Articles in PresS. J Neurophysiol (January 14, 2015). doi:10.1152/jn.00716.2014

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Acetylcholine excites neocortical

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pyramidal neurons via nicotinic receptors

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Abbreviated Title: Nicotinic responses of neocortical pyramidal neurons

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Tristan Hedrick and Jack Waters*

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Department of Physiology, Feinberg School of Medicine,

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Northwestern University, 303 E Chicago Ave, Chicago IL 60611

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* current address and address for correspondence:

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Jack Waters

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Allen Institute for Brain Science

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551 N 34th St

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Seattle WA 98103

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tel. 206-548-8439

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e-mail: [email protected]

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Keywords: acetylcholine, nicotinic acetylcholine receptor, neocortical pyramidal neurons

Copyright © 2015 by the American Physiological Society.

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ABSTRACT

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The neuromodulator acetylcholine shapes neocortical function during sensory

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perception, motor control, arousal, attention, learning and memory. Here we investigate

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the mechanisms by which ACh affects neocortical pyramidal neurons in adult mice.

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Stimulation of cholinergic axons activated muscarinic and nicotinic ACh receptors on

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pyramidal neurons in all cortical layers and in multiple cortical areas. Nicotinic receptor

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activation evoked short-latency, depolarizing postsynaptic potentials in many pyramidal

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neurons. Nicotinic receptor-mediated postsynaptic potentials promoted spiking of

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pyramidal neurons. The duration of the increase in spiking was membrane potential-

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dependent, with nicotinic receptor activation triggering persistent spiking lasting many

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seconds in neurons close to threshold. Persistent spiking was blocked by intracellular

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BAPTA, indicating that nAChR activation evoked persistent spiking via a long-lasting

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calcium-activated depolarizing current. We compared nicotinic PSPs in primary motor,

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prefrontal and visual cortices. The laminar pattern of nicotinic excitation was not

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uniform, but was broadly similar across areas, with stronger modulation in deep than

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superficial layers. Superimposed on this broad pattern were local differences, with

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nicotinic PSPs being particularly large and common in layer 5 of M1, but not layer 5 of

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PFC or V1. Hence, in addition to modulating the excitability of pyramidal neurons in all

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layers via muscarinic receptors, synaptically-released ACh preferentially increases the

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activity of deep-layer neocortical pyramidal neurons via nicotinic receptors, thereby

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adding laminar selectivity to the widespread enhancement of excitability mediated by

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muscarinic ACh receptors.

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INTRODUCTION

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The neuromodulator acetylcholine (ACh) shapes neocortical function during sensory

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perception (Metherate, 2004; Disney et al., 2007), motor control (Berg et al., 2005),

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arousal (Steriade, 2004; Jones, 2008), attention (Herrero, 2008; Parikh & Sarter,

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2008), learning (Kilgard, 2003; Ramanathan et al., 2009) and memory (Winkler et al.,

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1995). A decline in neocortical ACh has been tied to conditions such as depression

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(Dislaver, 1986), schizophrenia (Raedler & Tandon, 2006), Alzheimer’s disease

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(Whitehouse et al., 1982) and Parkinson's disease (Whitehouse et al., 1983).

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Most cholinergic axons in neocortex arise from nucleus basalis and other basal

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forebrain nuclei such as substantia innominata (Wainer & Mesulam, 1990). The

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cholinergic projection from basal forebrain plays a central role in shaping neocortical

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components of arousal, attention, learning, memory, sensory perception and motor

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control. For example, stimulation of vibrissal M1 evokes whisker movements that are

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enhanced by activation of basal forebrain (Berg et al., 2005) and basal forebrain lesions

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impair motor control (Garbawie & Whishaw, 2003) and motor map rearrangement

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during motor learning (Conner et al., 2003).

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ACh affects neocortical networks, in part by modulating the activity of pyramidal

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neurons. Pyramidal neurons express nicotinic and muscarinic ACh receptors (nAChRs

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and mAChRs) on their plasma membranes (van der Zee et al., 1992; Mrzljak et al., 1993),

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but ACh is thought to act on pyramidal neurons primarily via mAChRs. Activation of

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mAChRs evokes an initial hyperpolarization and subsequent slow depolarization of many

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cortical pyramidal neurons. The hyperpolarization results from activation of an SK-type

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potassium current, whereas the slow depolarization has been linked to a number of

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currents, including M-, AHP- and inward rectifier-type potassium currents and a non-

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specific cation current (Krnjevic, 1971; McCormick & Prince, 1985, 1986; McCormick &

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Williamson, 1989; Haj-Dahmane & Andrade, 1996; Delmas & Brown, 2005; Gulledge &

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Stuart, 2005; Carr & Surmeier, 2007; Zhang & Séguéla, 2010). There are reports of ACh

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activating nAChRs on pyramidal neurons (Roerig et al., 1997; Chu et al., 2000; Kassam

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et al., 2008; Zolles et al., 2009; Guillem et al., 2011; Poorthuis et al., 2012; but see also

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Vidal & Changeux, 1993; Gil et al., 1997; Porter et al., 1999). Few authors have studied

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nicotinic postsynaptic currents in neocortical pyramidal neurons (Roerig et al., 1997;

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Chu et al., 2000). Hence the functional roles of nAChRs on pyramidal neurons remain

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obscure.

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Here we studied how ACh affects pyramidal neurons, focusing on the role of nAChRs.

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To drive synaptic release of ACh, we expressed the light-activated protein

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channelrhodopsin-2 (ChR2) in cholinergic neurons in the basal forebrain, allowing us to

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selectively stimulate cholinergic axons in neocortex (Kalmbach et al., 2012). In contrast

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to many previous reports, we find that ACh excites pyramidal neurons via both mAChRs

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and nAChRs. Activation of nAChRs occurs with short latency, consistent with nAChRs

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being located at synapses between cholinergic axons and pyramidal neurons, and can

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evoke persistent spiking via a calcium-activated conductance. Direct nAChR-mediated

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effects occurred in pyramidal neurons in several neocortical areas and in all neocortical

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layers, indicating that direct excitation of pyramidal neurons via nAChRs can occur

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across neocortical layers and areas. However, laminar and regional differences in both

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the incidence and amplitude of the nAChR-mediated depolarization suggest regional

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differences in the modulation of neocortical networks by nAChRs.

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METHODS

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All experiments and procedures were approved by the Northwestern University

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Institutional Animal Care and Use Committee (IACUC).

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Two approaches were employed to selectively express ChR2 in cholinergic neurons:

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(1) stereotaxic injection of a floxed viral vector into the basal forebrain of ChAT-Cre

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mice, and (2) crossing ChAT-Cre and floxed ChR2 mouse lines.

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For experiments using virally-delivered ChR2, we used Tg(ChAT-Cre)60Gsat mice

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(GENSAT), which express Cre-recombinase on a choline-acetyltransferase (ChAT)

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promoter, resulting in Cre expression in cholinergic neurons throughout the brain. Into

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this mouse we injected adeno-associated virus with a double-floxed inverse open reading

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frame (EF1a-DIO-hChR2(H134R)eYFP, Virus Vector Core, University of North

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Carolina), which drives expression of ChR2-yellow fluorescent protein (ChR2-YFP) in

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infected neurons containing Cre. 400 nl of virus was injected into the basal forebrain at

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postnatal day 21 using stereotaxic coordinates (0.2 mm A-P, 1.7 mm M-L, 4.5 mm D-V).

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Three weeks after injection, ChR2 is expressed almost exclusively in cholinergic neurons

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and their axons in neocortex, with no adverse effects (Porter et al., 1999).

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In some experiments, selective expression of ChR2 in cholinergic neurons was

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achieved without the use of viral vectors by crossing ChAT-Cre (B6;129S6-

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Chattm1(cre)Lowl/J, Jax 006410) and floxed ChR2 (129S-Gt(ROSA)26Sortm32(CAG-

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COP4*H134R/EYFP)Hze/J,

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ChAT-ChR2(Ai32) mice. Cre+ mice were crossed with ChR2+/+ mice to yield Cre+ ChR2+/-

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offspring. These offspring were crossed to generate Cre+ ChR2+/+ mice, which were used

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for experiments. Results from ChAT-ChR2(Ai32) mice were similar to those from viral

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infections and results from these two approaches were pooled, unless otherwise noted.

Jax 012569, Ai32 (Madisen et al., 2012) mouse lines to produce

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Somatic and axonal labeling with ChR2-YFP was examined in fixed sections. Tissue

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was fixed by transcardial perfusion with 4% paraformaldehyde in phosphate buffer and

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100-200 µm thick coronal sections were cut using a vibrating microtome. In some

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sections, the YFP signal was enhanced with an anti-GFP primary (ab13970, 1:10,000,

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Abcam) and a fluorescent secondary antibody (613111, 1:750, Invitrogen). Images were

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acquired by widefield or 2-photon microscopy. Widefield images were acquired using a

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GFP filter set and a Hamamatsu Orca-285 camera. 2-photon images were acquired with

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880 nm illumination from a Coherent Chameleon Ultra II Ti:sapphire laser and a 505-

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545 nm emission filter.

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Whole-cell recordings were obtained from pyramidal neurons in 300 µm-thick acute

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parasagittal slices of primary motor cortex and coronal slices of prefrontal and visual

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cortices from postnatal day 33-61 mice of either sex. In virally-infected mice, recordings

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were obtained ~3 weeks after viral injection. Slices were prepared in ice-cold artificial

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cerebro-spinal fluid (ACSF; in mM): 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 20 NaHCO3, 5

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HEPES, 25 Glucose, 2 CaCl2, 1 MgCl2, pH 7.3, oxygenated with 95% O2/5% CO2. Whole-

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cell recording pipettes were 4–8 MΩ when filled with intracellular solution: 135 mM K

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gluconate, 4 mM KCl, 10 mM HEPES, 10 mM Na2-phosphocreatine, 4 mM Mg-ATP, 0.3

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mM Na2-GTP, 0.2% (w/v) biocytin, 10 µM alexa 594, pH 7.3.

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Recordings were obtained at 35-37 °C with a feedback circuit and temperature

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controller (TC-324B; Warner Instruments, Hamden, CT, USA). In experiments using

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calcium-free ACSF, 2 mM CaCl2 was replaced with 4 mM MgCl2 to give a final MgCl2

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concentration of 5 mM. ChR2 was activated by wide field illumination through a

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microscope objective (Olympus, x20/0.95 NA or x40/0.8 NA) using a blue light-emitting

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diode (LED; Thorlabs LEDC5 and LEDD1 or DC2100 driver). The maximum steady-state

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intensity was 20 mW/mm2. In some experiments (see figure 4E-G), illumination was

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restricted by closure or partial closure of the fluorescence field stop. The illumination

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area was measured offline by bleaching a thin, immobilized film of fluorescence on a

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microscope slide. Closure of the field stop had no effect on illumination intensity per unit

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area. In restricted illumination experiments, the soma was positioned in the center of the

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field of illumination unless noted otherwise.

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The amplitudes of voltage responses were calculated by subtracting the average

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baseline membrane potential for ≥1 s before illumination from the peak of the response.

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Voltage responses which failed to exceed 3 standard deviations of the baseline

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membrane potential were assigned an amplitude of 0 mV. During brief bursts of nAChR

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PSPs, the peak amplitude was calculated by subtracting the pre-burst membrane

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potential from the most depolarized potential during the burst.

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Local application of ACh was by pressure ejection of 100 µM ACh in ACSF from a

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glass pipette ~50 µm from the soma, 30 psi, Toohey Spritzer IIe (Toohey Company,

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Fairfield NJ).

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Mecamylamine hydrochloride and atropine were obtained from Sigma-Aldrich.

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Dihydro-β-erythroidine hydrobromide (DHβE), methyllycaconitine citrate,

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galanthamine hydrobromide and physostigmine hemisulfate were obtained from Tocris

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Bioscience. NBQX disodium salt, (R)-CPP, gabazine (SR95531), CGP 52432, tetrodotoxin

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citrate and (R,S)-MCPG were obtained from Ascent Scientific. In some experiments, we

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used (2R)-amino-5-phosphonopentanoic acid (AP5) to block NMDA receptors, instead of

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CPP. AP5 was obtained from Tocris.

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Statistical analyses were performed using Graphpad QuickCalcs online tool (http://www.graphpad.com/quickcalcs/) or in Graphpad Instat 3.06 (GraphPad

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Software Inc., La Jolla, CA). Continuous data (such pharmacology of the nAChR PSP)

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were analyzed with a two-tailed t-test, Kruskal-Wallis test or Mann-Whitney test and

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categorical data with Fisher's exact test.

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The kinetics and latency of nAChR PSPs were measured by fitting a sum of two

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exponentials to the mean of ten trials. In each trial the PSP was evoked with a single 2

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ms (or occasionally 5 ms) blue light illumination. The signal-to-noise ratio of our

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recordings was not sufficient to permit accurate measurement of the timing of the initial

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rise of the membrane potential of a nAChR PSP, which is smaller and slower than a

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glutamatergic PSP. To accurately estimate the point of initial rise we therefore measured

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the time from the start of illumination to 5% of the peak amplitude of the PSP. We would

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expect this method of latency measurement to overestimate the latency of each PSP by

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approximately 1 ms and we therefore corrected the latency of each PSP, by back-

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extrapolating the fit to the resting membrane potential preceding the stimulus.

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Pyramidal neurons were identified by their somatic shape and spiking pattern and by

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their large apical dendrites, which were visible by fluorescence microscopy once filled

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with indicator. Neurons were routinely filled with indicator and their dendrites

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examined by fluorescence microscopy. Neurons with truncated primary apical dendrites,

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lacking apical dendrites and with spiking patterns more typical of interneurons (narrow

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spikes, large after-hyperpolarization) were excluded from further analysis.

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Pyramidal cells were classified according to the laminar locations of their somata.

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Laminar borders were based on the Allen Brain Atlas and consistent with laminar

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variation in opacity of slices, which was visible under brightfield illumination. Distances

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from the pial surface of the slice were measured perpendicular to the pia. In primary

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motor cortex, cortical layers were: layer 2/3 150-500 µm, layer 5A 600-750 µm, layer 5B

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800-1200 µm, layer 6 >1300 µm. In prefrontal cortex, cortical layers were: layer 2/3

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100-300 µm, layer 5 330-550 µm, layer 6 >550-800 µm (Guillem et al., 2011). In visual

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cortex, cortical layers were: layer 2/3 100-275 µm, layer 5 450-750 µm, layer 6 >750 µm

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(Olivas et al., 2012; Petrof et al., 2012).

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Pyramidal neurons projecting to defined postsynaptic target tissues were labeled

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using fluorescent microspheres (RetroBeads, Lumafluor). Beads were injected into

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dorsolateral striatum (0 mm A-P, -2 mm M-L, 2.5 mm D-V), cervical spinal cord

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(between ~-0.5 mm M-L and the midline, ~1 mm D-V, at the level of the C2 vertebra) or

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ventral posteromedial thalamus (two sites: 1.6 mm A-P, 1.2 mm M-L, 3.25 mm D-V and

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1.6 mm A-P, 1.7 mm M-L, 3.5 mm D-V). Recordings were obtained 5-11 days after bead

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injection from M1 cortex contralateral to striatal and spinal injections and ipsilateral to

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thalamic injections.

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RESULTS

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To stimulate cholinergic axons entering neocortex, we used the blue light-activated

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membrane protein channelrhodopsin-2 (ChR2; Nagel et al., 2003). We expressed ChR2

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in cholinergic neurons in the basal forebrain (figure 1A-C), obtaining selectivity for

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cholinergic neurons with a ChAT-Cre mouse line and floxed virus or by crossing ChAT-

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Cre and floxed ChR2 mouse lines. We obtained whole-cell recordings from layer 5B

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pyramidal neurons in acute slices of primary motor cortex containing ChR2-labeled

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axons (figure 1D). Mean resting potential was -62 ± 1 mV (51 neurons). Widefield

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illumination of the slice with a blue LED evoked one or more of four voltage responses: a

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slow depolarization, a hyperpolarization, a medium depolarization, or a fast

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depolarization (figure 1F). All responses were absent in tetrodotoxin (500 nM TTX, 17

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neurons) and after removal of extracellular calcium (10 neurons), indicating that all four

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voltage responses were evoked by spikes, leading to vesicular release (figure 2).

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We used pharmacology to identify the receptors underlying each of the four voltage

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responses. The hyperpolarization and slow depolarization were mediated by muscarinic

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ACh receptors (mAChRs): both hyperpolarization and slow depolarization were

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eliminated by the mAChR antagonist atropine (1 µM, 140 of 141 neurons, figure 3), but

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not by the nAChR antagonist mecamylamine (100 µM, 11 of 13 neurons, figure 3) or by

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glutamate or GABA receptor antagonists (10 µM NBQX and 10 µM CPP; 1 µM gabazine

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and 3 µM CGP 52432; 14 of 16 neurons).

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The medium depolarization was mediated by nicotinic ACh receptors (nAChRs). It

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was eliminated by mecamylamine (100 µM, 12 of 12 neurons, P < 0.05, paired two-tailed

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t-test), enhanced 73 ± 21% by the nAChR allosteric potentiator and cholinesterase

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antagonist galanthamine (1 µM, 23 neurons, P < 0.05, paired two-tailed t-test) and 27 ±

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15% by the ACh esterase inhibitor physostigmine (0.5 µM, 4 neurons, P < 0.05, paired

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two-tailed t-test), and insensitive to antagonists of mAChRs, GluRs and GABA receptors

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(5 of 5, 7 of 7 and 8 of 8 neurons, respectively; figure 4). The medium depolarization was

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unaffected by the α7-nAChR antagonist methyllycaconitine (MLA, 10 nM, 12 of 12

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neurons) and eliminated by dihydro-β-erythroidine (DHβE, 10 µM, 16 of 16 neurons, P
0.05, Matt-Whitney test, 37 and 22 neurons from virally-infected and

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ChAT-ChR2(Ai32) mice, respectively). We therefore pooled results from the two

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techniques.

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All four postsynaptic responses could occur individually, but most pyramidal

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neurons exhibited a combination of responses mediated by two or more receptors (figure

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5B). Hence synaptically-released ACh activates pyramidal neurons via nAChRs and

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mAChRs, and often via both types of ACh receptor in the same neuron.

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Activation of pyramidal neuron mAChRs by synaptically-released ACh

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The peak amplitude of the hyperpolarization, evoked by brief illumination (≤10 x 5 ms at

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20 Hz), was 1.1 ± 0.5 mV and the decay time constant was 774 ± 73 ms (10 neurons). The

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slow depolarization displayed a peak amplitude of 2.0 ± 0.6 mV (6 neurons) and lasted

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several seconds. At resting membrane potentials, the hyperpolarization and slow

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depolarization were rare, occurring in only 7% (8 of 112) and 19% (21 of 112) of layer 5B

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pyramidal neurons, respectively.

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Many previous authors have reported activation of pyramidal neurons via mAChRs

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upon application of ACh to the soma, by pressure ejection from a nearby pipette

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(McCormick & Prince, 1985; McCormick & Williamson, 1989; Gulledge & Stuart, 2005;

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Gulledge et al., 2007), with relatively few authors reporting nAChR-mediated

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depolarization of pyramidal neurons (Roerig et al., 1997; Chu et al., 2000; Kassam et al.,

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2008; Zolles et al., 2009; Guillem et al., 2011; Poorthuis et al., 2013). Similar to many

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previous studies, we found that pressure ejection of ACh onto the soma evoked a

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mAChR-mediated hyperpolarization and slow depolarization (5 of 6 and 6 of 6 neurons,

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respectively; figure 6A), but no nAChR-mediated depolarization.

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One possible interpretation of our results might be that synaptically-released ACh

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activates primarily nAChRs and usually fails to activate mAChRs, whereas pressure

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ejection onto the soma activates a different population of receptors, primarily mAChRs.

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mAChR activation modulates primarily potassium conductances (McCormick, 1992) and

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the reversal potential for potassium is ~-90 mV. mAChR activation may therefore exert

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little effect on the membrane potential at rest: both mAChR-mediated hyperpolarization

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and slow depolarization are larger when the neuron is depolarized (McCormick & Prince,

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1986; Gulledge & Stuart, 2005). To maximize the effects of mAChR activation on the

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membrane potential and, therefore, the probability that we would observe activation of

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mAChRs, we depolarized neurons by somatic current injection (figure 6B). Brief stimuli

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(≤10 x 5 ms illumination at 20 Hz) evoked mAChR-mediated voltage responses in 79% of

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depolarized layer 5B pyramidal neurons (figure 6C), indicating that synaptically-released

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ACh activates mAChRs in the majority of layer 5B pyramidal neurons, but that the

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resulting hyperpolarization or depolarization from rest is often too small to be observed.

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Previous authors have also reported that ACh, applied by pressure application, more

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readily evokes postsynaptic mAChR-mediated responses in pyramidal neurons in deep-

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than in superficial-layers of the neocortex (McCormick & Prince, 1986; McCormick &

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Williamson, 1989; Gulledge et al., 2007). Similarly, we found that synaptically-released

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ACh more readily excites pyramidal neurons via mAChRs in deep than superficial layers

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of M1 cortex, with the proportion of pyramidal neurons that exhibited a mAChR-

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mediated slow depolarization or hyperpolarization ranging from 53% in layer 2/3 to 91%

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in layer 6 pyramidal neurons (figure 6C).

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We conclude that ACh released by a brief burst of cholinergic activity activates

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mAChRs on the majority of pyramidal neurons throughout primary motor cortex. In

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comparison to pressure application of ACh, activation of cholinergic synapses with brief

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bursts of stimuli provides relatively weak activation of mAChRs which often fails to affect

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the somatic membrane potential at rest.

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ACh release evokes a nicotinic PSP

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Brief illumination (≤10 x 5 ms at 20 Hz) in mAChR, GluR and GABAR antagonists

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evoked a nAChR-mediated medium depolarization in almost all (82 of 90) layer 5B

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pyramidal neurons at rest. We stimulated cholinergic axons with brief bursts of stimuli

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at 20 Hz, a stimulus designed to mimic the spiking of cholinergic basal forebrain

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neurons during paradoxical sleep and awake states, when cholinergic basal forebrain

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neurons spike in bursts, each of ~4-6 spikes at ~25 Hz (Manns et al., 2000; Lee et al.,

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2005). During such bursts, summation occurred at stimulus frequencies greater than ~5

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Hz. In our experiments summation may result, in part, from accumulation of calcium

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entering through ChR2 channels in the presynaptic terminal (Zhang & Oertner, 2007)

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and may therefore be an artifact of the optogenetic stimulus. Nonetheless, summation

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permitted us to evoke a substantial depolarization via nAChRs: a burst of stimuli at 20

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Hz frequently evoked a peak depolarization of 5-10 mV (figure 7A,B).

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We measured the latency and kinetics of the nAChR-mediated depolarization evoked

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by a single 2 ms illumination, in 1 µM atropine to inhibit mAChRs and GluR and GABAR

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antagonists to eliminate any indirect effects. In 15 of 15 neurons, 2 ms illumination

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evoked reproducible depolarizations (figure 7C), with a mean amplitude of 1.4 ± 0.2 mV

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(11 neurons). To the mean response we fit a sum of two exponentials (figure 7D) with

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mean rise and decay time constants of 28 ± 4 ms and 180 ± 23 ms (14 neurons). These

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relatively slow kinetics are comparable to those of PSPs mediated by α4β2-containing

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nAChRs in interneurons (Bell et al., 2011), but slower than PSPs mediated by α7-

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nAChRs (Frazier et al., 1998; Fedorov et al., 2012). These kinetics suggest that the

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medium depolarization is a PSP mediated by non-α7 nAChRs, consistent with the

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pharmacology of the medium depolarization, presented above.

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The latency of the nAChR PSP, measured from the start of illumination, was 5.9 ±

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0.8 ms (12 neurons). Compared to electrical stimulation, ChR2 drives relatively slow

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depolarization of the neuronal membrane, typically with rise and decay time constants of

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at least several milliseconds (Lin et al., 2009; Yizhar et al., 2011), which may be further

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elongated by the time constant of the membrane. The latency of the fast depolarization

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was 5.3 ± 0.5 ms (3 neurons). Hence the latency of the nicotinic PSP was only ~0.5 ms

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longer than that of a monosynaptic glutamatergic PSP, consistent with the medium

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depolarization being a monosynaptic PSP generated at synapses between cholinergic

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axons and pyramidal neurons.

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nAChRs appear to be distributed throughout the dendritic trees of cortical pyramidal

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neurons (van der Zee et al., 1992; Nakayama et al., 1995) but the locations of cholinergic

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synapses are unknown. To determine whether nAChR PSPs were evoked primarily by

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cholinergic synapses in the proximal or distal dendrites of layer 5B pyramidal neurons

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we measured nAChR PSPs during restricted illumination of the slice (figure 8A,B).

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Restricting illumination to a radius of less than ~300 µm around the soma was necessary

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to reduce the amplitude of the nAChR PSP and the amplitude was reduced by 50% when

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the radius of illumination was ~50 µm (figure 8C, 7 neurons). Illumination of the tuft

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dendrites failed to evoke a nAChR PSPs at the soma (figure 8B, 3 neurons). Hence the

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nAChRs which contribute to the somatic depolarization in our experiments are likely to

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be within 300 µm of the soma and many are probably located in the proximal 50 µm of

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the apical and basal arbor.

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nAChR activation can evoke persistent spiking

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We next addressed the functional consequences of nAChR activation. nAChR PSPs

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increased the spike rate of layer 5B pyramidal neurons in primary motor cortex,

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depolarized beyond threshold by somatic current injection (figure 9A). The increase was

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independent of initial spike rate (figure 9B), with 2-5 ms illumination increasing spike

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rate by 2.2 ± 0.1 Hz (6 neurons) and a brief burst (10 x 5 ms at 20 Hz) increasing spike

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rate by 3.1 ± 0.2 Hz (4 neurons). Hence, during spiking nAChR activation causes a linear,

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additive change in spike rate with no change in the slope of the input-output

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relationship. The elevated spike rate was maintained during repetitive stimulation and

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declined after cessation of the cholinergic stimulus with a decay time constant of 185 ±

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21 ms (figure 9C; 2 or 5 ms illumination; 6 neurons), which matches the decay of the

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underlying nAChR PSP.

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With the membrane depolarized from rest but subthreshold, cholinergic stimulation

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evoked persistent spiking. We evoked nAChR PSPs during long step current injections of

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increasing amplitude until the nAChR PSP evoked one or more spikes. A nAChR PSP

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reduced rheobase by 11.6 ± 3.7 pA (from 344 ± 26 pA to 337 ± 26 pA, 18 neurons, 5 ms

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illumination in the presence of atropine). However, under these conditions, a nAChR

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PSP typically evoked multiple spikes. The spike rate during the first second after

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stimulus onset was 3.0 ± 0.2 Hz (6 neurons) for a single 2-5 ms illumination and 7.1 ±

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1.2 Hz for a brief burst (10 x 5 ms at 20 Hz, 7 neurons). Spike rate declined slowly after

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the last nAChR PSP (figure 10A,B), but spiking typically continued until the holding

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current was removed, which was up to 4 seconds after the initial spike (figure 10A,B).

365

We defined persistent spiking as spiking that continued for at least 500 ms after the

366

end of the cholinergic stimulus. Using this definition, persistent spiking occurred in

367

every neuron (13 of 13 neurons), but in 6 of 13 neurons persistent spiking occurred on

368

some but not all trials. In trials in which persistent spiking failed to occur, the nAChR

369

PSP evoked only a single spike (mean 1.0 ± 0, 14 trials from 4 neurons, single 2-5 ms

370

illumination), whereas in trials in which persistent spiking occurred the nAChR PSP

371

evoked 7.5 ± 1.6 spikes (10 trials in the same 4 neurons with the same stimulus).

372

Comparing trials with and without persistent spiking, the resting membrane potential at

373

the start of the trial (-62.3 ± 1.9 and -63.2 ± 1.5 mV, respectively), the current injected to

374

depolarize the neuron (209 ± 20 and 268 ± 23 pA, respectively), and the membrane

375

potential immediately before the nAChR PSP (-48.0 ± 2.0 and -48.5 ± 1.8 mV,

376

respectively) and the threshold of the first spike (-35.2 ± 1.9 and -39.1 ± 1.9 mV,

377

respectively) were similar (18 and 14 trials respectively, each P > 0.05, paired 2-tailed t-

378

test). These measurements indicate that trial-to-trial variability in perisomatic

379

membrane potential, input resistance and spike threshold do not account for the

380

variability in persistent spiking, although they do not exclude a role for such changes in

381

the distal dendrite, as a result of ongoing synaptic activity, for example.

382

Persistent spiking required activation of nAChRs on pyramidal neurons, but not

383

glutamate or GABA receptors or mAChRs (figure 11). Persistent spiking occurred in the

384

presence of 10 µM NBQX, 10 µM CPP, 1 µM gabazine, 3 µM CGP and 1-10 µM atropine

385

(13 of 13 neurons). Subsequent addition of 100 µM mecamylamine (in the continued

386

presence of glutamate and GABA receptor antagonists and of atropine) blocked

387

persistent spiking (3 of 3 neurons, figure 11A) but 100 µM MCPG did not (3 of 3

388

neurons).

389

nAChR activation provides more than just the initial depolarization required to

390

initiate persistent spiking since in the absence of cholinergic stimulation, brief

391

depolarization failed to evoke persistent spiking (figure 11C). Furthermore, during

392

persistent spiking evoked by cholinergic stimulation, brief hyperpolarization of the

393

membrane inhibited persistent spiking, only for spiking to resume after the

394

hyperpolarizing pulse (figure 11D). Presumably, an additional depolarizing conductance

395

is activated (or hyperpolarizing conductance deactivated) by nAChR activation or by

396

another receptor co-activated with nAChRs and this additional conductance remains

397

active for several seconds, long after the decay of the nAChR PSP. The spike waveform

398

changed little during persistent spiking (figure 12), suggesting that this additional

399

current is unlikely to arise from one of the sodium or potassium conductances that shape

400

the spike waveform.

401

Persistent spiking was eliminated by 10 mM intracellular BAPTA. With or without

402

intracellular BAPTA, nAChR PSPs evoked 1 or more spikes (figure 11E), but in BAPTA

403

spiking did not continue after the decay of the underlying PSP. Without BAPTA spiking

404

continued until the holding current was removed, with the last spike 2155 ± 261 ms after

405

the start of a single 2-5 ms illumination and 3357 ± 169 ms after a burst of stimuli (10 x 5

406

ms at 20 Hz; 13 neurons). With BAPTA, spiking ended 185 ± 38 ms after a single

407

illumination and 445 ± 115 ms after a burst (3 neurons; single illumination and burst

408

each P < 0.05, unpaired 2-tailed t-test). As a result, cholinergic stimuli evoked fewer

409

spikes with BAPTA (single 5 ms illumination, 1.8 ± 0.1 spikes, 2 neurons; burst 3.3 ± 1.7

410

spikes, 3 neurons) than without BAPTA (single 5 ms illumination, 6.6 ± 0.8 spikes, 7

411

neurons; burst 21.7 ± 0.6 spikes, 7 neurons; single illumination and burst each P < 0.05,

412

unpaired 2-tailed t-test). Hence activation of cholinergic axons evokes persistent spiking

413

that requires activation of nAChRs and a calcium-activated current that does not affect

414

the spike waveform.

415

Our results indicate that ACh has both brief, additive and prolonged, non-linear

416

effects on the spiking of layer 5B pyramidal neurons, with neurons being particularly

417

sensitive to cholinergic activity when their membrane potentials are within ~10 mV of

418

spike threshold, such that a nAChR-mediated increase in spiking can be short-lived or

419

can be more dramatic, evoking spiking that persists for many seconds.

420 421

Cholinergic responses by projection target

422

In primary sensory neocortices, nAChRs are expressed by selected sub-populations of

423

presynaptic terminals. For example, ACh can enhance the transmission of sensory

424

information to neocortex via the activation of nAChRs on thalamocortical terminals in

425

primary somatosensory and visual cortices (Metherate, 2004; Disney et al., 2007; Gil et

426

al., 1997). Neuromodulators can also act selectively on different projection pathways out

427

of neocortex (Gaspar et al., 1995; Beique et al., 2007; Sheets et al., 2011; Avesar &

428

Gulledge, 2012; Gee et al., 2012; Seong & Carter, 2012). For example, in medial

429

prefrontal cortex mAChR activation by ACh has a greater effect on the excitability of

430

layer 5 pyramidal neurons that project to the pons than on neurons that project to

431

contralateral cortex (Dembrow et al., 2010) and nAChR activation evokes larger-

432

amplitude currents from corticothalamic layer 6 pyramidal neurons than from layer 6

433

pyramidal neurons that do not project to thalamus (Kassam et al., 2008). Might ACh

434

differentially modulate the output of motor cortex via expression of nACh receptors in

435

pyramidal neurons that project to some sub-cortical targets, but not pyramidal neurons

436

that project to other target tissues?

437

In motor cortex, descending axons of layer 5 pyramidal neurons project into the

438

pyramidal tract or to the contralateral striatum and these two pathways are mutually

439

exclusive (Shepherd, 2013). Within layer 6, the primary subcortical output is to thalamus

440

and layer 6 pyramidal neurons may therefore be divided into corticothalamic and non-

441

corticothalamic, or intracortical, neurons. We compared the incidence of nAChR- and

442

mAChR-mediated potentials in each of these subpopulations in motor cortex after

443

retrograde labeling of pyramidal neurons by injection of fluorescent beads into spinal

444

cord, contralateral striatum or ipsilateral thalamus (figure 13A). In slices, we identified

445

neurons with different projection targets by the somatic accumulation of fluorescent

446

beads (figure 13B).

447

Synaptically-released ACh frequently evoked nAChR PSPs in all four subpopulations of

448

deep layer M1 pyramidal neurons: corticospinal layer 5 pyramidal neurons;

449

corticostriatal layer 5 pyramidal neurons; corticothalamic layer 6 pyramidal neurons;

450

non-corticothalamic (intracortical) layer 6 pyramidal neurons (figure 13C). The

451

incidence of nAChR PSPs was not different between the two populations of layer 5

452

neurons nor between the two populations of layer 6 neurons (nAChR PSPs in 5 of 10

453

corticospinal layer 5 pyramidal neurons, 8 of 11 corticostriatal layer 5 pyramidal

454

neurons, P > 0.05, Fisher's exact test; nAChR PSPs in 8 of 11 corticothalamic layer 6

455

pyramidal neurons, 6 of 12 non-corticothalamic layer 6 pyramidal neurons, P > 0.05,

456

Fisher's exact test). Hence our experiments revealed no evidence for different

457

probabilities of nAChR PSPs in sub-populations of deep-layer pyramidal neurons with

458

different projection targets. However, the amplitudes of nAChR PSPs were greater in

459

layer 6 pyramidal neurons that projected to thalamus (9.5 ± 2.6 mV, 7 neurons) than in

460

layer 6 neurons that did not project to thalamus (6.0 ± 2.3 mV, 6 neurons; P < 0.05,

461

paired 2-tailed t-test). nAChR-mediated currents are larger in corticothalamic than non-

462

corticothalamic pyramidal neurons in prefrontal cortex (Kassam et al., 2008) and our

463

results suggest that this enhancement of nAChR responses in corticothalamic layer 6

464

pyramidal neurons extends to primary motor cortex.

465

The mAChR-mediated slow depolarization was also common in neurons from all four

466

projection-based populations of deep-layer pyramidal neurons (figure 13C; no difference

467

in incidence of slow depolarization between layer 5 or layer 6 subpopulations, P > 0.05,

468

Fisher's exact test). In contrast, the hyperpolarization displayed differential expression

469

by projection target (figure 13C), occurring often in both layer 5 projection-based

470

populations and in non-corticothalamic layer 6 pyramidal neurons (no difference in

471

incidence of hyperpolarization between layer 5 subpopulations, P > 0.05, Fisher's exact

472

test), but being completely absent from corticothalamic layer 6 pyramidal neurons

473

(different incidence of slow depolarization between layer 6 subpopulations, P < 0.05,

474

Fisher's exact test).

475 476

nAChR PSPs responses across layers and cortical areas

477

In primary motor cortex, nAChRs are expressed by pyramidal neurons throughout the

478

layers of neocortex (van der Zee et al., 1992; Nakayama et al., 1995; Duffy et al., 2009)

479

and cholinergic axons ramify through all layers (Wainer & Mesulam, 1990; Lysakowski et

480

al., 1989). Hence nAChR PSPs might be expected in pyramidal neurons in all layers. To

481

test this hypothesis, we determined the frequency with which synaptically-released ACh

482

evoked nAChR PSPs in pyramidal neurons in layers 2/3, 5, and 6 of primary motor,

483

prefrontal, and visual cortices.

484

In primary motor cortical slices with abundant ChR2-labeled axons in all neocortical

485

layers (figure 14A) and nAChR PSPs in layer 5B pyramidal neurons, cholinergic stimuli

486

(10 x 5 ms at 20 Hz) rarely evoked nAChR PSPs in layer 2/3 pyramidal neurons (4 of 21

487

layer 2/3 neurons; figure 14B; lower probability in layer 2/3 than in layer 5A, layer 5B, or

488

layer 6, P < 0.05 for each, Fisher's exact test). In layers 5A and 5B, nAChR PSPs were

489

common (6 of 8 layer 5A neurons, 82 of 90 layer 5B neurons; figure 14B; no difference in

490

probability between layers 5A and 5B, Fisher's exact test) and in layer 6, nAChR PSPs

491

were evoked in approximately half of neurons (16 of 32 neurons; figure 14B; greater

492

probability in layer 6 than in layer 2/3 and lower than in layer 5B, P < 0.05 for each,

493

Fisher's exact test). Hence in primary motor cortex, nAChR PSPs occur almost

494

exclusively in deep-layer pyramidal neurons.

495

In prefrontal and primary visual cortices (PFC and V1), the laminar pattern of nAChR

496

PSPs was different from that in primary motor cortex. In prefrontal cortex, cholinergic

497

stimuli (10 x 5 ms at 20 Hz) evoked nAChR PSPs in 2 of 6 layer 2/3 pyramidal neurons, 2

498

of 13 layer 5 pyramidal neurons and 5 of 8 layer 6 pyramidal neurons (figure 14B). Hence

499

nAChR PSPs were less common in all three layers of PFC than in layer 5B neurons in M1

500

(P < 0.05 for each layer, Fisher's exact test). Within PFC, nAChR PSPs were more

501

common in layer 6 than in more superficial layers (P < 0.05, Fisher's exact test). As

502

expected from previous studies (Kassam et al., 2008; Poorthuis et al., 2012; Bailey et al.,

503

2010), nAChR PSPs in layer 6 of prefrontal cortex arose from activation of non-α7

504

nAChRs, being unaffected by MLA and eliminated by DHβE (4 of 4 neurons). In visual

505

cortex, cholinergic stimuli (10 x 5 ms at 20 Hz) commonly evoked nAChR PSPs in

506

pyramidal neurons in all cortical layers (5 of 6 layer 2/3 neurons, 9 of 11 layer 5

507

pyramidal neurons, 8 of 9 layer 6 pyramidal neurons; figure 14B; no differences in

508

probability, Fisher's exact test).

509

The amplitudes of nAChR PSPs also differed across cortical layers and areas (figure

510

14C). The largest responses were observed in layer 5B of primary motor cortex

511

(maximum 18.3 mV), but the mean nAChR PSP amplitude was greatest in layer 6

512

pyramidal neurons (M1 6.06 ± 1.21 mV, maximum 16.2 mV; PFC 5.99 ± 2.86 mV,

513

maximum 15.9 mV; V1 2.76 ± 0.74 mV, maximum 5.8 mV; for M1, P < 0.05, Kruskal-

514

Wallis test; L5A different from L5B, L5B different from L6, each P < 0.05, Mann-

515

Whitney test). In all areas, mean peak amplitudes in layers 2-5 were between 1 and 2 mV,

516

with the exception of motor cortex (figure 14C), where responses in layer 5B were larger

517

than in layer 5A (P < 0.05, Mann-Whitney test) and larger than in layer 5 of prefrontal or

518

visual cortices (L5B of M1 14.21 ± 1.14 mV; amplitude larger in M1 L5B than PFC L5 and

519

V1 L5; P < 0.05, Kruskal-Wallis test).

520

To compare the effects of nAChR activation on pyramidal neurons in different layers

521

and areas, we multiplied the probability and amplitude of nAChR PSPs for each layer

522

(figure 14D). This analysis provides a measure of the overall effect of nAChR PSPs on

523

laminar excitability and reveals that, in all three cortical areas, the effects of nAChR

524

activation are greater in deep layers than in superficial layers. To summarize the average

525

effect of ACh via nAChRs across cortical areas, we plot the mean effect by layer by

526

averaging the effects in M1, PFC and V1 (figure 14E). Hence in these cortical areas there

527

is a general pattern of increased effectiveness of nAChR activation in deep layers, on

528

which is superimposed area-specific variations in laminar sensitivity to ACh.

529

Hence our results indicate that ACh, acting via nAChRs, can directly excite pyramidal

530

neurons in many cortical areas and layers. Our experiments reveal differences in the

531

nAChR-mediated responsiveness of pyramidal neurons between cortical areas and

532

neocortical layers. However, these local variations appear to operate within a more

533

general framework which is common to neocortical areas, in which ACh exerts greater

534

nAChR-mediated effects on deep-layer pyramidal neurons.

535

536

DISCUSSION

537

nAChRs on neocortical pyramidal neurons have proven difficult to activate in brain slice

538

preparations, limiting the study of nAChRs. We have overcome this barrier by expressing

539

channelrhodopsin in cholinergic axons and evoking ACh release in neocortical slices.

540

Our results indicate that ACh activates nAChRs on pyramidal neurons in multiple layers

541

and three cortical regions, probably via synapses between cholinergic axons and

542

pyramidal neurons. Hence cholinergic activation of pyramidal neurons via nAChRs is

543

common across neocortex.

544 545

Effects of ACh on pyramidal neurons

546

Several authors have reported nAChR-mediated responses from pyramidal neurons

547

(Roerig et al., 1997; Chu et al., 2000; Kassam et al., 2008; Zolles et al., 2009; Guillem et

548

al., 2011; Poorthuis et al., 2012), but in other studies no such responses were observed

549

(Vidal & Changeux, 1993; Gil et al., 1997; Porter et al., 1999) and in many the actions of

550

ACh were mediated by mAChRs (Krnjevic, 1971; McCormick & Prince, 1986; Haj-

551

Dahmane & Andrade, 1996; Gulledge & Stuart, 2005; Gulledge et al., 2007; McCormick,

552

1992; Schwindt et al., 1988; Giessel & Sabatini, 2010). The absence of nAChR responses

553

is puzzling as nAChRs are expressed in the dendrites of pyramidal neurons (van der Zee

554

et al., 1992; Nakayama et al., 1995; Duffy et al., 2009; Lubin et al., 1999; Levy & Aoki,

555

2002).

556

In most studies, ACh was applied by bulk perfusion or from a nearby pipette, which

557

results in a relatively slow, widespread increase in concentration. nAChR desensitization

558

during ACh application might be significant, reducing nAChR-mediated currents.

559

Furthermore, many mAChRs are located extrasynaptically (Mrzljak et al., 1998;

560

Yamasaki et al., 2010) and might be more strongly activated by applied ACh than by ACh

561

released from cholinergic axons. Our results suggest that ACh application favors

562

mAChR- over nAChR-mediated currents in pyramidal neurons and this weighting may

563

account for the paucity of nAChR-mediated responses in the literature.

564

ACh can act on interneurons in layer 1 of sensorimotor cortex via α7 nAChR-

565

mediated synaptic and non-α7 nAChR-mediated diffuse mechanisms (Bennett et al.,

566

2012) and there is a wider debate on whether ACh acts in neocortex primarily via

567

synaptic contacts or volume transmission (Sarter et al., 2009; Arroyo et al., 2014). The

568

short latency of nAChR-mediated responses in our experiments suggests that ACh forms

569

cholinergic synapses with pyramidal neurons, but via non-α7 nAChRs. We found no

570

evidence for an α7 nAChR-mediated effect or nAChR-mediated actions via volume

571

transmission, but our results do not exclude such responses in pyramidal neurons.

572

Although our recordings were from somata, there are nAChRs throughout the dendritic

573

trees of pyramidal neurons (van der Zee et al., 1992), and it therefore seems likely that

574

there are additional effects of ACh on the dendrites of pyramidal neurons.

575 576

Persistent spiking evoked by nAChRs

577

Our results indicate that nAChR activation can evoke persistent spiking when paired

578

with additional depolarization. Persistent spiking was prevented by intracellular BAPTA,

579

suggesting that a rise in intracellular calcium concentration is also required. Calcium

580

might enter through nAChRs or arise from a secondary source, such as voltage-activated

581

calcium channels or a transmitter co-released by cholinergic axons. Cholinergic axons

582

may co-release glutamate (Manns et al., 2001; Allen et al., 2006; Gritti et al., 2006;

583

Henny & Jones, 2008), but in our experiments persistent spiking was unaffected by

584

glutamate receptor antagonists. Other potential co-transmitters in the basal forebrain

585

include neurotensin, somatostatin, neuropeptide Y and galanin (Koliatsos et al., 1990).

586

nAChR-dependent persistent spiking has been reported in dopaminergic neurons in

587

the substantia nigra pars compacta and subthalamic nucleus (Yamashita & Isa, 2003a,

588

2003b), but not cortical neurons. In entorhinal, perirhinal, cingulate and somatosensory

589

cortices, persistent spiking can be evoked in excitatory neurons, but via mAChRs and a

590

rise in intracellular calcium concentration (Zhang & Séguéla, 2010; Egorov et al., 2002;

591

Navaroli et al., 2011; Rahman & Berger, 2011). Hence nAChR-dependent persistent

592

spiking shares common mechanistic elements with mAChR-dependent persistent

593

spiking, but is initiated by activation of nAChRs, not mAChRs.

594

In spiking neurons, nAChR PSPs evoked a brief and modest increase in spike rate. Why

595

was the effect during ongoing spiking not more prolonged and why was the resulting

596

spike rate typically lower than during nAChR-evoked persistent spiking? Presumably

597

ongoing spiking suppresses the current that underlies persistent spiking or the resulting

598

depolarization, perhaps by shunting the membrane.

599

Previous studies have revealed other mechanisms of prolonged spiking in pyramidal

600

neurons, particularly deep-layer pyramidal neurons. For example, subthreshold DC

601

current injection into the trunk of the apical dendrite can facilitate propagation of a

602

dendritic spike from the distal apical dendrite to the soma, resulting in a burst of spikes

603

(Larkum et al., 2001). Similarly, activation of glutamate receptors in the basal dendrites

604

can evoke a burst of spikes (Milojkovic et al., 2004; Milojkovic et al., 2007). Presumably

605

nAChR-persistent spiking and other mechanisms that can evoke prolonged spiking share

606

some common mechanistic elements and differ important ways. Further experiments

607

will be required to investigate these similarities and differences, but our results add

608

nAChR activation to the collection of identified mechanisms that can evoke prolonged

609

spiking from cortical pyramidal neurons.

610

In summary, nAChR activation increases the excitability of pyramidal neurons. The

611

increase in spiking can be modest and transient or profound and persistent, depending

612

on the membrane potential of the neuron. Hence ongoing synaptic drive to the

613

pyramidal neuron determines the strength and duration of the increase in spiking

614

evoked by ACh.

615 616

Laminar and regional variation in nAChR PSPs

617

α3, α4, α7 and β2 nAChR subunits are located on neocortical pyramidal neuron somata,

618

dendrites and spines (Disney et al., 2007; Nakayama et al., 1995; Duffy et al., 2009;

619

Lubin et al., 1999; Levy & Aoki, 2002; Wevers et al., 1994) and several studies describe

620

responses mediated by α4β2-, α7- and α5-containing nAChRs, the latter probably in

621

heteromeric assembly with α4 and β2 subunits (Kassam et al., 2008; Zolles et al., 2009;

622

Poorthuis et al., 2012).

623

Our results are generally consistent with these previous studies, but there are

624

contrasts. Layer 6 contains high expression of non-α7 nAChRs (Tribollet et al., 2004),

625

including α5 (Wada et al., 1990; Proulx et al., 2013) and α4 (Lein et al., 2007) subunits,

626

consistent with the high incidence of nAChR-mediated responses in layer 6 of PFC in

627

previous studies (Kassam et al., 2008; Poorthuis et al., 2012; Mailey et al., 2010). We

628

found that nAChR PSPs in layer 6 are common and of large amplitude in all three areas

629

studied, suggesting that the presence of α4/α5-mediated PSPs is a feature of layer 6

630

pyramidal neurons across cortical regions.

631

In layer 5, we found that nAChR PSPs are common in M1 and V1 and rare in PFC. In

632

M1, nAChR PSPs were mediated by non-α7 nAChRs. In contrast, Poorthuis et al., 2012

633

observed α7 nAChR-mediated responses in layer 5 pyramidal neurons in PFC. nAChR

634

PSPs that we observed originated from nAChRs in the proximal dendrites. Hence one

635

explanation for the lack of α7-mediated PSPs in our results might be that α7 nAChRs are

636

located primarily in the distal dendrites and that α7-mediated depolarization of the

637

distal dendrite failed to evoke depolarization at the soma in our experiments.

638

M1 is unusual in that the pyramidal neurons in layer 5B of M1 commonly display large-

639

amplitude nAChR PSPs, unlike layer 5 pyramidal neurons in PFC and V1. The α5 subunit

640

is not present in layer 5 (Wada et al., 1990). There is evidence for greater expression of

641

α4 subunits in deep than in superficial layers (Lein et al., 2007), but see also (Tribollet et

642

al., 2004), but this expression pattern is not unique to M1. Hence it is unclear which

643

nAChR subunit(s) underlie the unusually large nAChR PSPs in layer 5 of M1, but α5

644

subunits are unlikely to be involved.

645

In superficial layers, we found that pyramidal neurons in M1 and PFC rarely displayed

646

nAChR PSPs, consistent with results from PFC (Poorthuis et al., 2012), but that nAChR

647

PSPs are common in superficial pyramidal neurons in V1. Layer 2/3 contains dense non-

648

α7 labeling (Tribollet et al., 2004). Our results suggest that this dense labeling is from

649

interneurons and perhaps in the dendrites of deep-layer pyramidal neurons.

650

Our results extend our knowledge of pyramidal neuron nAChRs from PFC into M1 and

651

V1, revealing variation in laminar responsiveness to ACh between cortical regions;

652

presumably the responsiveness of pyramidal neurons is tuned to the unique demands of

653

each area. However, our results also reveal a general tendency for ACh to exert stronger

654

effects in deep than superficial layers, suggesting that preferential modulation of deep

655

layer pyramidal neurons via nAChRs is a general property of the actions of ACh in

656

neocortex.

657 658

nAChR-mediated modulation of neocortical circuits

659

How might the layer-selective effect of ACh influence the flow of excitation through

660

neocortex? The principal ascending excitatory drive to cortex is thalamocortical axons,

661

which contact pyramidal neurons primarily in layers 5B and 4 (5B and 3 in primary

662

motor cortex, which lacks layer 4 (Shepherd, 2009; Hooks et al., 2013). From layer 4,

663

information is passed by excitatory connections through layer 2/3 to layer 5 and to the

664

sub-cortical projection targets of neocortex. Hence there are two primary excitatory

665

pathways through neocortex: a short loop which connects thalamus with target

666

structures through layer 5 and a longer loop which includes layers 4 and 2/3

667

(Armstrong-James et al., 1992; de Kock et al., 2007; Petreanu et al., 2009;

668

Constantinople & Bruno, 2013).

669

ACh enhances activation of neocortical pyramidal neurons by ascending thalamic

670

drive. This enhancement arises from mAChR-mediated depolarization of pyramidal

671

neurons and enhanced glutamate release from thalamocortical terminals in layer 4

672

(Metherate, 2004; Disney et al., 2007; Gil et al., 1997). In addition, ACh activates non-

673

fast spiking, non-parvalbumin-expressing interneurons in layers 1 and 2/3 via non-α7

674

nAChRs (Porter et al., 1999; Gulledge et al., 2007; Letzkus et al., 2011; Arroyo et al.,

675

2012; Brombas et al., 2014). These interneurons inhibit parvalbumin- and somatostatin-

676

expressing interneurons that target the somata and dendrites of pyramidal neurons.

677

Hence nAChR-mediated inhibition of superficial interneurons reduces inhibition of

678

superficial pyramidal neurons (Letzkus et al., 2011; Brombas et al., 2014). Our results

679

indicate that ACh, again acting at nAChRs, directly promotes the spiking of deep-layer

680

pyramidal neurons. Hence ACh modulates cortical output by at least three different

681

nAChR-dependent mechanisms which enhance the responsiveness of neocortex to

682

incoming sensory drive: by increasing the release of glutamate in layer 4, by indirectly

683

enhancing the excitability of superficial pyramidal neurons and by directly enhancing the

684

excitability of deep-layer pyramidal neurons.

685

Why employ several mechanisms to modulate the excitability of pyramidal neurons

686

in cortex? Multiple mechanisms may offer circuit specificity and semi-independent

687

control. Multiple mechanisms of network modulation might allow ACh to independently

688

modulate the excitability of deep and superficial layer pyramidal neurons, thereby gating

689

the balance of sensory information processing in different cortical layers and controlling

690

the balance of information flowing through the long and short loops from thalamus to

691

the output structures of neocortex.

692

Acknowledgements:

693

We thank Becky Imhoff and Lauren Sybert for technical assistance. TH and JW

694

designed and performed the experiments, analyzed the results and wrote the manuscript.

695 696

Grants:

697

This work was supported by the National Institute of Mental Health (5T32MH067564

698

and 5R21MH085117), the National Institute of Neurological Disorders and Stroke

699

(1R01NS078067) and the Brain Research Foundation (BRF SG 2010-13).

700 701

Disclosures:

702

The authors declare no competing financial interests.

703

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704

Allen TGJ, Abogadie FC, Brown DA. Simultaneous release of glutamate and

705

acetylcholine from single magnocellular “cholinergic” basal forebrain neurons. J

706

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Figure legends

982

Figure 1. Stimulation of cholinergic axons evokes four responses in

983

pyramidal neurons

984

(A) Schematic of a coronal slice illustrating the expression of ChR2-YFP in cholinergic

985

neurons in the basal forebrainand their axonal projections to neocortex. CP: caudate

986

putamen, LV: lateral ventricle. (B) Image of ChR2-YFP labeled neurons in nucleus

987

basalis in a ChAT-ChR2(Ai32) mouse. Maximum intensity projection from a 2-photon z-

988

stack through a fixed section. (C) Image of ChR2-YFP labeled axons in primary motor

989

cortex in a ChAT-ChR2(Ai32) mouse. Maximum intensity projection from a 2-photon z-

990

stack through a fixed section. (D,E) Pyramidal neuron in layer 5B of primary motor

991

cortex, 3 weeks after virus injection. Neuron was filled with Alexa 594 during whole-cell

992

recording (D) and surrounding cholinergic axons expressing ChR2-YFP (E). Both images

993

are maximum intensity projections from 2-photon z-stacks. (F) Examples of voltage

994

recordings from four layer 5B pyramidal neurons at rest. Each voltage response evoked

995

by stimulation of cholinergic axons by widefield illumination of the slice with a blue LED

996

(stimulus: 10 x 5 ms at 20 Hz; blue bars). Example of slow depolarization includes a

997

preceding hyperpolarization. Examples of slow depolarization and fast hyperpolarization

998

were obtained from virus-injected mice. Examples of medium depolarization and fast

999

depolarization were obtained from ChAT-ChR2(Ai32) mice.

1000 1001

Figure 2. Light-evoked responses are eliminated by TTX and by removal of

1002

extracellular calcium

1003

Examples of the slow depolarization and hyperpolarization (A, neuron depolarized by DC

1004

current injection), medium depolarization (B, neuron at rest), and fast depolarization (C,

1005

neuron at rest) from three neurons. All responses were reversibly eliminated by removal

1006

of external calcium, and by addition of 500 nM TTX. Stimulus: 10 x 5 ms illumination at

1007

20 Hz. All examples were obtained from ChAT-ChR2(Ai32) mice.

1008 1009

Figure 3. Pharmacology of the hyperpolarization and slow depolarization

1010

(A) Example of sequential addition of 100 µM mecamylamine and 1 µM atropine to a

1011

neuron displaying a light-evoked hyperpolarization. Stimulus: 10 x 5 ms illumination at

1012

20 Hz. Neuron held close to threshold by DC current injection. Recording from a virus-

1013

injected mouse. (B) Example of sequential addition of 100 µM mecamylamine and 1 µM

1014

atropine to a neuron displaying a light-evoked slow depolarization. Stimulus: 10 x 5 ms

1015

illumination at 20 Hz. Neuron held close to threshold by DC current injection. Recording

1016

from a ChAT-ChR2(Ai32) mouse. (C) Probability of evoking a mAChR-mediated

1017

potential during constant depolarization by DC current injection. Control, 38 neurons;

1018

mec, 100 µM mecamylamine, 13 neurons; atr, 1 µM atropine, 141 neurons.

1019 1020

Figure 4. Pharmacology of the medium depolarization

1021

Example recordings from six neurons. Stimulus: 2-3 x 5 ms illumination at 20 Hz.

1022

Physo, 0.5 µM physostigmine; atr, 1 µM atropine; mec, 100 µM mecamylamine; gal, 1 µM

1023

galanthamine; NBQX/CPP, 10 µM NBQX and 10 µM CPP; GBZ / CGP, 1 µM gabazine

1024

and 3 µM CGP 52432; MLA, 10 nM methylylcaconitine; DHβE, 10 µM dihydro-β-

1025

erythroidine hydrobromide. All examples at resting membrane potential and from virus-

1026

injected mice. Summary: mean ± SEM, normalized to pre-drug amplitude. TTX, 7

1027

neurons; 0 Ca, 7 neurons; NBQX/CPP, 7 neurons; GBZ / CGP, 8 neurons;

1028

physostigmine, 4 neurons; atropine, 5 neurons; mecamylamine, 12 neurons;

1029

galanthamine, 23 neurons; MLA, 12 neurons; DHβE, 16 neurons. Asterisks denote

1030

significant effects (P