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Presynaptic nicotinic acetylcholine receptors enhance GABAergic synaptic transmission in rat periaqueductal gray neurons
European Journal of Pharmacology 640 (2010) 178–184
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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Pulmonary, Gastrointestinal and Urogenital Pharmacology
Presynaptic nicotinic acetylcholine receptors enhance GABAergic synaptic transmission in rat periaqueductal gray neurons Michiko Nakamura a, Il-Sung Jang a,b,⁎ a b
Department of Pharmacology, School of Dentistry, Kyungpook National University, Daegu, Republic of Korea Brain Science & Engineering Institute, Kyungpook National University, Daegu 700-412, Republic of Korea
a r t i c l e
i n f o
Article history: Received 20 January 2010 Received in revised form 30 March 2010 Accepted 24 April 2010 Available online 11 May 2010 Keywords: Presynaptic Nicotinic receptor mIPSC PAG Patch clamp Pain regulation
a b s t r a c t The periaqueductal gray (PAG) is a major component of the descending pain inhibitory pathway, which is related to central analgesia. In the present study, we have investigated the possible roles of presynaptic nicotinic acetylcholine receptors in GABAergic transmission onto PAG neurons. In acutely isolated rat PAG neurons, GABAergic miniature inhibitory postsynaptic currents (mIPSCs) were recorded by use of a wholecell patch clamp technique. Acetylcholine (30 μM) transiently increased both the frequency and amplitude of GABAergic mIPSCs. However, acetylcholine did not affect the GABA-induced membrane currents. This facilitatory action of acetylcholine disappeared in the presence of mecamylamine, a nonselective nicotinic receptor antagonist, and mimicked by nicotine, a nicotinic receptor agonist. The nicotine-induced increase in mIPSC frequency was completely blocked by dihydro-β-erythroidine, a selective β2-containing nicotinic receptor antagonist, but not methyllycaconitine or α-bungarotoxin, selective α7 nicotinic receptor antagonists. The results suggest that acetylcholine or nicotine acts presynaptic β2-containing nicotinic receptors, presumably α4β2 nicotinic receptors, to enhance spontaneous GABA release onto PAG neurons. The nicotine-induced increase in mIPSC frequency was completely occluded in the presence of Cd2+, a general voltage-dependent Ca2+ channels blocker, and in the absence of extracellular Ca2+ or Na+. The results suggest that presynaptic nicotinic receptors are less permeable to Ca2+, and that the activation of these receptors depolarizes GABAergic nerve terminals. In conclusion, presynaptic nicotinic receptors would temporally regulate the excitability of PAG neurons being not overexcited and eventually contribute to the cholinergic modulation of output from the PAG. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Nicotinic acetylcholine receptors are nonselective cation channels triggered by the binding of endogenous neurotransmitter acetylcholine. Nicotinic receptors have pentameric structures, which are homomeric or heteromeric combinations composed of α (α2–α10) and/or β (β2–β4) subunits, and they have different pharmacological and physiological properties based on the subunit composition. Although the subunit composition of nicotinic receptors varies among the brain region, both heteromeric α4β2 and homomeric α7 nicotinic receptors are abundantly distributed in the CNS (for review, Gotti et al., 2006). While nicotinic receptors are expressed at postsynaptic sides and contribute to the fast excitatory transmission via the influx of Na+ and Ca2+ in neuromuscular junction and ganglionic synapse, they are also widely expressed on presynaptic terminals in the CNS (Wonnacott, 1997; Vizi and Lendvai, 1999). The ⁎ Corresponding author. Department of Pharmacology, School of Dentistry, Kyungpook National University, 188-1, Samduk 2 ga-dong, Jung-gu, Daegu 700-412, Republic of Korea. Tel.: + 82 96 275 2268; fax: + 82 96 245 3172. E-mail address: [email protected] (I.-S. Jang). 0014-2999/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2010.04.057
activation of presynaptic nicotinic receptors increases the release probability of various neurotransmitters, such as GABA, glycine, glutamate, dopamine, noradrenalin and acetylcholine itself (Clarke and Reuben, 1996; Fu et al., 1998; Genzen and McGehee, 2003; Guo et al., 1998; Kiyosawa et al., 2001). Therefore, it has been suggested that presynaptic nicotinic receptors play a modulatory role in synaptic transmission. The midbrain periaqueductal gray (PAG) is involved in the various physiological functions including pain, fear and anxiety, vocalization, lordosis and cardiovascular control (for review, Behbehani, 1995; Millan, 2002). The PAG is also a major component of the descending pain inhibitory pathway, which is related to central analgesia, and is one of major target sites for the action of analgesics, such as opioids and cannnabinoids (Yaksh, 1997; Lichtman et al., 1996; Finn et al., 2003). The excitability of PAG neurons would be regulated by various neurotransmitters, such as GABA, glutamate, acetylcholine and so on, released from surrounding synapses projecting to the PAG. Among them, GABAergic input seems to be a pivotal regulating factor to maintain the excitability of PAG neurons, as the major intrinsic neural circuit within the PAG is a tonically active spontaneous GABAergic network and the inhibition of this network changes the intrinsic
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excitability of PAG neurons to modulate the output of the PAG (Behbehani et al., 1990; Ogawa et al., 1994). Therefore, the modulation of spontaneous GABAergic activity within the PAG would play a crucial role in the regulation of various functions. On the other hand, an immunohistochemical study has revealed that the PAG receives a dense projection of cholinergic fibers that arise from choline acetyltransferase-containing cells in the pontine tegmentum (Woolf et al., 1990). Although a recent study has shown that muscarinic receptors modulate GABAergic transmission onto PAG neurons (Lau and Vaughan, 2008), it is still unknown whether nicotinic receptors are expressed on GABAergic nerve terminals projecting to PAG neurons and whether their activation can regulate GABAergic transmission. In the present study, therefore, we have investigated the functional roles of nicotinic receptors in spontaneous GABAergic transmission in acutely isolated rat PAG neurons. 2. Materials and methods 2.1. Preparation All experiments complied with the guiding principles for the care and use of animals approved by the Council of the Physiological Society of Korea and the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and every effort was made to minimize both the number of animals used and their suffering. Sprague Dawley rats (11–14 d old) were decapitated under ketamine anesthesia (100 mg/kg, i. p.). The brain was dissected and transversely sliced at a thickness of 400 µm using a microslicer (VT1000S; Leica, Nussloch, Germany). Midbrain slices containing the PAG were kept in an incubation medium (see Solutions) saturated with 95% O2 and 5% CO2 at room temperature (22–24 ºC) for at least 1 h before the mechanical dissociation. For dissociation, slices were transferred into a 35 mm culture dish (Primaria 3801; Becton Dickinson, Rutherford, NJ, USA) containing a standard external solution (see Solutions), and the PAG region was identified under a binocular microscope (SMZ-1; Nikon, Tokyo, Japan). Details of the mechanical dissociation have been described previously (Rhee et al., 1999). Briefly, mechanical dissociation was accomplished using a custom-built vibration device and a fire-polished glass pipette oscillating at about 50–60 Hz (0.3–0.5 mm) on the surface of the PAG region. Slices were removed and the mechanically dissociated neurons were left for 15 min to allow the neurons to adhere to the bottom of the culture dish. 2.2. Electrical measurements All electrophysiological measurements were performed using conventional whole-cell patch recording mode at a holding potential (VH) of 0 mV (Axopatch 200B; Molecular Devices, Union City, CA, USA). Patch pipettes were made from borosilicate capillary glass (1.5 mm outer diameter, 0.9 mm inner diameter; G-1.5; Narishige, Tokyo, Japan) by use of a pipette puller (P-97; Sutter Instrument Co., Novato, CA, USA). The resistance of the recording pipettes filled with internal solution was 4–6 MΩ. The liquid junction potential and pipette capacitance were compensated for. Neurons were viewed under phase contrast on an inverted microscope (TE2000; Nikon). Membrane currents were filtered at 1 kHz, digitized at 4 kHz, and stored on a computer equipped with pCLAMP 10 (Molecular Devices). During the recordings, 10 mV hyperpolarizing step pulses (30 ms in duration) were periodically applied to monitor the access resistance. All experiments were performed at room temperature (22–25 °C). 2.3. Data analysis Spontaneous miniature inhibitory postsynaptic currents (mIPSCs) were counted and analyzed using the MiniAnalysis program (Synap-
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tosoft, Inc., Decatur, GA) as described previously (Jang et al., 2002). Briefly, mIPSCs were screened automatically using an amplitude threshold of 10 pA, and then visually accepted or rejected based upon the rise and decay times. Basal noise levels during voltage-clamp recordings were typically less than 8 pA. The average values of both the frequency and amplitude of mIPSCs during the control period (5– 10 min) or each drug condition (5–10 min) were calculated for each recording, and the frequency and amplitude of all the events during the agonist application (30 s or 3 min) were normalized to these values. The effects of these different conditions were quantified as a percentage increase in mIPSC frequency compared to the control values. The inter-event intervals and amplitudes of a large number of synaptic events obtained from the same neuron were examined by constructing cumulative probability distributions and compared using the Kolmogorov–Smirnov (K–S) test with Stat View software (SAS Institute, Inc., Cary, NC, USA). Numerical values are provided as the mean ± standard error of the mean (S.E.M.) using values normalized to the control. Significant differences in the mean amplitude and frequency were tested using Student's paired two-tailed t-test, using absolute values rather than normalized ones. Values of P b 0.05 were considered significant. 2.4. Solutions The ionic composition of the incubation medium consisted of (in mM) 124 NaCl, 3 KCl, 1.5 KH2PO4, 24 NaHCO3, 2 CaCl2, 1.3 MgSO4 and 10 glucose saturated with 95% O2 and 5% CO2. The pH was about 7.4– 7.5. The standard external solution was (in mM) 150 NaCl, 3 KCl, 2 CaCl2, 1 MgCl2, 10 glucose and 10 Hepes. The Ca2+-free external solution was (in mM) 150 NaCl, 3 KCl, 2 EGTA, 3 MgCl2, 10 glucose and 10 Hepes. The Na+-free external solution was (in mM) 150 N-methylD-glucamine-Cl, 3 KCl, 2 CaCl2, 1 MgCl2, 10 glucose and 10 Hepes. All these external solutions were adjusted to a pH of 7.4 with Tris-base. For recording mIPSCs, these standard external solutions routinely contained 300 nM tetrodotoxin (TTX), 10 μM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 20 μM DL-2-amino-5-phosphonovaleric acid (APV) to block voltage-dependent Na+ channels and ionotropic glutamate receptors, respectively. The ionic composition of the internal (pipette) solution was consisted of (in mM) 135 CsMeHSO3, 7 CsCl, 2 EGTA and 10 Hepes with a pH adjusted to 7.2 with Tris-base. 2.5. Drugs The drugs used in the present study were APV, TTX, CNQX, acetylcholine, nicotine, choline-Cl, 6-imino-3-(4-methoxyphenyl)-1 (6H)-pyridazinebutanoic acid HBr (SR95531), muscarine (from Sigma, St. Louis, MO, USA), dihydro-β-erythroidine (DHβE), methyllycaconitine (MLA), mecamylamine hydrochloride (MCA), α-bungarotoxin (from Tocris, Bristol, UK). All solutions containing drugs were applied using the ‘Y-tube system’ for rapid solution exchange (Murase et al., 1989). 3. Results 3.1. GABAergic mIPSCs in mechanically dissociated PAG neurons Previous comparative studies of the morphology of the PAG of rat, cat and monkey have shown considerable similarities in the types of neurons and their distributions within the PAG (Mantyh, 1982; Beitz and Shepard, 1985). Four major types of rat PAG neurons ranging between 10 and 35 μm in soma diameter have been identified based on their morphological properties; fusiform or bipolar neurons, multipolar neurons that have a very large number of dendrites, stellate cells that have 3–6 dendrites, and pyramidal-shaped neurons (Mantyh, 1982; Beitz and Shepard, 1985). After the mechanical
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dissociation of the PAG region, we found several kinds of neurons in soma diameter (≤ 15 μm) and shape (multipolar, bipolar and pyramidal-shaped). In the case of smaller bipolar or multipolar neurons (b10 μm in soma diameter), GABAergic synaptic events were hardly detected during electrophysiological recordings, so that it was hard to investigate the effect of acetylcholine on GABAergic synaptic transmission. In contrast, we could record abundant spontaneous synaptic events in bipolar and pyramidal-shaped neurons (10–15 μm in soma diameter), and there was no difference in the presynaptic response to acetylcholine or nicotine between bipolar and pyramidalshaped neurons. Therefore, we performed all electrophysiological recordings with these bipolar and pyramidal-shaped neurons. In the presence of 300 nM TTX, 10 μM CNQX and 20 μM APV, the spontaneous miniature currents were recorded from the mechanical dissociated PAG neurons at a VH of 0 mV. The observed spontaneous currents were completely and reversibly blocked by 10 μM SR95531, a selective GABAA receptor antagonist (Fig. 1A). The amplitude of spontaneous currents varied with the holding potentials (VH; Fig. 1B) and their reversal potential was −72.3 mV. This value was very similar to the theoretical Cl− equilibrium potential (ECl) of −78.7 mV, which was calculated from the Nernst equation using extracellular and intracellular Cl− concentrations ([Cl−]o ; 159 mM and [Cl−]i ; 7 mM, respectively). These results indicate that the spontaneous miniature currents are GABAergic mIPSCs mediated by GABAA receptors.
endogenous ligand of cholinergic receptors, on GABAergic mIPSCs. The application of acetylcholine (30 μM) during 3 min elicited a brief increase in the frequency of GABAergic mIPSCs and this increase was rapidly subsided to the control level within 40 s (Fig. 2A and B). In 11 neurons tested, acetylcholine increased both the mean mIPSC frequency to 351 ± 69% of the control (n = 11, P b 0.01) and the mean mIPSC amplitude (152 ± 21% of the control, n = 11, P b 0.01) (Fig. 2C insets). In addition, as shown in Fig. 2C, acetylcholine significantly shifted the distributions of inter-event interval and current amplitude of GABAergic mIPSCs to the left and right (P b 0.05, K–S test), respectively, indicating increases in the frequency and amplitude of GABAergic mIPSCs. An increase in mIPSC amplitude might result from the acetylcholine-mediated postsynaptic effects, such as an increase in the GABA sensitivity. However, this was not the case because acetylcholine (30 μM) did not affect 30 μM GABAinduced currents (101 ± 2% of the control, n = 6, P = 0.57, Fig. 2D). Taken together, these results suggest that acetylcholine acts presynaptically to increase spontaneous GABA release onto PAG neurons. 3.3. Facilitation of GABAergic mIPSCs mediated by presynaptic nicotinic receptors
To investigate whether functional nicotinic receptors exist on GABAergic presynaptic nerve terminals projecting to PAG neurons and their activation can modulate GABAergic synaptic transmission, we first tested the effect of exogenously applied acetylcholine, an
A previous study has shown that carbachol, a nonselective cholinergic agonist which can activate nicotinic and muscarinic receptors, decreases GABAergic transmission onto PAG neurons by activating presynaptic muscarinic receptors (Lau and Vaughan, 2008). Therefore, we further examined which muscarinic receptors also contribute to the acetylcholine-induced modulation of mIPSC frequency. To test this, we observed the effect of MCA, a nonselective nicotinic receptor antagonist, on the acetylcholine-induced increase in spontaneous GABA release. In 8 neurons, in which MCA effect was fully analyzed, the acetylcholine (30 μM)-induced initial increase in mIPSC
Fig. 1. GABAergic mIPSCs recorded from acutely isolated PAG neurons. A, A typical trace of GABAergic mIPSCs observed before, during and after the application of 10 μM SR95531 at a VH of 0 mV in the presence of 300 nM TTX, 10 μM CNQX and 20 μM APV. Insets represent GABAergic mIPSCs with an expanded time scale in each condition. Ba, Typical traces of GABAergic mIPSCs at various holding potentials (VH). b, A plot of the mean amplitude of mIPSCs at various VH values. The reversal potential was estimated to be − 72.3 mV using the Nernst equation, which was very similar to the theoretical ECl (− 78.8 mV). Each point was the mean and S.E.M. from 5 experiments.
Fig. 2. Effects of acetylcholine on GABAergic mIPSCs. A, A typical trace of GABAergic mIPSCs observed before, during and after application of 30 μM acetylcholine (ACh). Insets represent GABAergic mIPSCs with an expanded time scale in each condition. B, A time course of the acetylcholine-induced change in mIPSC frequency. Each point was the mean and S.E.M. from 11 experiments. C, Cumulative probability distributions for inter-event interval (a) and current amplitude (b) of GABAergic mIPSCs. 222 for control and 275 events for acetylcholine were plotted. Inset columns were the mean and S.E.M. from 11 experiments, respectively. *; P b 0.05, **; P b 0.01. D, Typical traces of GABA (30 μM)-induced membrane currents (IGABA) observed before, during and after the application of 30 μM acetylcholine.
3.2. Effect of acetylcholine on GABAergic mIPSCs
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frequency (during the first 30 s, 565 ± 135% of the control, n = 8, P b 0.01) was completely blocked by 10 μM MCA (during the first 30 s, 110 ± 21% to the MCA condition, n = 8, P = 0.94, Fig. 3A and B), suggesting that nicotinic receptors are involved in the acetylcholineinduced initial increase in mIPSC frequency. However, acetylcholine did not decrease GABAergic mIPSC frequency in the presence of MCA (Fig. 3A and B). In addition, muscarine (10 μM), a muscarinic receptor agonist, did not affect GABAergic mIPSC frequency (103± 10% to the condition, n = 6, P = 0.94, data not shown), indicating that the involvement of muscarinic receptors in the acetylcholine-induced modulation of spontaneous GABA release might be negligible. Next, we observed the effect of nicotine, a nicotinic receptor agonist, on GABAergic mIPSCs. Nicotine (3 μM, 30 s application) also increased both the mean mIPSC frequency to 620 ± 110% of the control (n = 10, P b 0.01) and the mean mIPSC amplitude (120± 7% to the control, n = 10, P b 0.05) (Fig. 3C and D insets). In addition, as shown in Fig. 3D, nicotine significantly shifted the distributions of inter-event interval and current amplitude to the left and right (P b 0.05, K–S test), respectively, indicating increases in the frequency and amplitude of GABAergic mIPSCs. The results suggest that function nicotinic receptors are expressed on GABAergic nerve terminals projecting to PAG neurons and that nicotinic but not muscarinic receptors are responsible for the cholinergic modulation of spontaneous GABAergic transmission in acutely isolated PAG neurons. In all subsequent pharmacological experiments, nicotine was used to activate presynaptic nicotinic receptors based on its selectivity for nicotinic receptors. 3.4. Subunit composition of presynaptic nicotinic receptors
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nicotine-induced increase in spontaneous GABA release. DHβE at a concentration of 1–10 μM is known to block β2-containing nicotinic receptors including α4β2 nicotinic receptors (Dickinson et al., 2008; Livingstone et al., 2009). In the presence of 10 μM DHβE, the nicotineinduced increase in mIPSC frequency was almost suppressed (133 ± 36% of the DHβE condition, n = 6, P = 0.99; Fig. 4Ab and B). We also observed the effect of MLA, a selective α7 nicotinic receptor antagonist, on the nicotine-induced increase in spontaneous GABA release. In the presence of 300 nM MLA, 3 μM nicotine still increased GABAergic mIPSC frequency (524 ± 154% to the MLA condition, n = 6, P b 0.01; Fig. 4Ac and B). In addition, nicotine (3 μM) still increased GABAergic mIPSC frequency even in the presence of 100 nM αbungarotoxin, a selective α7 nicotinic receptor antagonist (506 ± 117% to the MLA condition, n = 5, P b 0.05; Fig. 4Ad and B). Furthermore, choline (1 mM), which has a relatively higher sensitivity to α7 nicotinic receptors (Alkondon et al., 1997), had no facilitatory effect on GABAergic mIPSC frequency (Fig. 4B). Taken together, the results suggest that presynaptic nicotinic receptors at least contain β2 subunits rather than α7 subunits. 3.5. Ion permeability of presynaptic nicotinic receptors To elucidate the mechanisms underlying the nicotinic receptormediated facilitation of mIPSC frequency, we examined the effect of Ca2+-free (plus 2 mM EGTA) external solution on the nicotineinduced facilitation of mIPSC frequency. In the Ca2+-free external solution, both the frequency and amplitude of GABAergic mIPSCs were significantly reduced (30 ± 11% and 79 ± 8% of the control, n = 6, P b 0.05, respectively). This suggests indicate that GABAergic mIPSCs
In the mammalian brain, both heteromeric α4β2 and homomeric α7 nicotinic receptors are abundantly expressed (Gotti et al., 2006). Therefore, we examined the subunit composition of presynaptic nicotinic receptors expressed on GABAergic nerve terminals by use of pharmacological tools. We first observed the effect of DHβE on the
Fig. 3. Nicotinic receptors are responsible for the acetylcholine-induced increase in mIPSC frequency. A, A time course of the acetylcholine (30 μM)-induced change in mIPSC frequency in the presence of 10 μM MCA. Insets represent GABAergic mIPSCs with an expanded time scale in each condition. B, The acetylcholine-induced change in mIPSC frequency in the absence and presence of MCA. The frequency of GABAergic mIPSCs was calculated from the first 30 s period or entire (3 min) period during the acetylcholine application. Each column was the mean and S.E.M. from 8 experiments. *; P b 0.05, **; P b 0.01, n.s.; not significant. C, A typical trace of GABAergic mIPSCs observed before, during and after application of 3 μM nicotine (Nic). Insets represent GABAergic mIPSCs with an expanded time scale in each condition. D, Cumulative probability distributions for inter-event interval (a) and current amplitude (b) of GABAergic mIPSCs. 263 for control and 64 events for nicotine were plotted. Inset columns were the mean and S.E.M. from 10 experiments. *; P b 0.05, **; P b 0.01.
Fig. 4. The subunit composition of presynaptic nicotinic receptors on GABAergic nerve terminals. A, Typical traces of GABAergic mIPSCs observed before and during the application of 3 μM nicotine (Nic) in the control condition (a), in the presence of 10 μM DHβE (b), in the presence of 300 nM MLA (c), or in the presence of 100 nM αbungarotoxin (α-BgTx, d). B, Nicotine-induced changes in mIPSC frequency in each condition. Note that choline had no effect on mIPSC frequency. Each column was the mean and S.E.M. from 6 experiments. *; P b 0.05, **; P b 0.01, n.s.; not significant.
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Fig. 5. Presynaptic nicotinic receptors are less permeable to Ca2+. A, Typical traces of GABAergic mIPSCs observed before and during the application of 3 μM nicotine (Nic) in the control condition (a), in the Ca2+-free external solution (b), in the present of 100 μM Cd2+ (c), or in the Na+-free external solution (d). B, Nicotine-induced changes in mIPSC frequency in each condition. Each column was the mean and S.E.M. from 6 to 7 experiments. *; P b 0.05, **; P b 0.01, n.s.; not significant.
recorded from acutely isolated PAG neurons was, at least in part, dependent on extracellular Ca2+, and that mIPSCs observed in the Ca2+-free external solution might be classical miniature synaptic events, which are not dependent on the Ca2+ influx from the extracellular space (Scanziani et al., 1992; Capogna et al., 1993). In the Ca2+-free external solution, the facilitatory effect of nicotine on mIPSC frequency was completely occluded (115 ± 29% of the Ca2+free condition, n = 6, P = 0.99; Fig. 5Ab and Ba). We also tested the effect of Cd2+, a general VDCC blocker, on the nicotine-induced increase in mIPSC frequency. Cd2+ (100 µM) also decreased GABAergic mIPSC frequency (50 ± 9% of the control, n = 7, P b 0.01) but not amplitude (82 ± 9% of the control, n = 7, P = 0.08, Fig. 5Ac and B). In the presence of 100 µM Cd2+, the nicotineinduced increase in mIPSC frequency was completely attenuated (96 ± 9% of the Cd2+ condition, n = 7, P = 0.34, Fig. 5Ac and Bb). The results suggest that the nicotine-induced increase in spontaneous GABA release requires the Ca2+ influx from the extracellular space via presynaptic VDCCs. Finally, we examined the effect of Na+-free external solution on the nicotine-induced increase in mIPSC frequency. In the absence of extracellular Na+, nicotine again failed to increase the frequency of GABAergic mIPSCs (96 ± 9% of the Na+free control condition, n = 7, P = 0.34, Fig. 5Ad and Bc).
neurotransmitter release at a variety of central synapses (Clarke and Reuben, 1996; Fu et al., 1998; Genzen and McGehee, 2003; Guo et al., 1998; Kiyosawa et al., 2001). In the present study, we initially examined the effect of acetylcholine, an endogenous ligand of cholinergic receptors, on spontaneous GABA release onto acutely isolated PAG neurons. As acetylcholine can bind to both ionotropic nicotinic and metabotropic muscarinic receptors, these two receptor subtypes might be involved in the acetylcholine-induced modulation of spontaneous GABA release. However, several lines of evidence suggest that acetylcholine acts presynaptic nicotinic but not muscarinic receptors to increase spontaneous GABAergic transmission onto PAG neurons. First, acetylcholine significantly increased the frequency of GABAergic mIPSCs, indicating that acetylcholine acts presynaptically to change the probability of spontaneous GABA release. Although acetylcholine also increased the mean amplitude of mIPSCs, acetylcholine is likely to act presynaptically because acetylcholine had no effect on the sensitivity of postsynaptic GABAA receptors. An increase in mIPSC amplitude by acetylcholine might result from multivesicular release due to an increase in the intraterminal Ca2+ concentration ([Ca2+]terminal) (see also Sharma et al., 2008). Second, the acetylcholine-induced transient facilitation of GABAergic mIPSC frequency was completely blocked by MCA, a nonselective nicotinic receptor antagonist, and such an effect was closely mimicked by nicotine, a selective nicotinic receptor agonist. In addition, since acetylcholine had no effect on GABAergic mIPSC frequency after the blockade of nicotinic receptors with MCA, muscarinic receptors might be not involved in the acetylcholine-induced modulation of spontaneous GABA release. Third, the preparation used in this study should exclude any non-presynaptic actions, such as changes in the excitability of soma, because mechanically dissociated neurons retain functional cell-free presynaptic nerve terminals (for review, Akaike and Moorhouse, 2003). A recent study has shown that carbachol, a nonselective acetylcholine receptor agonist, suppresses GABAergic transmission in PAG neurons (Lau and Vaughan, 2008). However, the carbacholinduced presynaptic inhibition of GABAergic transmission would be largely mediated by the indirect M1/M3 muscarinic receptor-induced endocannabinoid signaling (Lau and Vaughan, 2008). In fact, the activation of M1 muscarinic receptors is known to produce endocannabinoids to inhibit neurotransmitter release (Ohno-Shosaku et al., 2003; Fukudome et al., 2004; Narushima et al., 2007). In contrast to these studies, our present results indicate that muscarinic receptors might be not involved in the acetylcholine-induced modulation of spontaneous GABA release onto PAG neurons, as muscarine had no effect on GABAergic mIPSC frequency. This discrepancy might be due to the preparation used, e.g., a slice preparation in the previous study (Lau and Vaughan, 2008) and isolated single neurons in the present study, because endocannabinoids, if any, produced by muscarinic receptor activation would be easily swept away during the drug application in isolated single neurons. Alternatively, acutely isolated PAG neurons used in the present study might be different from those used in the slice preparation, as several types of neurons have been identified in the PAG region. Further experiments combined with a morphological analysis would reveal the relationship between the cholinergic modulation of GABAergic transmission and neuronal types in the PAG region. 4.2. Presynaptic nicotinic receptors are less permeable to Ca2+
4. Discussion 4.1. Presynaptic nicotinic receptors facilitate spontaneous GABA release onto PAG neurons Previous studies have shown that nicotinic receptors are widely expressed on presynaptic terminals and their activation modulates
Despite of the large number of nicotinic receptor subunits, most of neuronal nicotinic receptors expressed in the CNS are heteromeric α4β2 or homomeric α7 nicotinic ones (for review, Gotti et al., 2006), and there are distinct differences in pharmacological and physiological properties between these two types of nicotinic receptors. For example, while α4β2 nicotinic receptors exhibit high affinity of
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nicotine (EC50; ∼ 15 μM) and low Ca2+ permeability (PCa/PNa; ∼ 1.5), α7 nicotinic receptors do low affinity of nicotine (EC50; ∼ 90 μM) and high Ca2+ permeability (PCa/PNa; N10) (Role and Berg, 1996; Giniatullin et al., 2005). In the present study, while DHβE, a selective antagonist of β2-containing nicotinic receptors (Dickinson et al., 2008; Livingstone et al., 2009), completely suppressed the nicotineinduced facilitation of spontaneous GABA release, MLA or αbungarotoxin, selective antagonists of α7-containing nicotinic receptors, did not block the nicotine action. In addition, choline, which is more sensitive to α7 than α4β2 nicotinic receptors (Alkondon et al., 1997), had no effect on GABAergic mIPSC frequency. These pharmacological properties indicate that presynaptic nicotinic receptors responsible for the nicotine-induced increase in spontaneous GABA release might be β2-containing, possibly α4β2 nicotinic receptors. However, further studies should be needed to elucidate the exact subunit composition of presynaptic nicotinic receptors as DHβE affects other subtypes such as α3β2 nicotinic receptors (Harvey and Luetje, 1996). Since nicotinic receptors are nonselective cation channels permeable to Na+ and Ca2+ (Role and Berg, 1996; Giniatullin et al., 2005), the activation of presynaptic nicotinic receptors is expected to enhance the probability of neurotransmitter release either by permitting Ca2+ influx passing through nicotinic receptors themselves or by eliciting a presynaptic depolarization, which subsequently activates presynaptic VDCCs. In the present study, the nicotine action on GABAergic mIPSCs was completely occluded by deleting extracellular Ca2+, indicating that the nicotine-induced increase in spontaneous GABA release requires the Ca2+ influx into nerve terminals from extracellular space. However, an increase in [Ca2+]terminal by nicotine might be not mediated by the direct Ca2+ influx passing through nicotinic receptors because the nicotineinduced increase in mIPSC frequency was eliminated by adding Cd2+, a general VDCC blocker. The results suggest that presynaptic nicotinic receptors are less permeable to Ca2+, and that the Na+ influx passing through nicotinic receptors directly depolarizes GABAergic nerve terminals to activate presynaptic VDCCs. This conclusion is consistent with our pharmacological data showing that presynaptic nicotinic receptors might be α4β2 nicotinic ones having low Ca2+ permeability, and that the facilitatory action of nicotine also completely disappeared in the Na+-free extracellular solution. In contrast to this idea, a previous study has shown that the activation of presynaptic α4β2 nicotinic receptors can increase spontaneous glycine release at spinal synapses even after the blockade of presynaptic VDCCs (Kiyosawa et al., 2001), suggesting that α4β2 nicotinic receptors expressed on glycinergic terminals are permeable to Ca2+. In the case of PAG neurons, even though presynaptic nicotinic receptors are permeable to Ca2+, the number of presynaptic nicotinic receptors expressed on single GABAergic nerve terminals might be too small so that an increase in [Ca2 + ]terminal via nicotinic receptors themselves cannot directly trigger spontaneous GABA release. 4.3. Physiological implications The PAG is closely involved in the descending pain inhibitory pathway, which is related to central analgesia (Yaksh, 1997; Lichtman et al., 1996; Finn et al., 2003), as the electrical stimulation of the PAG region is known to activate the descending inhibitory pathway to reduce pain (Reynolds, 1969; Monhemius et al., 2001). Considering that the major intrinsic neural circuit is a tonically active spontaneous GABAergic network within the PAG (Behbehani et al., 1990; Ogawa et al., 1994), GABAergic transmission would contribute to maintain the excitability of PAG neurons. For example, a behavioral study has revealed that the focal microinjection of bicuculline, a GABAA receptor antagonist, into the PAG abolishes the heat-evoked nociception (Sandkühler et al., 1989). In addition, the antinociceptive action of opioids is mainly mediated by decreasing GABAergic inhibition within the PAG (Moreau and Fields, 1986; Depaulis et al., 1987; Kalyuzhny
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et al., 1996). On the other hand, as the PAG receives innervations of cholinergic inputs from the pontine tegmentum (Woolf et al., 1990), the cholinergic system might be involved in the pain modulation by changing the excitability of PAG neurons. For example, Guimarães and Prado (1994), Guimarães et al. (2000) have reported that the microinjection of carbachol into the PAG produces analgesic actions, suggesting that cholinergic receptor-mediated mechanisms contribute to the modulation of nociceptive suppression in the PAG. Similarly, a recent study has shown that muscarinic receptors inhibit GABAergic transmission onto PAG neurons (Lau and Vaughan, 2008). In the present study, we have shown that functional nicotinic receptors, presumably α4β2 nicotinic receptors, are expressed on GABAergic nerve terminals projecting to PAG neurons, and that their activation transiently increases spontaneous GABA release. However, it is poorly known whether bipolar and pyramidal-shaped neurons used in this study are projection neurons innervating serotoninergic neurons or local interneurons, because there is little information available regarding the relationship between morphological cell types and their projection (see also Beitz, 1990). Although further study should be needed to elucidate the functional roles of presynaptic nicotinic receptors in the modulation of the descending pain inhibitory pathway, the present results suggest that presynaptic nicotinic receptors might temporally regulate the excitability of PAG neurons being not overexcited and eventually contribute to the cholinergic modulation of output from the PAG. Acknowledgements This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MEST) (Nos. R13-2008-009-01003-0 and KRF-2008-210-E00002). References Akaike, N., Moorhouse, A.J., 2003. Techniques: applications of the nerve-bouton preparation in neuropharmacology. Trends Pharmacol. Sci. 24, 44–47. Alkondon, M., Pereira, E.F., Cortes, W.S., Maelicke, A., Albuquerque, E.X., 1997. Choline is a selective agonist of alpha7 nicotinic acetylcholine receptors in the rat brain neurons. Eur. J. NeuroSci. 9, 2734–2742. Behbehani, M.M., 1995. Functional characteristics of the midbrain periaqueductal gray. Prog. Neurobiol. 46, 575–605. Behbehani, M.M., Jiang, M.R., Chandler, S.D., Ennis, M., 1990. The effect of GABA and its antagonists on midbrain periaqueductal gray neurons in the rat. Pain 40, 195–204. Beitz, A.J., 1990. Relationship of glutamate and aspartate to the periaqueductal grayraphe magnus projection: analysis using immunocytochemistry and microdialysis. J. Histochem. Cytochem. 38, 1755–1765. Beitz, A.J., Shepard, R.D., 1985. The midbrain periaqueductal gray in the rat. II. A Golgi analysis. J. Comp. Neurol. 237, 460–475. Capogna, M., Gahwiler, B.H., Thompson, S.M., 1993. Mechanism of µ-opioid receptormediated presynaptic inhibition in the rat hippocampus in vitro. J. Physiol. (Lond.) 470, 539–558. Clarke, P.B., Reuben, M., 1996. Release of [3H]-noradrenaline from rat hippocampal synaptosomes by nicotine: mediation by different nicotinic receptor subtypes from striatal [3H]-dopamine release. Br. J. Pharmacol. 117, 595–606. Depaulis, A., Morgan, M.M., Liebeskind, J.C., 1987. GABAergic modulation of the analgesic effects of morphine microinjected in the ventral periaqueductal gray matter of the rat. Brain Res. 436, 223–228. Dickinson, J.A., Kew, J.N., Wonnacott, S., 2008. Presynaptic α7- and β2-containing nicotinic acetylcholine receptors modulate excitatory amino acid release from rat prefrontal cortex nerve terminals via distinct cellular mechanisms. Mol. Pharmacol. 74, 348–359. Finn, D.P., Jhaveri, M.D., Beckett, S.R., Roe, C.H., Kendall, D.A., Marsden, C.A., Chapman, V., 2003. Effects of direct periaqueductal gray administration of a cannabinoid receptor agonist on nociceptive and aversive responses in rats. Neuropharmacology 45, 594–604. Fu, W.M., Liou, H.C., Chen, Y.H., 1998. Nerve terminal currents induced by autoreception of acetylcholine release. J. Neurosci. 18, 9954–9961. Fukudome, Y., Ohno-Shosaku, T., Matsui, M., Omori, Y., Fukaya, M., Tsubokawa, H., Taketo, M.M., Watanabe, M., Manabe, T., Kano, M., 2004. Two distinct classes of muscarinic action on hippocampal inhibitory synapses: M2-mediated direct suppression and M1/M3-mediated indirect suppression through endocannabinoid signalling. Eur. J. NeuroSci. 19, 2682–2692. Genzen, J.R., McGehee, D.S., 2003. Short- and long-term enhancement of excitatory transmission in the spinal cord dorsal horn by nicotinic acetylcholine receptors. Proc. Natl. Acad. Sci. U. S. A. 27 (100), 6807–6812.
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Contents lists available at ScienceDirect
European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Pulmonary, Gastrointestinal and Urogenital Pharmacology
Presynaptic nicotinic acetylcholine receptors enhance GABAergic synaptic transmission in rat periaqueductal gray neurons Michiko Nakamura a, Il-Sung Jang a,b,⁎ a b
Department of Pharmacology, School of Dentistry, Kyungpook National University, Daegu, Republic of Korea Brain Science & Engineering Institute, Kyungpook National University, Daegu 700-412, Republic of Korea
a r t i c l e
i n f o
Article history: Received 20 January 2010 Received in revised form 30 March 2010 Accepted 24 April 2010 Available online 11 May 2010 Keywords: Presynaptic Nicotinic receptor mIPSC PAG Patch clamp Pain regulation
a b s t r a c t The periaqueductal gray (PAG) is a major component of the descending pain inhibitory pathway, which is related to central analgesia. In the present study, we have investigated the possible roles of presynaptic nicotinic acetylcholine receptors in GABAergic transmission onto PAG neurons. In acutely isolated rat PAG neurons, GABAergic miniature inhibitory postsynaptic currents (mIPSCs) were recorded by use of a wholecell patch clamp technique. Acetylcholine (30 μM) transiently increased both the frequency and amplitude of GABAergic mIPSCs. However, acetylcholine did not affect the GABA-induced membrane currents. This facilitatory action of acetylcholine disappeared in the presence of mecamylamine, a nonselective nicotinic receptor antagonist, and mimicked by nicotine, a nicotinic receptor agonist. The nicotine-induced increase in mIPSC frequency was completely blocked by dihydro-β-erythroidine, a selective β2-containing nicotinic receptor antagonist, but not methyllycaconitine or α-bungarotoxin, selective α7 nicotinic receptor antagonists. The results suggest that acetylcholine or nicotine acts presynaptic β2-containing nicotinic receptors, presumably α4β2 nicotinic receptors, to enhance spontaneous GABA release onto PAG neurons. The nicotine-induced increase in mIPSC frequency was completely occluded in the presence of Cd2+, a general voltage-dependent Ca2+ channels blocker, and in the absence of extracellular Ca2+ or Na+. The results suggest that presynaptic nicotinic receptors are less permeable to Ca2+, and that the activation of these receptors depolarizes GABAergic nerve terminals. In conclusion, presynaptic nicotinic receptors would temporally regulate the excitability of PAG neurons being not overexcited and eventually contribute to the cholinergic modulation of output from the PAG. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Nicotinic acetylcholine receptors are nonselective cation channels triggered by the binding of endogenous neurotransmitter acetylcholine. Nicotinic receptors have pentameric structures, which are homomeric or heteromeric combinations composed of α (α2–α10) and/or β (β2–β4) subunits, and they have different pharmacological and physiological properties based on the subunit composition. Although the subunit composition of nicotinic receptors varies among the brain region, both heteromeric α4β2 and homomeric α7 nicotinic receptors are abundantly distributed in the CNS (for review, Gotti et al., 2006). While nicotinic receptors are expressed at postsynaptic sides and contribute to the fast excitatory transmission via the influx of Na+ and Ca2+ in neuromuscular junction and ganglionic synapse, they are also widely expressed on presynaptic terminals in the CNS (Wonnacott, 1997; Vizi and Lendvai, 1999). The ⁎ Corresponding author. Department of Pharmacology, School of Dentistry, Kyungpook National University, 188-1, Samduk 2 ga-dong, Jung-gu, Daegu 700-412, Republic of Korea. Tel.: + 82 96 275 2268; fax: + 82 96 245 3172. E-mail address: [email protected] (I.-S. Jang). 0014-2999/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2010.04.057
activation of presynaptic nicotinic receptors increases the release probability of various neurotransmitters, such as GABA, glycine, glutamate, dopamine, noradrenalin and acetylcholine itself (Clarke and Reuben, 1996; Fu et al., 1998; Genzen and McGehee, 2003; Guo et al., 1998; Kiyosawa et al., 2001). Therefore, it has been suggested that presynaptic nicotinic receptors play a modulatory role in synaptic transmission. The midbrain periaqueductal gray (PAG) is involved in the various physiological functions including pain, fear and anxiety, vocalization, lordosis and cardiovascular control (for review, Behbehani, 1995; Millan, 2002). The PAG is also a major component of the descending pain inhibitory pathway, which is related to central analgesia, and is one of major target sites for the action of analgesics, such as opioids and cannnabinoids (Yaksh, 1997; Lichtman et al., 1996; Finn et al., 2003). The excitability of PAG neurons would be regulated by various neurotransmitters, such as GABA, glutamate, acetylcholine and so on, released from surrounding synapses projecting to the PAG. Among them, GABAergic input seems to be a pivotal regulating factor to maintain the excitability of PAG neurons, as the major intrinsic neural circuit within the PAG is a tonically active spontaneous GABAergic network and the inhibition of this network changes the intrinsic
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excitability of PAG neurons to modulate the output of the PAG (Behbehani et al., 1990; Ogawa et al., 1994). Therefore, the modulation of spontaneous GABAergic activity within the PAG would play a crucial role in the regulation of various functions. On the other hand, an immunohistochemical study has revealed that the PAG receives a dense projection of cholinergic fibers that arise from choline acetyltransferase-containing cells in the pontine tegmentum (Woolf et al., 1990). Although a recent study has shown that muscarinic receptors modulate GABAergic transmission onto PAG neurons (Lau and Vaughan, 2008), it is still unknown whether nicotinic receptors are expressed on GABAergic nerve terminals projecting to PAG neurons and whether their activation can regulate GABAergic transmission. In the present study, therefore, we have investigated the functional roles of nicotinic receptors in spontaneous GABAergic transmission in acutely isolated rat PAG neurons. 2. Materials and methods 2.1. Preparation All experiments complied with the guiding principles for the care and use of animals approved by the Council of the Physiological Society of Korea and the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and every effort was made to minimize both the number of animals used and their suffering. Sprague Dawley rats (11–14 d old) were decapitated under ketamine anesthesia (100 mg/kg, i. p.). The brain was dissected and transversely sliced at a thickness of 400 µm using a microslicer (VT1000S; Leica, Nussloch, Germany). Midbrain slices containing the PAG were kept in an incubation medium (see Solutions) saturated with 95% O2 and 5% CO2 at room temperature (22–24 ºC) for at least 1 h before the mechanical dissociation. For dissociation, slices were transferred into a 35 mm culture dish (Primaria 3801; Becton Dickinson, Rutherford, NJ, USA) containing a standard external solution (see Solutions), and the PAG region was identified under a binocular microscope (SMZ-1; Nikon, Tokyo, Japan). Details of the mechanical dissociation have been described previously (Rhee et al., 1999). Briefly, mechanical dissociation was accomplished using a custom-built vibration device and a fire-polished glass pipette oscillating at about 50–60 Hz (0.3–0.5 mm) on the surface of the PAG region. Slices were removed and the mechanically dissociated neurons were left for 15 min to allow the neurons to adhere to the bottom of the culture dish. 2.2. Electrical measurements All electrophysiological measurements were performed using conventional whole-cell patch recording mode at a holding potential (VH) of 0 mV (Axopatch 200B; Molecular Devices, Union City, CA, USA). Patch pipettes were made from borosilicate capillary glass (1.5 mm outer diameter, 0.9 mm inner diameter; G-1.5; Narishige, Tokyo, Japan) by use of a pipette puller (P-97; Sutter Instrument Co., Novato, CA, USA). The resistance of the recording pipettes filled with internal solution was 4–6 MΩ. The liquid junction potential and pipette capacitance were compensated for. Neurons were viewed under phase contrast on an inverted microscope (TE2000; Nikon). Membrane currents were filtered at 1 kHz, digitized at 4 kHz, and stored on a computer equipped with pCLAMP 10 (Molecular Devices). During the recordings, 10 mV hyperpolarizing step pulses (30 ms in duration) were periodically applied to monitor the access resistance. All experiments were performed at room temperature (22–25 °C). 2.3. Data analysis Spontaneous miniature inhibitory postsynaptic currents (mIPSCs) were counted and analyzed using the MiniAnalysis program (Synap-
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tosoft, Inc., Decatur, GA) as described previously (Jang et al., 2002). Briefly, mIPSCs were screened automatically using an amplitude threshold of 10 pA, and then visually accepted or rejected based upon the rise and decay times. Basal noise levels during voltage-clamp recordings were typically less than 8 pA. The average values of both the frequency and amplitude of mIPSCs during the control period (5– 10 min) or each drug condition (5–10 min) were calculated for each recording, and the frequency and amplitude of all the events during the agonist application (30 s or 3 min) were normalized to these values. The effects of these different conditions were quantified as a percentage increase in mIPSC frequency compared to the control values. The inter-event intervals and amplitudes of a large number of synaptic events obtained from the same neuron were examined by constructing cumulative probability distributions and compared using the Kolmogorov–Smirnov (K–S) test with Stat View software (SAS Institute, Inc., Cary, NC, USA). Numerical values are provided as the mean ± standard error of the mean (S.E.M.) using values normalized to the control. Significant differences in the mean amplitude and frequency were tested using Student's paired two-tailed t-test, using absolute values rather than normalized ones. Values of P b 0.05 were considered significant. 2.4. Solutions The ionic composition of the incubation medium consisted of (in mM) 124 NaCl, 3 KCl, 1.5 KH2PO4, 24 NaHCO3, 2 CaCl2, 1.3 MgSO4 and 10 glucose saturated with 95% O2 and 5% CO2. The pH was about 7.4– 7.5. The standard external solution was (in mM) 150 NaCl, 3 KCl, 2 CaCl2, 1 MgCl2, 10 glucose and 10 Hepes. The Ca2+-free external solution was (in mM) 150 NaCl, 3 KCl, 2 EGTA, 3 MgCl2, 10 glucose and 10 Hepes. The Na+-free external solution was (in mM) 150 N-methylD-glucamine-Cl, 3 KCl, 2 CaCl2, 1 MgCl2, 10 glucose and 10 Hepes. All these external solutions were adjusted to a pH of 7.4 with Tris-base. For recording mIPSCs, these standard external solutions routinely contained 300 nM tetrodotoxin (TTX), 10 μM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 20 μM DL-2-amino-5-phosphonovaleric acid (APV) to block voltage-dependent Na+ channels and ionotropic glutamate receptors, respectively. The ionic composition of the internal (pipette) solution was consisted of (in mM) 135 CsMeHSO3, 7 CsCl, 2 EGTA and 10 Hepes with a pH adjusted to 7.2 with Tris-base. 2.5. Drugs The drugs used in the present study were APV, TTX, CNQX, acetylcholine, nicotine, choline-Cl, 6-imino-3-(4-methoxyphenyl)-1 (6H)-pyridazinebutanoic acid HBr (SR95531), muscarine (from Sigma, St. Louis, MO, USA), dihydro-β-erythroidine (DHβE), methyllycaconitine (MLA), mecamylamine hydrochloride (MCA), α-bungarotoxin (from Tocris, Bristol, UK). All solutions containing drugs were applied using the ‘Y-tube system’ for rapid solution exchange (Murase et al., 1989). 3. Results 3.1. GABAergic mIPSCs in mechanically dissociated PAG neurons Previous comparative studies of the morphology of the PAG of rat, cat and monkey have shown considerable similarities in the types of neurons and their distributions within the PAG (Mantyh, 1982; Beitz and Shepard, 1985). Four major types of rat PAG neurons ranging between 10 and 35 μm in soma diameter have been identified based on their morphological properties; fusiform or bipolar neurons, multipolar neurons that have a very large number of dendrites, stellate cells that have 3–6 dendrites, and pyramidal-shaped neurons (Mantyh, 1982; Beitz and Shepard, 1985). After the mechanical
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dissociation of the PAG region, we found several kinds of neurons in soma diameter (≤ 15 μm) and shape (multipolar, bipolar and pyramidal-shaped). In the case of smaller bipolar or multipolar neurons (b10 μm in soma diameter), GABAergic synaptic events were hardly detected during electrophysiological recordings, so that it was hard to investigate the effect of acetylcholine on GABAergic synaptic transmission. In contrast, we could record abundant spontaneous synaptic events in bipolar and pyramidal-shaped neurons (10–15 μm in soma diameter), and there was no difference in the presynaptic response to acetylcholine or nicotine between bipolar and pyramidalshaped neurons. Therefore, we performed all electrophysiological recordings with these bipolar and pyramidal-shaped neurons. In the presence of 300 nM TTX, 10 μM CNQX and 20 μM APV, the spontaneous miniature currents were recorded from the mechanical dissociated PAG neurons at a VH of 0 mV. The observed spontaneous currents were completely and reversibly blocked by 10 μM SR95531, a selective GABAA receptor antagonist (Fig. 1A). The amplitude of spontaneous currents varied with the holding potentials (VH; Fig. 1B) and their reversal potential was −72.3 mV. This value was very similar to the theoretical Cl− equilibrium potential (ECl) of −78.7 mV, which was calculated from the Nernst equation using extracellular and intracellular Cl− concentrations ([Cl−]o ; 159 mM and [Cl−]i ; 7 mM, respectively). These results indicate that the spontaneous miniature currents are GABAergic mIPSCs mediated by GABAA receptors.
endogenous ligand of cholinergic receptors, on GABAergic mIPSCs. The application of acetylcholine (30 μM) during 3 min elicited a brief increase in the frequency of GABAergic mIPSCs and this increase was rapidly subsided to the control level within 40 s (Fig. 2A and B). In 11 neurons tested, acetylcholine increased both the mean mIPSC frequency to 351 ± 69% of the control (n = 11, P b 0.01) and the mean mIPSC amplitude (152 ± 21% of the control, n = 11, P b 0.01) (Fig. 2C insets). In addition, as shown in Fig. 2C, acetylcholine significantly shifted the distributions of inter-event interval and current amplitude of GABAergic mIPSCs to the left and right (P b 0.05, K–S test), respectively, indicating increases in the frequency and amplitude of GABAergic mIPSCs. An increase in mIPSC amplitude might result from the acetylcholine-mediated postsynaptic effects, such as an increase in the GABA sensitivity. However, this was not the case because acetylcholine (30 μM) did not affect 30 μM GABAinduced currents (101 ± 2% of the control, n = 6, P = 0.57, Fig. 2D). Taken together, these results suggest that acetylcholine acts presynaptically to increase spontaneous GABA release onto PAG neurons. 3.3. Facilitation of GABAergic mIPSCs mediated by presynaptic nicotinic receptors
To investigate whether functional nicotinic receptors exist on GABAergic presynaptic nerve terminals projecting to PAG neurons and their activation can modulate GABAergic synaptic transmission, we first tested the effect of exogenously applied acetylcholine, an
A previous study has shown that carbachol, a nonselective cholinergic agonist which can activate nicotinic and muscarinic receptors, decreases GABAergic transmission onto PAG neurons by activating presynaptic muscarinic receptors (Lau and Vaughan, 2008). Therefore, we further examined which muscarinic receptors also contribute to the acetylcholine-induced modulation of mIPSC frequency. To test this, we observed the effect of MCA, a nonselective nicotinic receptor antagonist, on the acetylcholine-induced increase in spontaneous GABA release. In 8 neurons, in which MCA effect was fully analyzed, the acetylcholine (30 μM)-induced initial increase in mIPSC
Fig. 1. GABAergic mIPSCs recorded from acutely isolated PAG neurons. A, A typical trace of GABAergic mIPSCs observed before, during and after the application of 10 μM SR95531 at a VH of 0 mV in the presence of 300 nM TTX, 10 μM CNQX and 20 μM APV. Insets represent GABAergic mIPSCs with an expanded time scale in each condition. Ba, Typical traces of GABAergic mIPSCs at various holding potentials (VH). b, A plot of the mean amplitude of mIPSCs at various VH values. The reversal potential was estimated to be − 72.3 mV using the Nernst equation, which was very similar to the theoretical ECl (− 78.8 mV). Each point was the mean and S.E.M. from 5 experiments.
Fig. 2. Effects of acetylcholine on GABAergic mIPSCs. A, A typical trace of GABAergic mIPSCs observed before, during and after application of 30 μM acetylcholine (ACh). Insets represent GABAergic mIPSCs with an expanded time scale in each condition. B, A time course of the acetylcholine-induced change in mIPSC frequency. Each point was the mean and S.E.M. from 11 experiments. C, Cumulative probability distributions for inter-event interval (a) and current amplitude (b) of GABAergic mIPSCs. 222 for control and 275 events for acetylcholine were plotted. Inset columns were the mean and S.E.M. from 11 experiments, respectively. *; P b 0.05, **; P b 0.01. D, Typical traces of GABA (30 μM)-induced membrane currents (IGABA) observed before, during and after the application of 30 μM acetylcholine.
3.2. Effect of acetylcholine on GABAergic mIPSCs
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frequency (during the first 30 s, 565 ± 135% of the control, n = 8, P b 0.01) was completely blocked by 10 μM MCA (during the first 30 s, 110 ± 21% to the MCA condition, n = 8, P = 0.94, Fig. 3A and B), suggesting that nicotinic receptors are involved in the acetylcholineinduced initial increase in mIPSC frequency. However, acetylcholine did not decrease GABAergic mIPSC frequency in the presence of MCA (Fig. 3A and B). In addition, muscarine (10 μM), a muscarinic receptor agonist, did not affect GABAergic mIPSC frequency (103± 10% to the condition, n = 6, P = 0.94, data not shown), indicating that the involvement of muscarinic receptors in the acetylcholine-induced modulation of spontaneous GABA release might be negligible. Next, we observed the effect of nicotine, a nicotinic receptor agonist, on GABAergic mIPSCs. Nicotine (3 μM, 30 s application) also increased both the mean mIPSC frequency to 620 ± 110% of the control (n = 10, P b 0.01) and the mean mIPSC amplitude (120± 7% to the control, n = 10, P b 0.05) (Fig. 3C and D insets). In addition, as shown in Fig. 3D, nicotine significantly shifted the distributions of inter-event interval and current amplitude to the left and right (P b 0.05, K–S test), respectively, indicating increases in the frequency and amplitude of GABAergic mIPSCs. The results suggest that function nicotinic receptors are expressed on GABAergic nerve terminals projecting to PAG neurons and that nicotinic but not muscarinic receptors are responsible for the cholinergic modulation of spontaneous GABAergic transmission in acutely isolated PAG neurons. In all subsequent pharmacological experiments, nicotine was used to activate presynaptic nicotinic receptors based on its selectivity for nicotinic receptors. 3.4. Subunit composition of presynaptic nicotinic receptors
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nicotine-induced increase in spontaneous GABA release. DHβE at a concentration of 1–10 μM is known to block β2-containing nicotinic receptors including α4β2 nicotinic receptors (Dickinson et al., 2008; Livingstone et al., 2009). In the presence of 10 μM DHβE, the nicotineinduced increase in mIPSC frequency was almost suppressed (133 ± 36% of the DHβE condition, n = 6, P = 0.99; Fig. 4Ab and B). We also observed the effect of MLA, a selective α7 nicotinic receptor antagonist, on the nicotine-induced increase in spontaneous GABA release. In the presence of 300 nM MLA, 3 μM nicotine still increased GABAergic mIPSC frequency (524 ± 154% to the MLA condition, n = 6, P b 0.01; Fig. 4Ac and B). In addition, nicotine (3 μM) still increased GABAergic mIPSC frequency even in the presence of 100 nM αbungarotoxin, a selective α7 nicotinic receptor antagonist (506 ± 117% to the MLA condition, n = 5, P b 0.05; Fig. 4Ad and B). Furthermore, choline (1 mM), which has a relatively higher sensitivity to α7 nicotinic receptors (Alkondon et al., 1997), had no facilitatory effect on GABAergic mIPSC frequency (Fig. 4B). Taken together, the results suggest that presynaptic nicotinic receptors at least contain β2 subunits rather than α7 subunits. 3.5. Ion permeability of presynaptic nicotinic receptors To elucidate the mechanisms underlying the nicotinic receptormediated facilitation of mIPSC frequency, we examined the effect of Ca2+-free (plus 2 mM EGTA) external solution on the nicotineinduced facilitation of mIPSC frequency. In the Ca2+-free external solution, both the frequency and amplitude of GABAergic mIPSCs were significantly reduced (30 ± 11% and 79 ± 8% of the control, n = 6, P b 0.05, respectively). This suggests indicate that GABAergic mIPSCs
In the mammalian brain, both heteromeric α4β2 and homomeric α7 nicotinic receptors are abundantly expressed (Gotti et al., 2006). Therefore, we examined the subunit composition of presynaptic nicotinic receptors expressed on GABAergic nerve terminals by use of pharmacological tools. We first observed the effect of DHβE on the
Fig. 3. Nicotinic receptors are responsible for the acetylcholine-induced increase in mIPSC frequency. A, A time course of the acetylcholine (30 μM)-induced change in mIPSC frequency in the presence of 10 μM MCA. Insets represent GABAergic mIPSCs with an expanded time scale in each condition. B, The acetylcholine-induced change in mIPSC frequency in the absence and presence of MCA. The frequency of GABAergic mIPSCs was calculated from the first 30 s period or entire (3 min) period during the acetylcholine application. Each column was the mean and S.E.M. from 8 experiments. *; P b 0.05, **; P b 0.01, n.s.; not significant. C, A typical trace of GABAergic mIPSCs observed before, during and after application of 3 μM nicotine (Nic). Insets represent GABAergic mIPSCs with an expanded time scale in each condition. D, Cumulative probability distributions for inter-event interval (a) and current amplitude (b) of GABAergic mIPSCs. 263 for control and 64 events for nicotine were plotted. Inset columns were the mean and S.E.M. from 10 experiments. *; P b 0.05, **; P b 0.01.
Fig. 4. The subunit composition of presynaptic nicotinic receptors on GABAergic nerve terminals. A, Typical traces of GABAergic mIPSCs observed before and during the application of 3 μM nicotine (Nic) in the control condition (a), in the presence of 10 μM DHβE (b), in the presence of 300 nM MLA (c), or in the presence of 100 nM αbungarotoxin (α-BgTx, d). B, Nicotine-induced changes in mIPSC frequency in each condition. Note that choline had no effect on mIPSC frequency. Each column was the mean and S.E.M. from 6 experiments. *; P b 0.05, **; P b 0.01, n.s.; not significant.
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Fig. 5. Presynaptic nicotinic receptors are less permeable to Ca2+. A, Typical traces of GABAergic mIPSCs observed before and during the application of 3 μM nicotine (Nic) in the control condition (a), in the Ca2+-free external solution (b), in the present of 100 μM Cd2+ (c), or in the Na+-free external solution (d). B, Nicotine-induced changes in mIPSC frequency in each condition. Each column was the mean and S.E.M. from 6 to 7 experiments. *; P b 0.05, **; P b 0.01, n.s.; not significant.
recorded from acutely isolated PAG neurons was, at least in part, dependent on extracellular Ca2+, and that mIPSCs observed in the Ca2+-free external solution might be classical miniature synaptic events, which are not dependent on the Ca2+ influx from the extracellular space (Scanziani et al., 1992; Capogna et al., 1993). In the Ca2+-free external solution, the facilitatory effect of nicotine on mIPSC frequency was completely occluded (115 ± 29% of the Ca2+free condition, n = 6, P = 0.99; Fig. 5Ab and Ba). We also tested the effect of Cd2+, a general VDCC blocker, on the nicotine-induced increase in mIPSC frequency. Cd2+ (100 µM) also decreased GABAergic mIPSC frequency (50 ± 9% of the control, n = 7, P b 0.01) but not amplitude (82 ± 9% of the control, n = 7, P = 0.08, Fig. 5Ac and B). In the presence of 100 µM Cd2+, the nicotineinduced increase in mIPSC frequency was completely attenuated (96 ± 9% of the Cd2+ condition, n = 7, P = 0.34, Fig. 5Ac and Bb). The results suggest that the nicotine-induced increase in spontaneous GABA release requires the Ca2+ influx from the extracellular space via presynaptic VDCCs. Finally, we examined the effect of Na+-free external solution on the nicotine-induced increase in mIPSC frequency. In the absence of extracellular Na+, nicotine again failed to increase the frequency of GABAergic mIPSCs (96 ± 9% of the Na+free control condition, n = 7, P = 0.34, Fig. 5Ad and Bc).
neurotransmitter release at a variety of central synapses (Clarke and Reuben, 1996; Fu et al., 1998; Genzen and McGehee, 2003; Guo et al., 1998; Kiyosawa et al., 2001). In the present study, we initially examined the effect of acetylcholine, an endogenous ligand of cholinergic receptors, on spontaneous GABA release onto acutely isolated PAG neurons. As acetylcholine can bind to both ionotropic nicotinic and metabotropic muscarinic receptors, these two receptor subtypes might be involved in the acetylcholine-induced modulation of spontaneous GABA release. However, several lines of evidence suggest that acetylcholine acts presynaptic nicotinic but not muscarinic receptors to increase spontaneous GABAergic transmission onto PAG neurons. First, acetylcholine significantly increased the frequency of GABAergic mIPSCs, indicating that acetylcholine acts presynaptically to change the probability of spontaneous GABA release. Although acetylcholine also increased the mean amplitude of mIPSCs, acetylcholine is likely to act presynaptically because acetylcholine had no effect on the sensitivity of postsynaptic GABAA receptors. An increase in mIPSC amplitude by acetylcholine might result from multivesicular release due to an increase in the intraterminal Ca2+ concentration ([Ca2+]terminal) (see also Sharma et al., 2008). Second, the acetylcholine-induced transient facilitation of GABAergic mIPSC frequency was completely blocked by MCA, a nonselective nicotinic receptor antagonist, and such an effect was closely mimicked by nicotine, a selective nicotinic receptor agonist. In addition, since acetylcholine had no effect on GABAergic mIPSC frequency after the blockade of nicotinic receptors with MCA, muscarinic receptors might be not involved in the acetylcholine-induced modulation of spontaneous GABA release. Third, the preparation used in this study should exclude any non-presynaptic actions, such as changes in the excitability of soma, because mechanically dissociated neurons retain functional cell-free presynaptic nerve terminals (for review, Akaike and Moorhouse, 2003). A recent study has shown that carbachol, a nonselective acetylcholine receptor agonist, suppresses GABAergic transmission in PAG neurons (Lau and Vaughan, 2008). However, the carbacholinduced presynaptic inhibition of GABAergic transmission would be largely mediated by the indirect M1/M3 muscarinic receptor-induced endocannabinoid signaling (Lau and Vaughan, 2008). In fact, the activation of M1 muscarinic receptors is known to produce endocannabinoids to inhibit neurotransmitter release (Ohno-Shosaku et al., 2003; Fukudome et al., 2004; Narushima et al., 2007). In contrast to these studies, our present results indicate that muscarinic receptors might be not involved in the acetylcholine-induced modulation of spontaneous GABA release onto PAG neurons, as muscarine had no effect on GABAergic mIPSC frequency. This discrepancy might be due to the preparation used, e.g., a slice preparation in the previous study (Lau and Vaughan, 2008) and isolated single neurons in the present study, because endocannabinoids, if any, produced by muscarinic receptor activation would be easily swept away during the drug application in isolated single neurons. Alternatively, acutely isolated PAG neurons used in the present study might be different from those used in the slice preparation, as several types of neurons have been identified in the PAG region. Further experiments combined with a morphological analysis would reveal the relationship between the cholinergic modulation of GABAergic transmission and neuronal types in the PAG region. 4.2. Presynaptic nicotinic receptors are less permeable to Ca2+
4. Discussion 4.1. Presynaptic nicotinic receptors facilitate spontaneous GABA release onto PAG neurons Previous studies have shown that nicotinic receptors are widely expressed on presynaptic terminals and their activation modulates
Despite of the large number of nicotinic receptor subunits, most of neuronal nicotinic receptors expressed in the CNS are heteromeric α4β2 or homomeric α7 nicotinic ones (for review, Gotti et al., 2006), and there are distinct differences in pharmacological and physiological properties between these two types of nicotinic receptors. For example, while α4β2 nicotinic receptors exhibit high affinity of
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nicotine (EC50; ∼ 15 μM) and low Ca2+ permeability (PCa/PNa; ∼ 1.5), α7 nicotinic receptors do low affinity of nicotine (EC50; ∼ 90 μM) and high Ca2+ permeability (PCa/PNa; N10) (Role and Berg, 1996; Giniatullin et al., 2005). In the present study, while DHβE, a selective antagonist of β2-containing nicotinic receptors (Dickinson et al., 2008; Livingstone et al., 2009), completely suppressed the nicotineinduced facilitation of spontaneous GABA release, MLA or αbungarotoxin, selective antagonists of α7-containing nicotinic receptors, did not block the nicotine action. In addition, choline, which is more sensitive to α7 than α4β2 nicotinic receptors (Alkondon et al., 1997), had no effect on GABAergic mIPSC frequency. These pharmacological properties indicate that presynaptic nicotinic receptors responsible for the nicotine-induced increase in spontaneous GABA release might be β2-containing, possibly α4β2 nicotinic receptors. However, further studies should be needed to elucidate the exact subunit composition of presynaptic nicotinic receptors as DHβE affects other subtypes such as α3β2 nicotinic receptors (Harvey and Luetje, 1996). Since nicotinic receptors are nonselective cation channels permeable to Na+ and Ca2+ (Role and Berg, 1996; Giniatullin et al., 2005), the activation of presynaptic nicotinic receptors is expected to enhance the probability of neurotransmitter release either by permitting Ca2+ influx passing through nicotinic receptors themselves or by eliciting a presynaptic depolarization, which subsequently activates presynaptic VDCCs. In the present study, the nicotine action on GABAergic mIPSCs was completely occluded by deleting extracellular Ca2+, indicating that the nicotine-induced increase in spontaneous GABA release requires the Ca2+ influx into nerve terminals from extracellular space. However, an increase in [Ca2+]terminal by nicotine might be not mediated by the direct Ca2+ influx passing through nicotinic receptors because the nicotineinduced increase in mIPSC frequency was eliminated by adding Cd2+, a general VDCC blocker. The results suggest that presynaptic nicotinic receptors are less permeable to Ca2+, and that the Na+ influx passing through nicotinic receptors directly depolarizes GABAergic nerve terminals to activate presynaptic VDCCs. This conclusion is consistent with our pharmacological data showing that presynaptic nicotinic receptors might be α4β2 nicotinic ones having low Ca2+ permeability, and that the facilitatory action of nicotine also completely disappeared in the Na+-free extracellular solution. In contrast to this idea, a previous study has shown that the activation of presynaptic α4β2 nicotinic receptors can increase spontaneous glycine release at spinal synapses even after the blockade of presynaptic VDCCs (Kiyosawa et al., 2001), suggesting that α4β2 nicotinic receptors expressed on glycinergic terminals are permeable to Ca2+. In the case of PAG neurons, even though presynaptic nicotinic receptors are permeable to Ca2+, the number of presynaptic nicotinic receptors expressed on single GABAergic nerve terminals might be too small so that an increase in [Ca2 + ]terminal via nicotinic receptors themselves cannot directly trigger spontaneous GABA release. 4.3. Physiological implications The PAG is closely involved in the descending pain inhibitory pathway, which is related to central analgesia (Yaksh, 1997; Lichtman et al., 1996; Finn et al., 2003), as the electrical stimulation of the PAG region is known to activate the descending inhibitory pathway to reduce pain (Reynolds, 1969; Monhemius et al., 2001). Considering that the major intrinsic neural circuit is a tonically active spontaneous GABAergic network within the PAG (Behbehani et al., 1990; Ogawa et al., 1994), GABAergic transmission would contribute to maintain the excitability of PAG neurons. For example, a behavioral study has revealed that the focal microinjection of bicuculline, a GABAA receptor antagonist, into the PAG abolishes the heat-evoked nociception (Sandkühler et al., 1989). In addition, the antinociceptive action of opioids is mainly mediated by decreasing GABAergic inhibition within the PAG (Moreau and Fields, 1986; Depaulis et al., 1987; Kalyuzhny
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et al., 1996). On the other hand, as the PAG receives innervations of cholinergic inputs from the pontine tegmentum (Woolf et al., 1990), the cholinergic system might be involved in the pain modulation by changing the excitability of PAG neurons. For example, Guimarães and Prado (1994), Guimarães et al. (2000) have reported that the microinjection of carbachol into the PAG produces analgesic actions, suggesting that cholinergic receptor-mediated mechanisms contribute to the modulation of nociceptive suppression in the PAG. Similarly, a recent study has shown that muscarinic receptors inhibit GABAergic transmission onto PAG neurons (Lau and Vaughan, 2008). In the present study, we have shown that functional nicotinic receptors, presumably α4β2 nicotinic receptors, are expressed on GABAergic nerve terminals projecting to PAG neurons, and that their activation transiently increases spontaneous GABA release. However, it is poorly known whether bipolar and pyramidal-shaped neurons used in this study are projection neurons innervating serotoninergic neurons or local interneurons, because there is little information available regarding the relationship between morphological cell types and their projection (see also Beitz, 1990). Although further study should be needed to elucidate the functional roles of presynaptic nicotinic receptors in the modulation of the descending pain inhibitory pathway, the present results suggest that presynaptic nicotinic receptors might temporally regulate the excitability of PAG neurons being not overexcited and eventually contribute to the cholinergic modulation of output from the PAG. Acknowledgements This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MEST) (Nos. R13-2008-009-01003-0 and KRF-2008-210-E00002). References Akaike, N., Moorhouse, A.J., 2003. Techniques: applications of the nerve-bouton preparation in neuropharmacology. Trends Pharmacol. Sci. 24, 44–47. Alkondon, M., Pereira, E.F., Cortes, W.S., Maelicke, A., Albuquerque, E.X., 1997. Choline is a selective agonist of alpha7 nicotinic acetylcholine receptors in the rat brain neurons. Eur. J. NeuroSci. 9, 2734–2742. Behbehani, M.M., 1995. Functional characteristics of the midbrain periaqueductal gray. Prog. Neurobiol. 46, 575–605. Behbehani, M.M., Jiang, M.R., Chandler, S.D., Ennis, M., 1990. The effect of GABA and its antagonists on midbrain periaqueductal gray neurons in the rat. Pain 40, 195–204. Beitz, A.J., 1990. Relationship of glutamate and aspartate to the periaqueductal grayraphe magnus projection: analysis using immunocytochemistry and microdialysis. J. Histochem. Cytochem. 38, 1755–1765. Beitz, A.J., Shepard, R.D., 1985. The midbrain periaqueductal gray in the rat. II. A Golgi analysis. J. Comp. Neurol. 237, 460–475. Capogna, M., Gahwiler, B.H., Thompson, S.M., 1993. Mechanism of µ-opioid receptormediated presynaptic inhibition in the rat hippocampus in vitro. J. Physiol. (Lond.) 470, 539–558. Clarke, P.B., Reuben, M., 1996. Release of [3H]-noradrenaline from rat hippocampal synaptosomes by nicotine: mediation by different nicotinic receptor subtypes from striatal [3H]-dopamine release. Br. J. Pharmacol. 117, 595–606. Depaulis, A., Morgan, M.M., Liebeskind, J.C., 1987. GABAergic modulation of the analgesic effects of morphine microinjected in the ventral periaqueductal gray matter of the rat. Brain Res. 436, 223–228. Dickinson, J.A., Kew, J.N., Wonnacott, S., 2008. Presynaptic α7- and β2-containing nicotinic acetylcholine receptors modulate excitatory amino acid release from rat prefrontal cortex nerve terminals via distinct cellular mechanisms. Mol. Pharmacol. 74, 348–359. Finn, D.P., Jhaveri, M.D., Beckett, S.R., Roe, C.H., Kendall, D.A., Marsden, C.A., Chapman, V., 2003. Effects of direct periaqueductal gray administration of a cannabinoid receptor agonist on nociceptive and aversive responses in rats. Neuropharmacology 45, 594–604. Fu, W.M., Liou, H.C., Chen, Y.H., 1998. Nerve terminal currents induced by autoreception of acetylcholine release. J. Neurosci. 18, 9954–9961. Fukudome, Y., Ohno-Shosaku, T., Matsui, M., Omori, Y., Fukaya, M., Tsubokawa, H., Taketo, M.M., Watanabe, M., Manabe, T., Kano, M., 2004. Two distinct classes of muscarinic action on hippocampal inhibitory synapses: M2-mediated direct suppression and M1/M3-mediated indirect suppression through endocannabinoid signalling. Eur. J. NeuroSci. 19, 2682–2692. Genzen, J.R., McGehee, D.S., 2003. Short- and long-term enhancement of excitatory transmission in the spinal cord dorsal horn by nicotinic acetylcholine receptors. Proc. Natl. Acad. Sci. U. S. A. 27 (100), 6807–6812.
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