- Email: [email protected]
Nicotinic acetylcholine receptor-mediated synaptic potentials in rat neocortex
Brain Research 887 (2000) 399–405 www.elsevier.com / locate / bres
Research report
Nicotinic acetylcholine receptor-mediated synaptic potentials in rat neocortex a,b a ,1 a, Z.G. Chu , F.M. Zhou , J.J. Hablitz * a
Department of Neurobiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA b Department of Psychology, University of Alabama at Birmingham, Birmingham, AL 35294, USA Accepted 3 October 2000
Abstract In the neocortex, fast excitatory synaptic transmission can typically be blocked by using excitatory amino acid (EAA) receptor antagonists. In recordings from layer II / III neocortical pyramidal neurons, we observed an evoked excitatory postsynaptic potential (EPSP) or current (EPSC) in the presence of EAA receptor antagonists (40–100 mM D-APV120 mM CNQX, or 5 mM kynurenic acid) plus the GABAA -receptor antagonist bicuculline (BIC, 20 mM). This EAA-antagonist resistant EPSC was observed in about 70% of neurons tested. It had a duration of approximately 20 ms and an amplitude of 61.566.8 pA at 270 mV (n535). The EAA-antagonist resistant EPSC current–voltage relation was linear and reversed near 0 mV (n523). The nonselective nicotinic acetylcholine receptor (nAChR) antagonists dihydro-b-erythroidine (DHbE, 100 mM) or mecamylamine (50 mM) reduced EPSC amplitudes by 42 (n520) and 33% (n59), respectively. EPSC kinetics were not significantly changed by either antagonist. Bath application of 10 mM neostigmine, a potent acetylcholinesterase inhibitor, prolonged the EPSC decay time. EAA-antagonist resistant EPSCs were observed in the presence of antagonists of metabotropic glutamate, serotonergic (5-HT 3 ) and purinergic (P2) receptors. The EAA-antagonist resistant EPSC appears to be due in part to activation of postsynaptic nAChRs. These results suggest the existence of functional synaptic nAChRs on pyramidal neurons in rat neocortex. 2000 Elsevier Science B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters, and receptors Topic: Acetycholine receptors: nicotinic Keywords: EPSP; Nicotine; Acetylcholine; Rat; Neocortex; Electrophysiology
1. Introduction Excitatory synaptic transmission in the neocortex is principally mediated by excitatory amino acid (EAA) receptors. Fast and slow components of excitatory postsynaptic potentials (EPSPs) are mediated by a-amino-3hydroxy-5-methylisoxazole-4-proprionic acid (AMPA) / kainate (KA) and N-methyl-D-aspartate (NMDA) receptors, respectively [31,34,35]. When 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX) and D-2-amino-5-phosphonvaleric acid (D-APV), respective blockers of AMPA / KA and NMDA receptors, are applied to in vitro brain slices, *Corresponding author. Tel.: 11-205-934-0742; fax: 11-205-9346571. E-mail address: [email protected] (J.J. Hablitz). 1 Present address: Division of Neuroscience, Baylor College of Medicine, Houston, TX 77030, USA.
excitatory transmission is generally thought to be blocked [2]. In the hippocampus, monosynaptic biphasic inhibitory postsynaptic potentials (IPSPs) are evoked in the presence of CNQX and D-APV [8]. GABAA - and GABA B -receptor antagonists block the early and late components of the IPSPs, respectively [8]. Similarly, monosynaptic inhibitory postsynaptic currents (IPSCs) are seen in neocortex after EAA-receptor blockade [4,39]. Under these conditions, fast IPSCs are blocked by bicuculline in sensorimotor cortex [4]. Our preliminary studies indicated that bicuculline (BIC) unmasks a fast EPSP resistant to EAA-receptor antagonists in rat frontal cortex [41]. The nature of the receptor mediating this EAA-antagonist resistant EPSP has not been established. Considerable evidence indicates that acetylcholine (ACh) is a synaptic neurotransmitter in the neocortex [17]. Intracellular studies in vitro have indicated that the postsynaptic effects of ACh are predominantly mediated by
0006-8993 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 00 )03076-6
400
Z.G. Chu et al. / Brain Research 887 (2000) 399 – 405
muscarinic receptors [22]. The predominant role of neuronal nicotinic (n) AChRs on synaptic transmission appears to be modulation of neurotransmitter release at presynaptic sites [1,14,24,38]. nAChRs can also regulate transmitter release at dopaminergic, serotonergic, GABAergic, and adrenergic synapses [21]. Rapid nAChR-mediated synaptic excitation of rat neocortical pyramidal neurons in vitro has not been reported [23,25,27,36] although excitations in response to iontophoretic applications of nicotine have been observed in vivo [18,32]. Roerig et al., [29] however, recorded spontaneous and evoked nAChR-mediated synaptic events in developing ferret visual cortex. We therefore examined whether nAChRs contributed to the EAA-antagonist resistant EPSCs in rat frontal neocortex.
2. Materials and methods Slices of frontal neocortex were prepared from Sprague– Dawley rats 16–60 days of age, using techniques described previously [7]. In brief, animals of either sex were anesthetized with ketamine (100 mg / kg) and decapitated. The brains were quickly removed and immersed in icecold oxygenated saline for 30–60 s. Six to eight coronal slices of frontal neocortex were cut on a Vibratome (300 mm thick for patch clamp and 400–500 mm for intracellular recordings). Slices were placed in a storage chamber and incubated at room temperature (21–238C) for at least 1 h. The normal extracellular bath solution contained (in mM): 125 NaCl, 3.5 KCl, 2.5 CaCl 2 , 1.3 MgSO 4 , 26 NaHCO 3 , and 10 D-glucose. It was bubbled with a mixture of 95% O 2 and 5% CO 2 to attain a steady-state level of oxygenation and maintain a pH of 7.4. Pipettes for wholecell patch-clamp recording were filled with a solution containing (in mM): 10 KCl, 125 K-gluconate, 0.5 EGTA, 10 HEPES, 2 MgATP, 0.2 NaGTP. The pH was adjusted to 7.3 with 1 M NaOH and osmolarity was adjusted to 270 mOsm with sucrose. Patch pipettes (3–4 MVs) were prepared from Garner KG-33 glass capillaries using a Narishige Model PP-83 puller. Intracellular microelectrodes were filled with 4 M potassium acetate and had resistances from 50 to 80 MV. Whole-cell patch clamp and intracellular recordings were obtained from layer II / III pyramidal neurons. Neurons were visualized in slices using a Zeiss Axioskop FS microscope equipped with Nomarski optics, a 403 water immersion lens and infrared illumination. Pyramidal cells were identified by their depth below the pial surface, presence of a prominent apical dendrite and regular firing properties. We have shown previously that such criteria unambiguously distinguish pyramidal cells from interneurons [40]. Electrical signals were recorded using an Axopatch-200A or Axoclamp 1A amplifier (Axon Instruments) for whole-cell and intracellular recordings, respec-
tively. Data was digitized, and analyzed off-line. Digitization and analysis of the records were achieved utilizing pClamp software (Axon Instruments). A bipolar stimulating electrode was placed in cortex at a distance of 150– 200 mm below the recording site. Constant-current pulses (100 ms, 50–600 mA) were used to evoke synaptic responses. Drugs used in the present study were obtained from the following sources: (1)-a-methyl-4-carboxyphenylglycine (MCPG) and kynurenic acid were from Tocris Neuramin; 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and D(2)2amino-5-phosphonovaleric acid (D-APV) were purchased from Cambridge Research Biochemicals; ketamine, dihydro-b-erythroidine (DHbE), mecamylamine, QX-314, suramin, MDL7222 and metropramide were purchased from RBI. Bicuculline methiodide and picrotoxin were obtained from Sigma. Compounds were prepared as stock solutions in water (except for CNQX which was dissolved in DMSO; final DMSO concentration,0.05%), stored at 2208C and diluted in the physiological saline just prior to recording. All compounds were bath applied, and each neuron served as its own control. Paired t-tests or a one-way analysis of variance (ANOVA) was used to test significance. A significance level of 0.05 was used. Data are presented as mean6S.E.M.
3. Results
3.1. Bicuculline unmasks a fast EPSP resistant to ionotropic glutamate receptor antagonists The synaptic response of a neocortical pyramidal cell, under control conditions, to electrical stimulation is shown in Fig. 1A. At the resting potential of 285 mV (Fig. 1A, lower) the response consisted of an EPSP–IPSP complex. Upon depolarization to 270 mV (Fig. 1A, upper), an EPSP, early IPSP and late IPSP were seen, as described previously [34,35]. Addition of 20–100 mM D-APV and 20 mM CNQX, antagonists of NMDA and AMPA / KA receptors, respectively, blocked the EPSP leaving IPSPs relatively unaffected (Fig. 1B). Subsequent addition of BIC (10–20 mM) or picrotoxin (100 mM) to the D-APV and CNQX containing saline, blocked the early IPSP. In the presence of these three antagonists, a novel, depolarizing component (Fig. 1C) was unmasked. This was followed by a late IPSP. The depolarizing synaptic potential was observed in 70% of the 22 neurons examined under these conditions. The latency to onset of the EAA-antagonist resistant EPSP was 260.3 ms (n522). The amplitude was up to 10 mV with a mean of 4.261 mV at a membrane potential of 270 mV. Single action potentials were often triggered at or above 260 mV by the EAA-antagonist resistant EPSP (Fig. 1C). The duration of this EPSP was 2062.8 ms at 270 mV (n535). The EAA-antagonist resistant EPSP
Z.G. Chu et al. / Brain Research 887 (2000) 399 – 405
401
Fig. 1. Blockade of EAA- and GABAA -receptors unmasks a novel EPSP. A: synaptic potentials recorded at two membrane potentials under control conditions. B: responses after bath application of APV1CNQX. EPSPs were blocked by application of EAA antagonists but biphasic inhibitory synaptic potentials were still observed. C: BIC was added in addition to APV and CNQX. A depolarizing potential was observed which could trigger an action potential.
displayed anomalous voltage dependence, with amplitude increasing upon membrane depolarization. In fifteen neurons where EPSP voltage-dependence was assessed, input resistance, measured by passing 20.2 nA hyperpolarizing current pulses, also increased with depolarization from 1463 MVs at 290 mV to 3668 MVs at 260 mV (n515). The anomalous EPSP voltage dependence is likely attributable to this change in input resistance [35]. The EAA-antagonist resistant EPSP was further characterized using whole-cell voltage-clamp recordings to explore the biophysical properties of the underlying synaptic current. In all subsequent experiments, synaptic currents were recorded in the presence of 40 mM D-APV plus 20 mM CNQX or 5 mM kynurenic acid and 20 mM BIC. The amplitude of the EAA-antagonist resistant EPSC was graded with stimulus intensity, as shown in Fig. 2A and B. Peak amplitude of EAA-antagonist resistant EPSCs in response to the strongest stimulation was 61.566.8 pA (n535) at a holding potential 270 mV. Examples of EAA-antagonist resistant EPSCs evoked at different holding potentials are shown in Fig. 2C. The amplitude of the response decreased with depolarization and reversed near 0 mV (n56). A current–voltage plot is shown in Fig. 2D. The underlying synaptic current shows a linear current– voltage relationship without the region of negative slope conductance characteristic of NMDA receptor-mediated responses.
3.2. Effects of other receptor antagonists Glutamate receptor subunits exhibit distinct differences
in sensitivity to competitive antagonists. The EAA-antagonist resistant EPSC observed in the presence of CNQX, D-APV and BIC could represent an EPSC insensitive to these antagonists at the concentrations employed. Alternatively, it could represent activation of other neurotransmitter receptors since EPSPs mediated by 5-HT 3 receptors [33], ATP receptors [10] and mGluRs [28] have been described. To test for EAA-receptors insensitive to CNQX and D-APV, ketamine (100 mM; n53) or kynurenic acid (3–5 mM; n55) were applied in the presence of CNQX and D-APV. No change in the EAA-antagonist resistant response was observed. 5-HT 3 receptor-mediated rapid synaptic transmission has been reported in rat amygdala slices [33]. Even though 5-HT positive nerve terminals have been found in rat cortex, their role in cortical synaptic transmission is unknown. We examined the effects of the selective 5-HT 3 antagonists MDL7222 (0.1–4 mM) and metropramide (4 mM) on EAA-antagonist resistant EPSPs. These 5-HT 3 antagonists were not effective in reducing the occurrence of EAA-antagonist resistant EPSPs. It has been reported that ATP can mediate a fast EPSC in the medial habenula [10] and purinergic receptor antagonists such as suramin can block fast EPSPs in cultured ganglion cells [11]. However, in the present experiments, bath application of 50–100 mM suramin had no effect on the ability to record EAA-antagonist resistant EPSCs. Similarly, EAA-antagonist resistant EPSCs were examined before and during the bath application of 500 mM MCPG, a nonselective mGluR antagonist. MCPG
402
Z.G. Chu et al. / Brain Research 887 (2000) 399 – 405
Fig. 2. Whole-cell voltage clamp recordings of EAA-antagonist resistant EPSCs. A: superimposed examples of EPSCs evoked by stimuli of increasing intensity. B: plot of response amplitude as a function of stimulus strength. Bath solution contained 5 mM kynurenate and 10 mM BIC. C and D. The current–voltage relationship of the EAA-antagonist resistant EPSC. The EAA-antagonist resistant EPSCs were recorded using whole-cell patch clamp technique. CsCl-based intracellular solution with QX-314. The cells were voltage-clamped at 270 mV, and then stepped from 280 mV to 130 mV in 10 increments. C: specimen traces at membrane potentials from 280 mV to 130 mV. The numbers beside each trace indicate the potential at which the membrane was voltage-clamped. D: I–V relationship for EAA-antagonist resistant EPSCs. I–V relations were relatively linear. A linear regression line fitted to the data indicated a reversal potential of 24.77 mV. Each data point is an average of 3–4 individual responses from each of six cells. The value expressed in the Y-axis is mean6S.E.M.
(500 mM) had no effect on the EAA-antagonist resistant EPSCs (n56).
3.3. Sensitivity to nAChR antagonists and AChE inhibitors Spontaneous and evoked nicotinic cholinergic synaptic potentials have been reported in developing ferret visual cortex [29]. To test if the EAA-antagonist resistant evoked EPSCs observed here were mediated by nAChRs, the nonselective nAChR antagonists DHbE (100 mM) and mecamylamine (50 mM) were bath applied. As shown in Fig. 3, DHbE reduced EAA-antagonist resistant EPSCs. This effect was reversible upon wash. In 20 cells, 100 mM DHbE reduced EPSC amplitude by 4263.59% (Fig. 3). Mecamylamine (50 mM) also significantly reduced EPSC amplitude, by 3362.75% (n59). These results suggest that functional synaptic nAChRs are present on pyramidal
neurons in rat frontal neocortex and mediate, at least in part, a fast EPSC. At the vertebrate neuromuscular junction, it is well known that anticholinesterases prolong the time course of ACh action [16]. We reasoned that if the EAA-antagonist resistant EPSC was mediated in part by nAChRs, cholinesterase inhibitors should prolong the EPSC time course. Fig. 4A shows EPSCs before and after bath application of 10 mM neostigmine, a potent AChE inhibitor. It can be seen that EPSC amplitude was not affected but the decay time was prolonged. This is apparent when the traces are superimposed (Fig. 4A, right). To quantify these changes, exponential curves were fitted to the decay phase of the EPSC. Decay time constants were significantly enhanced in the presence of neostigmine (Fig. 4B) (8.5360.25 versus 11.6560.42 ms) whereas amplitudes were not significantly different (51.8165.36 versus 52.6160.68 pA; n528).
Z.G. Chu et al. / Brain Research 887 (2000) 399 – 405
403
Fig. 3. Effects of the nAchR antagonist DHbE and mecamylamine on EAA-antagonist resistant EPSCs. A: EPSCs evoked at different stimulation intensities before (control) and during perfusion with DHbE (100 mM). The EPSC was reduced by bath application of DHbE. B: relationship between EPSC amplitude and stimulation intensity under control and during bath application of DHbE (100 mM). C: superimposed traces of evoked EPSCs taken from a pyramidal neuron before, during, and after application of DHbE. D: plot of averaged EPSC amplitude expressed as percent of control. DHbE significantly (P,0.05) decreased the amplitude. Each bar represents the mean6S.E.M. of 20 cells. E: sample traces of evoked EAA-antagonist resistant EPSC recorded before (control), during 50 mM mecamylamine and after washout. F: bar graph showing the mean effect of mecamylamine. Mecamylamine significantly (P,0.05) reduced the residual EPSC amplitude. Each bar represents the mean1S.E.M. of 9 cells.
4. Discussion The present study provides evidence that, in the presence of bicuculline and high concentrations of ionotropic glutamate receptor antagonists, functional excitatory synaptic transmission still remains in neocortical pyramidal neurons. The evoked EAA-antagonist resistant EPSC appears to be mediated in part by nAChRs. It had a reversal potential indicative of a non-specific cation channel, was reduced by nAChR antagonists and was prolonged by AChE inhibitors. Synaptic potentials mediated by nAChRs have been reported in developing ferret visual cortex [29] but not in other studies of postsynaptic ACh responses in neocortex [22,27,36]. Although several groups have used EAA-receptor antagonists to study directly evoked monosynaptic IPSPs
[4,39], GABAA -receptor antagonists were not generally employed. Furthermore, as shown in the present study, EAA-antagonist resistant EPSC amplitude, like that of other synaptic potentials, was stimulus intensity-dependent. Weak stimuli did not evoke detectable EPSCs. We were initially trying to isolate directly evoked GABA B -mediated late IPSPs by recording in the presence of EAA-receptor antagonists and BIC. Relatively strong stimulation is needed to evoke late IPSPs and this may have enhanced detection of the EAA-antagonist resistant EPSCs. EAA-antagonist resistant EPSCs were routinely observed. Careful mapping with the stimulation electrode revealed EPSCs in about 70% of recorded neurons. The source of synaptically released ACh, the presumed mechanism underlying the novel EPSC, is unclear. Although the cortex receives a diffuse cholinergic innervation from the
404
Z.G. Chu et al. / Brain Research 887 (2000) 399 – 405
Fig. 4. Acetylcholinesterase inhibition prolongs EAA-antagonist resistant EPSCs. A: evoked residual EPSCs before (control) and during perfusion with neostigmine (10 mM). Right panel shows superimposed traces under control conditions and after bath application of neostigmine. B: bar graph showing the effects of neostigmine on decay time constant of EAAantagonist resistant EPSCs (n528). Neostigmine significantly prolonged the EPSC decay time constant. C: bar graph showing that neostigmine had no significant effect on the amplitude of EAA-antagonist resistant EPSCs.
basal forebrain and midbrain [30], there is little convincing evidence for cholinergic neurons within the neocortex (but see [9]). It is hypothesized that cholinergic afferents remain functional in in vitro slice preparations and can be activated, as shown in the hippocampus [6]. The concentrations of nAChR antagonists employed in the present study were high, but similar to those used previously in ferret cortex [29]. It is not possible to infer the nature of the subunits involved. It is known from in situ hybridization studies that a3, a4, a5, a7 and b2 subunits are present in rat neocortex [20,37]. a7 subunits are known from functional studies to be involved with modulation of glutamate release (see review by MacDermott et al. [21]). The function of the other subunits is unknown although a4, a5 and b2 subunits may underlie nicotinic excitation of neocortical interneurons [27]. The present results suggest some or all of these subunits may also mediate synaptic excitation of neocortical pyramidal cells. The current–voltage relation of the EAA antagonist resistant EPSC is consistent with a response mediated by a non-specific cation channel. However, neuronal nAChRs typically show inwardly rectifying current–voltage relationships [5]. The failure to observe such rectification in the present study may result from the EAA-antagonist resistant EPSC being a mixed response. nAChR-antago-
nists only partially blocked the EPSCs. The nature of the unblocked component is unclear. mGluRs are clearly not involved since recordings of EAA-antagonist resistant EPSCs were not affected by bath application of the mGluR antagonist MCPG. Antagonists of 5HT 3 and purinergic receptors did not affect the occurrence of EAA-antagonist resistant EPSCs. However, the effects of these agents on responses recorded in the presence of nAChR-antagonists were not made. Such studies may identify other excitatory transmitter systems active in neocortex. The functional role of nAChR-mediated EPSCs in rat neocortex is unknown. In the presence of AMPA / KA and NMDA receptor antagonists, bath application of 4-aminopyridine (4-AP) induces spontaneous field potential discharges in normal [3] and dysplastic neocortex [15]. These discharges are blocked by BIC and are attributed to the synchronous bursting of inhibitory interneurons. The mechanisms underlying this synchronization, especially in neocortex are unclear. In the hippocampus, it has been suggested that an inward bicarbonate current underlies the depolarizing GABA response [19,26]. The present results, coupled with reports of nAChR-mediated postsynaptic potentials in hippocampal interneurons [12,13], suggest that other excitatory transmitter systems may contribute to the synchronization of GABAergic interneurons.
Acknowledgements This work was supported by NIH grants NS18145 and NS22373.
References [1] M. Alkondon, E.S. Rocha, A. Maelicke, E.X. Albuquerque, Diversity of nicotinic acetycholine receptors in rat brain. V. a-bungarotoxinsensitive nicotinic receptors in olfactory bulb neurons and presynaptic modulation of glutamate release, J. Pharmacol. Exp. Ther. 278 (1996) 1460–1471. [2] M. Andreasen, J.D.C. Lambert, M. Skovgaard Jensen, Direct demonstration of an N-methyl-D-aspartate receptor mediated component of excitatory synaptic transmission in area CA1 of the rat hippocampus, Neurosci. Lett. 93 (1998) 61–66. [3] J.A. Aram, H.B. Michaelson, R.K.S. Wong, Synchronized GABAergic IPSPs recorded in the neocortex after blockade of synaptic transmission mediated by excitatory amino acids, J. Neurophysiol. 65 (1991) 1034–1041. [4] B.D. Bennett, J.R. Huguenard, D.A. Prince, Adrenergic modulation of GABAA receptor-mediated inhibition in rat sensorimotor cortex, J. Neurophysiol. 79 (1998) 937–946. [5] R. Bonfante-Cabarcas, K.L. Swanson, M. Alkondon, E.X. Albuquerque, Diversity of nicotinic acetylcholine receptors in rat hippocampal neurons. IV. Regulation by external Ca 11 of alpha-bungarotoxin-sensitive receptor function and of rectification induced by internal Mg 11 , J. Pharmacol. Exp. Ther. 277 (1996) 432–444. [6] A.E. Cole, R.A. Nicoll, Acetylcholine mediates a slow synaptic potential in hippocampus pyramidal cells, Science 221 (1993) 1299–1301. [7] Z.G. Chu, J.J. Hablitz, Activation of group I mGluRs increases
Z.G. Chu et al. / Brain Research 887 (2000) 399 – 405
[8]
[9]
[10]
[11] [12]
[13]
[14]
[15] [16]
[17] [18]
[19]
[20]
[21]
[22]
[23]
[24]
spontaneous IPSC frequency in rat neocortex, J. Neurophysiol. 80 (1998) 621–627. C.H. Davies, S.N. Davies, G.L. Collingridge, Paired-pulse depression of monosynaptic GABA-mediated inhibitory postsynaptic responses in rat hippocampus, J. Physiol. (Lond.) 424 (1991) 513– 531. ´ M. del Mar Arroyo-Jimenez, J.-P. Bourgeois, L.M. Marubio, A.-M. ´ J.-P. Changeux, Le Sourd, O.P. Ottersen, R. Rinvik, A. Fairen, Ultrastructural localization of the a4-subunit of the neuronal acetylcholine nicotinic receptor in the rat substantia nigra, J. Neurosci. 19 (1999) 6475–6487. F.A. Edwards, A.J. Gibb, D. Colquhoun, ATP receptor-mediated synaptic currents in the central nervous system, Nature 359 (1992) 144–147. R.J. Evans, V. Derkach, A. Surprenant, ATP mediates fast synaptic transmission in mammalian neurons, Nature 357 (1992) 503–505. C.J. Frazier, A.V. Buhler, J.L. Weiner, T.V. Dunwiddie, Synaptic potentials mediated via a-bungarotoxin-sensitive nicotinic acetylcholine receptors in rat hippocampal interneurons, J. Neurosci. 18 (1998) 8228–8235. C.J. Frazier, Y.D. Rollins, C. Breese, S. Leonard, R. Freedman, T.V. Dunwiddie, Acetylcholine activates an a-bungarotoxin-sensitive nicotinic current in rat hippocampal interneurons, but not pyramidal cells, J. Neurosci. 18 (1998) 1187–1195. R. Gray, A.R.K. Rajan, M. Yakehiro, J. Dani, Hippocampal synaptic transmission enhanced by low concentrations of nicotine, Nature 383 (1996) 713–716. J.J. Hablitz, T. DeFazio, Excitability changes in freeze-induced neocortical microgyria, Epilepsy Res. 32 (1998) 75–82. B. Katz, R. Miledi, The binding of acetylcholine to receptors and its removal from the synaptic cleft, J. Physiol. (Lond.) 231 (1973) 549–574. K. Krnjevic, Chemical nature of synaptic transmission, Physiol. Rev. 54 (1974) 418–540. Y. Lamour, P. Dutar, A. Jobert, Excitatory effect of acetylcholine on different types of neurons in the first somatosensory neocortex of the rat: laminar distribution and pharmacological characteristics, Neuroscience 7 (1982) 1483–1504. K. Lamsa, K. Kaila, Ionic mechanisms of spontaneous GABAergic events in rat hippocampal slices exposed to 4-aminopryidine, J. Neurophysiol. 78 (1997) 2582–2591. C. Lena, J.-P. Changeux, Pathological mutations of nicotinic receptors and nicotine-based therapies for brain disorders, Curr. Opin. Neurobiol. 7 (1997) 674–682. A.B. MacDermott, L.W. Role, S.A. Siegelbaum, Presynaptic ionotropic receptors and the control of transmitter release, Ann. Rev. Neurosci. 22 (1999) 443–485. D.A. McCormick, D.A. Prince, Mechanisms of action of acetylcholine in the guinea-pig cerebral cortex in vitro, J. Physiol. (Lond.) 375 (1986) 169–194. D.A. McCormick, D.A. Prince, Acetylcholine causes rapid nicotinic excitation in the medial habenular nucleus of guinea pig, in vitro, J. Neurosci. 7 (1987) 742–752. D.S. McGehee, L.W. Role, Presynaptic ionotropic receptors, Curr. Opin. Neurobiol. 6 (1996) 342–349.
405
[25] A.R. McQuiston, D.V. Madison, Nicotinic receptor activation excites distinct subtypes of interneurons in the rat hippocampus, J. Neurosci. 19 (1999) 2887–2896. [26] K.L. Perkins, R.K.S. Wong, Ionic basis of the postsynaptic depolarizing GABA response in hippocampal pyramidal cells, J. Neurophysiol. 76 (1996) 3886–3894. [27] J.T. Porter, B. Cauli, K. Tsuzuki, B. Lambolez, J. Rossier, E. Audinat, Selective excitation of subtypes of neocortical interneurons by nicotinic receptors, J. Neurosci. 19 (1999) 5228–5235. [28] L.D. Pozzo-Miller, J.J. Petrozzino, J.A. Connor, G-protein-coupled receptors mediate a fast excitatory postsynaptic current in CA3 pyramidal neurons in hippocampal slices, J. Neurosci. 15 (1995) 8320–8330. [29] B. Roerig, D.A. Nelson, L.C. Katz, Fast synaptic signaling by nicotinic acetylcholine and serotonin 5-HT 3 receptors in developing visual cortex, J. Neurosci. 17 (1997) 8353–8362. [30] C.C.D. Shute, P.R. Lewis, The ascending cholinergic reticular system: neocortical, olfactory and subcortical projections, Brain 90 (1967) 497–520. [31] P. Stern, F.A. Edwards, B. Sakmann, Fast and slow components of unitary EPSCs on stellate cells elicited by focal stimulation in slices of rat visual cortex, J. Physiol (Lond.) 449 (1992) 247–278. [32] T.W. Stone, Cholinergic mechanisms in the rat somatosensory cerebral cortex, J. Physiol. (Lond.) 225 (1972) 485–499. [33] S. Sugita, K.Z. Shen, R.A. North, 5-hydroxytryptamine is a fast excitatory transmitter at 5-HT3 receptors in rat amygdala, Neuron 8 (1992) 199–203. [34] B. Sutor, J.J. Hablitz, Excitatory postsynaptic potentials in rat neocortical neurons in vitro. I. Electrophysiological evidence for two distinct EPSPs, J. Neurophysiol. 61 (1989) 607–620. [35] B. Sutor, J.J. Hablitz, Excitatory postsynaptic potentials in rat neocortical neurons in vitro. II. Involvement of N-methyl-D-aspartate receptors in the generation of EPSPs, J. Neurophysiol. 61 (1989) 621–634. [36] C. Vidal, J.-P. Changeux, Nicotinic and muscarinic modulations of excitatory synaptic transmission in the rat prefrontal cortex in vitro, Neuroscience 56 (1993) 23–32. [37] E. Wada, K. Wada, J. Boulter, E. Deneris, S. Heinemann, J. Patrick, L.W. Swanson, Distribution of alpha 2, alpha 3, alpha 4, and beta 2 neuronal nicotinic receptor subunit mRNAs in the central nervous system: a hybridization histochemical study in the rat, J. Comp. Neurol. 284 (1989) 314–335. [38] S. Wonnacott, Presynaptic nicotinic ACh receptors, Trends Neurosci. 20 (1997) 92–98. [39] Z. Xiang, J.R. Huguenard, D.A. Prince, GABAA receptor-mediated currents in interneurons and pyramidal cells of rat visual cortex, J. Physiol. (Lond.) 506 (1998) 715–730. [40] F.M. Zhou, J.J. Hablitz, Morphological properties of rat neocortical layer I neurons examined using intracellular staining, J. Comp. Neurol. 376 (1996) 198–213. [41] F.M. Zhou, J.J. Hablitz, Bicuculline unmasks a fast EPSP resistant to glutamatergic antagonists in rat neocortex, Soc. Neurosci. Abstr. 19 (1993) 469.
Research report
Nicotinic acetylcholine receptor-mediated synaptic potentials in rat neocortex a,b a ,1 a, Z.G. Chu , F.M. Zhou , J.J. Hablitz * a
Department of Neurobiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA b Department of Psychology, University of Alabama at Birmingham, Birmingham, AL 35294, USA Accepted 3 October 2000
Abstract In the neocortex, fast excitatory synaptic transmission can typically be blocked by using excitatory amino acid (EAA) receptor antagonists. In recordings from layer II / III neocortical pyramidal neurons, we observed an evoked excitatory postsynaptic potential (EPSP) or current (EPSC) in the presence of EAA receptor antagonists (40–100 mM D-APV120 mM CNQX, or 5 mM kynurenic acid) plus the GABAA -receptor antagonist bicuculline (BIC, 20 mM). This EAA-antagonist resistant EPSC was observed in about 70% of neurons tested. It had a duration of approximately 20 ms and an amplitude of 61.566.8 pA at 270 mV (n535). The EAA-antagonist resistant EPSC current–voltage relation was linear and reversed near 0 mV (n523). The nonselective nicotinic acetylcholine receptor (nAChR) antagonists dihydro-b-erythroidine (DHbE, 100 mM) or mecamylamine (50 mM) reduced EPSC amplitudes by 42 (n520) and 33% (n59), respectively. EPSC kinetics were not significantly changed by either antagonist. Bath application of 10 mM neostigmine, a potent acetylcholinesterase inhibitor, prolonged the EPSC decay time. EAA-antagonist resistant EPSCs were observed in the presence of antagonists of metabotropic glutamate, serotonergic (5-HT 3 ) and purinergic (P2) receptors. The EAA-antagonist resistant EPSC appears to be due in part to activation of postsynaptic nAChRs. These results suggest the existence of functional synaptic nAChRs on pyramidal neurons in rat neocortex. 2000 Elsevier Science B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters, and receptors Topic: Acetycholine receptors: nicotinic Keywords: EPSP; Nicotine; Acetylcholine; Rat; Neocortex; Electrophysiology
1. Introduction Excitatory synaptic transmission in the neocortex is principally mediated by excitatory amino acid (EAA) receptors. Fast and slow components of excitatory postsynaptic potentials (EPSPs) are mediated by a-amino-3hydroxy-5-methylisoxazole-4-proprionic acid (AMPA) / kainate (KA) and N-methyl-D-aspartate (NMDA) receptors, respectively [31,34,35]. When 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX) and D-2-amino-5-phosphonvaleric acid (D-APV), respective blockers of AMPA / KA and NMDA receptors, are applied to in vitro brain slices, *Corresponding author. Tel.: 11-205-934-0742; fax: 11-205-9346571. E-mail address: [email protected] (J.J. Hablitz). 1 Present address: Division of Neuroscience, Baylor College of Medicine, Houston, TX 77030, USA.
excitatory transmission is generally thought to be blocked [2]. In the hippocampus, monosynaptic biphasic inhibitory postsynaptic potentials (IPSPs) are evoked in the presence of CNQX and D-APV [8]. GABAA - and GABA B -receptor antagonists block the early and late components of the IPSPs, respectively [8]. Similarly, monosynaptic inhibitory postsynaptic currents (IPSCs) are seen in neocortex after EAA-receptor blockade [4,39]. Under these conditions, fast IPSCs are blocked by bicuculline in sensorimotor cortex [4]. Our preliminary studies indicated that bicuculline (BIC) unmasks a fast EPSP resistant to EAA-receptor antagonists in rat frontal cortex [41]. The nature of the receptor mediating this EAA-antagonist resistant EPSP has not been established. Considerable evidence indicates that acetylcholine (ACh) is a synaptic neurotransmitter in the neocortex [17]. Intracellular studies in vitro have indicated that the postsynaptic effects of ACh are predominantly mediated by
0006-8993 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 00 )03076-6
400
Z.G. Chu et al. / Brain Research 887 (2000) 399 – 405
muscarinic receptors [22]. The predominant role of neuronal nicotinic (n) AChRs on synaptic transmission appears to be modulation of neurotransmitter release at presynaptic sites [1,14,24,38]. nAChRs can also regulate transmitter release at dopaminergic, serotonergic, GABAergic, and adrenergic synapses [21]. Rapid nAChR-mediated synaptic excitation of rat neocortical pyramidal neurons in vitro has not been reported [23,25,27,36] although excitations in response to iontophoretic applications of nicotine have been observed in vivo [18,32]. Roerig et al., [29] however, recorded spontaneous and evoked nAChR-mediated synaptic events in developing ferret visual cortex. We therefore examined whether nAChRs contributed to the EAA-antagonist resistant EPSCs in rat frontal neocortex.
2. Materials and methods Slices of frontal neocortex were prepared from Sprague– Dawley rats 16–60 days of age, using techniques described previously [7]. In brief, animals of either sex were anesthetized with ketamine (100 mg / kg) and decapitated. The brains were quickly removed and immersed in icecold oxygenated saline for 30–60 s. Six to eight coronal slices of frontal neocortex were cut on a Vibratome (300 mm thick for patch clamp and 400–500 mm for intracellular recordings). Slices were placed in a storage chamber and incubated at room temperature (21–238C) for at least 1 h. The normal extracellular bath solution contained (in mM): 125 NaCl, 3.5 KCl, 2.5 CaCl 2 , 1.3 MgSO 4 , 26 NaHCO 3 , and 10 D-glucose. It was bubbled with a mixture of 95% O 2 and 5% CO 2 to attain a steady-state level of oxygenation and maintain a pH of 7.4. Pipettes for wholecell patch-clamp recording were filled with a solution containing (in mM): 10 KCl, 125 K-gluconate, 0.5 EGTA, 10 HEPES, 2 MgATP, 0.2 NaGTP. The pH was adjusted to 7.3 with 1 M NaOH and osmolarity was adjusted to 270 mOsm with sucrose. Patch pipettes (3–4 MVs) were prepared from Garner KG-33 glass capillaries using a Narishige Model PP-83 puller. Intracellular microelectrodes were filled with 4 M potassium acetate and had resistances from 50 to 80 MV. Whole-cell patch clamp and intracellular recordings were obtained from layer II / III pyramidal neurons. Neurons were visualized in slices using a Zeiss Axioskop FS microscope equipped with Nomarski optics, a 403 water immersion lens and infrared illumination. Pyramidal cells were identified by their depth below the pial surface, presence of a prominent apical dendrite and regular firing properties. We have shown previously that such criteria unambiguously distinguish pyramidal cells from interneurons [40]. Electrical signals were recorded using an Axopatch-200A or Axoclamp 1A amplifier (Axon Instruments) for whole-cell and intracellular recordings, respec-
tively. Data was digitized, and analyzed off-line. Digitization and analysis of the records were achieved utilizing pClamp software (Axon Instruments). A bipolar stimulating electrode was placed in cortex at a distance of 150– 200 mm below the recording site. Constant-current pulses (100 ms, 50–600 mA) were used to evoke synaptic responses. Drugs used in the present study were obtained from the following sources: (1)-a-methyl-4-carboxyphenylglycine (MCPG) and kynurenic acid were from Tocris Neuramin; 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and D(2)2amino-5-phosphonovaleric acid (D-APV) were purchased from Cambridge Research Biochemicals; ketamine, dihydro-b-erythroidine (DHbE), mecamylamine, QX-314, suramin, MDL7222 and metropramide were purchased from RBI. Bicuculline methiodide and picrotoxin were obtained from Sigma. Compounds were prepared as stock solutions in water (except for CNQX which was dissolved in DMSO; final DMSO concentration,0.05%), stored at 2208C and diluted in the physiological saline just prior to recording. All compounds were bath applied, and each neuron served as its own control. Paired t-tests or a one-way analysis of variance (ANOVA) was used to test significance. A significance level of 0.05 was used. Data are presented as mean6S.E.M.
3. Results
3.1. Bicuculline unmasks a fast EPSP resistant to ionotropic glutamate receptor antagonists The synaptic response of a neocortical pyramidal cell, under control conditions, to electrical stimulation is shown in Fig. 1A. At the resting potential of 285 mV (Fig. 1A, lower) the response consisted of an EPSP–IPSP complex. Upon depolarization to 270 mV (Fig. 1A, upper), an EPSP, early IPSP and late IPSP were seen, as described previously [34,35]. Addition of 20–100 mM D-APV and 20 mM CNQX, antagonists of NMDA and AMPA / KA receptors, respectively, blocked the EPSP leaving IPSPs relatively unaffected (Fig. 1B). Subsequent addition of BIC (10–20 mM) or picrotoxin (100 mM) to the D-APV and CNQX containing saline, blocked the early IPSP. In the presence of these three antagonists, a novel, depolarizing component (Fig. 1C) was unmasked. This was followed by a late IPSP. The depolarizing synaptic potential was observed in 70% of the 22 neurons examined under these conditions. The latency to onset of the EAA-antagonist resistant EPSP was 260.3 ms (n522). The amplitude was up to 10 mV with a mean of 4.261 mV at a membrane potential of 270 mV. Single action potentials were often triggered at or above 260 mV by the EAA-antagonist resistant EPSP (Fig. 1C). The duration of this EPSP was 2062.8 ms at 270 mV (n535). The EAA-antagonist resistant EPSP
Z.G. Chu et al. / Brain Research 887 (2000) 399 – 405
401
Fig. 1. Blockade of EAA- and GABAA -receptors unmasks a novel EPSP. A: synaptic potentials recorded at two membrane potentials under control conditions. B: responses after bath application of APV1CNQX. EPSPs were blocked by application of EAA antagonists but biphasic inhibitory synaptic potentials were still observed. C: BIC was added in addition to APV and CNQX. A depolarizing potential was observed which could trigger an action potential.
displayed anomalous voltage dependence, with amplitude increasing upon membrane depolarization. In fifteen neurons where EPSP voltage-dependence was assessed, input resistance, measured by passing 20.2 nA hyperpolarizing current pulses, also increased with depolarization from 1463 MVs at 290 mV to 3668 MVs at 260 mV (n515). The anomalous EPSP voltage dependence is likely attributable to this change in input resistance [35]. The EAA-antagonist resistant EPSP was further characterized using whole-cell voltage-clamp recordings to explore the biophysical properties of the underlying synaptic current. In all subsequent experiments, synaptic currents were recorded in the presence of 40 mM D-APV plus 20 mM CNQX or 5 mM kynurenic acid and 20 mM BIC. The amplitude of the EAA-antagonist resistant EPSC was graded with stimulus intensity, as shown in Fig. 2A and B. Peak amplitude of EAA-antagonist resistant EPSCs in response to the strongest stimulation was 61.566.8 pA (n535) at a holding potential 270 mV. Examples of EAA-antagonist resistant EPSCs evoked at different holding potentials are shown in Fig. 2C. The amplitude of the response decreased with depolarization and reversed near 0 mV (n56). A current–voltage plot is shown in Fig. 2D. The underlying synaptic current shows a linear current– voltage relationship without the region of negative slope conductance characteristic of NMDA receptor-mediated responses.
3.2. Effects of other receptor antagonists Glutamate receptor subunits exhibit distinct differences
in sensitivity to competitive antagonists. The EAA-antagonist resistant EPSC observed in the presence of CNQX, D-APV and BIC could represent an EPSC insensitive to these antagonists at the concentrations employed. Alternatively, it could represent activation of other neurotransmitter receptors since EPSPs mediated by 5-HT 3 receptors [33], ATP receptors [10] and mGluRs [28] have been described. To test for EAA-receptors insensitive to CNQX and D-APV, ketamine (100 mM; n53) or kynurenic acid (3–5 mM; n55) were applied in the presence of CNQX and D-APV. No change in the EAA-antagonist resistant response was observed. 5-HT 3 receptor-mediated rapid synaptic transmission has been reported in rat amygdala slices [33]. Even though 5-HT positive nerve terminals have been found in rat cortex, their role in cortical synaptic transmission is unknown. We examined the effects of the selective 5-HT 3 antagonists MDL7222 (0.1–4 mM) and metropramide (4 mM) on EAA-antagonist resistant EPSPs. These 5-HT 3 antagonists were not effective in reducing the occurrence of EAA-antagonist resistant EPSPs. It has been reported that ATP can mediate a fast EPSC in the medial habenula [10] and purinergic receptor antagonists such as suramin can block fast EPSPs in cultured ganglion cells [11]. However, in the present experiments, bath application of 50–100 mM suramin had no effect on the ability to record EAA-antagonist resistant EPSCs. Similarly, EAA-antagonist resistant EPSCs were examined before and during the bath application of 500 mM MCPG, a nonselective mGluR antagonist. MCPG
402
Z.G. Chu et al. / Brain Research 887 (2000) 399 – 405
Fig. 2. Whole-cell voltage clamp recordings of EAA-antagonist resistant EPSCs. A: superimposed examples of EPSCs evoked by stimuli of increasing intensity. B: plot of response amplitude as a function of stimulus strength. Bath solution contained 5 mM kynurenate and 10 mM BIC. C and D. The current–voltage relationship of the EAA-antagonist resistant EPSC. The EAA-antagonist resistant EPSCs were recorded using whole-cell patch clamp technique. CsCl-based intracellular solution with QX-314. The cells were voltage-clamped at 270 mV, and then stepped from 280 mV to 130 mV in 10 increments. C: specimen traces at membrane potentials from 280 mV to 130 mV. The numbers beside each trace indicate the potential at which the membrane was voltage-clamped. D: I–V relationship for EAA-antagonist resistant EPSCs. I–V relations were relatively linear. A linear regression line fitted to the data indicated a reversal potential of 24.77 mV. Each data point is an average of 3–4 individual responses from each of six cells. The value expressed in the Y-axis is mean6S.E.M.
(500 mM) had no effect on the EAA-antagonist resistant EPSCs (n56).
3.3. Sensitivity to nAChR antagonists and AChE inhibitors Spontaneous and evoked nicotinic cholinergic synaptic potentials have been reported in developing ferret visual cortex [29]. To test if the EAA-antagonist resistant evoked EPSCs observed here were mediated by nAChRs, the nonselective nAChR antagonists DHbE (100 mM) and mecamylamine (50 mM) were bath applied. As shown in Fig. 3, DHbE reduced EAA-antagonist resistant EPSCs. This effect was reversible upon wash. In 20 cells, 100 mM DHbE reduced EPSC amplitude by 4263.59% (Fig. 3). Mecamylamine (50 mM) also significantly reduced EPSC amplitude, by 3362.75% (n59). These results suggest that functional synaptic nAChRs are present on pyramidal
neurons in rat frontal neocortex and mediate, at least in part, a fast EPSC. At the vertebrate neuromuscular junction, it is well known that anticholinesterases prolong the time course of ACh action [16]. We reasoned that if the EAA-antagonist resistant EPSC was mediated in part by nAChRs, cholinesterase inhibitors should prolong the EPSC time course. Fig. 4A shows EPSCs before and after bath application of 10 mM neostigmine, a potent AChE inhibitor. It can be seen that EPSC amplitude was not affected but the decay time was prolonged. This is apparent when the traces are superimposed (Fig. 4A, right). To quantify these changes, exponential curves were fitted to the decay phase of the EPSC. Decay time constants were significantly enhanced in the presence of neostigmine (Fig. 4B) (8.5360.25 versus 11.6560.42 ms) whereas amplitudes were not significantly different (51.8165.36 versus 52.6160.68 pA; n528).
Z.G. Chu et al. / Brain Research 887 (2000) 399 – 405
403
Fig. 3. Effects of the nAchR antagonist DHbE and mecamylamine on EAA-antagonist resistant EPSCs. A: EPSCs evoked at different stimulation intensities before (control) and during perfusion with DHbE (100 mM). The EPSC was reduced by bath application of DHbE. B: relationship between EPSC amplitude and stimulation intensity under control and during bath application of DHbE (100 mM). C: superimposed traces of evoked EPSCs taken from a pyramidal neuron before, during, and after application of DHbE. D: plot of averaged EPSC amplitude expressed as percent of control. DHbE significantly (P,0.05) decreased the amplitude. Each bar represents the mean6S.E.M. of 20 cells. E: sample traces of evoked EAA-antagonist resistant EPSC recorded before (control), during 50 mM mecamylamine and after washout. F: bar graph showing the mean effect of mecamylamine. Mecamylamine significantly (P,0.05) reduced the residual EPSC amplitude. Each bar represents the mean1S.E.M. of 9 cells.
4. Discussion The present study provides evidence that, in the presence of bicuculline and high concentrations of ionotropic glutamate receptor antagonists, functional excitatory synaptic transmission still remains in neocortical pyramidal neurons. The evoked EAA-antagonist resistant EPSC appears to be mediated in part by nAChRs. It had a reversal potential indicative of a non-specific cation channel, was reduced by nAChR antagonists and was prolonged by AChE inhibitors. Synaptic potentials mediated by nAChRs have been reported in developing ferret visual cortex [29] but not in other studies of postsynaptic ACh responses in neocortex [22,27,36]. Although several groups have used EAA-receptor antagonists to study directly evoked monosynaptic IPSPs
[4,39], GABAA -receptor antagonists were not generally employed. Furthermore, as shown in the present study, EAA-antagonist resistant EPSC amplitude, like that of other synaptic potentials, was stimulus intensity-dependent. Weak stimuli did not evoke detectable EPSCs. We were initially trying to isolate directly evoked GABA B -mediated late IPSPs by recording in the presence of EAA-receptor antagonists and BIC. Relatively strong stimulation is needed to evoke late IPSPs and this may have enhanced detection of the EAA-antagonist resistant EPSCs. EAA-antagonist resistant EPSCs were routinely observed. Careful mapping with the stimulation electrode revealed EPSCs in about 70% of recorded neurons. The source of synaptically released ACh, the presumed mechanism underlying the novel EPSC, is unclear. Although the cortex receives a diffuse cholinergic innervation from the
404
Z.G. Chu et al. / Brain Research 887 (2000) 399 – 405
Fig. 4. Acetylcholinesterase inhibition prolongs EAA-antagonist resistant EPSCs. A: evoked residual EPSCs before (control) and during perfusion with neostigmine (10 mM). Right panel shows superimposed traces under control conditions and after bath application of neostigmine. B: bar graph showing the effects of neostigmine on decay time constant of EAAantagonist resistant EPSCs (n528). Neostigmine significantly prolonged the EPSC decay time constant. C: bar graph showing that neostigmine had no significant effect on the amplitude of EAA-antagonist resistant EPSCs.
basal forebrain and midbrain [30], there is little convincing evidence for cholinergic neurons within the neocortex (but see [9]). It is hypothesized that cholinergic afferents remain functional in in vitro slice preparations and can be activated, as shown in the hippocampus [6]. The concentrations of nAChR antagonists employed in the present study were high, but similar to those used previously in ferret cortex [29]. It is not possible to infer the nature of the subunits involved. It is known from in situ hybridization studies that a3, a4, a5, a7 and b2 subunits are present in rat neocortex [20,37]. a7 subunits are known from functional studies to be involved with modulation of glutamate release (see review by MacDermott et al. [21]). The function of the other subunits is unknown although a4, a5 and b2 subunits may underlie nicotinic excitation of neocortical interneurons [27]. The present results suggest some or all of these subunits may also mediate synaptic excitation of neocortical pyramidal cells. The current–voltage relation of the EAA antagonist resistant EPSC is consistent with a response mediated by a non-specific cation channel. However, neuronal nAChRs typically show inwardly rectifying current–voltage relationships [5]. The failure to observe such rectification in the present study may result from the EAA-antagonist resistant EPSC being a mixed response. nAChR-antago-
nists only partially blocked the EPSCs. The nature of the unblocked component is unclear. mGluRs are clearly not involved since recordings of EAA-antagonist resistant EPSCs were not affected by bath application of the mGluR antagonist MCPG. Antagonists of 5HT 3 and purinergic receptors did not affect the occurrence of EAA-antagonist resistant EPSCs. However, the effects of these agents on responses recorded in the presence of nAChR-antagonists were not made. Such studies may identify other excitatory transmitter systems active in neocortex. The functional role of nAChR-mediated EPSCs in rat neocortex is unknown. In the presence of AMPA / KA and NMDA receptor antagonists, bath application of 4-aminopyridine (4-AP) induces spontaneous field potential discharges in normal [3] and dysplastic neocortex [15]. These discharges are blocked by BIC and are attributed to the synchronous bursting of inhibitory interneurons. The mechanisms underlying this synchronization, especially in neocortex are unclear. In the hippocampus, it has been suggested that an inward bicarbonate current underlies the depolarizing GABA response [19,26]. The present results, coupled with reports of nAChR-mediated postsynaptic potentials in hippocampal interneurons [12,13], suggest that other excitatory transmitter systems may contribute to the synchronization of GABAergic interneurons.
Acknowledgements This work was supported by NIH grants NS18145 and NS22373.
References [1] M. Alkondon, E.S. Rocha, A. Maelicke, E.X. Albuquerque, Diversity of nicotinic acetycholine receptors in rat brain. V. a-bungarotoxinsensitive nicotinic receptors in olfactory bulb neurons and presynaptic modulation of glutamate release, J. Pharmacol. Exp. Ther. 278 (1996) 1460–1471. [2] M. Andreasen, J.D.C. Lambert, M. Skovgaard Jensen, Direct demonstration of an N-methyl-D-aspartate receptor mediated component of excitatory synaptic transmission in area CA1 of the rat hippocampus, Neurosci. Lett. 93 (1998) 61–66. [3] J.A. Aram, H.B. Michaelson, R.K.S. Wong, Synchronized GABAergic IPSPs recorded in the neocortex after blockade of synaptic transmission mediated by excitatory amino acids, J. Neurophysiol. 65 (1991) 1034–1041. [4] B.D. Bennett, J.R. Huguenard, D.A. Prince, Adrenergic modulation of GABAA receptor-mediated inhibition in rat sensorimotor cortex, J. Neurophysiol. 79 (1998) 937–946. [5] R. Bonfante-Cabarcas, K.L. Swanson, M. Alkondon, E.X. Albuquerque, Diversity of nicotinic acetylcholine receptors in rat hippocampal neurons. IV. Regulation by external Ca 11 of alpha-bungarotoxin-sensitive receptor function and of rectification induced by internal Mg 11 , J. Pharmacol. Exp. Ther. 277 (1996) 432–444. [6] A.E. Cole, R.A. Nicoll, Acetylcholine mediates a slow synaptic potential in hippocampus pyramidal cells, Science 221 (1993) 1299–1301. [7] Z.G. Chu, J.J. Hablitz, Activation of group I mGluRs increases
Z.G. Chu et al. / Brain Research 887 (2000) 399 – 405
[8]
[9]
[10]
[11] [12]
[13]
[14]
[15] [16]
[17] [18]
[19]
[20]
[21]
[22]
[23]
[24]
spontaneous IPSC frequency in rat neocortex, J. Neurophysiol. 80 (1998) 621–627. C.H. Davies, S.N. Davies, G.L. Collingridge, Paired-pulse depression of monosynaptic GABA-mediated inhibitory postsynaptic responses in rat hippocampus, J. Physiol. (Lond.) 424 (1991) 513– 531. ´ M. del Mar Arroyo-Jimenez, J.-P. Bourgeois, L.M. Marubio, A.-M. ´ J.-P. Changeux, Le Sourd, O.P. Ottersen, R. Rinvik, A. Fairen, Ultrastructural localization of the a4-subunit of the neuronal acetylcholine nicotinic receptor in the rat substantia nigra, J. Neurosci. 19 (1999) 6475–6487. F.A. Edwards, A.J. Gibb, D. Colquhoun, ATP receptor-mediated synaptic currents in the central nervous system, Nature 359 (1992) 144–147. R.J. Evans, V. Derkach, A. Surprenant, ATP mediates fast synaptic transmission in mammalian neurons, Nature 357 (1992) 503–505. C.J. Frazier, A.V. Buhler, J.L. Weiner, T.V. Dunwiddie, Synaptic potentials mediated via a-bungarotoxin-sensitive nicotinic acetylcholine receptors in rat hippocampal interneurons, J. Neurosci. 18 (1998) 8228–8235. C.J. Frazier, Y.D. Rollins, C. Breese, S. Leonard, R. Freedman, T.V. Dunwiddie, Acetylcholine activates an a-bungarotoxin-sensitive nicotinic current in rat hippocampal interneurons, but not pyramidal cells, J. Neurosci. 18 (1998) 1187–1195. R. Gray, A.R.K. Rajan, M. Yakehiro, J. Dani, Hippocampal synaptic transmission enhanced by low concentrations of nicotine, Nature 383 (1996) 713–716. J.J. Hablitz, T. DeFazio, Excitability changes in freeze-induced neocortical microgyria, Epilepsy Res. 32 (1998) 75–82. B. Katz, R. Miledi, The binding of acetylcholine to receptors and its removal from the synaptic cleft, J. Physiol. (Lond.) 231 (1973) 549–574. K. Krnjevic, Chemical nature of synaptic transmission, Physiol. Rev. 54 (1974) 418–540. Y. Lamour, P. Dutar, A. Jobert, Excitatory effect of acetylcholine on different types of neurons in the first somatosensory neocortex of the rat: laminar distribution and pharmacological characteristics, Neuroscience 7 (1982) 1483–1504. K. Lamsa, K. Kaila, Ionic mechanisms of spontaneous GABAergic events in rat hippocampal slices exposed to 4-aminopryidine, J. Neurophysiol. 78 (1997) 2582–2591. C. Lena, J.-P. Changeux, Pathological mutations of nicotinic receptors and nicotine-based therapies for brain disorders, Curr. Opin. Neurobiol. 7 (1997) 674–682. A.B. MacDermott, L.W. Role, S.A. Siegelbaum, Presynaptic ionotropic receptors and the control of transmitter release, Ann. Rev. Neurosci. 22 (1999) 443–485. D.A. McCormick, D.A. Prince, Mechanisms of action of acetylcholine in the guinea-pig cerebral cortex in vitro, J. Physiol. (Lond.) 375 (1986) 169–194. D.A. McCormick, D.A. Prince, Acetylcholine causes rapid nicotinic excitation in the medial habenular nucleus of guinea pig, in vitro, J. Neurosci. 7 (1987) 742–752. D.S. McGehee, L.W. Role, Presynaptic ionotropic receptors, Curr. Opin. Neurobiol. 6 (1996) 342–349.
405
[25] A.R. McQuiston, D.V. Madison, Nicotinic receptor activation excites distinct subtypes of interneurons in the rat hippocampus, J. Neurosci. 19 (1999) 2887–2896. [26] K.L. Perkins, R.K.S. Wong, Ionic basis of the postsynaptic depolarizing GABA response in hippocampal pyramidal cells, J. Neurophysiol. 76 (1996) 3886–3894. [27] J.T. Porter, B. Cauli, K. Tsuzuki, B. Lambolez, J. Rossier, E. Audinat, Selective excitation of subtypes of neocortical interneurons by nicotinic receptors, J. Neurosci. 19 (1999) 5228–5235. [28] L.D. Pozzo-Miller, J.J. Petrozzino, J.A. Connor, G-protein-coupled receptors mediate a fast excitatory postsynaptic current in CA3 pyramidal neurons in hippocampal slices, J. Neurosci. 15 (1995) 8320–8330. [29] B. Roerig, D.A. Nelson, L.C. Katz, Fast synaptic signaling by nicotinic acetylcholine and serotonin 5-HT 3 receptors in developing visual cortex, J. Neurosci. 17 (1997) 8353–8362. [30] C.C.D. Shute, P.R. Lewis, The ascending cholinergic reticular system: neocortical, olfactory and subcortical projections, Brain 90 (1967) 497–520. [31] P. Stern, F.A. Edwards, B. Sakmann, Fast and slow components of unitary EPSCs on stellate cells elicited by focal stimulation in slices of rat visual cortex, J. Physiol (Lond.) 449 (1992) 247–278. [32] T.W. Stone, Cholinergic mechanisms in the rat somatosensory cerebral cortex, J. Physiol. (Lond.) 225 (1972) 485–499. [33] S. Sugita, K.Z. Shen, R.A. North, 5-hydroxytryptamine is a fast excitatory transmitter at 5-HT3 receptors in rat amygdala, Neuron 8 (1992) 199–203. [34] B. Sutor, J.J. Hablitz, Excitatory postsynaptic potentials in rat neocortical neurons in vitro. I. Electrophysiological evidence for two distinct EPSPs, J. Neurophysiol. 61 (1989) 607–620. [35] B. Sutor, J.J. Hablitz, Excitatory postsynaptic potentials in rat neocortical neurons in vitro. II. Involvement of N-methyl-D-aspartate receptors in the generation of EPSPs, J. Neurophysiol. 61 (1989) 621–634. [36] C. Vidal, J.-P. Changeux, Nicotinic and muscarinic modulations of excitatory synaptic transmission in the rat prefrontal cortex in vitro, Neuroscience 56 (1993) 23–32. [37] E. Wada, K. Wada, J. Boulter, E. Deneris, S. Heinemann, J. Patrick, L.W. Swanson, Distribution of alpha 2, alpha 3, alpha 4, and beta 2 neuronal nicotinic receptor subunit mRNAs in the central nervous system: a hybridization histochemical study in the rat, J. Comp. Neurol. 284 (1989) 314–335. [38] S. Wonnacott, Presynaptic nicotinic ACh receptors, Trends Neurosci. 20 (1997) 92–98. [39] Z. Xiang, J.R. Huguenard, D.A. Prince, GABAA receptor-mediated currents in interneurons and pyramidal cells of rat visual cortex, J. Physiol. (Lond.) 506 (1998) 715–730. [40] F.M. Zhou, J.J. Hablitz, Morphological properties of rat neocortical layer I neurons examined using intracellular staining, J. Comp. Neurol. 376 (1996) 198–213. [41] F.M. Zhou, J.J. Hablitz, Bicuculline unmasks a fast EPSP resistant to glutamatergic antagonists in rat neocortex, Soc. Neurosci. Abstr. 19 (1993) 469.