JN-00010-2004.R1

1

Functional nicotinic acetylcholine receptors on subplate neurons in neonatal rat somatosensory cortex

Ileana L. Hanganu & Heiko J. Luhmann

Institute of Physiology & Pathophysiology, Johannes-Gutenberg University,

55128 Mainz, Germany

38 pages, 5 figures

Corresponding author: Ileana L. Hanganu

Institute of Physiology & Pathophysiology

Johannes-Gutenberg University

Duesbergweg 6

D-55128 Mainz

Germany

phone: +49 6131 3926081

fax: +49 6131 3926071

e-mail: hanganu@uni-mainz.de

Running title: Nicotinic receptors on subplate neurons

Articles in PresS. J Neurophysiol (March 3, 2004). 10.1152/jn.00010.2004

Copyright © 2004 by the American Physiological Society.

JN-00010-2004.R1

2

The establishment of cortical synaptic circuits during early development requires the

presence of subplate neurons (SPn), a heterogeneous population of neurons capable

to integrate and process synaptic information from the thalamus, cortical plate and

neighboring SPn. An accumulation of cholinergic afferents and nicotinic acetylcholine

receptors (nAChR) has been documentated in the subplate around birth. To assess

the developmental role of the cholinergic innervation onto SPn, we used whole-cell

patch-clamp recordings of visually identified and biocytin-labeled SPn in neonatal rat

somatosensory cortex. Functional nAChR were present in 92% of the investigated

SPn. Activation of postsynaptic nAChR by local application of agonists elicited a brief

membrane depolarization associated with a barrage of action potentials and large

inward currents reversing around 0 mV. According to our pharmacological data

excitation of SPn is mediated by α4β2 receptors. In contrast, functional α7 nAChR

could not be identified on SPn. Activation of nAChR affected neither the spontaneous

synaptic activity of SPn nor the synaptic connections between thalamus and SPn and

within subplate. Nicotine, at concentrations reaching the developing brain by

maternal smoking, induced a severe desensitization of nAChR and an increase in the

baseline noise. These results indicate that nAChR-mediated excitation of SPn may

stabilize the developing synaptic circuits and suggest the involvement of nAChR

located on SPn in the fetal tobacco syndrome.

JN-00010-2004.R1

3

Nicotinic acetylcholine receptors (nAChR) are involved in a variety of brain functions,

including memory formation, anxiety and addiction (Levin and Simon 1998; Perry et

al. 1999; Jones et al. 1999). They play a critical role in the adult as well as in the

developing nervous system by mediating excitatory neurotransmission

postsynaptically and by modulating the release of various neurotransmitters

presynaptically (Role and Berg 1996). The prevalent functional nAChR subtypes in

the mammalian brain are either homomeric, being assembled of α7 subunits or

heteromeric, containing mostly α4 and β2 subunits (Le Novere et al. 2002). The

cholinergic innervation has been proposed to serve trophic functions and influence

brain development, mostly by modulating Ca2+-dependent proliferation,

differentiation, apoptosis and survival processes (Lipton and Kater 1989; Role and

Berg 1996; Pugh and Margiotta 2000). Moreover, nicotine as an exogenous agonist

and the psychoactive component of tobacco is likely to interact with nAChR and

affect their function. Maternal smoking associated with the fetal tobacco syndrome

alters normal brain development by disturbing cell proliferation and differentiation

(Slotkin 1998).

The major cholinergic innervation originating from the nucleus basalis of

Meynert (Mesulam et al. 1983) enters the cerebral cortex around birth (Hohmann and

Berger-Sweeney 1998) and is initially confined to the subplate (Candy et al. 1985;

Kostovic 1986; Mechawar and Descarries 2001), a transient synapse-rich layer of

neurons located directly under the cortical plate (Kostovic and Rakic 1980). The

subplate neurons (SPn) express different morphologies and neurotransmitter profiles,

are actively involved in the pathfinding of corticofugal and corticopetal axonal

projections (Rakic 1977; McConnell et al. 1989; Ghosh and Shatz 1992b) and

receive distinct functional synaptic inputs from the thalamus, cortical plate and

subplate (Friauf et al. 1990; Hanganu et al. 2001; Hanganu et al. 2002).

JN-00010-2004.R1

4

The presence of a dense cholinergic innervation to the subplate raises the

question whether SPn express functional nAChR. Accumulation of α7 and α4 nAChR

subunits at the level of the subplate has been reported previously (Fuchs 1989;

Ostermann et al. 1995; Broide et al. 1996; Csillik et al. 2002). However, functional

nAChR are assembled from multiple subunits and therefore detection of a receptor

subunit mRNA or protein does not necessarily proof the presence of functional

receptor-channel complexes. The status of nAChR on SPn and the question whether

they play an active role in cortical development is still unknown. In the present study,

we characterize for the first time the functional status of nAChR on SPn by

performing whole-cell patch-clamp recordings on visually identified and biocytin-

labeled SPn in neonatal rat cortical slices. Since nAChR are also the target for

exogenous nicotine in the fetal tobacco syndrome (Lambers and Clark 1996),

alterations of receptor function by nicotine concentrations similar to those found

during maternal smoking were investigated. We demonstrate that functional nAChR

are present and directly excite SPn during the neonatal period of the rat. The strong

desensitization of nAChR in the presence of behaviorally relevant concentrations of

nicotine suggests a possible role of SPn in the development of pathophysiological

disturbances associated with the early tobacco exposure.

MATERIALS AND METHODS

Slice preparation and identification of SPn

All experiments were conducted in accordance with the national laws for the use of

animals in research and approved by the local ethical committee. Brain slices were

prepared as described previously (Hanganu et al. 2001; Hanganu et al. 2002).

Briefly, 0- to 4-days-old Wistar rats were anesthetized by hypothermia and

decapitated. Whole-brain coronal slices (400 µm thick) including the primary

JN-00010-2004.R1

5

somatosensory cortex were cut on a vibroslicer (Pelco 101, TPI, St. Louis, MO and

HR2, SIGMANN Elektronik, Hüffenhardt, Germany) and separated into two

hemispheres. Slices were maintained > 1 hr at 33 °C in a storage chamber before

transferring them into the submerge recording chamber. During preparation and

recording procedures, slices were maintained in artificial cerebrospinal fluid (ACSF)

containing (in mM) 124 NaCl, 26 NaHCO3, 3 KCl, 1.6 CaCl2, 1.8 MgCl2, 1.3 NaH2PO4

and 20 D-glucose, pH 7.4, after equilibration with 95% O2 and 5% CO2 (osmolarity

333 mOsm).

The SPn were visualized using infrared differential interference contrast (DIC)

videomicroscopy (Dodt and Zieglgänsberger 1990). SPn were identified by their

location, morphology and electrophysiological properties. Only neurons located

between the cell-dense cortical plate with radially oriented neurons and the cell-

sparse white matter were investigated. The exact morphological classification of

previously recorded SPn was performed after biocytin-staining. Neurons were

excluded from data analysis if their morphological properties did not correspond to

those reported previously (Friauf et al. 1990; Hanganu et al. 2002) or/and if their

electrophysiological properties did not fulfill the criteria reported for SPn (resting

membrane potential [RMP] negative to –40 mV and input resistance [Rin] >500 MΩ)

(Hanganu et al. 2001; Hanganu et al. 2002).

Electrophysiological recordings

The videomicroscopic setup consisted of an upright microscope with DIC optics

(Axioskop, Zeiss, Oberkochen, Germany) and a CCD camera (C5405, Hamamatsu,

Japan). The video image was contrast-enhanced by a video-processor (C2741,

Hamamatsu), visualized on a video-monitor and digitized online using a frame

grabber card (Screen machine II, Fast, Munich, Germany). Whole-cell patch-clamp

JN-00010-2004.R1

6

recordings were performed according to the procedure described by Stuart et al.

(1993). All recordings were performed at 32-33° C. Recording electrodes (8-15 MΩ)

were pulled from borosilicate glass tubing (Science Products, Hofheim, Germany) on

a vertical puller (PP83, Narishige, Tokyo, Japan) and filled with standard electrode

solution containing (in mM): 117 K-gluconate, 13 KCl, 1 CaCl2, 2 MgCl2, 11 EGTA, 10

K-HEPES, 2 NaATP and 0.5 NaGTP. For the investigation of GABAA receptor-

mediated synaptic currents K-gluconate was replaced by 117 mM KCl. Both

electrode solutions were adjusted to pH 7.4 with 1 M KOH and to an osmolarity of

306 mOsm with sucrose.

Capacitance artifacts and series resistance were minimized using the built-in

circuitry of the discontinuous voltage-clamp/current-clamp amplifier (SEC05L, npi

elektronik, Tamm, Germany). The signals were amplified and low-pass filtered at 3

kHz, visualized on an oscilloscope (TDS210, Tektronix, Beaverton, OR), digitized

online with an AD/DA board (ITC16, Heka, Lambrecht/Pfalz, Germany) and recorded

and processed with WinTida software 4.11 (Heka). The bathing solution was

connected to the ground via a chlorided silver wire. All potentials were corrected for

liquid junction potentials with -10 mV for the gluconate-based electrode solution

(Mienville and Pesold 1999; Kilb and Luhmann 2000) and -4 mV for the high chloride

electrode solution (Marty and Neher 1995). The RMP was measured immediately

after obtaining the whole-cell configuration. For the determination of the Rin,

hyperpolarizing 2 s long current pulses were applied from a holding potential of -70

mV.

A bipolar tungsten electrode (5 MΩ, FHC, Bowdoinham, ME, USA) was used

for the selective electrical stimulation of the thalamocortical afferents (TA), while the

stimulus-evoked postsynaptic currents (PSCs) were recorded in SPn. The stimulation

of TA was performed as described previously (Hanganu et al. 2002). For stimulation

JN-00010-2004.R1

7

of the subplate a horizontal cut was performed directly below the white matter to

eliminate possible inputs from subcortical regions and the stimulation electrode was

placed within the subplate, laterally to the investigated cell. In all experiments the

duration of the electrical stimulus was 70 µs. The intensity of the stimuli varied

between 20 and 50 V. Unless otherwise noted, stimuli were delivered at 0.05 Hz.

Five stimulus-evoked responses were recorded under control conditions and after

drug application.

Data analysis

Agonist-induced currents and spontaneous postsynaptic currents (sPSCs) were

analyzed using the Mini Analysis Program (Synaptosoft, Leonia, NJ, USA). The

sPSCs were captured using a threshold-crossing detector set above the noise level.

Events that did not show a typical sPSC waveform were rejected manually and by

optimal settings of the program parameters. The evoked PSCs and agonist-induced

potentials were analyzed using WinTida Software (Heka). sPSCs, evoked PSCs and

agonist-induced currents and potentials were analyzed in their peak amplitude, 10-

90% rise-time and decay-time. The decay-time constant (τ) was calculated by fitting a

single or double exponential function to currents using a simplex algorithm. To

determine the current-voltage relationship of the cholinergic currents, voltage steps of

20 mV between –130 and +10 mV were applied. Root mean square (RMS) noise

values were calculated using Mini Analysis Software as derivation of the sampled

baseline current from the mean. For the evaluation of the agonist-binding constant

the data points were fitted by the equation I/Imax= (ch/IC50h)/(1+(ch/IC50

h)) with c =

agonist concentration, IC50 = concentration required for half-maximal response and

h = Hill coefficient using a least square algorithm.

JN-00010-2004.R1

8

Data are presented as mean ± standard error of the mean (SEM). For

statistical analyses the two-tailed Student’s t-test and one-way ANOVA test were

used. Significance levels of p<0.05, p<0.01 and p<0.001 were considered.

Pharmacological procedures

All substances were purchased from Merck with the exception of R(-)-3-(2-

carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP), 6-cyano-7-nitroquinoxaline-

2,3-dione (CNQX), carbamylcholine chloride (carbachol), (-)nicotine tartrate, choline

chloride, methyllycaconitine citrate (MLA), dihydro-β-erythroidine hydrobromide

(DHβE), N-methyl-4-(3-pyridinyl)-3-buten-1-amine hemigalactarate (RJR-2403),

kynurenic acid (KYNA), mecamylamine hydrochloride, neostigmine bromide and

tetrodotoxin citrate (TTX) which were from Sigma-Aldrich (Taufkirchen, Germany)

and atropine sulfate which was from RBI (Natick, MA, USA). Stock solutions of these

drugs were prepared as follows: TTX, CPP, nicotine, carbachol, choline, RJR-2403,

DHβE, KYNA, mecamylamine, neostigmine and MLA in distilled water and CNQX in

dimethylsulfoxide (DMSO). Stock solutions were stored at –20° C and diluted to their

final concentration in ACSF on the day of experiment. The maximal concentration of

DMSO in the superfusate was 0.1%. Receptor antagonists (CPP, CNQX, MLA,

DHβE, KYNA, mecamylamine) and TTX were bath-applied at a rate of 3-4 ml/min

and superfused for at least 5 min before initiating the experiment. Slices were

incubated for at least 6 min with neostigmine before starting the experiment. Unless

otherwise noted, cholinergic agonists (carbachol, nicotine, RJR-2403, choline) were

applied focally under visual control by pressure ejection (100-300 ms at 0.4 bar) from

a glass application pipette (4-6 MΩ) using a fast application system (PDES 02T, npi

elektronik). The application pipette was positioned 5-20 µm from the cell soma. In

JN-00010-2004.R1

9

some experiments, local application of carbachol was shorten to the minimum

pressure pulse width allowed by the device to induce reliable responses (20 ms). In

addition, long focal application of carbachol for >5 s or bath application of nicotine for

>4 min was performed.

Histology and morphological analyses

In all experiments 0.5% biocytin (Sigma-Aldrich) was included in the patch electrode

solution for later morphological identification of the recorded cells. The staining

protocol for biocytin was described previously (Schröder and Luhmann 1997). Slices

were fixed in 4% buffered paraformaldehyde solution for at least 24 h, rinsed and

incubated for 60 min with 0.5% H2O2 and 0.8% Triton X-100 to inhibit endogenous

peroxidases. An overnight incubation with an avidin-coupled peroxidase (ABC kit,

Vectorlab, Burlinghame, CA) was followed by incubation in 0.5 mM

diaminobenzidine. The reaction product was intensified with 0.5% OsO4. Finally,

slices were dehydrated and embedded in Durcopan (Fluka, Buchs, Switzerland).

Biocytin-stained neurons were analyzed in their somatodendritic properties using an

Axioskop microscope (Zeiss). The morphology of a few biocytin-filled SPn was

reconstructed with Neurolucida software (Microbrightfield, Colchester, VT, USA) and

analyzed with the Neuroexplorer software package (Microbrightfield).

RESULTS

Whole-cell recordings were performed from 115 SPn in somatosensory cortical slices

from P0-4 Wistar rats. As reported previously (Hanganu et al. 2001; Hanganu et al.

2002), the location of the subplate, the appearance of SPn under video-assisted

Nomarski microscopy (Fig. 1A), their firing pattern and their morphological properties

after histological processing for biocytin served as criteria to identify the SPn. The 86

JN-00010-2004.R1

10

biocytin-stained SPn revealed an extensive arborization of dendritic processes (Fig.

1B). According to previously reported criteria concerning the variable shape of the

soma and orientation of the primary dendrites (Hanganu et al. 2002), 27 SPn were

classified as horizontal bitufted, 7 SPn as horizontal monotufted, 22 SPn as

multipolar, 17 SPn as inverted pyramid, 12 SPn as tripolar and 1 SPn as vertical.

The passive and active membrane properties of SPn were similar to those

reported previously (Luhmann et al. 1999; Hanganu et al. 2001; Hanganu et al.

2002). Using gluconate-based electrode solution, the RMP and the Rin were –53.4 ±

0.6 mV and 995.1 ± 36 MΩ (n=98), respectively. Similar values were obtained when

using a chloride-based electrode solution (RMP –52.5 ± 1.2 mV and Rin 894 ± 58.5

MΩ, n=17). All SPn were capable to fire one or repetitive overshooting action

potentials in response to sustained depolarization by intracellular current injection.

Functional postsynaptic nicotinic acetylcholine receptors on SPn

Local pressure application of cholinergic agonists directly onto the recorded cell body

by a glass micropipette (Fig. 1A) elicited excitatory responses in 92% of the 91

investigated SPn. The remaining 7 SPn showed no response to application of

cholinergic agonists. No correlation between the morphology and responsiveness to

nAChR activation could be observed. To eliminate potential artifacts due to the

pressure ejection of agonists, control recordings were performed by applying the

extracellular solution (n=5). Under these conditions, no changes in the membrane

potential or the quality of the seal were observed. The presence of functional nAChR

on SPn was assessed using the cholinergic agonist carbachol (100 µM). To ensure a

complete block of muscarinic receptors activated by carbachol, all experiments were

performed in the presence of relatively high concentrations (10 µM) of the

competitive antagonist atropine (Margiotta et al. 1987). In current-clamp mode,

JN-00010-2004.R1

11

carbachol caused a transient membrane depolarization from –70 mV to –54 ± 1.8 mV

(n=4), sufficient to trigger a short-lasting barrage of action potentials (Fig. 2A). When

recordings were performed in voltage-clamp mode, carbachol induced a large inward

current with a mean peak amplitude of 81.2 ± 10 pA (n=33) (Fig. 2B). The decay

kinetic of the carbachol-induced currents varied from cell to cell, but was generally

described by the sum of two exponential functions with time constants of 0.8 ± 0.2 ms

and 2.7 ± 0.2 ms, similar to values reported for nAChR-mediated responses during

embryonic development (Atluri et al. 2001). Carbachol-elicited responses showed

essentially no rundown and could be elicited repeatedly for >40 min with no

significant reduction in the amplitude. To ensure a complete recovery, carbachol

applications were usually performed at 30 s intervals, although 10 s intervals were

equally effective. Carbachol-elicited currents exhibited little or no desensitization

when the agonist was applied for longer times ranging from 10 to 60 s (Fig. 2C). The

amplitude, but not the kinetics of the carbachol-induced currents was dependent on

the agonist concentration (10 µM, 14.3 ± 7.4 pA, n=3; 50 µM, 36.3 ± 4 pA, n=4; 100

µM, 81.2 ± 10 pA, n=33).

Local application of the nAChR agonist nicotine (100 µM) excited SPn by

eliciting inward currents with a mean amplitude of 32.9 ± 5.7 pA (n=5) (Fig. 2D). The

decay-times of the nicotine-elicited currents (25.5 ± 3.4 s, n=5) were significantly

(p<0.001) longer than those of the carbachol-induced currents and a second

application of nicotine, even after 45 min, produced a much smaller response as

compared to the initial exposure. A similar pharmacodynamic response pattern

(tachyphylaxis) to nicotine has been reported previously for nAChR in rats (Wong

and Gallagher 1989; Chessell and Humphrey 1995) and humans (Benowitz et al.

1989). Therefore, activation of nAChR in subsequent experiments was performed by

carbachol in the presence of atropine.

JN-00010-2004.R1

12

As previously reported for nicotinic receptor channels (Mathie et al. 1990;

Bertrand et al. 1999), carbachol-induced currents reversed at around 0 mV and

showed a strong inward rectification in all 5 investigated SPn. Little or no current was

seen at positive holding potentials (Fig. 3A).

In the brain, nAChR may act both postsynaptically by directly exciting the

neurons and presynaptically by modulating neurotransmitter release (McGehee and

Role 1995; Gray et al. 1996; Role & Berg 1996). To detect the location and function

of nAChR on SPn, carbachol-elicited responses were recorded under control

conditions as well as in the presence of the AMPA/kainate receptor antagonist CNQX

(10 µM), the NMDA receptor antagonist CPP (20 µM) and TTX (1 µM). Under this

condition, local application of carbachol elicited a postsynaptic inward current (26.3 ±

6 pA; n=5) and a membrane depolarization (-53.8 ± 1.6 mV, n=3) that was similar to

that obtained under control conditions (31.8 ± 4.9 pA, n=5; -54 ± 1.8 mV, n=3) (Fig.

3B). When carbachol was bath applied for 30 s (n=7), the induced currents persisted

in the presence of TTX, CPP and CNQX. The reduction in peak amplitude of the

inward current from 61.6 ± 9.1 pA to 49.6 ± 6.1 pA (n=7) was not significant. These

results suggest that carbachol-induced responses are mediated by a direct activation

of postsynaptic nAChR and not by a presynaptic facilitation of glutamate release.

To decide whether endogenous acetylcholine (ACh) release could activate

functional nAChR, the effects of the nAChR antagonist mecamylamine (10 µM) on

the spontaneous synaptic activity in 8 SPn was investigated. Mecamylamine

significantly (p<0.05) decreased the sPSCs frequency from 0.33 ± 0.06 Hz to 0.22 ±

0.06 Hz in 6 SPn, but had no effect in the two remaining neurons. The amplitude of

the sPSCs was not affected. As reported previously (Hanganu et al. 2001), SPn

show fast, AMPA receptor-mediated sPSCs and slow, NMDA receptor-mediated

sPSCs. Although mecamylamine decreased the frequency of both populations of

JN-00010-2004.R1

13

events, only the frequency of the fast sPSCs was significantly (p<0.05) reduced from

0.3 ± 0.06 Hz to 0.18 ± 0.07 Hz (n=6). In addition, a similar decrease in sPSCs

frequency from 0.15 ± 0.01 to 0.11 ± 0.02 was observed in 3 out of 4 investigated

SPn when bath application of mecamylamine was preceded by incubation of the

slices with the AChesterase inhibitor neostigmine (10 µM). These results suggest that

endogenously released ACh acts on the functional nAChR of the SPn.

Functional subtypes of nAChR on SPn

To analyze the subunit composition of the functional nAChR, receptor subtype-

selective agonists and antagonists were used. Bath application of dehydro-β-

erythroidine (DHβE), a selective antagonist of the α4β2 subtype of nAChR (Alkondon

and Albuquerque 1993), completely abolished carbachol-induced currents in all 10

investigated SPn (Fig. 4A). A low concentration of this antagonist (500 nM) was

applied to prevent partial blockade of other nAChR subtypes reported to be achieved

with DHβE at higher concentrations (1-10 µM) (Alkondon et al. 1999). In addition, the

nicotine-derived compound RJR-2403, an agonist with selectivity for the α4β2

subtype of nAChR (Papke et al. 2000) depolarized SPn in a similar manner as

carbachol. Local application of RJR-2403 (30 µM) elicited a long-lasting membrane

depolarization to –49.2 ± 5 mV (n=4) in all 4 investigated SPn and this depolarization

was accompanied by a barrage of action potentials in 3 out of 4 SPn (Fig. 4B). RJR-

2403-elicited responses showed similar amplitudes and rise-times as compared to

carbachol-induced responses, but their decay-times were significantly (p<0.05)

longer (Fig. 4B inset). The longer decay-times may result from a slower washout of

the α4β2 nAChR agonist, but relative few data concerning the kinetic of the RJR-

2403-elicited responses in the slice are currently available.

JN-00010-2004.R1

14

To assess the presence of functional homomeric α7 nAChR on SPn, the

selective α7 nAChR antagonist methyllycaconitine (MLA) (Palma et al. 1996) and the

α7 nAChR agonist choline (Albuquerque et al. 1998) were used. Bath application of

MLA (50 nM) had no significant effect on the amplitude (control: 113.8 ± 10.2 pA,

MLA: 92.1 ± 15.8 pA, n=6) or the kinetic of carbachol-induced currents (Fig. 4C).

Moreover, local application of choline (10 mM) elicited no response in any of the 5

investigated SPn. Another series of experiments also failed to demonstrate α7

nAChR-mediated responses. The brain metabolite kynurenic acid (KYNA) is known

to act as an antagonist at the glycine site of the NMDA receptor (Stone 1972) and

inhibit α7 nAChR (Hilmas et al. 2001). Bath application of 10 µM KYNA in the

presence of the NMDA receptor antagonist CPP (10 µM) had no effect on the

amplitude or the kinetic of carbachol-induced responses (Fig. 4D). Since the fast

desensitization kinetic of the α7 nAChR may preclude the identification of the α7

nAChR-mediated responses, the duration of the carbachol application was reduced

from 100 ms to 20 ms. Under these conditions, carbachol-induced responses with a

peak amplitude ranging from 6 to 33 pA could be recorded in all 3 investigated SPn

and were completely blocked by bath application of DHβE. Taking together, these

results indicate that the α4β2 nAChR mediate the excitation of SPn, whereas α7 are

not involved.

Pathophysiological effects of low concentrations of nicotine

Maternal nicotine gains access to the fetal compartment via the placenta, arrives

more slowly and acts for a longer time in the immature brain (Benowitz et al. 1989;

Slotkin 1998). To better mimic the pathophysiological conditions associated with fetal

exposure through maternal smoking, behaviorally relevant concentrations of nicotine

JN-00010-2004.R1

15

were bath applied on SPn. The concentration of nicotine in the venous blood after

smoking of several cigarettes ranges from 60 to 300 nM (Benowitz et al. 1989) and in

the arterial blood, which better represents the level of nicotine in the brain, reaches

∼600 nM (Henningfield et al. 1993). In addition, the developing brain tends to have

higher nicotine concentrations than the smoking mother because of its higher lipid

content and the lower clearance of nicotine from the fetal compartment (Lambers and

Clark 1996; TA Slotkin, personal communication). In the present study, SPn were

exposed to nicotine at bath concentrations ranging from 10 nM to 1 µM. Although,

little or no detectable changes in the membrane potential were induced by nicotine,

the amplitude of carbachol-induced currents was decreased in the presence of

nicotine in all 14 investigated SPn. Bath application of 100 nM and 500 nM nicotine

applied for 5-10 min reduced the maximal amplitude of carbachol-induced currents

by 31.4 ± 5.4% (n=4, p<0.05) and 66.6 ± 12% (n=6, p<0.005), respectively (Fig. 5A).

This effect was accompanied by a slight decrease in the decay-time of the currents

(control: 2.7 ± 0.6 s, 500 nM nicotine: 0.7 ± 0.2 s, p<0.05). A more consistent

reduction of the carbachol-induced currents by 86.4 ± 7.7% was obtained after

application of 1 µM nicotine. In all investigated SPn, desensitization of the nAChR on

SPn in the presence of nicotine was accompanied by a significant (p<0.01) decrease

in Rin by 37.1 ± 6.9% (n=4) and in the membrane time constant by 44.7 ± 6.3% (n=4).

Because the half-life of nicotine in the body is about 2 h (Benowitz et al. 1989),

the brain of smokers experiences long exposure to nicotine. When SPn were

exposed to 500 nM nicotine for >20 min, carbachol-induced currents were almost

abolished (Fig. 5A, inset), indicating that a greater fraction of the nAChR

desensitizes. Recovery from desensitization was not achieved even after 60 min

washout. Similar results were reported for nAChR present on midbrain dopamine

neurons (Pidoplichko et al. 1997).

JN-00010-2004.R1

16

Behaviorally relevant concentrations of nicotine affected also the baseline

current noise in SPn. A significant (p<0.05) increase in the variance of the baseline

current to 278.7 ± 45.1% (n=4) was obtained using 500 nM nicotine, whereas the

effects induced by 50 nM, 100 nM and 1 µM nicotine were not significantly different

(Fig. 5B). These results indicate that after long-lasting exposure similar to that

experienced after tobacco exposure, the majority of α4β2 nAChR present on SPn are

desensitized by concentrations of nicotine that are too low to activate the receptor

directly. However, a small fraction of the total number of nAChR stochastically

opened in response to bath application of nicotine, producing an increase in the

baseline current.

SPn receive glutamatergic synaptic inputs from the thalamus, CP and

subplate (Hanganu et al. 2002). Therefore we studied the effects of carbachol and

low concentrations of nicotine on spontaneous as well as on evoked glutamatergic

synaptic activity. In agreement with our previous data (Hanganu et al. 2001), 90% of

the SPn showed glutamatergic spontaneous postsynaptic currents (sPSCs) at a

frequency of 0.19 ± 0.02 Hz (n=19 cells) and with an amplitude of 15.9 ± 0.6 pA

(n=19 cells). Carbachol (100 µM) failed to modify the frequency, amplitude and

kinetics of the sPSCs. When nicotine at bath concentrations ranging between 50 nM

and 1 µM was applied, neither the amplitude nor the kinetics of glutamatergic sPSCs

was significantly modified (Fig. 5C). Since fast sPSCs are mediated by AMPA

receptors and slow sPSCs by NMDA receptors (Hanganu et al. 2001), and since

neither carbachol nor nicotine elicited significant changes in the kinetics of these

sPSCs, our observations suggest that neither AMPA nor NMDA receptor-mediated

sPSCs were affected by activation of nAChR. In addition, in 5 out of 5 investigated

SPn glutamatergic PSCs evoked by electrical stimulation of the thalamocortical

afferents were not affected by nicotine in their amplitude (control: 28 ± 9.4 pA,

JN-00010-2004.R1

17

nicotine: 26.6 ± 5.6 pA), rise-time (control: 5.5 ± 1.4 ms, nicotine: 5.3 ± 0.8 ms) or

decay-time (control: 36.6 ± 1.9 ms, nicotine: 37.8 ± 2.6 ms) (Fig. 5D).

A functional intrinsic GABAergic synaptic circuit is present within the subplate

(Hanganu et al. 2002). To assess the interactions between nAChR and the local

GABAergic synaptic circuit within the subplate, the effects of nAChR activation on

spontaneous as well as on evoked GABAergic activity were investigated. Long-

lasting GABAergic sPSCs (decay-time 58.3 ± 8.3 ms) could be recorded at a low-

frequency (0.09 ± 0.04 Hz) in all 11 investigated SPn using chloride-based electrode

solution and an extracellular solution containing 10 µM CNQX and 10 µM CPP to

block glutamatergic synaptic transmission. Bath application of nicotine at

concentrations ranging from 100 nM to 1 µM did not affect the amplitude or the

kinetics of the sPSCs (Fig. 5C). Moreover, GABAergic PSCs evoked by electrical

stimulation of the subplate were also not affected by nicotine (Fig. 5E).

These findings suggest that neither glutamatergic synaptic transmission of the

thalamocortical input nor GABAergic synaptic interactions between SPn are affected

by carbachol or nicotine at behaviorally relevant concentrations.

DISCUSSION

An accumulation of cholinergic afferents (Kostovic et al. 1989; Kiss and Patel 1992;

Mechawar and Descarries 2001) and nAChR subunits (Bina et al. 1995; Broide et al.

1996; Csillik et al. 2002) has been previously demonstrated in the subplate, but the

receptor assembly and function is largely unknown. The present in vitro

electrophysiological study in neonatal rat somatosensory cortex demonstrates and

characterizes for the first time functional nAChR on SPn, presumably mediating the

excitation induced by endogenously released ACh. Activation of nAChR by local

pressure application of cholinergic agonists excited the large majority of the

JN-00010-2004.R1

18

investigated SPn. Cholinergic excitation seems to be mediated exclusively by

postsynaptic α4β2 nAChR. Neither spontaneous synaptic activity nor the

glutamatergic synaptic input from the thalamus or the GABAergic synaptic input from

other SPn seem to be modulated by nAChR. Our results further demonstrate that

nicotine, reaching the developing brain by maternal smoking or by early postnatal

exposure, causes a severe desensitization of nAChR on SPn. These data indicate

that SPn and immature cortical circuits involving SPn may be critically modified by

low doses of nicotine during pre- and neonatal development.

Expression of functional nAChR on SPn

The large majority of the investigated SPn were excited by nAChR agonists,

indicating that the expression of functional nAChR is a common property of SPn.

Since a large number of SPn corresponding to all previously described morphological

types was investigated, it is unlikely that a particular subtype of SPn was excluded

from the present study.

Activation of nAChR excited the SPn to fire action potentials and mediated

fast, slowly desensitizing inward currents reversing near 0 mV. Our observations

strongly suggest that the nAChR on SPn are located exclusively postsynaptically, in

contrast to the presynaptic role of nAChR reported previously (McGehee and Role

1995; Gray et al. 1996; Aramakis and Metherate 1998).

Although the exact subunit composition of the receptors is not known (see

also Footnote), the pharmacological tools used in the present study offer the best

currently available specificity for the nAChR subtypes. Several lines of evidence

indicate that the nAChR on SPn are assembled from α4 and β2 subunits. The

presence of the α4 subunit at the level of the subplate was shown by

immunoperoxidase labeling (H Schröder, unpublished observation). The presence of

JN-00010-2004.R1

19

functional α4β2 nAChR on SPn is also supported by the slow desensitization rate of

the responses to nicotinic agonists, the blockade of the nicotinic responses by DHβE

and the efficiency of RJR-2403 to mimic the carbachol-induced responses.

Coexpression of additional subunits, like α5, α3 or β4, as described in heterologous

expression systems (Ramirez-Latorre et al. 1996; Conroy and Berg 1998) cannot be

excluded, but seems unlikely, since neither α3 nor β4 subunits mRNA have been

detected in the subplate (Zoli et al. 1995).

In contrast to the previously reported accumulation of α7 mRNA and α-

bungarotoxin binding sites in the subplate (Naeff et al. 1992; Bina et al. 1995; Broide

et al. 1995; Csillik et al. 2002), MLA did not affect cholinergic responses and choline

did not elicit α7 nAChR-mediated currents in SPn. This discrepancy between a

molecularbiological demonstration and a lack of a functional proof for the presence of

α7 nAChR is not unique for SPn. Previous studies (Frazier et al. 1998; Radcliffe and

Dani 1998) suggested that the number of α7 subunit-binding sites exceeds the

number of functional α7 nAChR. Neither in situ hybridization experiments nor the α-

bungarotoxin binding in sections and lysates of brain tissue are capable to distinguish

clearly between receptors located intracellularly or at the membrane surface. The

presence of intracellular pools of subunits which are not assembled into functional

was reported for neuronal nAChR (Jacob et al. 1986). The mechanisms of receptor

trafficking to the membrane and their assembling depend on cell- or receptor-specific

factors (Dineley and Patrick 2000; Wang et al. 2002), which differ for heteromeric and

homomeric receptors (Wang et al. 2002). The possibility that rapid desensitization of

α7 nAChR (Aramakis and Metherate 1998) masks the functional detection of these

receptors on SPn cannot completely be ignored, but, according to our data, seems to

be unlikely. A decrease of the application pulse duration to 20 ms did not induce an

JN-00010-2004.R1

20

α7 nAChR-mediated response. However, previous studies identified α7 nAChR-

mediated responses by using slow bath perfusion of agonists (Gil et al. 1997).

Moreover, fast application of the α7 nAChR agonist choline, which has the advantage

that receptor desensitization is much less pronounced compared with nicotine (Vogt

and Regehr 2001), also failed to elicit α7 nAChR-mediated responses in SPn.

Physiological significance of the nAChR on SPn during cortical development

The neurotransmitter acetylcholine plays an important role in the manifestation and

modulation of activity-dependent processes in the developing brain (Feller 2002). In

the immature cerebral cortex, the cholinergic system has a prominent modulatory

influence on a number of structural and functional modifications underlying age-

dependent synaptic plasticity (Bear and Singer 1986; Hohmann and Berger-Sweeney

1998; Aramakis et al. 2000; Ego-Stengel et al. 2001). Both the endogenous Ach

release and the presence of functional nAChR on SPn indicate that the cholinergic

system may modulate immature cortical neurons and circuits transiently expressed

during early development. The cholinergic projection from the nucleus basalis of

Meynert innervates the subplate (Mesulam et al. 1983) and upon activation causes a

pronounced excitation of the SPn. Since SPn are key elements in the development of

cortical afferents, efferents and in the establishment of the columnar architecture

(McConnell et al. 1989; Ghosh et al. 1990; Ghosh and Shatz 1992a; Kanold et al.

2003), the functional modulation of SPn by the cholinergic input may also profoundly

influence these developmental processes. A simultaneous activation of the

subthreshold glutamatergic input from the thalamus (Hanganu et al. 2002) and the

excitatory cholinergic input mediated by nAChR may cause a suprathreshold

activation of SPn and a Hebb-like stabilization of immature cortical synapses and

JN-00010-2004.R1

21

circuits. These connections may then form the template for the generation of the

mature cortical circuits and architecture.

Besides modulating the activity-dependent refinement of early cortical circuits

involving SPn, the nAChR may also influence the fate of this transient neuronal

population. In rodents, a subpopulation of SPn disappears by apoptosis, whereas

other SPn seem to survive and transform themselves into other cell types within the

white matter or cortical layer VIb (Woo et al. 1991). A lack of nicotinic binding sites

has been previously associated with an increased neuronal degeneration (Zoli et al.

1999) and a neuroprotective action resulting from activation of α4β2 nAChR was

reported for neonatal brain insults (Laudenbach et al. 2002). The data indicate that

nAChR may regulate the survival of SPn in developing cerebral cortex.

Pathophysiological role of nAChR during early cortical development

Since the rat cerebral cortex is relatively immature at birth when compared at the

cellular level with the cortex of a full-term human infant (Romijn et al. 1991), the

findings of the present study concerning the action of nicotine on the newborn rat

SPn can be compared with the situation of the prenatal human cortex. Maternal

smoking during pregnancy has been reported to cause tobacco-induced abortions,

premature deliveries, low birth weight and an increased incidence for sudden infant

death syndrome (Lambers and Clark 1996; Slotkin 1998). The cellular mechanisms

of these processes are not completely understood, but exposure to nicotine during

prenatal stages may disturb cell division and neuronal differentiation.

Since nicotine at concentrations experienced by smokers caused a

pronounced desensitization of the α4β2 nAChR in SPn, exogenous nicotine may

profoundly influence cortical development. The desensitization of nAChR on SPn

precludes the neuro-trophic and -modulatory function of acetylcholine in the cortex.

JN-00010-2004.R1

22

Moreover, this desensitization may up-regulate the number of nAChR as shown to

occur in vivo (Flores et al. 1992; Shacka and Robinson 1998; Buisson and Bertrand

2001). To which extent the desensitization of nAChR may prevent the excessive

stimulation and the potentially excitotoxic death of SPn remains to be elucidated.

In conclusion, the present electrophysiological data demonstrate that SPn

express functional nAChR which are assembled most likely from α4 and β2 subunits

and shape the excitability of immature synaptic circuits present in the cortex around

birth. The nAChR are desensitized by long exposure to nicotine at concentrations

similar to those found in cigarette smokers.

Text Footnotes

According to the current status of the nomenclature for nicotinic acetylcholine

receptors (Lukas et al., 1999), the exact subunit composition of the α7 and α4β2

nAChR is not known.

Acknowledgments

We are most thankful to Beate Krumm for excellent technical assistance, Drs.

Werner Kilb and Till Opatz for helpful discussions and Dr. Jochen Staiger for

competent help in Neurolucida cell reconstruction. This project was supported by

DFG grant Lu375/4-1.

JN-00010-2004.R1

23

Reference List

Albuquerque EX, Pereira EF, Braga MF and Alkondon M. Contribution of nicotinic

receptors to the function of synapses in the central nervous system: the action of

choline as a selective agonist of alpha 7 receptors. J Physiol Paris 92: 309-316,

1998.

Alkondon M and Albuquerque EX. Diversity of nicotinic acetylcholine receptors in

rat hippocampal neurons. I. Pharmacological and functional evidence for distinct

structural subtypes. J Pharmacol Exp Ther 265: 1455-1473, 1993.

Alkondon M, Pereira EF, Eisenberg HM and Albuquerque EX. Choline and

selective antagonists identify two subtypes of nicotinic acetylcholine receptors that

modulate GABA release from CA1 interneurons in rat hippocampal slices. J Neurosci

19: 2693-2705, 1999.

Aramakis VB, Hsieh CY, Leslie FM and Metherate R. A critical period for nicotine-

induced disruption of synaptic development in rat auditory cortex. J Neurosci 20:

6106-6116, 2000.

Aramakis VB and Metherate R. Nicotine selectively enhances NMDA receptor-

mediated synaptic transmission during postnatal development in sensory neocortex.

J Neurosci 18: 8485-8495, 1998.

Atluri P, Fleck MW, Shen Q, Mah SJ, Stadfelt D, Barnes W, Goderie SK, Temple

S and Schneider AS. Functional nicotinic acetylcholine receptor expression in stem

JN-00010-2004.R1

24

and progenitor cells of the early embryonic mouse cerebral cortex. Dev Biol 240:

143-156, 2001.

Bear MF and Singer W. Modulation of visual cortical plasticity by acetylcholine and

noradrenaline. Nature 320: 172-176, 1986.

Benowitz NL, Porchet H and Jacob P, III. Nicotine dependence and tolerance in

man: pharmacokinetic and pharmacodynamic investigations. Prog Brain Res 79:

279-287, 1989.

Bertrand D, Ballivet M and Rungger D. Activation and blocking of neuronal

nicotinic acetylcholine receptor reconstituted in Xenopus oocytes. Proc Natl Acad Sci

U S A 87: 1993-1997, 1990.

Bina KG, Guzman P, Broide RS, Leslie FM, Smith MA and O'Dowd DK.

Localization of alpha 7 nicotinic receptor subunit mRNA and alpha- bungarotoxin

binding sites in developing mouse somatosensory thalamocortical system. J Comp

Neurol 363: 321-332, 1995.

Broide RS, O'Connor LT, Smith MA, Smith JA and Leslie FM. Developmental

expression of alpha 7 neuronal nicotinic receptor messenger RNA in rat sensory

cortex and thalamus. Neuroscience 67: 83-94, 1995.

Broide RS, Robertson RT and Leslie FM. Regulation of alpha7 nicotinic

acetylcholine receptors in the developing rat somatosensory cortex by

thalamocortical afferents. J Neurosci 16: 2956-2971, 1996.

JN-00010-2004.R1

25

Buisson B and Bertrand D. Chronic exposure to nicotine upregulates the human

(alpha)4((beta)2 nicotinic acetylcholine receptor function. J Neurosci 21: 1819-1829,

2001.

Candy JM, Perry EK, Perry RH, Bloxham CA, Thompson J, Johnson M, Oakley

AE and Edwardson JA. Evidence for the early prenatal development of cortical

cholinergic afferents from the nucleus of Meynert in the human foetus. Neurosci Lett

61: 91-95, 1985.

Chessell IP and Humphrey PP. Nicotinic and muscarinic receptor-evoked

depolarizations recorded from a novel cortical brain slice preparation.

Neuropharmacology 34: 1289-1296, 1995.

Conroy WG and Berg DK. Nicotinic receptor subtypes in the developing chick brain:

appearance of a species containing the alpha4, beta2, and alpha5 gene products.

Mol Pharmacol 53: 392-401, 1998.

Csillik AE, Okuno E, Csillik B, Knyihar E and Vecsei L. Expression of kynurenine

aminotransferase in the subplate of the rat and its possible role in the regulation of

programmed cell death. Cereb Cortex 12: 1193-1201, 2002.

Dineley KT and Patrick JW. Amino acid determinants of alpha 7 nicotinic

acetylcholine receptor surface expression. J Biol Chem 275: 13974-13985, 2000.

Dodt H-U and Zieglgänsberger W. Visualizing unstained neurons in living brain

slices by infrared DIC-videomicroscopy. Brain Res 537: 333-336, 1990.

JN-00010-2004.R1

26

Ego-Stengel B, Shulz DE, Haidarliu S, Sosnik R and Ahissar E. Acetylcholine-

dependent induction and expression of functional plasticity in the barrel cortex of the

adult rat. J Neurophysiol 86: 422-437, 2001.

Feller MB. The role of nAChR-mediated spontaneous retinal activity in visual system

development. J Neurobiol 53: 556-567, 2002.

Flores CM, Rogers SW, Pabreza LA, Wolfe BB and Kellar KJ. A subtype of

nicotinic cholinergic receptor in rat brain is composed of alpha 4 and beta 2 subunits

and is up-regulated by chronic nicotine treatment. Mol Pharmacol 41: 31-37, 1992.

Frazier CJ, Rollins YD, Breese CR, Leonard S, Freedman R and Dunwiddie TV.

Acetylcholine activates an alpha-bungarotoxin-sensitive nicotinic current in rat

hippocampal interneurons, but not pyramidal cells. J Neurosci 18: 1187-1195, 1998.

Friauf E, McConnell SK and Shatz CJ. Functional synaptic circuits in the subplate

during fetal and early postnatal development of cat visual cortex. J Neurosci 10:

2601-2613, 1990.

Fuchs JL. [125I]alpha-bungarotoxin binding marks primary sensory area developing

rat neocortex. Brain Res 501: 223-234, 1989.

Ghosh A, Antonini A, McConnell SK and Shatz CJ. Requirements of subplate

neurons in the formation of thalamocortical connections. Nature 347: 179-181, 1990.

Ghosh A and Shatz CJ. Involvement of subplate neurons in the formation of ocular

dominance columns. Science 255: 1441-1443, 1992.

JN-00010-2004.R1

27

Ghosh A and Shatz CJ. Pathfinding and target selection by developing

geniculocortical axons. J Neurosci 12: 39-55, 1992.

Gil Z, Connors BW and Amitai Y. Differential regulation of neocortical synapses by

neuromodulators and activity. Neuron 19: 679-686, 1997.

Gray R, Rajan AS, Radcliffe KA, Yakehiro M and Dani JA. Hippocampal synaptic

transmission enhanced by low concentrations of nicotine. Nature 383: 713-716,

1996.

Hanganu IL, Kilb W and Luhmann HJ. Spontaneous synaptic activity of subplate

neurons in neonatal rat somatosensory cortex. Cereb Cortex 11: 400-410, 2001.

Hanganu IL, Kilb W and Luhmann HJ. Functional synaptic projections onto

subplate neurons in neonatal rat somatosensory cortex. J Neurosci 22: 7165-7176,

2002.

Henningfield JE, Stapleton JM, Benowitz NL, Grayson RF and London ED.

Higher levels of nicotine in arterial than in venous blood after cigarette smoking. Drug

Alcohol Depend 33: 23-29, 1993.

Hilmas C, Pereira EF, Alkondon M, Rassoulpour A, Schwarcz R and

Albuquerque EX. The brain metabolite kynurenic acid inhibits alpha7 nicotinic

receptor activity and increases non-alpha7 nicotinic receptor expression:

physiopathological implications. J Neurosci 21: 7463-7473, 2001.

JN-00010-2004.R1

28

Hohmann CF and Berger-Sweeney J. Cholinergic regulation of cortical

development and plasticity. New twists to an old story. Perspect Dev Neurobiol 5:

401-425, 1998.

Jacob MH, Lindstrom JM and Berg DK. Surface and intracellular distribution of a

putative neuronal nicotinic acetylcholine receptor. J Cell Biol 103: 205-214, 1986.

Jones S, Sudweeks S and Yakel JL. Nicotinic receptors in the brain: correlating

physiology with function. Trends Neurosci 22: 555-561, 1999.

Kanold PO, Kara P, Reid RC and Shatz CJ. Role of subplate neurons in functional

maturation of visual cortical columns. Science 301: 521-525, 2003.

Kilb W and Luhmann HJ. Characterization of a hyperpolarization-activated inward

current in Cajal-Retzius cells in rat neonatal neocortex. J Neurophysiol 84: 1681-1691,

2000.

Kiss J and Patel AJ. Development of the cholinergic fibres innervating the cerebral

cortex of the rat. Int J Dev Neurosci 10: 153-170, 1992.

Kostovic I. Prenatal development of nucleus basalis complex and related fiber

systems in man: a histochemical study. Neuroscience 17: 1047-1077, 1986.

Kostovic I, Lukinovic N, Judas M, Bogdanovic N, Mrzljak L, Zecevic N and

Kubat M. Structural basis of the developmental plasticity in the human cerebral

cortex: the role of the transient subplate zone. Metab Brain Dis 4: 17-23, 1989.

JN-00010-2004.R1

29

Kostovic I and Rakic P. Cytology and time of origin of interstitial neurons in the

white matter in infant and adult human and monkey telencephalon. J Neurocytol 9:

219-242, 1980.

Lambers DS and Clark KE. The maternal and fetal physiologic effects of nicotine.

Semin Perinatol 20: 115-126, 1996.

Laudenbach V, Medja F, Zoli M, Rossi FM, Evrard P, Changeux JP and

Gressens P. Selective activation of central subtypes of the nicotinic acetylcholine

receptor has opposite effects on neonatal excitotoxic brain injuries. FASEB J 16:

423-425, 2002.

Le Novere N, Corringer PJ and Changeux JP. The diversity of subunit composition

in nAChRs: evolutionary origins, physiologic and pharmacologic consequences. J

Neurobiol 53: 447-456, 2002.

Levin ED and Simon BB. Nicotinic acetylcholine involvement in cognitive function in

animals. Psychopharmacology (Berl) 138: 217-230, 1998.

Lipton SA and Kater SB. Neurotransmitter regulation of neuronal outgrowth,

plasticity and survival. Trends Neurosci 12: 265-270, 1989.

Luhmann HJ, Schubert D, Kötter R and Staiger JF. Cellular morphology and

physiology of the perinatal rat cerebral cortex. Dev Neurosci 21: 298-309, 1999.

Lukas RJ, Changeux JP, Le Novere N, Albuquerque EX, Balfour DJ, Berg DK,

Bertrand D, Chiappinelli VA, Clarke PB, Collins AC, Dani JA, Grady SR, Kellar

JN-00010-2004.R1

30

KJ, Lindstrom JM, Marks MJ, Quik M, Taylor PW and Wonnacott S. International

Union of Pharmacology. XX. Current status of the nomenclature for nicotinic

acetylcholine receptors and their subunits. Pharmacol Rev 51: 397-401, 1999.

Margiotta JF, Berg DK and Dionne VE. Cyclic AMP regulates the proportion of

functional acetylcholine receptors on chicken ciliary ganglion neurons. Proc Natl

Acad Sci U S A 84: 8155-8159, 1987.

Marty A and Neher E. Tight-seal whole-cell recordings. In: Single-channel recording,

edited by Sakmann B and Neher E. New York: Plenum, 1995, p. 31-52.

Mathie A, Colquhoun D and Cull-Candy SG. Rectification of currents activated by

nicotinic acetylcholine receptors in rat sympathetic ganglion neurones. J Physiol 427:

625-655, 1990.

McConnell SK, Ghosh A and Shatz CJ. Subplate neurons pioneer the first axon

pathway from the cerebral cortex. Science 245: 978-982, 1989.

McGehee DS and Role LW. Physiological diversity of nicotinic acetylcholine

receptors expressed by vertebrate neurons. Annu Rev Physiol 57: 521-546, 1995.

Mechawar N and Descarries L. The cholinergic innervation develops early and

rapidly in the rat cerebral cortex: A quantitative immunocytochemical study.

Neuroscience 108: 555-567, 2001.

JN-00010-2004.R1

31

Mesulam MM, Mufson EJ, Wainer BH and Levey AI. Central cholinergic pathways

in the rat: an overview based on an alternative nomenclature (Ch1-Ch6).

Neuroscience 10: 1185-1201, 1983.

Mienville JM and Pesold C. Low resting potential and postnatal upregulation of

NMDA receptors may cause Cajal-Retzius cell death. J Neurosci 19: 1636-1646,

1999.

Naeff B, Schlumpf M and Lichtensteiger W. Pre- and postnatal development of

high-affinity [3H]nicotine binding sites in rat brain regions: An autoradiographic study.

Dev Brain Res 68: 163-174, 1992.

Ostermann CH, Grunwald J, Wevers A, Lorke DE, Reinhardt S, Maelicke A and

Schröder H. Cellular expression of alpha 4 subunit mRNA of the nicotinic

acetylcholine receptor in the developing rat telencephalon. Neurosci Lett 192: 21-24,

1995.

Palma E, Bertrand S, Binzoni T and Bertrand D. Neuronal nicotinic alpha 7

receptor expressed in Xenopus oocytes presents five putative binding sites for

methyllycaconitine. J Physiol 491 ( Pt 1): 151-161, 1996.

Papke RL, Webster JC, Lippiello PM, Bencherif M and Francis MM. The

activation and inhibition of human nicotinic acetylcholine receptor by RJR-2403

indicate a selectivity for the alpha4beta2 receptor subtype. J Neurochem 75: 204-

216, 2000.

JN-00010-2004.R1

32

Perry E, Walker M, Grace J and Perry R. Acetylcholine in mind: a neurotransmitter

correlate of consciousness? Trends in Neurosciences 22: 273-280, 1999.

Pidoplichko VI, DeBiasi M, Williams JT and Dani JA. Nicotine activates and

desensitizes midbrain dopamine neurons. Nature 390: 401-404, 1997.

Pugh PC and Margiotta JF. Nicotinic acetylcholine receptor agonists promote

survival and reduce apoptosis of chick ciliary ganglion neurons. Mol Cell Neurosci 15:

113-122, 2000.

Radcliffe KA and Dani JA. Nicotinic stimulation produces multiple forms of

increased glutamatergic synaptic transmission. J Neurosci 18: 7075-7083, 1998.

Rakic P. Prenatal development of the visual system in rhesus monkey. Philos Trans

R Soc Lond B Biol Sci 278: 245-260, 1977.

Ramirez-Latorre J, Yu CR, Qu X, Perin F, Karlin A and Role L. Functional

contributions of alpha5 subunit to neuronal acetylcholine receptor channels. Nature

380: 347-351, 1996.

Role LW and Berg DK. Nicotinic receptors in the development and modulation of

CNS synapses. Neuron 16: 1077-1085, 1996.

Romijn HJ, Hofman MA and Gramsbergen A. At what age is the developing

cerebral cortex of the rat comparable to that of the full-term newborn human baby?

Early Human Devel 26: 61-67, 1991.

JN-00010-2004.R1

33

Schröder R and Luhmann HJ. Morphology, electrophysiology and pathophysiology

of supragranular neurons in rat primary somatosensory cortex. Eur J Neurosci 9:

163-176, 1997.

Shacka JJ and Robinson SE. Exposure to prenatal nicotine transiently increases

neuronal nicotinic receptor subunit alpha7, alpha4 and beta2 messenger RNAs in the

postnatal rat brain. Neuroscience 84: 1151-1161, 1998.

Slotkin TA. Fetal nicotine or cocaine exposure: which one is worse? J Pharmacol

Exp Ther 285: 931-945, 1998.

Stone TW. Cholinergic mechanisms in the rat somatosensory cerebral cortex. J

Physiol 225: 485-499, 1972.

Stuart GJ, Dodt H-U and Sakmann B. Patch-clamp recordings from the soma and

dendrites of neurons in brain slices using infrared video microscopy. Pflügers Arch

423: 511-518, 1993.

Vogt KE and Regehr WG. Cholinergic modulation of excitatory synaptic

transmission in the CA3 area of the hippocampus. J Neurosci 21: 75-83, 2001.

Wang JM, Zhang L, Yao Y, Viroonchatapan N, Rothe E and Wang ZZ. A

transmembrane motif governs the surface trafficking of nicotinic acetylcholine

receptors. Nat Neurosci 5: 963-970, 2002.

Wong LA and Gallagher JP. A direct nicotinic receptor-mediated inhibition recorded

intracellularly in vitro. Nature 341: 439-442, 1989.

JN-00010-2004.R1

34

Woo TU, Beale JM and Finlay BL. Dual fate of subplate neurons in a rodent. Cereb

Cortex 1: 433-443, 1991.

Zoli M, Le Novere N, Hill JA, Jr. and Changeux JP. Developmental regulation of

nicotinic ACh receptor subunit mRNAs in the rat central and peripheral nervous

systems. J Neurosci 15: 1912-1939, 1995.

Zoli M, Picciotto MR, Ferrari R, Cocchi D and Changeux JP. Increased

neurodegeneration during ageing in mice lacking high-affinity nicotine receptors.

EMBO J 18: 1235-1244, 1999.

JN-00010-2004.R1

35

Legends

Figure 1: Morphology of SPn and position of the local application pipette.

A. Videomicroscopic image of a P4 SPn (black arrow) visualized with the local

application pipette (white arrow). B. Neurolucida reconstruction of the biocytin-

stained SPn shown in A. The neuron displayed the typical morphology of a horizontal

bitufted SPn with a large fusiform soma, primary dendrites oriented parallel to the pial

surface and an extensively arborized dendritic tree.

Figure 2: Responses of SPn to local application of nAChR agonists. A. nAChR-

induced depolarization recorded from a current-clamped P4 SPn in response to local

application of carbachol (100 µM). Note that the carbachol-induced depolarization

was of sufficient magnitude to trigger action potentials. Inset, carbachol-induced

action potentials displayed at a larger scale. B. Carbachol-elicited currents recorded

in voltage-clamp modus from the same P4 SPn as displayed in A. The decay-time of

the current could be described by the sum of two exponential functions (τ1=0.4 s,

τ2=2.3 s). C. nAChR-mediated currents recorded after prolonged local application of

100 µM carbachol. Carbachol-elicited currents maintained in a steady-state level in

the presence of the agonist. Inset, the increased baseline current was not

accompanied by an increase in the spontaneous synaptic activity. D. nAChR-

mediated current recorded from a voltage-clamped P2 SPn in response to local

application of nicotine (100 µM). Note the slow kinetic of the current (τ=31.8 s) as

compared to the carbachol-induced current in C. All recordings were performed in the

presence of 10 µM atropine to prevent activation of the muscarinic receptors and the

holding potential was adjusted to –70 mV. Local application of carbachol or nicotine

marked by arrows was performed for 200 ms (A, B, D).

JN-00010-2004.R1

36

Figure 3. A. Current-voltage relationship of nAChR-mediated responses. Plot of the

peak-amplitude of carbachol-induced currents as a function of the holding potential.

Data show average ± SEM from 5 SPn. Inset, carbachol-induced currents recorded

from a P3 SPn. Each current trace corresponds to a single application of the agonist.

B. Bar diagram displaying the effects of synaptic activity blockade on carbachol-

induced currents. Peak responses to local and bath application of carbachol were

recorded in the presence of 10 µM CNQX, 10 µM CPP and 1 µM TTX. Data were

averaged from 8 SPn (local application) and 7 SPn (bath application) and normalized

to control values. Inset, nAChR-mediated currents elicited by local or bath application

of carbachol and recorded in a voltage-clamped P1 and P2 SPn, respectively under

control conditions (black trace) and after blocking synaptic transmission (gray trace).

All recordings were performed at holding potential of –70 mV. Control recordings

were performed in the presence of 10 µM atropine.

Figure 4. Pharmacological identification of functional nAChR subtypes on SPn.

A. Carbachol-induced responses recorded in a voltage-clamped P3 SPn before

(black trace) and during bath application of 10 µM DHßE (gray trace). B. nAChR-

mediated response elicited in a current-clamped P1 SPn by local application of RJR-

2403 (30 µM). Note the long-lasting decay phase of the response. Inset, amplitude,

rise-time and decay-time of the RJR-2403-elicited responses averaged from 4 SPn

and normalized to the data recorded under control conditions. C. Carbachol-induced

responses recorded in a voltage-clamped P2 SPn before (black trace) and in the

presence of 50 nM MLA (gray trace). Inset, amplitude of the carbachol-elicited

currents averaged from 6 SPn under control conditions (black bar) and in the

presence of MLA (gray bar). D. Carbachol-induced responses recorded in a current-

JN-00010-2004.R1

37

clamped P2 SPn under control conditions (black trace) and during bath application of

10 µM kynurenic acid (KYNA, gray trace). Control recordings were performed in

atropine (A, B, C) or in 10 µM CPP and 10 µM atropine (D). Local pressure

application of agonists is marked by arrow and all recordings were performed at a

holding potential of –70 mV.

Figure 5. Effects of behaviorally relevant concentrations of nicotine on SPn.

A. Dose-response relationship for nicotine-induced inhibition of carbachol-induced

responses. The peak amplitude of the carbachol-induced currents in the presence of

nicotine was normalized to that of the control and plotted against log concentrations

of nicotine. Values were obtained from four to six SPn. The solid line passing through

the symbols represents the best fit of the data to a Hill equation. Inset, consecutive

carbachol-induced responses recorded from a P3 SPn under control conditions and

under prolonged application of 500 nM nicotine. The time interval between the

displayed responses was 30 s under control conditions and 120 s in the presence of

nicotine. B. Effects of nicotine on baseline current noise of SPn. The baseline noise

was calculated for current traces recorded under control conditions (black squares)

and in the presence of nicotine concentrations ranging from 50 nM to1 µM (gray

squares). Mean values were obtained from four to six SPn. Inset, current traces

recorded under control conditions (black traces) and in the presence of various

nicotine concentrations (gray traces). Note the significant increase in the baseline

noise in the presence of 500 nM nicotine. C. Effects of nicotine on glutamatergic

(black squares) and GABAergic (gray squares) spontaneous synaptic activity of SPn.

The frequency of sPSCs was expressed as number of events pro 24 s-bin and the

values were averaged from 4 SPn before and during nicotine (500 nM) application.

D. Effects of nicotine on thalamocortical synaptic function. The PSCs were evoked by

JN-00010-2004.R1

38

electrical stimulation of the thalamocortical afferents at 0.05 Hz and recorded under

control conditions (black trace) and in the presence of 500 nM nicotine (gray trace)

from a P2 SPn. E. Effects of nicotine on GABAergic synaptic interactions between

SPn. The PSCs were evoked by electrical stimulation of the subplate at 0.05 Hz and

recorded under control conditions (black trace) and in the presence of 500 nM

nicotine (gray trace) from a P0 SPn. All control recordings were performed in the

presence of 10 µM atropine and GABAergic synaptic activity was recorded using

high-chloride electrode solution and extracellular solution containing 10 µM CNQX

and 10 µM CPP.

10 µm

Dorsal

Ventral

50 µm

Dorsal

Ventral

A

B

Nicotinic receptors on SPnHanganu et al

Fig. 1

30 pA10 s

15 mV10 s

A B

D

40 pA10 s

Carbachol

Carbachol

Carbachol

30 pA5 s

Nicotine

C

15 mV0.5 s

-70 mV

Nicotinic receptors on SPnHanganu et al

Fig. 2

50 pA500 ms

-150 -100 -50 50

50

-50

-100

-150

10

-10

-30

-50

-70

-90

-110

-130

I(pA

)

V(mV)

50 pA3 s

Carbachol

B

A

0

20

40

60

80

100

Rela

tive

am

plit

ude

(%)

20 pA5 s

Local Carbachol

LocalCarbachol

BathCarbachol

Nicotinic receptors on SPnHanganu et al

Fig. 3

50 pA50 s

Bath Carbachol

20 pA2 s

30 pA4 s

10 mV3 s

Amplitude Rise-time Decay-time0

100

200

300

400

500

%ofcontr

ol

Carbachol

Carbachol

Control

+DH E *

20 mV5 s

RJR-2403

Control

+MLA

A B

C

Control

+KYNA

D

Carbachol

0

100

20

60

80

40

120

Control MLA

Am

plit

ude

(pA

)

140

Nicotinic receptors on SPnHanganu et al

Fig. 4

GABAergic sPSCs

80

60

40

20

2000Variance

ofth

ebaselin

enois

e(p

A)

15 pA50 ms

30 pA100 ms

Control

Nicotine

Control

Nicotine

400 600 800 1000

2.5

2

1.5

1

0.5

Nicotine [nM]

A B

D E

*

100

10-6

10-5

10-7

10-8

10-4

Rela

tive

am

plit

ude

(%ofth

econtr

ol)

IC = 239 nM

n = 1.2350

H

Nicotine [M]

Time (s)

Num

ber

ofsP

SC

s/2

4s

2

6

4

8

0120 240 300

0.5 µM nicotine

Glutamatergic sPSCs

540420

Nicotine 0.5 µM

80 pA10 s

30 s 120 s

25 pA5 s

0.05

1

0.50.1

Control Nicotine

C

Nicotinic receptors on SPnHanganu et al

Fig. 5

]