Acetylcholine excites neocortical

21 The neuromodulator acetylcholine shapes neocortical function during sensory 22 perception, motor control, arousal, attention, learning and memory. Here we investigate 23 the mechanisms by which ACh affects neocortical pyramidal neurons in adult mice. 24 Stimulation of cholinergic axons activated muscarinic and nicotinic ACh receptors on 25 pyramidal neurons in all cortical layers and in multiple cortical areas. Nicotinic receptor 26 activation evoked short-latency, depolarizing postsynaptic potentials in many pyramidal 27 neurons. Nicotinic receptor-mediated postsynaptic potentials promoted spiking of 28 pyramidal neurons. The duration of the increase in spiking was membrane potential29 dependent, with nicotinic receptor activation triggering persistent spiking lasting many 30 seconds in neurons close to threshold. Persistent spiking was blocked by intracellular 31 BAPTA, indicating that nAChR activation evoked persistent spiking via a long-lasting 32 calcium-activated depolarizing current. We compared nicotinic PSPs in primary motor, 33 prefrontal and visual cortices. The laminar pattern of nicotinic excitation was not 34 uniform, but was broadly similar across areas, with stronger modulation in deep than 35 superficial layers. Superimposed on this broad pattern were local differences, with 36 nicotinic PSPs being particularly large and common in layer 5 of M1, but not layer 5 of 37 PFC or V1. Hence, in addition to modulating the excitability of pyramidal neurons in all 38 layers via muscarinic receptors, synaptically-released ACh preferentially increases the 39 activity of deep-layer neocortical pyramidal neurons via nicotinic receptors, thereby 40 adding laminar selectivity to the widespread enhancement of excitability mediated by 41 muscarinic ACh receptors. 42 INTRODUCTION 43 The neuromodulator acetylcholine (ACh) shapes neocortical function during sensory 44 perception (Metherate, 2004; Disney et al., 2007), motor control (Berg et al., 2005), 45 arousal (Steriade, 2004; Jones, 2008), attention (Herrero, 2008; Parikh & Sarter, 46 2008), learning (Kilgard, 2003; Ramanathan et al., 2009) and memory (Winkler et al., 47 1995). A decline in neocortical ACh has been tied to conditions such as depression 48 (Dislaver, 1986), schizophrenia (Raedler & Tandon, 2006), Alzheimer’s disease 49 (Whitehouse et al., 1982) and Parkinson's disease (Whitehouse et al., 1983). 50 Most cholinergic axons in neocortex arise from nucleus basalis and other basal 51 forebrain nuclei such as substantia innominata (Wainer & Mesulam, 1990). The 52 cholinergic projection from basal forebrain plays a central role in shaping neocortical 53 components of arousal, attention, learning, memory, sensory perception and motor 54 control. For example, stimulation of vibrissal M1 evokes whisker movements that are 55 enhanced by activation of basal forebrain (Berg et al., 2005) and basal forebrain lesions 56 impair motor control (Garbawie & Whishaw, 2003) and motor map rearrangement 57 during motor learning (Conner et al., 2003). 58 ACh affects neocortical networks, in part by modulating the activity of pyramidal 59 neurons. Pyramidal neurons express nicotinic and muscarinic ACh receptors (nAChRs 60 and mAChRs) on their plasma membranes (van der Zee et al., 1992; Mrzljak et al., 1993), 61 but ACh is thought to act on pyramidal neurons primarily via mAChRs. Activation of 62 mAChRs evokes an initial hyperpolarization and subsequent slow depolarization of many 63 cortical pyramidal neurons. The hyperpolarization results from activation of an SK-type 64 potassium current, whereas the slow depolarization has been linked to a number of 65 currents, including M-, AHPand inward rectifier-type potassium currents and a non66 specific cation current (Krnjevic, 1971; McCormick & Prince, 1985, 1986; McCormick & 67 Williamson, 1989; Haj-Dahmane & Andrade, 1996; Delmas & Brown, 2005; Gulledge & 68 Stuart, 2005; Carr & Surmeier, 2007; Zhang & Séguéla, 2010). There are reports of ACh 69 activating nAChRs on pyramidal neurons (Roerig et al., 1997; Chu et al., 2000; Kassam 70 et al., 2008; Zolles et al., 2009; Guillem et al., 2011; Poorthuis et al., 2012; but see also 71 Vidal & Changeux, 1993; Gil et al., 1997; Porter et al., 1999). Few authors have studied 72 nicotinic postsynaptic currents in neocortical pyramidal neurons (Roerig et al., 1997; 73 Chu et al., 2000). Hence the functional roles of nAChRs on pyramidal neurons remain 74 obscure. 75 Here we studied how ACh affects pyramidal neurons, focusing on the role of nAChRs. 76 To drive synaptic release of ACh, we expressed the light-activated protein 77 channelrhodopsin-2 (ChR2) in cholinergic neurons in the basal forebrain, allowing us to 78 selectively stimulate cholinergic axons in neocortex (Kalmbach et al., 2012). In contrast 79 to many previous reports, we find that ACh excites pyramidal neurons via both mAChRs 80 and nAChRs. Activation of nAChRs occurs with short latency, consistent with nAChRs 81 being located at synapses between cholinergic axons and pyramidal neurons, and can 82 evoke persistent spiking via a calcium-activated conductance. Direct nAChR-mediated 83 effects occurred in pyramidal neurons in several neocortical areas and in all neocortical 84 layers, indicating that direct excitation of pyramidal neurons via nAChRs can occur 85 across neocortical layers and areas. However, laminar and regional differences in both 86 the incidence and amplitude of the nAChR-mediated depolarization suggest regional 87 differences in the modulation of neocortical networks by nAChRs. 88 METHODS 89 All experiments and procedures were approved by the Northwestern University 90 Institutional Animal Care and Use Committee (IACUC). 91 Two approaches were employed to selectively express ChR2 in cholinergic neurons: 92 (1) stereotaxic injection of a floxed viral vector into the basal forebrain of ChAT-Cre 93 mice, and (2) crossing ChAT-Cre and floxed ChR2 mouse lines. 94 For experiments using virally-delivered ChR2, we used Tg(ChAT-Cre)60Gsat mice 95 (GENSAT), which express Cre-recombinase on a choline-acetyltransferase (ChAT) 96 promoter, resulting in Cre expression in cholinergic neurons throughout the brain. Into 97 this mouse we injected adeno-associated virus with a double-floxed inverse open reading 98 frame (EF1a-DIO-hChR2(H134R)eYFP, Virus Vector Core, University of North 99 Carolina), which drives expression of ChR2-yellow fluorescent protein (ChR2-YFP) in 100 infected neurons containing Cre. 400 nl of virus was injected into the basal forebrain at 101 postnatal day 21 using stereotaxic coordinates (0.2 mm A-P, 1.7 mm M-L, 4.5 mm D-V). 102 Three weeks after injection, ChR2 is expressed almost exclusively in cholinergic neurons 103 and their axons in neocortex, with no adverse effects (Porter et al., 1999). 104 In some experiments, selective expression of ChR2 in cholinergic neurons was 105 achieved without the use of viral vectors by crossing ChAT-Cre (B6;129S6106 Chattm1(cre)Lowl/J, Jax 006410) and floxed ChR2 (129S-Gt(ROSA)26Sortm32(CAG107 COP4*H134R/EYFP)Hze/J, Jax 012569, Ai32 (Madisen et al., 2012) mouse lines to produce 108 ChAT-ChR2(Ai32) mice. Cre+ mice were crossed with ChR2+/+ mice to yield Cre+ ChR2+/109 offspring. These offspring were crossed to generate Cre+ ChR2+/+ mice, which were used 110 for experiments. Results from ChAT-ChR2(Ai32) mice were similar to those from viral 111 infections and results from these two approaches were pooled, unless otherwise noted. 112 Somatic and axonal labeling with ChR2-YFP was examined in fixed sections. Tissue 113 was fixed by transcardial perfusion with 4% paraformaldehyde in phosphate buffer and 114 100-200 μm thick coronal sections were cut using a vibrating microtome. In some 115 sections, the YFP signal was enhanced with an anti-GFP primary (ab13970, 1:10,000, 116 Abcam) and a fluorescent secondary antibody (613111, 1:750, Invitrogen). Images were 117 acquired by widefield or 2-photon microscopy. Widefield images were acquired using a 118 GFP filter set and a Hamamatsu Orca-285 camera. 2-photon images were acquired with 119 880 nm illumination from a Coherent Chameleon Ultra II Ti:sapphire laser and a 505120 545 nm emission filter. 121 Whole-cell recordings were obtained from pyramidal neurons in 300 μm-thick acute 122 parasagittal slices of primary motor cortex and coronal slices of prefrontal and visual 123 cortices from postnatal day 33-61 mice of either sex. In virally-infected mice, recordings 124 were obtained ~3 weeks after viral injection. Slices were prepared in ice-cold artificial 125 cerebro-spinal fluid (ACSF; in mM): 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 20 NaHCO3, 5 126 HEPES, 25 Glucose, 2 CaCl2, 1 MgCl2, pH 7.3, oxygenated with 95% O2/5% CO2. Whole127 cell recording pipettes were 4–8 MΩ when filled with intracellular solution: 135 mM K 128 gluconate, 4 mM KCl, 10 mM HEPES, 10 mM Na2-phosphocreatine, 4 mM Mg-ATP, 0.3 129 mM Na2-GTP, 0.2% (w/v) biocytin, 10 μM alexa 594, pH 7.3. 130 Recordings were obtained at 35-37 °C with a feedback circuit and temperature 131 controller (TC-324B; Warner Instruments, Hamden, CT, USA). In experiments using 132 calcium-free ACSF, 2 mM CaCl2 was replaced with 4 mM MgCl2 to give a final MgCl2 133 concentration of 5 mM. ChR2 was activated by wide field illumination through a 134 microscope objective (Olympus, x20/0.95 NA or x40/0.8 NA) using a blue light-emitting 135 diode (LED; Thorlabs LEDC5 and LEDD1 or DC2100 driver). The maximum steady-state 136 intensity was 20 mW/mm2. In some experiments (see figure 4E-G), illumination was 137 restricted by closure or partial closure of the fluorescence field stop. The illumination 138 area was measured offline by bleaching a thin, immobilized film of fluorescence on a 139 microscope slide. Closure of the field stop had no effect on illumination intensity per unit 140 area. In restricted illumination experiments, the soma was positioned in the center of the 141 field of illumination unless noted otherwise. 142 The amplitudes of voltage responses were calculated by subtracting the average 143 baseline membrane potential for ≥1 s before illumination from the peak of the response. 144 Voltage responses which failed to exceed 3 standard deviations of the baseline 145 membrane potential were assigned an amplitude of 0 mV. During brief bursts of nAChR 146 PSPs, the peak amplitude was calculated by subtracting the pre-burst membrane 147 potential from the most depolarized potential during the burst. 148 Local application of ACh was by pressure ejection of 100 μM ACh in ACSF from a 149 glass pipette ~50 μm from the soma, 30 psi, Toohey Spritzer IIe (Toohey Company, 150 Fairfield NJ). 151 Mecamylamine hydrochloride and atropine were obtained from Sigma-Aldrich. 152 Dihydro-β-erythroidine hydrobromide (DHβE), methyllycaconitine citrate, 153 galanthamine hydrobromide and physostigmine hemisulfate were obtained from Tocris 154 Bioscience. NBQX disodium salt, (R)-CPP, gabazine (SR95531), CGP 52432, tetrodotoxin 155 citrate and (R,S)-MCPG were obtained from Ascent Scientific. In some experiments, we 156 used (2R)-amino-5-phosphonopentanoic acid (AP5) to block NMDA receptors, instead of 157 CPP. AP5 was obtained from Tocris. 158 Statistical analyses were performed using Graphpad QuickCalcs online tool 159 (http://www.graphpad.com/quickcalcs/) or in Graphpad Instat 3.06 (GraphPad 160 Software Inc., La Jolla, CA). Continuous data (such pharmacology of the nAChR PSP) 161 were analyzed with a two-tailed t-test, Kruskal-Wallis test or Mann-Whitney test and 162 categorical data with Fisher's exact test. 163 The kinetics and latency of nAChR PSPs were measured by fitting a sum of two 164 exponentials to the mean of ten trials. In each trial the PSP was evoked with a single 2 165 ms (or occasionally 5 ms) blue light illumination. The signal-to-noise ratio of our 166 recordings was not sufficient to permit accurate measurement of the timing of the initial 167 rise of the membrane potential of a nAChR PSP, which is smaller and slower than a 168 glutamatergic PSP. To accurately estimate the point of initial rise we therefore measured 169 the time from the start of illumination to 5% of the peak amplitude of the PSP. We would 170 expect this method of latency measurement to overestimate the latency of each PSP by 171 approximately 1 ms and we therefore corrected the latency of each PSP, by back172 extrapolating the fit to the resting membrane potential preceding the stimulus. 173 Pyramidal neurons were identified by their somatic shape and spiking pattern and by 174 their large apical dendrites, which were visible by fluorescence microscopy once filled 175 with indicator. Neurons were routinely filled with indicator and their dendrites 176 examined by fluorescence microscopy. Neurons with truncated primary apical dendrites, 177 lacking apical dendrites and with spiking patterns more typical of interneurons (narrow 178 spikes, large after-hyperpolarization) were excluded from further analysis. 179 Pyramidal cells were classified according to the laminar locations of their somata. 180 Laminar borders were based on the Allen Brain Atlas and consistent with laminar 181 variation in opacity of slices, which was visible under brightfield illumination. Distances 182 from the pial surface of the slice were measured perpendicular to the pia. In primary 183 motor cortex, cortical layers were: layer 2/3 150-500 μm, layer 5A 600-750 μm, layer 5B 184 800-1200 μm, layer 6 >1300 μm. In prefrontal cortex, cortical layers were: layer 2/3 185 100-300 μm, layer 5 330-550 μm, layer 6 >550-800 μm (Guillem et al., 2011). In visual 186 cortex, cortical layers were: layer 2/3 100-275 μm, layer 5 450-750 μm, layer 6 >750 μm 187 (Olivas et al., 2012; Petrof et al., 2012). 188 Pyramidal neurons projecting to defined postsynaptic target tissues were labeled 189 using fluorescent microspheres (RetroBeads, Lumafluor). Beads were injected into 190 dorsolateral striatum (0 mm A-P, -2 mm M-L, 2.5 mm D-V), cervical spinal cord 191 (between ~-0.5 mm M-L and the midline, ~1 mm D-V, at the level of the C2 vertebra) or 192 ventral posteromedial thalamus (two sites: 1.6 mm A-P, 1.2 mm M-L, 3.25 mm D-V and 193 1.6 mm A-P, 1.7 mm M-L, 3.5 mm D-V). Recordings were obtained 5-11 days after bead 194 injection from M1 cortex contralateral to striatal and spinal injections and ipsilateral to 195 thalamic injections. 196 RESULTS 197 To stimulate cholinergic axons entering neocortex, we used the blue light-activated 198 membrane protein channelrhodopsin-2 (ChR2; Nagel et al., 2003). We expressed ChR2 199 in cholinergic neurons in the basal forebrain (figure 1A-C), obtaining selectivity for 200 cholinergic neurons with a ChAT-Cre mouse line and floxed virus or by crossing ChAT201 Cre and floxed ChR2 mouse lines. We obtained whole-cell recordings from layer 5B 202 pyramidal neurons in acute slices of primary motor cortex containing ChR2-labeled 203 axons (figure 1D). Mean resting potential was -62 ± 1 mV (51 neurons). Widefield 204 illumination of the slice with a blue LED evoked one or more of four voltage responses: a 205 slow depolarization, a hyperpolarization, a medium depolarization, or a fast 206 depolarization (figure 1F). All responses were absent in tetrodotoxin (500 nM TTX, 17 207 neurons) and after removal of extracellular calcium (10 neurons), indicating that all four 208 voltage responses were evoked by spikes, leading to vesicular release (figure 2). 209 We used pharmacology to identify the receptors underlying each of the four voltage 210 responses. The hyperpolarization and slow depolarization were mediated by muscarinic 211 ACh receptors (mAChRs): both hyperpolarization and slow depolarization were 212 eliminated by the mAChR antagonist atropine (1 μM, 140 of 141 neurons, figure 3), but 213 not by the nAChR antagonist mecamylamine (100 μM, 11 of 13 neurons, figure 3) or by 214 glutamate or GABA receptor antagonists (10 μM NBQX and 10 μM CPP; 1 μM gabazine 215 and 3 μM CGP 52432; 14 of 16 neurons). 216 The medium depolarization was mediated by nicotinic ACh receptors (nAChRs). It 217 was eliminated by mecamylamine (100 μM, 12 of 12 neurons, P < 0.05, paired two-tailed 218 t-test), enhanced 73 ± 21% by the nAChR allosteric potentiator and cholinesterase 219 antagonist galanthamine (1 μM, 23 neurons, P < 0.05, paired two-tailed t-test) and 27 ± 220 15% by the ACh esterase inhibitor physostigmine (0.5 μM, 4 neurons, P < 0.05, paired 221 two-tailed t-test), and insensitive to antagonists of mAChRs, GluRs and GABA receptors 222 (5 of 5, 7 of 7 and 8 of 8 neurons, respectively; figure 4). The medium depolarization was 223 unaffected by the α7-nAChR antagonist methyllycaconitine (MLA, 10 nM, 12 of 12 224 neurons) and eliminated by dihydro-β-erythroidine (DHβE, 10 μM, 16 of 16 neurons, P < 225 0.05, paired two-tailed t-test, figure 4), which has high affinity for α4β2-containing 226 nAChRs. Hence the medium depolarization is mediated by non-α7 nAChRs, probably 227 α4β2-containing nAChRs. 228 The fast depolarization was mediated by ionotropic glutamate receptors (GluRs): the 229 fast depolarization was not inhibited by nAChR and mAChR nor by GABA receptor 230 antagonists (each P < 0.05, paired two-tailed t-test), but was eliminated by ionotropic 231 GluR antagonists (figure 5A). GluR-mediated fast depolarizations were observed only in 232 a subset of ChAT-ChR2(Ai32) mice and not in virally-infected mice. Furthermore, for 233 each ChAT-ChR2(Ai32) mouse we found that GluR-mediated fast depolarizations were 234 obtained in either all or no neurons. We recorded from multiple neurons in slices from 235 25 mice. In 13 of these 25 mice, we observed GluR-mediated fast depolarizations in every 236 neuron; 12 of 25 mice exhibited no GluR-mediated fast depolarization in any recording. 237 Hence it is likely that the GluR-mediated fast depolarization results from expression of 238 ChR2 in non-cholinergic neurons in a subset of ChAT-ChR2(Ai32) mice. To eliminate 239 GluR-mediated responses, we included GluR receptor-antagonists in all remaining 240 experiments with ChAT-ChR2(Ai32) mice. The presence of GluR-mediated responses in 241 some ChAT-ChR2(Ai32) mice is the only difference we observed between genetic and 242 viral methods of driving ChR2 expression in cholinergic neurons: in other respects, the 243 two methods of driving ChR2 expression were equivalent (no difference in amplitudes of 244 nAChR PSPs, P > 0.05, Matt-Whitney test, 37 and 22 neurons from virally-infected and 245 ChAT-ChR2(Ai32) mice, respectively). We therefore pooled results from the two 246 techniques. 247 All four postsynaptic responses could occur individually, but most pyramidal 248 neurons exhibited a combination of responses mediated by two or more receptors (figure 249 5B). Hence synaptically-released ACh activates pyramidal neurons via nAChRs and 250 mAChRs, and often via both types of ACh receptor in the same neuron. 251 252 Activation of pyramidal neuron mAChRs by synaptically-released ACh 253 The peak amplitude of the hyperpolarization, evoked by brief illumination (≤10 x 5 ms at 254 20 Hz), was 1.1 ± 0.5 mV and the decay time constant was 774 ± 73 ms (10 neurons). The 255 slow depolarization displayed a peak amplitude of 2.0 ± 0.6 mV (6 neurons) and lasted 256 several seconds. At resting membrane potentials, the hyperpolarization and slow 257 depolarization were rare, occurring in only 7% (8 of 112) and 19% (21 of 112) of layer 5B 258 pyramidal neurons, respectively. 259 Many previous authors have reported activation of pyramidal neurons via mAChRs 260 upon application of ACh to the soma, by pressure ejection from a nearby pipette 261 (McCormick & Prince, 1985; McCormick & Williamson, 1989; Gulledge & Stuart, 2005; 262 Gulledge et al., 2007), with relatively few authors reporting nAChR-mediated 263 depolarization of pyramidal neurons (Roerig et al., 1997; Chu et al., 2000; Kassam et al., 264 2008; Zolles et al., 2009; Guillem et al., 2011; Poorthuis et al., 2013). Similar to many 265 previous studies, we found that pressure ejection of ACh onto the soma evoked a 266 mAChR-mediated hyperpolarization and slow depolarization (5 of 6 and 6 of 6 neurons, 267 respectively; figure 6A), but no nAChR-mediated depolarization. 268 One possible interpretation of our results might be that synaptically-released ACh 269 activates primarily nAChRs and usually fails to activate mAChRs, whereas pressure 270 ejection onto the soma activates a different population of receptors, primarily mAChRs. 271 mAChR activation modulates primarily potassium conductances (McCormick, 1992) and 272 the reversal potential for potassium is ~-90 mV. mAChR activation may therefore exert 273 little effect on the membrane potential at rest: both mAChR-mediated hyperpolarization 274 and slow depolarization are larger when the neuron is depolarized (McCormick & Prince, 275 1986; Gulledge & Stuart, 2005). To maximize the effects of mAChR activation on the 276 membrane potential and, therefore, the probability that we would observe activation of 277 mAChRs, we depolarized neurons by somatic current injection (figure 6B). Brief stimuli 278 (≤10 x 5 ms illumination at 20 Hz) evoked mAChR-mediated voltage responses in 79% of 279 depolarized layer 5B pyramidal neurons (figure 6C), indicating that synaptically-released 280 ACh activates mAChRs in the majority of layer 5B pyramidal neurons, but that the 281 resulting hyperpolarization or depolarization from rest is often too small to be observed. 282 Previous authors have also reported that ACh, applied by pressure application, more 283 readily evokes postsynaptic mAChR-mediated responses in pyramidal neurons in deep284 than in superficial-layers of the neocortex (McCormick & Prince, 1986; McCormick & 285 Williamson, 1989; Gulledge et al., 2007). Similarly, we found that synaptically-released 286 ACh more readily excites pyramidal neurons via mAChRs in deep than superficial layers 287 of M1 cortex, with the proportion of pyramidal neurons that exhibited a mAChR288 mediated slow depolarization or hyperpolarization ranging from 53% in layer 2/3 to 91% 289 in layer 6 pyramidal neurons (figure 6C). 290 We conclude that ACh released by a brief burst of cholinergic activity activates 291 mAChRs on the majority of pyramidal neurons throughout primary motor cortex. In 292 comparison to pressure application of ACh, activation of cholinergic synapses with brief 293 bursts of stimuli provides relatively weak activation of mAChRs which often fails to affect 294 the somatic membrane potential at rest. 295 296 ACh release evokes a nicotinic PSP 297 Brief illumination (≤10 x 5 ms at 20 Hz) in mAChR, GluR and GABAR antagonists 298 evoked a nAChR-mediated medium depolarization in almost all (82 of 90) layer 5B 299 pyramidal neurons at rest. We stimulated cholinergic axons with brief bursts of stimuli 300 at 20 Hz, a stimulus designed to mimic the spiking of cholinergic basal forebrain 301 neurons during paradoxical sleep and awake states, when cholinergic basal forebrain 302 neurons spike in bursts, each of ~4-6 spikes at ~25 Hz (Manns et al., 2000; Lee et al., 303 2005). During such bursts, summation occurred at stimulus frequencies greater than ~5 304 Hz. In our experiments summation may result, in part, from accumulation of calcium 305 entering through ChR2 channels in the presynaptic terminal (Zhang & Oertner, 2007) 306 and may therefore be an artifact of the optogenetic stimulus. Nonetheless, summation 307 permitted us to evoke a substantial depolarization via nAChRs: a burst of stimuli at 20 308 Hz frequently evoked a peak depolarization of 5-10 mV (figure 7A,B). 309 We measured the latency and kinetics of the nAChR-mediated depolarization evoked 310 by a single 2 ms illumination, in 1 μM atropine to inhibit mAChRs and GluR and GABAR 311 antagonists to eliminate any indirect effects. In 15 of 15 neurons, 2 ms illumination 312 evoked reproducible depolarizations (figure 7C), with a mean amplitude of 1.4 ± 0.2 mV 313 (11 neurons). To the mean response we fit a sum of two exponentials (figure 7D) with 314 mean rise and decay time constants of 28 ± 4 ms and 180 ± 23 ms (14 neurons). These 315 relatively slow kinetics are comparable to those of PSPs mediated by α4β2-containing 316 nAChRs in interneurons (Bell et al., 2011), but slower than PSPs mediated by α7317 nAChRs (Frazier et al., 1998; Fedorov et al., 2012). These kinetics suggest that the 318 medium depolarization is a PSP mediated by non-α7 nAChRs, consistent with the 319 pharmacology of the medium depolarization, presented above. 320 The latency of the nAChR PSP, measured from the start of illumination, was 5.9 ± 321 0.8 ms (12 neurons). Compared to electrical stimulation, ChR2 drives relatively slow 322 depolarization of the neuronal membrane, typically with rise and decay time constants of 323 at least several milliseconds (Lin et al., 2009; Yizhar et al., 2011), which may be further 324 elongated by the time constant of the membrane. The latency of the fast depolarization 325 was 5.3 ± 0.5 ms (3 neurons). Hence the latency of the nicotinic PSP was only ~0.5 ms 326 longer than that of a monosynaptic glutamatergic PSP, consistent with the medium 327 depolarization being a monosynaptic PSP generated at synapses between cholinergic 328 axons and pyramidal neurons. 329 nAChRs appear to be distributed throughout the dendritic trees of cortical pyramidal 330 neurons (van der Zee et al., 1992; Nakayama et al., 1995) but the locations of cholinergic 331 synapses are unknown. To determine whether nAChR PSPs were evoked primarily by 332 cholinergic synapses in the proximal or distal dendrites of layer 5B pyramidal neurons 333 we measured nAChR PSPs during restricted illumination of the slice (figure 8A,B). 334 Restricting illumination to a radius of less than ~300 μm around the soma was necessary 335 to reduce the amplitude of the nAChR PSP and the amplitude was reduced by 50% when 336 the radius of illumination was ~50 μm (figure 8C, 7 neurons). Illumination of the tuft 337 dendrites failed to evoke a nAChR PSPs at the soma (figure 8B, 3 neurons). Hence the 338 nAChRs which contribute to the somatic depolarization in our experiments are likely to 339 be within 300 μm of the soma and many are probably located in the proximal 50 μm of 340 the apical and basal arbor. 341 342 nAChR activation can evoke persistent spiking 343 We next addressed the functional consequences of nAChR activation. nAChR PSPs 344 increased the spike rate of layer 5B pyramidal neurons in primary motor cortex, 345 depolarized beyond threshold by somatic current injection (figure 9A). The increase was 346 independent of initial spike rate (figure 9B), with 2-5 ms illumination increasing spike 347 rate by 2.2 ± 0.1 Hz (6 neurons) and a brief burst (10 x 5 ms at 20 Hz) increasing spike 348 rate by 3.1 ± 0.2 Hz (4 neurons). Hence, during spiking nAChR activation causes a linear, 349 additive change in spike rate with no change in the slope of the input-output 350 relationship. The elevated spike rate was maintained during repetitive stimulation and 351 declined after cessation of the cholinergic stimulus with a decay time constant of 185 ± 352 21 ms (figure 9C; 2 or 5 ms illumination; 6 neurons), which matches the decay of the 353 underlying nAChR PSP. 354 With the membrane depolarized from rest but subthreshold, cholinergic stimulation 355 evoked persistent spiking. We evoked nAChR PSPs during long step current injections of 356 increasing amplitude until the nAChR PSP evoked one or more spikes. A nAChR PSP 357 reduced rheobase by 11.6 ± 3.7 pA (from 344 ± 26 pA to 337 ± 26 pA, 18 neurons, 5 ms 358 illumination in the presence of atropine). However, under these conditions, a nAChR 359 PSP typically evoked multiple spikes. The spike rate during the first second after 360 stimulus onset was 3.0 ± 0.2 Hz (6 neurons) for a single 2-5 ms illumination and 7.1 ± 361 1.2 Hz for a brief burst (10 x 5 ms at 20 Hz, 7 neurons). Spike rate declined slowly after 362 the last nAChR PSP (figure 10A,B), but spiking typically continued until the holding 363 current was removed, which was up to 4 seconds after the initial spike (figure 10A,B). 364 We defined persistent spiking as spiking that continued for at least 500 ms after the 365 end of the cholinergic stimulus. Using this definition, persistent spiking occurred in 366 every neuron (13 of 13 neurons), but in 6 of 13 neurons persistent spiking occurred on 367 some but not all trials. In trials in which persistent spiking failed to occur, the nAChR 368 PSP evoked only a single spike (mean 1.0 ± 0, 14 trials from 4 neurons, single 2-5 ms 369 illumination), whereas in trials in which persistent spiking occurred the nAChR PSP 370 evoked 7.5 ± 1.6 spikes (10 trials in the same 4 neurons with the same stimulus). 371 Comparing trials with and without persistent spiking, the resting membrane potential at 372 the start of the trial (-62.3 ± 1.9 and -63.2 ± 1.5 mV, respectively), the current injected to 373 depolarize the neuron (209 ± 20 and 268 ± 23 pA, respectively), and the membrane 374 potential immediately before the nAChR PSP (-48.0 ± 2.0 and -48.5 ± 1.8 mV, 375 respectively) and the threshold of the first spike (-35.2 ± 1.9 and -39.1 ± 1.9 mV, 376 respectively) were similar (18 and 14 trials respectively, each P > 0.05, paired 2-tailed t377 test). These measurements indicate that trial-to-trial variability in perisomatic 378 membrane potential, input resistance and spike threshold do not account for the 379 variability in persistent spiking, although they do not exclude a role for such changes in 380 the distal dendrite, as a result of ongoing synaptic activity, for example. 381 Persistent spiking required activation of nAChRs on pyramidal neurons, but not 382 glutamate or GABA receptors or mAChRs (figure 11). Persistent spiking occurred in the 383 presence of 10 μM NBQX, 10 μM CPP, 1 μM gabazine, 3 μM CGP and 1-10 μM atropine 384 (13 of 13 neurons). Subsequent addition of 100 μM mecamylamine (in the continued 385 presence of glutamate and GABA receptor antagonists and of atropine) blocked 386 persistent spiking (3 of 3 neurons, figure 11A) but 100 μM MCPG did not (3 of 3 387 neurons). 388 nAChR activation provides more than just the initial depolarization required to 389 initiate persistent spiking since in the absence of cholinergic stimulation, brief 390 depolarization failed to evoke persistent spiking (figure 11C). Furthermore, during 391 persistent spiking evoked by cholinergic stimulation, brief hyperpolarization of the 392 membrane inhibited persistent spiking, only for spiking to resume after the 393 hyperpolarizing pulse (figure 11D). Presumably, an additional depolarizing conductance 394 is activated (or hyperpolarizing conductance deactivated) by nAChR activation or by 395 another receptor co-activated with nAChRs and this additional conductance remains 396 active for several seconds, long after the decay of the nAChR PSP. The spike waveform 397 changed little during persistent spiking (figure 12), suggesting that this additional 398 current is unlikely to arise from one of the sodium or potassium conductances that shape 399 the spike waveform. 400 Persistent spiking was eliminated by 10 mM intracellular BAPTA. With or without 401 intracellular BAPTA, nAChR PSPs evoked 1 or more spikes (figure 11E), but in BAPTA 402 spiking did not continue after the decay of the underlying PSP. Without BAPTA spiking 403 continued until the holding current was removed, with the last spike 2155 ± 261 ms after 404 the start of a single 2-5 ms illumination and 3357 ± 169 ms after a burst of stimuli (10 x 5 405 ms at 20 Hz; 13 neurons). With BAPTA, spiking ended 185 ± 38 ms after a single 406 illumination and 445 ± 115 ms after a burst (3 neurons; single illumination and burst 407 each P < 0.05, unpaired 2-tailed t-test). As a result, cholinergic stimuli evoked fewer 408 spikes with BAPTA (single 5 ms illumination, 1.8 ± 0.1 spikes, 2 neurons; burst 3.3 ± 1.7 409 spikes, 3 neurons) than without BAPTA (single 5 ms illumination, 6.6 ± 0.8 spikes, 7 410 neurons; burst 21.7 ± 0.6 spikes, 7 neurons; single illumination and burst each P < 0.05, 411 unpaired 2-tailed t-test). Hence activation of cholinergic axons evokes persistent spiking 412 that requires activation of nAChRs and a calcium-activated current that does not affect 413 the spike waveform. 414 Our results indicate that ACh has both brief, additive and prolonged, non-linear 415 effects on the spiking of layer 5B pyramidal neurons, with neurons being particularly 416 sensitive to cholinergic activity when their membrane potentials are within ~10 mV of 417 spike threshold, such that a nAChR-mediated increase in spiking can be short-lived or 418 can be more dramatic, evoking spiking that persists for many seconds. 419 420 Cholinergic responses by projection target 421 In primary sensory neocortices, nAChRs are expressed by selected sub-populations of 422 presynaptic terminals. For example, ACh can enhance the transmission of sensory 423 information to neocortex via the activation of nAChRs on thalamocortical terminals in 424 primary somatosensory and visual cortices (Metherate, 2004; Disney et al., 2007; Gil et 425 al., 1997). Neuromodulators can also act selectively on different projection pathways out 426 of neocortex (Gaspar et al., 1995; Beique et al., 2007; Sheets et al., 2011; Avesar & 427 Gulledge, 2012; Gee et al., 2012; Seong & Carter, 2012). For example, in medial 428 prefrontal cortex mAChR activation by ACh has a greater effect on the excitability of 429 layer 5 pyramidal neurons that project to the pons than on neurons that project to 430 contralateral cortex (Dembrow et al., 2010) and nAChR activation evokes larger431 amplitude currents from corticothalamic layer 6 pyramidal neurons than from layer 6 432 pyramidal neurons that do not project to thalamus (Kassam et al., 2008). Might ACh 433 differentially modulate the output of motor cortex via expression of nACh receptors in 434 pyramidal neurons that project to some sub-cortical targets, but not pyramidal neurons 435 that project to other target tissues? 436 In motor cortex, descending axons of layer 5 pyramidal neurons project into the 437 pyramidal tract or to the contralateral striatum and these two pathways are mutually 438 exclusive (Shepherd, 2013). Within layer 6, the primary subcortical output is to thalamus 439 and layer 6 pyramidal neurons may therefore be divided into corticothalamic and non440 corticothalamic, or intracortical, neurons. We compared the incidence of nAChRand 441 mAChR-mediated potentials in each of these subpopulations in motor cortex after 442 retrograde labeling of pyramidal neurons by injection of fluorescent beads into spinal 443 cord, contralateral striatum or ipsilateral thalamus (figure 13A). In slices, we identified 444 neurons with different projection targets by the somatic accumulation of fluorescent 445 beads (figure 13B). 446 Synaptically-released ACh frequently evoked nAChR PSPs in all four subpopulations of 447 deep layer M1 pyramidal neurons: corticospinal layer 5 pyramidal neurons; 448 corticostriatal layer 5 pyramidal neurons; corticothalamic layer 6 pyramidal neurons; 449 non-corticothalamic (intracortical) layer 6 pyramidal neurons (figure 13C). The 450 incidence of nAChR PSPs was not different between the two populations of layer 5 451 neurons nor between the two populations of layer 6 neurons (nAChR PSPs in 5 of 10 452 corticospinal layer 5 pyramidal neurons, 8 of 11 corticostriatal layer 5 pyramidal 453 neurons, P > 0.05, Fisher's exact test; nAChR PSPs in 8 of 11 corticothalamic layer 6 454 pyramidal neurons, 6 of 12 non-corticothalamic layer 6 pyramidal neurons, P > 0.05, 455 Fisher's exact test). Hence our experiments revealed no evidence for different 456 probabilities of nAChR PSPs in sub-populations of deep-layer pyramidal neurons with 457 different projection targets. However, the amplitudes of nAChR PSPs were greater in 458 layer 6 pyramidal neurons that projected to thalamus (9.5 ± 2.6 mV, 7 neurons) than in 459 layer 6 neurons that did not project to thalamus (6.0 ± 2.3 mV, 6 neurons; P < 0.05, 460 paired 2-tailed t-test). nAChR-mediated currents are larger in corticothalamic than non461 corticothalamic pyramidal neurons in prefrontal cortex (Kassam et al., 2008) and our 462 results suggest that this enhancement of nAChR responses in corticothalamic layer 6 463 pyramidal neurons extends to primary motor cortex. 464 The mAChR-mediated slow depolarization was also common in neurons from all four 465 projection-based populations of deep-layer pyramidal neurons (figure 13C; no difference 466 in incidence of slow depolarization between layer 5 or layer 6 subpopulations, P > 0.05, 467 Fisher's exact test). In contrast, the hyperpolarization displayed differential expression 468 by projection target (figure 13C), occurring often in both layer 5 projection-based 469 populations and in non-corticothalamic layer 6 pyramidal neurons (no difference in 470 incidence of hyperpolarization between layer 5 subpopulations, P > 0.05, Fisher's exact 471 test), but being completely absent from corticothalamic layer 6 pyramidal neurons 472 (different incidence of slow depolarization between layer 6 subpopulations, P < 0.05, 473 Fisher's exact test). 474 475 nAChR PSPs responses across layers and cortical areas 476 In primary motor cortex, nAChRs are expressed by pyramidal neurons throughout the 477 layers of neocortex (van der Zee et al., 1992; Nakayama et al., 1995; Duffy et al., 2009) 478 and cholinergic axons ramify through all layers (Wainer & Mesulam, 1990; Lysakowski et 479 al., 1989). Hence nAChR PSPs might be expected in pyramidal neurons in all layers. To 480 test this hypothesis, we determined the frequency with which synaptically-released ACh 481 evoked nAChR PSPs in pyramidal neurons in layers 2/3, 5, and 6 of primary motor, 482 prefrontal, and visual cortices. 483 In primary motor cortical slices with abundant ChR2-labeled axons in all neocortical 484 layers (figure 14A) and nAChR PSPs in layer 5B pyramidal neurons, cholinergic stimuli 485 (10 x 5 ms at 20 Hz) rarely evoked nAChR PSPs in layer 2/3 pyramidal neurons (4 of 21 486 layer 2/3 neurons; figure 14B; lower probability in layer 2/3 than in layer 5A, layer 5B, or 487 layer 6, P < 0.05 for each, Fisher's exact test). In layers 5A and 5B, nAChR PSPs were 488 common (6 of 8 layer 5A neurons, 82 of 90 layer 5B neurons; figure 14B; no difference in 489 probability between layers 5A and 5B, Fisher's exact test) and in layer 6, nAChR PSPs 490 were evoked in approximately half of neurons (16 of 32 neurons; figure 14B; greater 491 probability in layer 6 than in layer 2/3 and lower than in layer 5B, P < 0.05 for each, 492 Fisher's exact test). Hence in primary motor cortex, nAChR PSPs occur almost 493 exclusively in deep-layer pyramidal neurons. 494 In prefrontal and primary visual cortices (PFC and V1), the laminar pattern of nAChR 495 PSPs was different from that in primary motor cortex. In prefrontal cortex, cholinergic 496 stimuli (10 x 5 ms at 20 Hz) evoked nAChR PSPs in 2 of 6 layer 2/3 pyramidal neurons, 2 497 of 13 layer 5 pyramidal neurons and 5 of 8 layer 6 pyramidal neurons (figure 14B). Hence 498 nAChR PSPs were less common in all three layers of PFC than in layer 5B neurons in M1 499 (P < 0.05 for each layer, Fisher's exact test). Within PFC, nAChR PSPs were more 500 common in layer 6 than in more superficial layers (P < 0.05, Fisher's exact test). As 501 expected from previous studies (Kassam et al., 2008; Poorthuis et al., 2012; Bailey et al., 502 2010), nAChR PSPs in layer 6 of prefrontal cortex arose from activation of non-α7 503 nAChRs, being unaffected by MLA and eliminated by DHβE (4 of 4 neurons). In visual 504 cortex, cholinergic stimuli (10 x 5 ms at 20 Hz) commonly evoked nAChR PSPs in 505 pyramidal neurons in all cortical layers (5 of 6 layer 2/3 neurons, 9 of 11 layer 5 506 pyramidal neurons, 8 of 9 layer 6 pyramidal neurons; figure 14B; no differences in 507 probability, Fisher's exact test). 508 The amplitudes of nAChR PSPs also differed across cortical layers and areas (figure 509 14C). The largest responses were observed in layer 5B of primary motor cortex 510 (maximum 18.3 mV), but the mean nAChR PSP amplitude was greatest in layer 6 511 pyramidal neurons (M1 6.06 ± 1.21 mV, maximum 16.2 mV; PFC 5.99 ± 2.86 mV, 512 maximum 15.9 mV; V1 2.76 ± 0.74 mV, maximum 5.8 mV; for M1, P < 0.05, Kruskal513 Wallis test; L5A different from L5B, L5B different from L6, each P < 0.05, Mann514 Whitney test). In all areas, mean peak amplitudes in layers 2-5 were between 1 and 2 mV, 515 with the exception of motor cortex (figure 14C), where responses in layer 5B were larger 516 than in layer 5A (P < 0.05, Mann-Whitney test) and larger than in layer 5 of prefrontal or 517 visual cortices (L5B of M1 14.21 ± 1.14 mV; amplitude larger in M1 L5B than PFC L5 and 518 V1 L5; P < 0.05, Kruskal-Wallis test). 519 To compare the effects of nAChR activation on pyramidal neurons in different layers 520 and areas, we multiplied the probability and amplitude of nAChR PSPs for each layer 521 (figure 14D). This analysis provides a measure of the overall effect of nAChR PSPs on 522 laminar excitability and reveals that, in all three cortical areas, the effects of nAChR 523 activation are greater in deep layers than in superficial layers. To summarize the average 524 effect of ACh via nAChRs across cortical areas, we plot the mean effect by layer by 525 averaging the effects in M1, PFC and V1 (figure 14E). Hence in these cortical areas there 526 is a general pattern of increased effectiveness of nAChR activation in deep layers, on 527 which is superimposed area-specific variations in laminar sensitivity to ACh. 528 Hence our results indicate that ACh, acting via nAChRs, can directly excite pyramidal 529 neurons in many cortical areas and layers. Our experiments reveal differences in the 530 nAChR-mediated responsiveness of pyramidal neurons between cortical areas and 531 neocortical layers. However, these local variations appear to operate within a more 532 general framework which is common to neocortical areas, in which ACh exerts greater 533 nAChR-mediated effects on deep-layer pyramidal neurons. 534 535 DISCUSSION 536 nAChRs on neocortical pyramidal neurons have proven difficult to activate in brain slice 537 preparations, limiting the study of nAChRs. We have overcome this barrier by expressing 538 channelrhodopsin in cholinergic axons and evoking ACh release in neocortical slices. 539 Our results indicate that ACh activates nAChRs on pyramidal neurons in multiple layers 540 and three cortical regions, probably via synapses between cholinergic axons and 541 pyramidal neurons. Hence cholinergic activation of pyramidal neurons via nAChRs is 542 common across neocortex. 543 544 Effects of ACh on pyramidal neurons 545 Several authors have reported nAChR-mediated responses from pyramidal neurons 546 (Roerig et al., 1997; Chu et al., 2000; Kassam et al., 2008; Zolles et al., 2009; Guillem et 547 al., 2011; Poorthuis et al., 2012), but in other studies no such responses were observed 548 (Vidal & Changeux, 1993; Gil et al., 1997; Porter et al., 1999) and in many the actions of 549 ACh were mediated by mAChRs (Krnjevic, 1971; McCormick & Prince, 1986; Haj550 Dahmane & Andrade, 1996; Gulledge & Stuart, 2005; Gulledge et al., 2007; McCormick, 551 1992; Schwindt et al., 1988; Giessel & Sabatini, 2010). The absence of nAChR responses 552 is puzzling as nAChRs are expressed in the dendrites of pyramidal neurons (van der Zee 553 et al., 1992; Nakayama et al., 1995; Duffy et al., 2009; Lubin et al., 1999; Levy & Aoki, 554 2002). 555 In most studies, ACh was applied by bulk perfusion or from a nearby pipette, which 556 results in a relatively slow, widespread increase in concentration. nAChR desensitization 557 during ACh application might be significant, reducing nAChR-mediated currents. 558 Furthermore, many mAChRs are located extrasynaptically (Mrzljak et al., 1998; 559 Yamasaki et al., 2010) and might be more strongly activated by applied ACh than by ACh 560 released from cholinergic axons. Our results suggest that ACh application favors 561 mAChRover nAChR-mediated currents in pyramidal neurons and this weighting may 562 account for the paucity of nAChR-mediated responses in the literature. 563 ACh can act on interneurons in layer 1 of sensorimotor cortex via α7 nAChR564 mediated synaptic and non-α7 nAChR-mediated diffuse mechanisms (Bennett et al., 565 2012) and there is a wider debate on whether ACh acts in neocortex primarily via 566 synaptic contacts or volume transmission (Sarter et al., 2009; Arroyo et al., 2014). The 567 short latency of nAChR-mediated responses in our experiments suggests that ACh forms 568 cholinergic synapses with pyramidal neurons, but via non-α7 nAChRs. We found no 569 evidence for an α7 nAChR-mediated effect or nAChR-mediated actions via volume 570 transmission, but our results do not exclude such responses in pyramidal neurons. 571 Although our recordings were from somata, there are nAChRs throughout the dendritic 572 trees of pyramidal neurons (van der Zee et al., 1992), and it therefore seems likely that 573 there are additional effects of ACh on the dendrites of pyramidal neurons. 574 575 Persistent spiking evoked by nAChRs 576 Our results indicate that nAChR activation can evoke persistent spiking when paired 577 with additional depolarization. Persistent spiking was prevented by intracellular BAPTA, 578 suggesting that a rise in intracellular calcium concentration is also required. Calcium 579 might enter through nAChRs or arise from a secondary source, such as voltage-activated 580 calcium channels or a transmitter co-released by cholinergic axons. Cholinergic axons 581 may co-release glutamate (Manns et al., 2001; Allen et al., 2006; Gritti et al., 2006; 582 Henny & Jones, 2008), but in our experiments persistent spiking was unaffected by 583 glutamate receptor antagonists. Other potential co-transmitters in the basal forebrain 584 include neurotensin, somatostatin, neuropeptide Y and galanin (Koliatsos et al., 1990). 585 nAChR-dependent persistent spiking has been reported in dopaminergic neurons in 586 the substantia nigra pars compacta and subthalamic nucleus (Yamashita & Isa, 2003a, 587 2003b), but not cortical neurons. In entorhinal, perirhinal, cingulate and somatosensory 588 cortices, persistent spiking can be evoked in excitatory neurons, but via mAChRs and a 589 rise in intracellular calcium concentration (Zhang & Séguéla, 2010; Egorov et al., 2002; 590 Navaroli et al., 2011; Rahman & Berger, 2011). Hence nAChR-dependent persistent 591 spiking shares common mechanistic elements with mAChR-dependent persistent 592 spiking, but is initiated by activation of nAChRs, not mAChRs. 593 In spiking neurons, nAChR PSPs evoked a brief and modest increase in spike rate. Why 594 was the effect during ongoing spiking not more prolonged and why was the resulting 595 spike rate typically lower than during nAChR-evoked persistent spiking? Presumably 596 ongoing spiking suppresses the current that underlies persistent spiking or the resulting 597 depolarization, perhaps by shunting the membrane. 598 Previous studies have revealed other mechanisms of prolonged spiking in pyramidal 599 neurons, particularly deep-layer pyramidal neurons. For example, subthreshold DC 600 current injection into the trunk of the apical dendrite can facilitate propagation of a 601 dendritic spike from the distal apical dendrite to the soma, resulting in a burst of spikes 602 (Larkum et al., 2001). Similarly, activation of glutamate receptors in the basal dendrites 603 can evoke a burst of spikes (Milojkovic et al., 2004; Milojkovic et al., 2007). Presumably 604 nAChR-persistent spiking and other mechanisms that can evoke prolonged spiking share 605 some common mechanistic elements and differ important ways. Further experiments 606 will be required to investigate these similarities and differences, but our results add 607 nAChR activation to the collection of identified mechanisms that can evoke prolonged 608 spiking from cortical pyramidal neurons. 609 In summary, nAChR activation increases the excitability of pyramidal neurons. The 610 increase in spiking can be modest and transient or profound and persistent, depending 611 on the membrane potential of the neuron. Hence ongoing synaptic drive to the 612 pyramidal neuron determines the strength and duration of the increase in spiking 613 evoked by ACh. 614 615 Laminar and regional variation in nAChR PSPs 616 α3, α4, α7 and β2 nAChR subunits are located on neocortical pyramidal neuron somata, 617 dendrites and spines (Disney et al., 2007; Nakayama et al., 1995; Duffy et al., 2009; 618 Lubin et al., 1999; Levy & Aoki, 2002; Wevers et al., 1994) and several studies describe 619 responses mediated by α4β2-, α7and α5-containing nAChRs, the latter probably in 620 heteromeric assembly with α4 and β2 subunits (Kassam et al., 2008; Zolles et al., 2009; 621 Poorthuis et al., 2012). 622 Our results are generally consistent with these previous studies, but there are 623 contrasts. Layer 6 contains high expression of non-α7 nAChRs (Tribollet et al., 2004), 624 including α5 (Wada et al., 1990; Proulx et al., 2013) and α4 (Lein et al., 2007) subunits, 625 consistent with the high incidence of nAChR-mediated responses in layer 6 of PFC in 626 previous studies (Kassam et al., 2008; Poorthuis et al., 2012; Mailey et al., 2010). We 627 found that nAChR PSPs in layer 6 are common and of large amplitude in all three areas 628 studied, suggesting that the presence of α4/α5-mediated PSPs is a feature of layer 6 629 pyramidal neurons across cortical regions. 630 In layer 5, we found that nAChR PSPs are common in M1 and V1 and rare in PFC. In 631 M1, nAChR PSPs were mediated by non-α7 nAChRs. In contrast, Poorthuis et al., 2012 632 observed α7 nAChR-mediated responses in layer 5 pyramidal neurons in PFC. nAChR 633 PSPs that we observed originated from nAChRs in the proximal dendrites. Hence one 634 explanation for the lack of α7-mediated PSPs in our results might be that α7 nAChRs are 635 located primarily in the distal dendrites and that α7-mediated depolarization of the 636 distal dendrite failed to evoke depolarization at the soma in our experiments. 637 M1 is unusual in that the pyramidal neurons in layer 5B of M1 commonly display large638 amplitude nAChR PSPs, unlike layer 5 pyramidal neurons in PFC and V1. The α5 subunit 639 is not present in layer 5 (Wada et al., 1990). There is evidence for greater expression of 640 α4 subunits in deep than in superficial layers (Lein et al., 2007), but see also (Tribollet et 641 al., 2004), but this expression pattern is not unique to M1. Hence it is unclear which 642 nAChR subunit(s) underlie the unusually large nAChR PSPs in layer 5 of M1, but α5 643 subunits are unlikely to be involved. 644 In superficial layers, we found that pyramidal neurons in M1 and PFC rarely displayed 645 nAChR PSPs, consistent with results from PFC (Poorthuis et al., 2012), but that nAChR 646 PSPs are common in superficial pyramidal neurons in V1. Layer 2/3 contains dense non647 α7 labeling (Tribollet et al., 2004). Our results suggest that this dense labeling is from 648 interneurons and perhaps in the dendrites of deep-layer pyramidal neurons. 649 Our results extend our knowledge of pyramidal neuron nAChRs from PFC into M1 and 650 V1, revealing variation in laminar responsiveness to ACh between cortical regions; 651 presumably the responsiveness of pyramidal neurons is tuned to the unique demands of 652 each area. However, our results also reveal a general tendency for ACh to exert stronger 653 effects in deep than superficial layers, suggesting that preferential modulation of deep 654 layer pyramidal neurons via nAChRs is a general property of the actions of ACh in 655 neocortex. 656 657 nAChR-mediated modulation of neocortical circuits 658 How might the layer-selective effect of ACh influence the flow of excitation through 659 neocortex? The principal ascending excitatory drive to cortex is thalamocortical axons, 660 which contact pyramidal neurons primarily in layers 5B and 4 (5B and 3 in primary 661 motor cortex, which lacks layer 4 (Shepherd, 2009; Hooks et al., 2013). From layer 4, 662 information is passed by excitatory connections through layer 2/3 to layer 5 and to the 663 sub-cortical projection targets of neocortex. Hence there are two primary excitatory 664 pathways through neocortex: a short loop which connects thalamus with target 665 structures through layer 5 and a longer loop which includes layers 4 and 2/3 666 (Armstrong-James et al., 1992; de Kock et al., 2007; Petreanu et al., 2009; 667 Constantinople & Bruno, 2013). 668 ACh enhances activation of neocortical pyramidal neurons by ascending thalamic 669 drive. This enhancement arises from mAChR-mediated depolarization of pyramidal 670 neurons and enhanced glutamate release from thalamocortical terminals in layer 4 671 (Metherate, 2004; Disney et al., 2007; Gil et al., 1997). In addition, ACh activates non672 fast spiking, non-parvalbumin-expressing interneurons in layers 1 and 2/3 via non-α7 673 nAChRs (Porter et al., 1999; Gulledge et al., 2007; Letzkus et al., 2011; Arroyo et al., 674 2012; Brombas et al., 2014). These interneurons inhibit parvalbuminand somatostatin675 expressing interneurons that target the somata and dendrites of pyramidal neurons. 676 Hence nAChR-mediated inhibition of superficial interneurons reduces inhibition of 677 superficial pyramidal neurons (Letzkus et al., 2011; Brombas et al., 2014). Our results 678 indicate that ACh, again acting at nAChRs, directly promotes the spiking of deep-layer 679 pyramidal neurons. Hence ACh modulates cortical output by at least three different 680 nAChR-dependent mechanisms which enhance the responsiveness of neocortex to 681 incoming sensory drive: by increasing the release of glutamate in layer 4, by indirectly 682 enhancing the excitability of superficial pyramidal neurons and by directly enhancing the 683 excitability of deep-layer pyramidal neurons. 684 Why employ several mechanisms to modulate the excitability of pyramidal neurons 685 in cortex? Multiple mechanisms may offer circuit specificity and semi-independent 686 control. Multiple mechanisms of network modulation might allow ACh to independently 687 modulate the excitability of deep and superficial layer pyramidal neurons, thereby gating 688 the balance of sensory information processing in different cortical layers and controlling 689 the balance of information flowing through the long and short loops from thalamus to 690 the output structures of neocortex. 691 Acknowledgements: 692 We thank Becky Imhoff and Lauren Sybert for technical assistance. TH and JW 693 designed and performed the experiments, analyzed the results and wrote the manuscript. 694 695 Grants: 696 This work was supported by the National Institute of Mental Health (5T32MH067564 697 and 5R21MH085117), the National Institute of Neurological Disorders and Stroke 698 (1R01NS078067) and the Brain Research Foundation (BRF SG 2010-13). 699 700 Disclosures: 701 The authors declare no competing financial interests. 702 References 703 Allen TGJ, Abogadie FC, Brown DA. 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