in both A and B. (Top) Forskolin 10 WM (A) or 50 WM Br-cAMP (B) facilitated TTX-insensitive enhancement of GABAergic events by nicotine (1 WM); (bottom) cumulative distributions of GABAergic event intervals. In the presence of 0.5 WM TTX+10 WM forskolin (A) or 50 WM Br-cAMP (B), nicotine (1 WM) decreased the spontaneous GABAergic event interval after 0.5 min and 4 min of exposure. (A) Number of GABAergic events was 64 under control, 116 and 135 at 0.5 min or 4 min nicotine application, respectively. (B) Number of GABAergic events was 71 under control, 107 and 139 at 0.5 min or 4 min nicotine application, respectively. A and B are from di¡erent neurons.
agents that are known to increase intracellular cAMP. Nicotine increases intracellular cAMP and activates protein kinase A in adrenal medullary cells (Anderson et al., 1992; Marley et al., 1995). Activation of protein kinase A has been shown to positively modulate VDCCs (Gray et al., 1998; Solem et al., 1997; Hell et al., 1995; Pfei¡erLinn and Lasater, 1998; Fukuda et al., 1996). Therefore, a cAMP-dependent cascade may be involved in the TTXinsensitive enhancement of GABA release by nicotine in the SPL. Protein kinase A activation also phosphorylates the nAChR itself (Vijayaraghavan et al., 1990; Huganir et al., 1986), which could up-regulate nicotinic receptor function and contribute to the observed TTX-insensitive enhancement of GABA release. In summary, a possible sequence of events underlying the TTX-insensitive actions of nicotine in the SPL can be advanced. First, nicotine activates high a⁄nity nAChRs
located on GABAergic neurons that form synapses with SPL neurons. Cation £ux through these activated nAChRs produces local membrane depolarizations that activate nearby high threshold VDCCs. The resulting increase in intracellular Ca2þ leads to enhanced activity of protein kinase A. Protein kinase A then phosphorylates VDCCs and/or nAChRs, thereby further enhancing the entry of Ca2þ into the terminal region, which causes the observed increase in release of GABA at synapses on SPL neurons. This enhanced GABA release occurs at a concentration of nicotine (100 nM) that is regularly attained by human cigarette smokers.
Acknowledgements(This research was supported by Grant NS17574 from the National Institute of Neurological Disorders and Stroke to V.A.C.
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Gao, J., Mathias, R.T., Cohen, I.S., Wang, Y., Sun, X., Baldo, G.J., 1999. Activation of PLC increases Naþ -Kþ pump current in ventricular myocytes from guinea pig heart. P£u«gers Arch. 437, 643^652. Gray, P.C., Johnson, B.D., Westenbroek, R.E., Yates, J.R., III, Scheuer, T., Catterall, W.A., Murphy, B.J., 1998. Primary structure and function of an kinase anchoring protein associated with calcium channels. Neuron 20, 1017^1026. Gray, R., Rajan, A.S., Radcli¡e, K.A., Yakehiro, M., Dani, J.A., 1996. Hippocampal synaptic transmission enhanced by low concentration of nicotine. Nature 383, 713^716. Guo, J.Z., Tredway, T.L., Chiappinelli, V.A., 1998. Glutamate and GABA release are enhanced by di¡erent subtypes of presynaptic nicotinic receptors in the lateral geniculate nucleus. J. Neurosci. 18, 1963^1969. Hell, J.W., Yaokoyama, C.T., Breeze, L.J., Chavkin, C., Catterall, W.A., 1995. Phosphorylation of presynaptic and postsynaptic calcium channels by cAMP-dependent protein kinase in hippocampal neurons. EMBO J. 14, 3036^3044. Huganir, R.L., Delcour, A.H., Greengard, P., Hess, G.P., 1986. Phosphorylation of the nicotinic acetylcholine receptor regulates its rate of desensitization. Nature 321, 774^776. Lee, A.D., Gulve, E.A., Chen, M., Schluter, J., Holloszy, J.O., 1995. E¡ects of Ca2þ ionophore ionomycin on insulin-stimulated and basal glucose transport in muscle. Am. J. Physiol. 268, r997^1002. Le¤na, C., Changeux, J.-P., 1997. Role of Ca2þ ions in nicotinic facilitation of GABA release in mouse thalamus. J. Neurosci. 17, 576^585. Le¤na, C., Changeux, J.-P., Mulle, C., 1993. Evidence for ‘preterminal’ nicotinic receptors on GABAergic axons in the rat interpeduncular nucleus. J. Neurosci. 13, 2680^2688. Marley, P.D., Thomson, K.A., Bralow, R.A., 1995. Protein kinase A and nicotinic activation of bovine adrenal tyrosine hydroxylase. Br. J. Pharmacol. 114, 1687^1693. McGehee, D.S., Heath, M.J.S., Gelbar, S., Devay, P., Role, L.W., 1995. Nicotine enhancement of fast excitatory synaptic transmission in CNS by presynaptic receptors. Science 269, 1692^1696. McMahon, L.L., Yoon, K.-W., Chiappinelli, V.A., 1994a. Electrophysiological evidence for presynaptic nicotinic receptors in the avian ventral lateral geniculate nucleus. J. Neurophysiol. 71, 826^829. McMahon, L.L., Yoon, K.-W., Chiappinelli, V.A., 1994b. Nicotinic receptor activation facilitates GABAergic neurotransmission in the avian lateral spiriform nucleus. Neuroscience 59, 689^698. Nong, Y., Sorenson, E.M., Chiappinelli, V.A., 1999. Fast excitatory nicotinic transmission in the chick lateral spiriform nucleus. J. Neurosci. 19, 7804^7811. Peng, Y., 1996. Ryanodine-sensitive component of calcium transients evoked by nerve ¢ring at presynaptic nerve terminals. J. Neurosci. 16, 6703^ 6712. Pfei¡er-Linn, C.L., Lasater, E.M., 1998. Multiple second-messenger system modulation of voltage-activated calcium currents in teleost retinal horizontal cells. J. Neurophysol. 80, 377^388. Role, L.W., Berg, D.K., 1996. Nicotinic receptors in the development and modulation of CNS synapses. Neuron 16, 1077^1085. Sauve, D.M., Anderson, H.J., Ray, J.M., James, W.M., Roberge, M., 1999. Phosphorylation-induced rearrangement of the histone H3 NH2 terminal domain during mitotic chromosome condensation. J. Cell Biol. 145, 225^235. Solem, M., McMahon, T., Messing, R.O., 1997. Protein kinase A regulates inhibition of N- and P/Q-type calcium channels by ethanol in PC12 cells. J. Pharmacol. Exp. Ther. 282, 1487^1495. Tredway, T.L., Guo, J.Z., Chiappinelli, V.A., 1999. N-type voltage-dependent calcium channels mediate the nicotinic enhancement of GABA release in chick brain. J. Neurophysiol. 81, 447^454. Ullian, E.M., Sargent, P.B., 1995. Pronounced cellular diversity and extrasynaptic location of nicotinic acetylcholine receptor subunit immunoreactivities in the chicken pretectum. J. Neurosci. 15, 7012^7023. Vijayaraghavan, S., Schmid, H.A., Halvorsen, S.W., Berg, D.K., 1990. Cyclic AMP-dependent phosphorylation of a neuronal acetylcholine receptor alpha-type subunit. J. Neurosci. 10, 3255^3262. Wang, J.H., Kelly, P.T., 1997. Attenuation of paired-pulse facilitation associated with synaptic potentiation mediated by postsynaptic mechanisms. J. Neurophysiol. 78, 2707^2716. Wonnacott, S., 1997. Presynaptic nicotinic ACh receptors. Trends Neurosci. 20, 92^98. Yang, C., Tsao, H.L., Chiu, C.T., Fan, L.W., Yu, S.M., 1996. Regulation of 5-hydroxytryptamine-induced calcium mobilization by cAMPelevating agents in cultured canine tracheal smooth muscle cells. P£u«gers Arch. 432, 708^716. Yum, L., Wolf, K.M., Chiappinelli, V.A., 1996. Nicotinic acetylcholine receptors in separate brain regions exhibit di¡erent a⁄nities for methyllycaconitine. Neuroscience 72, 545^555. Zhu, P.J., Chiappinelli, V.A., 1999. Nicotine modulates GABAergic transmission in the brain. J. Neurophysiol. 82, 3041^3045. (Accepted 18 June 2002)
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Neuroscience Vol. 115, No. 1, pp. 137^144, 2002 Published by Elsevier Science Ltd on behalf of IBRO Printed in Great Britain 0306-4522 / 02 $22.00+0.00
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NICOTINIC RECEPTORS MEDIATE INCREASED GABA RELEASE IN BRAIN THROUGH A TETRODOTOXIN-INSENSITIVE MECHANISM DURING PROLONGED EXPOSURE TO NICOTINE P. J. ZHU and V. A. CHIAPPINELLI Department of Pharmacology, The George Washington University Medical Center, Washington, DC 20037, USA
Abstract0The e¡ects of nicotine on the spontaneous release of GABA from nerve terminals in the chick lateral spiriform nucleus were examined using whole cell patch-clamp recording in brain slices. Exposure to 1 WM nicotine produced an early immediate increase in the frequency of spontaneous postsynaptic GABAergic currents. This e¡ect was blocked in the presence of 0.5 WM tetrodotoxin. However, a prolonged application of 0.1^1 WM nicotine ( s 3 min) caused a tetrodotoxin-insensitive increase in the frequency of spontaneous GABAergic currents. This late tetrodotoxin-insensitive e¡ect was blocked by the nicotinic antagonists dihydro-L-erythroidine (30 WM) and mecamylamine (10 WM), but not by methyllycaconitine (50^100 nM), indicating that activation of high a⁄nity nicotine receptors was mainly responsible for this e¡ect. This enhancement was inhibited by the high threshold Ca2þ channel blocker Cd2þ (100 WM), but not by dantrolene or ryanodine. The tetrodotoxin-insensitive enhancement of the frequency of GABA currents by nicotine was reduced by inhibition of cAMP-dependent protein kinase with HA1004 (30 WM), but not by inhibition of protein kinase C with staurosporine (1 WM), and was facilitated by forskolin (10 WM) or bromo-cAMP (50 WM). The results indicate that nicotine-enhanced GABA release can operate through both tetrodotoxin-sensitive and -insensitive mechanisms in a single brain region and that a second messenger cascade may be involved in the tetrodotoxin-insensitive enhancement by nicotine. Published by Elsevier Science Ltd on behalf of IBRO. Key words: chick lateral spiriform nucleus, cadmium, second messenger cascade.
TTX-sensitive responses are thought to be mediated primarily by receptors located at some distance from the release sites, and hence activation of sodium channels is required to transmit the nAChR-initiated depolarization to the transmitter release sites (Le¤na et al., 1993; McMahon et al., 1994b). The nAChRs mediating these TTX-sensitive responses have been called ‘preterminal’ receptors, in contrast to the ‘presynaptic’ nAChRs involved in TTX-insensitive enhancement of transmitter release. Since this terminology is based on functional studies, it does not provide a precise cellular location for the nAChRs involved in the responses (see Wonnacott, 1997). Nicotinic agonists can increase the frequency of spontaneous postsynaptic GABAergic currents by activating preterminal nAChRs (Le¤na et al., 1993; McMahon et al., 1994b) or presynaptic nAChRs (McMahon et al., 1994a; Le¤na and Changeux, 1997). In the chick SPL, a TTXsensitive nicotinic enhancement of GABA release is mediated by nAChRs with a high a⁄nity for nicotine (McMahon et al., 1994b; Yum et al., 1996). Immunohistochemical studies (Nong et al., 1999) reveal numerous nAChR-positive ¢bers in the chick SPL nucleus, many of which could be located on presynaptic terminals. The aim of this study was to further investigate synaptic mechanisms of nicotine enhancement of transmitter release in chick SPL neurons. Our results indicate that nicotine enhancement of GABA release involves both TTX-sensitive and TTX-insensitive mechanisms. The TTX-insensitive enhancement is only observed after pro-
Numerous subtypes of nicotinic acetylcholine receptors (nAChRs) are present in the CNS and can be distinguished both pharmacologically and functionally (Role and Berg, 1996). Some somatodendritic nAChRs have a postsynaptic functional role in fast synaptic transmission in the rat hippocampus and chick lateral spiriform nucleus (SPL) (Alkondon et al., 1998; Nong et al., 1999). Many other central nAChRs have a presynaptic functional role and mediate enhanced release of various neurotransmitters from nerve terminals. This enhancement of transmitter release has been further divided into two groups, based on sensitivity of the nAChRmediated responses to the sodium channel blocker tetrodotoxin (TTX). Responses that are insensitive to blockade by TTX are generally assumed to be mediated by nAChRs located nearby transmitter release sites (McMahon et al., 1994a; Gray et al., 1996). In contrast,
*Corresponding author. Present address: Laboratory of Molecular and Cellular Neurobiology, NIAAA/NIH, 12420 Parklawn Drive, Park Building/Room 118, Rockville, MD 20852, USA. Tel.: +1-301443-8164; fax: +1-301-480-6882. E-mail address: [email protected] (P. J. Zhu). Abbreviations : K-BgTx, K-bungarotoxin; BIC, bicuculline ; DHLE, dihydro-L-erythroidine; EGTA, ethylene glycol-bis(2-aminoethylether)-N,N,NP,NP-tetraacetic acid; HEPES, N-(2-hydroxyethyl)piperazine-NP-(2-ethanesulfonic acid); MLA, methyllycaconitine ; nAChR, nicotinic acetylcholine receptor ; SPL, lateral spiriform nucleus; TTX, tetrodotoxin; VDCC, voltage-dependent calcium channel. 137
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longed exposure to nicotine and it appears that a second messenger cascade is involved in this process.
EXPERIMENTAL PROCEDURES
Brain slices (400 Wm) containing the SPL were prepared from White Leghorn chick embryos (18 days of incubation). Slices were cut with a Vibroslicer in cold bu¡er bubbled with 95% O2 /5% CO2 . The composition of the external recording bu¡er was (in mM): 126 NaCl, 2.5 KCl, 2.5 CaCl2 , 1.3 MgCl2 , 1.2 Na2 HPO4 , 25 NaHCO3 and 10 glucose. The slice was continuously perfused (4 ml/min) at room temperature (23‡C) in a recording chamber as previously described (Zhu and Chiappinelli, 1999). The SPL neurons were visualized with a
Zeiss Axioskop ¢xed stage upright microscope at 400U for voltage clamp recording. Patch pipettes were made by a two-stage microelectrode puller and had resistances of 4^7 M6 after ¢lling with internal solution containing (in mM): 140 KCl, 10 HEPES, 5.5 EGTA, 2.0 MgCl2 , 2.0 Mg-ATP, and 5 QX314 (added to block fast sodium channels). Conventional patch-clamp techniques were used with an Axoclamp-2A or Axopatch 200B ampli¢er. Holding potential was 360 to 370 mV. The spontaneous postsynaptic currents were recorded with a video cassette recorder. The data were sampled by pClamp7 software through a Digidata 1200 interface. Drugs were applied by bath perfusion using manually controlled valves. TTX was pre-applied for 2^ 3 min and then co-applied with nicotine. Likewise, other antagonists were also pre-applied (15 min) and then co-applied with nicotine when examining their e¡ects on the responses to the agonist. A nicotine-free period of at least 15 min separated consecutive nicotine applications. This was su⁄cient time for
Fig. 1. Identi¢cation of TTX-sensitive and TTX-insensitive enhancements of spontaneous GABAergic events by nicotine. (Aa) Nicotine 1 WM rapidly increased the frequency of spontaneous GABAergic events and produced an inward postsynaptic current. (Ab) In the presence of 0.5 WM TTX, nicotine still produced a sustained inward current, but synaptic event frequency was only increased after several minutes of exposure to nicotine. (Ac) The e¡ect of TTX on nicotinic enhancement was reversible after 90 min of washing out TTX. (B) TTX-sensitive and TTX-insensitive enhancements of synaptic events are shown in detail. (C) The e¡ects of TTX on cumulative distributions of GABAergic event interval. (a) Before TTX, nicotine rapidly increased the frequency of spontaneous GABA events; (b) in the presence of 0.5 WM TTX, nicotine increased GABA event frequency after 4 min exposure, but not after 0.5 min. (c) Recovery of the rapid response to nicotine was observed after washing out TTX. Vh was 370 mV.
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Fig. 2. DHLE (30 WM), a competitive nicotinic receptor antagonist, blocked TTX-insensitive enhancement of spontaneous GABA events induced by nicotine. (A) All traces were obtained in the presence of 0.5 WM TTX. Nicotine (1 WM) produced a delayed enhancement of spontaneous GABAergic currents (left); this response was blocked by DHLE (middle). Recovery of the response to nicotine appears after washing out DHLE (right). (B) Plots of the cumulative distributions of GABA event interval. The reduced spontaneous event interval produced by 4 min exposure to nicotine (a) was prevented by DHLE (b), and recovered partially after 1.5 h washing out DHLE (c). All recordings were from the same neuron and Vh was 370 mV.
full recovery of TTX-sensitive responses. Spontaneous events were analyzed as described previously by McMahon et al. (1994a), using MINI Ver.1.2 software package. Our detection threshold was set at a di/dt of 5 pA/ms, with minimum and maximum rise times set at 0.1 and 10 ms, respectively. The minimal acceptable amplitude for a spontaneous event was 7 pA. The ¢gures were plotted with SigmaPlot. Drugs were obtained as follows: (3)-nicotine bitartrate and bicuculline methiodide (BIC) from Sigma (St. Louis, MO, USA); TTX from Calbiochem (San Diego, CA, USA) and other drugs were from RBI/Sigma (Natick, MA, USA). Data are expressed as mean S S.E.M.; di¡erences between means were examined by the paired t-test unless otherwise indicated, with P 6 0.05 indicating signi¢cance.
RESULTS
Perfusion of slices with arti¢cial cerebrospinal £uid containing the nicotinic agonist nicotine (1 WM) caused an immediate enhancement in the frequency of spontaneous currents and a steady-state inward current in SPL neurons (Fig. 1Aa). These spontaneous GABAergic inward currents were completely blocked by the GABAA receptor antagonist, BIC (McMahon et al., 1994b). In 31 cells tested, nicotine (1 WM) increased the frequency of spontaneous currents by 410 S 43% (mean S S.E.M.) and increased the holding current by 3116 S 6 pA from the control level of 377 S 9 pA. TTX-insensitive enhancement of spontaneous synaptic currents The immediate enhancement of spontaneous GABAer-
gic currents produced by nicotine (Fig. 1Aa) was blocked in the presence of 0.5 WM TTX (Fig. 1Ab). However, nicotine caused a delayed, TTX-insensitive enhancement of spontaneous currents after it was perfused for a prolonged time (usually s 3 min, Fig. 1Ab). To determine the magnitude of the TTX-sensitive and TTX-insensitive nicotinic responses, 60 s sampling at 0.5 min exposure to nicotine was used to assess early immediate responses (which were TTX-sensitive) and unless otherwise indicated, sampling at 4 min was done to assess the e¡ects of prolonged exposure to nicotine (Fig. 1B, C). In 18 cells tested in the presence of TTX (0.5 WM), brief exposure to nicotine (sampling started at 0.5 min exposure) had no signi¢cant e¡ect on the frequency of spontaneous GABAergic current events (di¡ered from control by 10 S 12%; P s 0.05), whereas prolonged exposure to nicotine (4 min) produced a 168 S 21% (P 6 0.001) increase in the frequency of spontaneous GABAergic events. TTX did not prevent nicotine-induced sustained inward current in SPL neurons (inward shift by 148 S 13 pA from the control value of 380 S 9 pA) due to direct activation of nAChRs located on the SPL neuron’s soma/dendrites. Nicotine increased baseline current noise at both 0.5 min and 4 min after nicotine application, consistent with increased nAChR channel activities. A lower concentration of nicotine was also found to increase the frequency of GABAergic spontaneous currents in a TTX-insensitive manner. After 5 min exposure to nicotine (100 nM), the frequency of spontaneous GABAergic events recorded in SPL neurons was increased by 41 S 10% (P 6 0.01, n = 10) with an inward shift of the holding current by 11 S 2 pA.
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Fig. 3. Mecamylamine, but not MLA, blocked the TTX-insensitive increase in the frequency of GABAergic currents. (A) Traces (top) were recorded in the presence of 0.5 WM TTX and 10 WM mecamylamine, an open channel blocker of nAChRs. Nicotine (1 WM) did not produce detectable changes in the intervals of spontaneous GABAergic currents under these conditions. Plots (bottom) show the cumulative distributions of event intervals with and without nicotine for the traces above. GABAergic event numbers were 169 in control, and 164 and 160 in the presence of nicotine at 0.5 min and 4 min, respectively. (B) In the presence of 0.5 WM TTX and 50 nM MLA, a blocker of K7-containing nAChRs, nicotine (1 WM) still produced a delayed enhancement of the frequency of spontaneous GABAergic currents. Plots (bottom) of cumulative distributions show that nicotine decreases the spontaneous event interval only at 4 min of exposure. GABAergic event numbers were 81 in control, and 90 and 188 in the presence of nicotine at 0.5 min and 4 min, respectively.
Pharmacology of the TTX-insensitive component Dihydro-L-erythroidine (DHLE, 30 WM), a competitive nAChR antagonist, blocked the TTX-insensitive enhancement of spontaneous responses induced by nicotine (Fig. 2). In seven cells tested, nicotine (1 WM) for 4 min had no signi¢cant e¡ect on spontaneous GABA events in the presence of DHLE (di¡ered from control by 8 S 4%, P s 0.05). An inward shift in membrane current of 34 S 7 pA (n = 7) was produced by nicotine in the presence of DHLE, which was signi¢cantly smaller (P 6 0.01, group t-test) than the inward shift produced by nicotine alone in the presence of TTX (di¡erence of 3148 S 13 pA, n = 18). The non-competitive nAChR blocker mecamylamine (10 WM), an open channel blocker, also abolished this TTX-insensitive enhancement by nicotine (di¡ered from control by 310 S 7%, n = 6, P s 0.1, Fig. 3A). Mecamylamine fully blocked the nicotine-induced inward shift of holding current (difference of 7 S 4 pA, P s 0.05, n = 6). The SPL expresses a diversity of nAChRs. About 90% of SPL neurons exhibit immunoreactivity to the K2 and/ or K4 subunits, components of high a⁄nity nicotine receptors, and 20% of neurons are immunoreactive to the K7 subunit (Ullian and Sargent, 1995). Presynaptic
K7-containing nAChRs sensitive to K-bungarotoxin (K-BgTx) have been reported to enhance transmitter release in brain (McGehee et al., 1995; Gray et al., 1996; Guo et al., 1998). We used methyllycaconitine (MLA), a blocker of K7-containing nAChRs (Alkondon et al., 1992), to examine if this type of receptor mediates the TTX-insensitive enhancement of GABAergic events by nicotine. The nicotine-induced enhancement of spontaneous GABAergic events persisted in the presence of 50^100 nM MLA (Fig. 3B), which is su⁄cient to block typical K7-containing nAChRs (Alkondon et al., 1992). In seven cells tested in the presence of TTX (0.5 WM) and MLA (50^100 nM), 4 min perfusion with 1 WM nicotine increased spontaneous events by 66 S 22% (P 6 0.01) and caused a 3122 S 18 pA increase in holding current (P 6 0.01). These results indicate that the presynaptic, TTX-insensitive nicotine enhancement of spontaneous GABAergic events in the SPL is mediated primarily by high a⁄nity nicotine receptors. Role of high threshold voltage-dependent calcium channels We next examined whether the TTX-insensitive enhancement of GABAergic currents by nicotine was due to (1) release of Ca2þ from intracellular Ca2þ stores,
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Fig. 4. Blockade of high threshold calcium channels by Cd2þ , but not inhibition of Ca2þ release by ryanodine and dantrolene, prevented TTX-insensitive enhancement of spontaneous GABAergic events by nicotine. (A) (Top) Ryanodine/dantrolene (10/10 WM) did not prevent TTX-insensitive enhancement of spontaneous GABAergic currents by nicotine (1 WM). Plots of data show a decreased GABAergic event interval at 4 min (bottom). GABAergic event numbers were 80 for control, and 89 and 175 at 0.5 or 4 min nicotine application, respectively. (B) Traces (top) were recorded in the presence of 0.5 WM TTX and 100 WM Cd2þ . Nicotine (1 WM) did not have a signi¢cant e¡ect on spontaneous GABAergic event interval (bottom). GABAergic event numbers were 48 for control, and 58 and 60 at 0.5 or 4 min nicotine application, respectively.
(2) depolarization of neurons and subsequent opening of high threshold calcium channels or (3) Ca2þ £ux directly through nAChRs. In the presence of ryanodine (10 WM) and dantrolene (10 WM), 1 WM nicotine (4 min exposure) still produced a 103 S 33% increase (P 6 0.05, n = 6) in the frequency of spontaneous GABAergic events (Fig. 4A) which was not signi¢cantly di¡erent from prolonged nicotine plus TTX (P s 0.05, group t-test). In contrast, Cd2þ (100 WM), a blocker of high threshold calcium channels, prevented the enhancement of spontaneous events following prolonged application of nicotine (Fig. 4B), but did not suppress the inward shift of holding current in SPL neurons. In eight cells tested, 4 min exposure to nicotine produced a 3227 S 25 pA increase in holding current (P 6 0.001), but had no signi¢cant e¡ect (di¡ered from control by 8 S 10%, P s 0.4) on the frequency of GABAergic spontaneous events.
presence of 1 WM staurosporine (P 6 0.01, Fig. 5B). However, 30 WM HA1004, a blocker of cAMP-dependent protein kinase (Yang et al., 1996; Lee et al., 1995), prevented the nicotine-induced enhancement (differed from control by 34.4 S 5.6% after 4 min exposure to nicotine, P s 0.3, n = 7, Fig. 5A). Furthermore, treatment with forskolin (10 WM) or bromo-cAMP (50 WM), both of which should increase intracellular cAMP, facilitated nicotine-induced TTX-insensitive enhancement of GABAergic currents (Fig. 6). In the presence of TTX and either forskolin or bromo-cAMP, a 0.5 min exposure to nicotine produced an increase in the frequency of spontaneous GABAergic events of 66 S 20% (P 6 0.03, n = 5) and 32 S 2.2% (P 6 0.001, n = 5), respectively.
DISCUSSION
The role of second messenger cascades in the TTX-insensitive enhancement of spontaneous currents When SPL neurons were exposed to nicotine in the presence of TTX, the induced sustained inward current preceded the TTX-insensitive enhancement of GABAergic events (Fig. 1). This delay suggests that a second messenger cascade might be involved in the TTX-insensitive increase in GABA events. Staurosporine, an inhibitor of protein kinase C (Sauve et al., 1999; Freyssenet et al., 1999; Gao et al., 1999), did not inhibit the TTXinsensitive enhancement. In three cells tested, 4 min exposure to nicotine produced a 210 S 6% increase in the frequency of spontaneous GABAergic events in the
The present study demonstrates that both TTX-sensitive and TTX-insensitive enhancements of spontaneous GABAergic currents are produced by nicotine in SPL neurons. The TTX-insensitive enhancement of spontaneous GABAergic events was produced only after prolonged exposure to nicotine. The data indicate that in the SPL, nAChRs are di¡erentially distributed on neurons and may have distinct roles in modulating synaptic function. The blockade of high threshold calcium channels abolished the TTX-insensitive enhancement. Furthermore, this delayed enhancement was suppressed following the inhibition of cAMP-dependent protein kinase and was facilitated by agents that increase the intra-
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(McMahon et al., 1994b) and as presynaptic receptors. The preterminal nAChRs mediate an early immediate enhancement of GABA release (McMahon et al., 1994b, and present study), while activation of presynaptic nAChRs is responsible for a delayed and more persistent enhancement of transmitter release. High threshold voltage-dependent calcium channels (VDCCs)
Fig. 5. HA1004 (30 WM), but not staurosporine (1 WM), suppressed TTX-insensitive enhancement of spontaneous GABAergic current by nicotine. All recordings were obtained in the presence of 0.5 WM TTX. (A) (Top) The normal delayed enhancement of GABAergic currents by 1 WM nicotine was suppressed by the cAMP/cGMP-dependent protein kinase inhibitor, HA1004. (Bottom) Cumulative distributions of GABAergic event intervals. (B) Staurosporine (1 WM) did not signi¢cantly alter the TTX-insensitive enhancement produced by 1 WM nicotine. Similar arrangement to A: (top) chart recording trace ; (bottom) cumulative distribution of GABAergic event intervals. A and B are from di¡erent neurons.
Two distinct mechanisms have been proposed for nicotine-induced enhancement of transmitter release. In the ¢rst, enhanced release is due to an increased Ca2þ in£ux directly through open nAChR channels, such as K7-containing nAChRs having a high permeability for Ca2þ (McGehee et al., 1995; Gray et al., 1996). In the second mechanism, enhanced release is due to Ca2þ in£ux through VDCCs that open following the depolarization initiated by activated nAChRs. In a previous study (Tredway et al., 1999), functional high threshold N-type VDCCs were required for the immediate, TTXsensitive enhancement of GABA release produced by nicotine in the SPL. In the present study the delayed, TTX-insensitive enhancement of GABA release by nicotine was also dependent on functional calcium channels. Furthermore, Cd2þ (100 WM) blocked this enhancement without reducing the nicotine-mediated postsynaptic inward current in SPL neurons, suggesting that Cd2þ did not directly alter nAChR function. Thus, the nicotine-induced TTX-insensitive enhancement of GABA release in the present study appears to be due to secondary activation of VDCCs following depolarization of GABAergic terminals by activated nAChRs. Release of Ca2þ from intracellular stores has been shown to modulate transmitter release (Cochilla and Alford, 1998; Wang and Kelly, 1997; Peng, 1996). However, in the SPL it appears that the release of Ca2þ from intracellular stores does not play a major role in the TTX-insensitive enhancement of GABA release by nicotine, since neither ryanodine nor dantrolene prevented the enhancement of GABA release. Second messenger cascade
cellular level of cAMP, indicating a second messenger cascade was likely involved. Presynaptic nAChRs Nicotinic receptors at presynaptic sites can modulate synaptic transmission by regulating transmitter release. A well-established example is nicotine-induced enhancement of excitatory transmission in the rat hippocampus, which is TTX-insensitive. The enhancement is blocked by K-BgTx or 10 nM MLA, indicating that the nAChRs responsible are K7-containing (McGehee et al., 1995; Gray et al., 1996). In contrast, the presynaptic enhancement of GABA release by nicotine in the SPL was mediated primarily through activation of non-K7-containing nAChRs, since they were blocked by DHLE, but not by 100 nM MLA. Therefore in the SPL, high a⁄nity nicotine receptors function both as preterminal receptors
A striking feature of the TTX-insensitive enhancement of GABA release is that it is delayed by several minutes after nicotine exposure. One explanation for this delay could be that the number of nAChRs on the presynaptic terminals is low, and activation of these receptors may not immediately depolarize the terminal su⁄ciently to open voltage-dependent Ca2þ channels. A problem with this explanation is that the nicotine-induced postsynaptic current reaches steady-state before the enhancement of spontaneous GABA currents begins. Further, prolonged application of nicotine may actually reduce, not enhance, nicotinic channel function due to desensitization. A second and more likely explanation for the delay would be that second messenger pathways are involved. In the present study, TTX-insensitive enhancement of GABA release by nicotine was reduced by an inhibitor of cAMP-dependent protein kinase and facilitated by
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Fig. 6. E¡ects of forskolin and Br-cAMP on TTX-insensitive enhancement of GABAergic events produced by nicotine. Similar arrangement in both A and B. (Top) Forskolin 10 WM (A) or 50 WM Br-cAMP (B) facilitated TTX-insensitive enhancement of GABAergic events by nicotine (1 WM); (bottom) cumulative distributions of GABAergic event intervals. In the presence of 0.5 WM TTX+10 WM forskolin (A) or 50 WM Br-cAMP (B), nicotine (1 WM) decreased the spontaneous GABAergic event interval after 0.5 min and 4 min of exposure. (A) Number of GABAergic events was 64 under control, 116 and 135 at 0.5 min or 4 min nicotine application, respectively. (B) Number of GABAergic events was 71 under control, 107 and 139 at 0.5 min or 4 min nicotine application, respectively. A and B are from di¡erent neurons.
agents that are known to increase intracellular cAMP. Nicotine increases intracellular cAMP and activates protein kinase A in adrenal medullary cells (Anderson et al., 1992; Marley et al., 1995). Activation of protein kinase A has been shown to positively modulate VDCCs (Gray et al., 1998; Solem et al., 1997; Hell et al., 1995; Pfei¡erLinn and Lasater, 1998; Fukuda et al., 1996). Therefore, a cAMP-dependent cascade may be involved in the TTXinsensitive enhancement of GABA release by nicotine in the SPL. Protein kinase A activation also phosphorylates the nAChR itself (Vijayaraghavan et al., 1990; Huganir et al., 1986), which could up-regulate nicotinic receptor function and contribute to the observed TTX-insensitive enhancement of GABA release. In summary, a possible sequence of events underlying the TTX-insensitive actions of nicotine in the SPL can be advanced. First, nicotine activates high a⁄nity nAChRs
located on GABAergic neurons that form synapses with SPL neurons. Cation £ux through these activated nAChRs produces local membrane depolarizations that activate nearby high threshold VDCCs. The resulting increase in intracellular Ca2þ leads to enhanced activity of protein kinase A. Protein kinase A then phosphorylates VDCCs and/or nAChRs, thereby further enhancing the entry of Ca2þ into the terminal region, which causes the observed increase in release of GABA at synapses on SPL neurons. This enhanced GABA release occurs at a concentration of nicotine (100 nM) that is regularly attained by human cigarette smokers.
Acknowledgements(This research was supported by Grant NS17574 from the National Institute of Neurological Disorders and Stroke to V.A.C.
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