ORIGINAL ARTICLE

Functional expression and axonal transport of a7 nAChRsby peptidergic nociceptors of rat dorsal root ganglion

Irina Shelukhina • Renate Paddenberg •

Wolfgang Kummer • Victor Tsetlin

Received: 11 November 2013 / Accepted: 19 March 2014

� Springer-Verlag Berlin Heidelberg 2014

Abstract In recent pain studies on animal models, a7

nicotinic acetylcholine receptor (nAChR) agonists dem-

onstrated analgesic, anti-hyperalgesic and anti-inflamma-

tory effects, apparently acting through some peripheral

receptors. Assuming possible involvement of a7 nAChRs

on nociceptive sensory neurons, we investigated the mor-

phological and neurochemical features of the a7 nAChR-

expressing subpopulation of dorsal root ganglion (DRG)

neurons and their ability to transport a7 nAChR axonally.

In addition, a7 receptor activity and its putative role in pain

signal neurotransmitter release were studied. Medium-

sized a7 nAChR-expressing neurons prevailed, although

the range covered all cell sizes. These cells accounted for

one-fifth of total medium and large DRG neurons and

\5 % of small ones. 83.2 % of a7 nAChR-expressing

DRG neurons were peptidergic nociceptors (CGRP-im-

munopositive), one half of which had non-myelinated

C-fibers and the other half had myelinated Ad- and likely

Aa/b-fibers, whereas 15.2 % were non-peptidergic C-fiber

nociceptors binding isolectin B4. All non-peptidergic and a

third of peptidergic a7 nAChR-bearing nociceptors

expressed TRPV1, a capsaicin-sensitive noxious stimulus

transducer. Nerve crush experiments demonstrated that

CGRPergic DRG nociceptors axonally transported a7

nAChRs both to the spinal cord and periphery. a7 nAChRs

in DRG neurons were functional as their specific agonist

PNU282987 evoked calcium rise enhanced by a7-selective

positive allosteric modulator PNU120596. However, a7

nAChRs do not modulate neurotransmitter CGRP and

glutamate release from DRG neurons since nicotinic

ligands affected neither their basal nor provoked levels,

showing the necessity of further studies to elucidate the

true role of a7 nAChRs in those neurons.

Keywords Sensory ganglia � Nicotinic acetylcholine

receptor � Nociception � Nociceptor � Calcitonin gene-

related peptide

Introduction

a7 Nicotinic acetylcholine receptors (nAChRs) are ligand-

gated homopentameric cation channels formed by five

identical a7 subunits. Less frequently they combine a

heteropentameric a7b2 receptor subtype (Cuevas et al.

2000; Wu and Lukas 2011; Murray et al. 2012). Homo-

pentameric a7 nAChR is characterized by high calcium

permeability, very fast and complete desensitization at high

agonist concentrations, and sensitivity to selective agonists

such as PNU282987 and to competitive antagonists such as

long-chain a-neurotoxins, e.g. a-bungarotoxin (Bgt)

(Tsetlin et al. 2009; Wu and Lukas 2011; Gundisch and

Eibl 2011). a7 nAChRs are widespread in the nervous

system, the majority have a presynaptic localization and

modulate neurotransmitter release, but some are postsyn-

aptic and mediate fast synaptic transmission or activate

downstream signaling (Gotti et al. 2009; Berg and Conroy

2002; Marchi and Grilli 2010). Activation of a7 nAChRs in

CNS improves learning, memory and attention deficiency

(Thomsen et al. 2010) and relieves pain (Damaj et al. 2000;

Abdin et al. 2006; Medhurst et al. 2008). In addition,

pain animal model studies have shown analgesic,

I. Shelukhina (&) � V. Tsetlin

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry

RAS, Miklukho-Maklaya str., 16/10, 117997 Moscow, Russia

e-mail: ner-neri@yandex.ru

R. Paddenberg � W. Kummer

Institute for Anatomy and Cell Biology, Justus-Liebig-

University, 35385 Giessen, Germany

123

Brain Struct Funct

DOI 10.1007/s00429-014-0762-4

anti-hyperalgesic and anti-inflammatory effects of a7

agonists acting through unidentified peripheral sites (Gurun

et al. 2009; Pacini et al. 2010; Wang et al. 2005; Feuerbach

et al. 2009; Rowley et al. 2010). Partially these effects

might be mediated by a7 nAChRs on immune cells through

cholinergic anti-inflammatory reflex (Huston 2012; Wang

et al. 2003; Nizri and Brenner 2011). Another prominent

peripheral target for a7 agonists is sensory axons of dorsal

root ganglion (DRG). Although the presence of different

nAChRs in DRG was shown by autoradiography, RT-PCR,

histochemical and electrophysiological studies (Sucher

et al. 1990; Boyd et al. 1991; Genzen et al. 2001; Haber-

berger et al. 2004; Shelukhina et al. 2009; Rau et al. 2005;

Ninkovic and Hunt 1983), it is not yet clear what types of

sensory modalities are perceived by DRG neurons

expressing a7 nAChRs and what direct function these

receptors served for.

DRG neurons are a diverse population including non-

nociceptors responding to non-damaging, low-intensity

stimuli (touch and proprioception) and nociceptors reacting

to high-intensity, potentially damaging stimuli (pain sen-

sation) (Lawson 2002). Two distinct differentiation path-

ways lead to the formation of peptidergic nociceptors

expressing calcitonin gene-related peptide (CGRP) and

substance P, and non-peptidergic nociceptors binding iso-

lectin B4 (IB4) (Bennett et al. 1996; Molliver et al. 1997;

Woolf and Ma 2007). The most extensively studied trans-

ducer of noxious stimuli is TRPV1, a non-selective cation

channel highly permeable to Ca2? and sensitive to tem-

peratures above 43 �C, acidification and capsaicin (Tomi-

naga et al. 1998; Caterina et al. 1997; Brenneis et al. 2013;

Wetsel 2011).

The present study was focused on the first level of the

pain pathway—DRG and sensory axons. Based on histo-

chemical classification we characterized the a7 nAChR-

expressing subpopulation of DRG neurons, specifically,

their cell size distribution and sensory modalities. We also

examined their ability to transport a7 nAChR axonally to

the spinal cord and periphery. In addition, the receptor

activity as a ligand-gated calcium channel and its role in

neurotransmitter CGRP and glutamate release were studied.

Materials and methods

Animals

Animal care and animal experiments were performed fol-

lowing the current version of the German Law on the

Protection of Animals as well as the NIH ‘‘principles of

laboratory animal care’’. Adult (3–5 months old) Wistar

rats of either sex were used. They were kept under a 12-h

light–dark cycle with free access to chow and water. Rats

were deeply anesthetized by inhalation of isoflurane

(Baxter Deutschland GmbH, Unterschleissheim, Ger-

many), DRGs and spinal cord from segmental levels

T1–T12 and L1–L5 were excised immediately or after

transcardial perfusion with 250 ml fixative solution [4 %

paraformaldehyde in 0.1 M phosphate buffer (PB, pH

7.4)]. For histochemistry the tissues were shock frozen in

melting isopentane or paraformaldehyde-fixed by immer-

sion in the fixative solution for 2 h at room temperature,

repeated washing with phosphate buffered saline (PBS) for

6–8 h and overnight incubation in 18 % w/v sucrose in PB

at 4 �C. Freshly dissected tissues from animals not per-

fused with fixative solution were used in functional studies.

Histochemistry

The histochemical procedure was based on the protocol

published earlier (Shelukhina et al. 2009) with some

modifications. Tissues were sectioned at a thickness of

10 lm with a cryostat. Sections from shock-frozen tissue

were fixed with isopropanol for 10 min at 4 �C or, alter-

natively, with 4 % paraformaldehyde for 10 min at 21 �C,

rinsed with PBS and distilled water and air dried for 1 h.

Sections from paraformaldehyde-fixed tissue were dried

without any pretreatment. Unspecific protein binding sites

were saturated for 1 h with PBS containing 100 ml/l nor-

mal horse serum, 10 g/l BSA and 5 ml/l Tween 20 and

were further pre-incubated for 1–2 h in buffer A (1 g/l

BSA and 150 mmol/l NaCl in PBS). Blocking controls

were run simultaneously by adding 100-fold excess of

unlabeled a-cobratoxin (CTX) or a-neurotoxin II (NTII) to

buffer A (Table 1). Then, a mixture of primary antibodies

and/or Alexa Fluor 488-conjugated aBgt (Alexa-aBgt;

Invitrogen, Molecular Probes, OR, USA) was added to the

slides to reach final concentrations as specified in Table 1.

Biotinylated IB4 (1:100, Vector Laboratories, USA) mixed

with Alexa-aBgt was applied in the same way. After

overnight incubation the sections were washed with PBS

and processed with appropriate combinations of secondary

reagents (Table 1). Cell nuclei were visualized by incu-

bation with 1 lg/ml 40,6-diamidino-20-phenylindole-dihy-

drochloride (DAPI; Sigma-Aldrich, Germany) for 20 min.

Further, the sections were washed with PBS, fixed with

4 % paraformaldehyde for 10 min, rinsed with PBS again

and coverslipped in carbonate-buffered glycerol at pH 8.6.

The slides were evaluated by epifluorescence microscopy

(Axioplan2, Zeiss, Germany) using appropriate filter com-

binations. Preabsorption of rabbit anti-CGRP antibodies

(Table 1) with the corresponding peptide (Acris Antibodies

GmbH, Germany) (1 lg of peptide in 50 ll of antibody final

dilution) or omission of any antibody (Table 1) abolished the

immunoreactivity. Substitution of goat anti-CGRP antibodies

for the rabbit anti-CGRP-antibodies gave the same typical

Brain Struct Funct

123

punctate staining of the perinuclear region (Zhang et al. 1994;

Guo et al. 1999) and the same percentage of co-localization

with Alexa-aBgt.

Analysis of cell size distribution and percentage of his-

tochemically specified neuron populations was done on

neuronal section profiles containing a nucleus visualized by

DAPI labeling. Mean cell diameter was measured as the

mean of the shortest and longest axes of the neuronal section

profile. Data obtained from lumbar and thoracic DRGs were

pooled since there were no significant differences in their

size distributions (Kolmogorov–Smirnov test, p [ 0.2).

Nerve crush experiment

DRG together with ventral, dorsal roots and spinal nerve

and a piece of sciatic nerve were dissected. Both the roots

and the nerves were crushed with forceps. The preparations

were incubated for 12 h at 37 �C in Hepes-Ringer buffer

(5.6 mM KCl, 136.4 mM NaCl, 1 mM MgCl2, 2.2 mM

CaCl2, 11 mM glucose, 10 mM HEPES, pH 7.4) bubbled

with carbogen (95 % O2/5 % CO2). Then the preparations

were shock frozen and sectioned longitudinally for histo-

chemical procedures.

Primary cell culture

The culturing procedure was performed as described earlier

(Nassenstein et al. 2010; Gold 2012) with some

modifications. Isolated DRGs were incubated in an enzyme

mixture [2 mg/ml collagenase type 1 and 2 mg/ml dispase

II (Sigma-Aldrich, Germany)] in calcium-, magnesium-free

Hanks’ balanced salt solution (Invitrogen, USA) for

15 min at 37 �C and neurons were dissociated by tritur-

ation with a glass Pasteur pipette. This step was repeated

three times, and then cells were washed by centrifugation

(three times at 1,000g for 2 min) and suspended in L15

medium (Invitrogen, USA) containing 10 % fetal bovine

serum (FBS). The cell suspension was transferred onto

poly-D-lysine/laminin (Sigma-Aldrich, Germany)-coated

coverslips or tissue culture 24-well plates (cells obtained

from two ganglia/well). After the suspended neurons had

adhered for 2 h, the neuron-attached coverslips or plates

were flooded with the L-15 medium (10 % FBS) and used

after 20–24 h in culture.

Calcium imaging

The intracellular calcium concentration ([Ca2?]i) mea-

surements were performed on primary cultures of DRG

neurons. Measurements were done in oxygenated Locke’s

buffer (pH 7.4) containing (in mM): NaHCO3 14.3,

NaH2PO4 1.2, KCl 5.6, NaCl 136, MgCl2 1.2, CaCl2 2.2, D-

glucose 10 at constant temperature 34–36 �C. The cells

were loaded for 30 min with 4 lM Fura-2 AM (Invitrogen,

USA) in L-15 medium containing 20 % FBS, placed in a

recording chamber (2 ml bath volume) and washed for

Table 1 Toxins, primary antibodies and secondary reagents used in this work

Toxin Conjugate Host Concentration Source

aBgt Alexa Fluor 488 Bungarus multicinctus 12.5 nM Invitrogen, USA

CTX Naja kaouthia 1.25 lM Own (Kukhtina et al. 2000)

NTII Naja oxiana 1.25 lM Own (Utkin et al. 2001)

Primary antibody, antigen Host Dilution Source

CGRP Goat 1:4,000 Biotrend, Germany

CGRP Rabbit 1:3,200 Peninsula Laboratories, USA

MBP Sheep 1:2,000 Chemicon, USA

NF200 Rabbit 1:600 Sigma-Aldrich, Germany

NF-L Mouse 1:600 Invitrogen, USA

PGP 9.5 Rabbit 1:5,000 Biotrend, Germany

TH Rabbit 1:400 Biotrend, Germany

TRPV1 Rabbit 1:2,000 Chemicon, USA

Reagent Conjugate Host Dilution Source

Anti-goat IgG Texas Red Donkey 1:400 Dianova, Germany

Anti-rabbit IgG Biotin Donkey 1:200 Amersham, UK

Anti-sheep IgG Biotin Donkey 1:400 Amersham, UK

Anti-mouse IgG Biotin Goat 1:200 Jackson Immunoresearch, USA

Streptavidin AMCA 1:200 Dianova, Germany

Brain Struct Funct

123

20 min by infusion of Locke’s buffer. Fura-2 was excited

at 340 and 380 nm wavelengths (k), and fluorescence was

collected at k[ 420 nm. Each cell was tracked indepen-

dently, and the fluorescence intensity ratio of 340/380 nm

at the beginning of the experiment was set to 100 % and

the relative changes were recorded. In each experiment, the

cells were exposed to nicotinic agonist epibatidine

(10-5 M) or PNU282987 (10-6 M) followed by capsaicin

(0.5 lM) and KCl (50 mM) applications. In some experi-

ments, cells were pre-incubated with nicotinic positive

allosteric modulator PNU120596 (10-6 M) or antagonists

CTX (10-6–10-7 M, Table 1) and mecamylamine

(10-4 M) before agonist application. All ligands except

CTX (Table 1) were purchased from Sigma-Aldrich,

Germany.

Neurotransmitter release

CGRP

For CGRP release studies, primary DRG neuronal culture

and whole ganglia were used. Whole DRGs were placed

into plate wells and repeatedly incubated at 35–37 �C with

the oxygenated Locke’s buffer, pH 7.4, for 15 min. Cell

cultures underwent the same procedures after aspiration of

growth medium and washing with oxygenated Locke’s

buffer, but incubation intervals were decreased to 10 min.

In order to exclude CGRP released due to plating and

initial handling of the preparations, we discarded the first

four incubation solutions and collected the fifth for ana-

lyzing basal CGRP release. Then the specimens were

exposed to successive incubation with the buffer alone or

buffer containing nicotinic drugs. Similarly, next incuba-

tion with 500 nM capsaicin was performed to evoke CGRP

release in the presence or absence of nicotinic drugs. The

supernatants were collected at 10 min intervals for neuro-

nal cultures and at 15 min intervals for ganglia, and ana-

lyzed for CGRP content by radioimmunoassay (RIA,

Phoenix Pharmaceuticals, USA) and by enzyme immuno-

assay [CGRP (rat) EIA kit, Bertin Pharma, France]

according to the manufacturer’s protocol. At the end of the

experiment, DRG cell cultures were exposed to 2 N acetic

acid for 10 min to determine the remaining intracellular

peptide content. Aliquots of this incubation were diluted

ten times prior to RIA of CGRP content.

The reliability of EIA was checked using CGRP

knockout and wild-type mouse DRGs. Extracts from DRGs

were made according to the protocol given by Bracci-

Laudieroa et al. (2002). Briefly, ten DRGs were placed in

0.25 ml of 1 M acetic acid and boiled for 5 min. Subse-

quently, the DRGs were disrupted in a ball mill. After

centrifugation (20 min, 960g, 4 �C) the supernatants were

collected, lyophilized and stored at -80 �C. Immediately

before starting the CGRP EIA, the pellets were solubilised

in 100 ll of EIA buffer. Samples were analyzed at a 1:100

dilution.

Glutamate

Freshly dissected spinal cords (T1–T12, L1–L5) were

sectioned at a thickness of 300 lm with a vibratome, then

slices were manually divided into dorsal and ventral parts.

Dorsal parts of spinal cord slices were placed into plate

wells and underwent the same protocol as for CGRP

release from whole ganglia with minor modifications.

Instead of capsaicin, 50 mM KCl solution was utilized to

evoke neurotransmitter release, and after stimulation the

slices were exposed for 15 min to oxygenated Locke’s

buffer alone to re-establish basal release. Glutamate con-

tent in supernatants was measured by high-performance

liquid chromatography (HPLC) of its phthaldialdehyde

(OPA, Sigma-Aldrich, Germany) derivative. The chro-

matograph system included binary HPLC pump Waters

1525 and Waters 2487 absorbance detector (Waters Corp.,

USA). The analytical column used was a reverse-phase

Phenomenex Nucleosil C18 (250 9 4.6 mm, 5 lm particle

size). Chromatographic separation was carried out at room

temperature in linear gradient of methanol (0–40 % for

15 min, 40–95 % for 1 min, 95–0 % for 1 min) in

12.5 mM phosphate buffer, pH 6.8, at a flow rate of 0.7 ml/

min and the detection was performed at 340 nm. The level

of glutamate was calculated by comparing the peak areas

with those of standards [glutamic acid (Sigma-Aldrich,

Germany), 125 pmol–2 nmol]. Using this method, the

minimal detectable amount of glutamate was 125 pmol.

Statistical analysis

Statistical analyses of normally distributed data were made

by one-way ANOVA for multiple comparisons or by Stu-

dent’s t test, when only two sets of data were compared.

For analysis of other data, Kruskal–Wallis one-way

ANOVA on ranks with Dunn’s post test or Kolmogorov–

Smirnov test was performed. p \ 0.05 was considered

significant.

Results

Specificity of labeling of DRG neuronal a7 nAChR-

expressing subpopulation

At the first step we showed that could reliably visualize a7

nAChR expressed by rat lumbar and thoracic DRG neurons

using a histochemical protocol with Alexa-aBgt, validated

on a7 nAChR knockout mouse tissues (Shelukhina et al.

Brain Struct Funct

123

2009). Positive neurons exhibited labeling of their cell

membrane as well as granular perinuclear labeling (Fig. 1a,

b) similar to that obtained for mouse DRGs (Shelukhina

et al. 2009). Double labeling with Alexa-aBgt and an

antibody against a neuronal cytoplasmic protein [protein

gene product 9.5 (PGP 9.5) (Thompson et al. 1983)]

showed that all a7 nAChR-positive cells were neurons (112

cells, n = 4) (Fig. 1c) and did not reveal any a7 nAChR-

positive glial satellite cells. The specificity of a7 nAChR

histochemical labeling was confirmed by competition of

Alexa-aBgt with a-CTX (Fig. 1f), capable of blocking

both muscle-type and a7 nAChRs, and by a lack of com-

petition for a-NTII specific for muscle-type nAChR

(Fig. 1e).

Morphological and neurochemical classification of a7

nAChR-expressing DRG neurons

DRG neurons are grossly divided into two major classes:

(1) Neurons with small to medium, neurofilament-poor

somata and thin non-myelinated C-fibers and (2) neurons

with medium to large, neurofilament-rich somata and thinly

(Ad) or thickly (Aa/b) myelinated A-fibers, respectively

(Lawson 2002). DRG neuronal soma size and fiber type are

known to correlate with its sensory modality. Generally,

the number of nociceptors decreases from small to large

DRG neurons (C [ Ad[ Aa/b), and the proportion of

non-nociceptors increases in parallel (C \ Ad\ Aa/b)

(Djouhri and Lawson 2004). On this basis, we investigated

cell size and neurofilament expression of a7 nAChR-

expressing neurons.

Alexa-aBgt-binding neurons formed an individual sub-

population with a significant *5 lm shift of their soma

diameter median to larger values relative to the total pop-

ulation of DRG neurons (Fig. 2a, b). Medium-sized a7

nAChR-expressing neurons prevailed, although the range

covered all cell sizes except the smallest group (15–20 lm)

(Fig. 2b). Alexa-aBgt-binding cells accounted for one-fifth

of all medium-sized and large DRG neurons, and\5 % of

small somata (Fig. 2c). Double labeling showed that 46 %

of a7 nAChR-expressing cells were neurofilament-immu-

nopositive (287/624 cells, n = 6, Fig. 3A(a), B). Conse-

quently, one half of the a7 nAChR-expressing population

consisted of small- to medium-sized, neurofilament-poor

DRG neurons with presumably thin, non-myelinated

C-fibers, and the other of medium-sized to large neurofil-

ament-rich cells with thick, myelinated Ad- and Aa/b-

axons.

To obtain more clues as to their potential involvement in

nociception, we applied two neurochemical markers widely

used to define two major classes of nociceptors: peptidergic

(CGRP-immunopositive) and non-peptidergic (IB4-binding)

Fig. 1 Detection of a7 nAChRs in rat DRG cryostat slices with

Alexa-aBgt (green). Alexa-aBgt-binding DRG neurons exhibit (a, b,

d) granular perinuclear labeling (arrowhead) and labeling of the cell

membrane (arrow), and (c) nerve fiber labeling. b Simultaneous

staining with antibody to cytoplasm marker PGP 9.5 (red) revealed

neuronal section profiles. c Neurofilament- (red) and a7 nAChR-

containing (green) nerve fibers localize next to each other, but are

separate. The specificity of a7 nAChR detection was confirmed by

(f) inhibition of Alexa-aBgt binding with a 100-fold excess of a-

CTX, capable of blocking both muscle-type and a7 nAChRs, and by

(e) a lack of inhibition with a-NTII specific for muscle-type nAChR.

Bars 40 lm

Brain Struct Funct

123

cells (Lawson et al. 2002; Woolf and Ma 2007; Fang et al.

2006; Djouhri and Lawson 2004). In accordance with

previous studies (Lawson et al. 2002) where CGRP was

shown to be expressed in small to large nociceptive neu-

rons with C-, Ad- and Aa/b-fibers, in our material CGRP-

positive cells were 12.5–57 lm in diameter (Fig. 4). IB4-

binding neurons were smaller (12.5–42 lm, Fig. 4)

including small C-fiber nociceptors exclusively (Fang et al.

2006). Double-labeling experiments demonstrated that the

vast majority of a7 nAChR-expressing DRG neurons were

peptidergic, whereas only a small fraction was non-pepti-

dergic nociceptors, as 83 % of Alexa-aBgt-labeled neurons

co-expressed CGRP [459/552 cells, n = 6, Fig. 3A(b), B)]

and only 15 % (127/838 cells, n = 6, Fig. 3A(c), B) bound

IB4.

Next, we investigated co-expression of TRPV1, a

transducer of some noxious stimuli (Tominaga et al. 1998;

Caterina et al. 1997; Wetsel 2011; Brenneis et al. 2013),

with a7 nAChR in DRG. This receptor is expressed in C-

and Ad-fiber nociceptors (Mitchell et al. 2010; Brenneis

et al. 2013; Kobayashi et al. 2005): in 49.2–59 % of small

to medium peptidergic and in 50–67 % of small non-pep-

tidergic neurons of adult rat DRGs (Guo et al. 1999; Price

and Flores 2007). In our conditions, 64.8 % (362/559 cells,

15–42 lm in diameter, n = 4) of peptidergic and 61.2 %

(336/549 cells, 14–40 lm, n = 4) of non-peptidergic DRG

neurons contained TRPV1 (Fig. 4). 28 % of Alexa-aBgt-

binding cells (347/1242 cells, n = 10) were TRPV1-im-

munolabeled [Fig. 3A(d), B]. Triple staining showed that,

among a7 nAChR-expressing neurons, almost all (92 %,

105/114 cells, n = 5) non-peptidergic (binding IB4) and a

third (29.7 %, 77/259 cells, n = 4) of peptidergic (CGRP-

positive) neurons were TRPV1 positive (Fig. 3C).

In sum, the presented histochemical data showed that a7

nAChR-expressing DRG neurons were primarily small to

large peptidergic nociceptive neurons, one half of which

had C-fibers and the other had A-fibers, which most likely

included both Ad- and Aa/b-fiber types. A small part of

Alexa-aBgt-binding cells was non-peptidergic IB4-binding

C-fiber nociceptors. Almost all non-peptidergic and a third

of peptidergic a7 nAChR-containing neurons expressed a

receptor responsive to noxious stimuli, i.e., TRPV1.

Axonal localization and transport of a7 nAChR

Alexa-aBgt-labeled varicose nerve fibers were observed in

DRG sections without additional intervention to block

axonal transport (Fig. 1c). There are two possible origins

for these axons: (1) DRG neurons and (2) postganglionic

noradrenergic sympathetic neurons which are known to

innervate DRGs (Kummer 1994) and to express a7 nAC-

hRs (Cuevas et al. 2000; Si and Lee 2003). Since these

A

B

C

Fig. 2 Size distribution of neurons in rat DRGs. The plots represent

the size distribution of (a) total (402 cells, n = 5) and b Alexa-aBgt-

labeled neurons (309 cells, n = 7) present in the ganglia; c relative

frequencies of labeled neurons within each size class. Size distribu-

tions of total and aBgt-binding populations were significantly

different according to Kolmogorov–Smirnov test, p \ 0.05. The

relative frequency (rf) was calculated with a formula rfi = (li/

ti) 9 8.5 %, where rfi is the relative frequency of the size group i, li is

the frequency of the size group i within labeled neurons, ti is the

frequency of the size group i within total neurons and 8.5 % is the

average percentage of labeled among total cells in DRGs (218/2553

cells, n = 5)

Brain Struct Funct

123

Alexa-aBgt-positive fibers were not immunoreactive (data

not shown) to tyrosine hydroxylase (TH) and the rate-

limiting enzyme of noradrenaline synthesis, and since

nerve fibers containing TH were thinner and generally

localized in the DRG capsule (Kummer 1994), these in-

traganglionic a7 nAChR-positive axons are unlikely of

sympathetic origin. They were immunonegative to both

heavy (NF200, Fig. 1c) and light (NF-L, data not shown)

components of neurofilament as well, leading us to assume

that most of them were non-myelinated, sensory C-fibers.

To increase the local level of intra-axonal a7 nAChR

and to demonstrate its directed transport in DRG neurons, a

complex nerve crush experiment was performed. It inclu-

ded dorsal root and both sciatic and spinal nerve crushing

to test transport in central and peripheral branches of axons

of DRG neurons, respectively. Since the sciatic and spinal

nerves do not consist of afferent fibers exclusively and also

contain efferent fibers, we performed crushing of ventral

root to monitor axonal transport in efferent motoneurons

separately.

A notable anterograde accumulation of Alexa-aBgt-

binding receptors was revealed at the peripheral (i.e.,

ganglionic) side of a dorsal root crush and at the central

(i.e., ganglionic) side of spinal and sciatic nerve crushes,

where a weak retrograde accumulation was demonstrated

as well (Fig. 5a–c, f). However, we did not find any aBgt-

binding sites in crushed ventral roots (Fig. 5d), in contrast

to clearly observed proximal accumulation of CGRP

expressed by efferent motoneurons (Gibson et al. 1984)

(Fig. 5e). This indicated that the axonal anterograde

transport in motoneurons functioned normally (Kashihara

et al. 1989), but did not contribute to a7 nAChR accu-

mulation observed in sciatic and spinal nerves. In addition

to extensive co-localisation of a7 nAChRs and CGRP in

Fig. 3 Double (a, b) and triple (c) labeling of rat DRG cryostat

sections with Alexa-aBgt (Green) and antibodies (red) to NF200 (a),

CGRP (b) and TRPV1 (d) as well as with biotinylated IB4 [(c), red].

Histograms (B, C) demonstrate percentage of double- and triple-

labeled cells among a7 nAChR-expressing (Alexa-aBgt-binding)

neurons. At least 4 (up to 10) independent experiments were carried

out for each pair (three) of substances. Bars 40 lm

Fig. 4 Comparison of mean diameters of CGRP-, IB4-, TRPV1- or

double-positive rat DRG neurons with Alexa-aBgt-binding ones.

Neuron diameter profiles were compared by Kruskal–Wallis one-way

ANOVA on ranks, Dunn’s post test, p \ 0.05, 309–726 cells per

condition, n = 4–7

Brain Struct Funct

123

cell bodies of DRG neurons (Fig. 3) we showed their joint

anterograde transport by some afferent axons in dorsal root

and in spinal and sciatic nerves (Fig. 5f). We did not reveal

if these axons were myelinated because immunoreactivity

to histochemical markers NF200 and myelin basic protein

(MBP) significantly decreased near the crush point. It was

earlier demonstrated that afferent neurons react to axotomy

by down-regulating neurofilament expression (Fornaro

et al. 2008). Collectively, the obtained results demonstrated

a strong anterograde and weak retrograde transport of a7

nAChRs in both central and peripheral processes of DRG

neurons, particularly, CGRPergic.

Calcium responses to a7 nAChR activation

Calcium entry through a7 nAChR activates a variety of

biological processes in neurons including, but not restricted

to, neurotransmitter release, cell signaling, and gene

expression (Shen and Yakel 2009; Uteshev 2012; Dajas-

Bailador and Wonnacott 2004). The scheme of our

experiment included from one to three applications of

nicotinic drugs followed by capsaicin administration to

distinguish C- and Ad-fiber nociceptive neurons (Brenneis

et al. 2013; Mitchell et al. 2010; Ringkamp et al. 2001) and

ended by addition of potassium chloride at high concen-

tration to confirm the excitability of the investigated cell.

To activate exclusively a7 nAChRs, their highly potent

selective agonist PNU282987 (10-6 M) (Bodnar et al.

2005) was used. It induced a small and fast-decaying cal-

cium rise in 5.8 % of cultured DRG neurons (31/537 cells,

n = 13) (Fig. 6). This response was amplified by pre-

treatment with the a7 nAChR-specific positive allosteric

modulator PNU120596 (10-6 M, Fig. 6), which cannot

initiate nicotinic channel opening, but decreases its

desensitization rate and increases agonist-evoked calcium

flux (Hurst et al. 2005). Under these conditions, 12 % of

Fig. 5 Accumulation a7

nAChR (Alexa-aBgt binding;

green) after blockade of axonal

transport by nerve crush.

a Dorsal root, b, f spinal nerve,

c sciatic nerve and d, e ventral

root. The sites of crushes are

indicated with arrows;

P peripheral, C central side of

the crush. f CGRP (red) is co-

transported with a7 nAChRs

(green) centrally (ganglionic

side) to a crush of a spinal nerve

(yellow, arrowheads), but in

ventral root (e) CGRP (red)

transport was revealed in the

absence of (d) a7 nAChR

(green) accumulation. Bars

40 lm

Brain Struct Funct

123

DRG neurons (30/249 cells, n = 6) showed an a7 nAChR-

mediated calcium response, with most of them were being

capsaicin-sensitive C- and Ad-fiber nociceptors (23/30

cells, n = 6), Fig. 6a).

Further, we used non-selective nicotinic agonist epi-

batidine to produce calcium responses through both ho-

mopentameric a7 nAChR and heteropentameric nAChR

subtypes and to compare their form, amplitude and number

with those evoked by the a7 nAChR-specific agonist.

Epibatidine (10-5 M) induced a rapid calcium rise in 33 %

(280/851 cells, n = 18) of DRG neurons, half of which

were nociceptive capsaicin-sensitive (55 %, 154/280 cells,

n = 18) (Fig. 6). This response was due to activation of

nAChRs as it was abolished by preincubation with the

neuronal nicotinic receptor antagonist mecamylamine

(10-4 M) (Fig. 6a, 0/204 cells, n = 4). In the presence of

a7-specific antagonist CTX (10-6 M) epibatidine provoked

[Ca2?]i increase in 30.6 % cells (139/454 cells, n = 9)

demonstrating that a7 nAChR could be rarely found in

isolation and is mainly co-expressed with other nAChR

subtypes in DRG neurons (Fig. 6a). The amplitude and

character of the epibatidine-evoked responses varied sig-

nificantly indicating involvement of different nAChR

types, along with amplification of calcium signal through a

number of mechanisms (Dajas-Bailador and Wonnacott

2004; Ween et al. 2010) (Fig. 6b–d). PNU282987 induced

a small and fast-decaying calcium response in comparison

with most epibatidine-evoked ones (Fig. 6b–d). It can be

explained by the typical fast and complete desensitization

of a7 nAChR rather than by different potency of two

agonists applied, since the pretreatment with the positive

allosteric modulator increased the amplitude of a7 nAChR-

mediated responses considerably, but they remained fast-

decaying. All these data show that along with other nAChR

subtypes which are not in the focus of this work, adult rat

DRG neurons express typical calcium-permeable func-

tional a7 nAChR.

Neurotransmitter release

CGRP

Before neurotransmitter release studies, we confirmed the

specificity of a commercially available enzyme immuno-

assay. We could detect 101.3 ± 0.5 pg of CGRP/DRG in

Fig. 6 Intracellular calcium response of rat DRG neurons to nicotinic

ligands: agonists epibatidine (EPI, 10-5 M) and PNU282987 (PNU,

10-6 M), antagonists a-CTX (10-6 M) and mecamylamine (MEC,

10-4 M), positive allosteric modulator PNU120596 (PAM, 10-6 M),

and to capsaicin (CPS, 0.5 lM). a Percentage of reactive neurons,

b calcium response curves, c amplitude and d extent of calcium rise

Brain Struct Funct

123

wild-type mouse, while measured concentration of CGRP

in knockout mouse tissue was at background level.

The study of putative a7 nAChR role in neurotrans-

mitter CGRP and glutamate release from DRG neurons

included three test systems: primary DRG neuronal cell

culture, whole ganglia and dorsal part of spinal cord slices

where these neurons terminate. Basal CGRP release from

DRG cell culture within 10 min was 69 ± 9.4 pg/ml

(Fig. 7a, n = 5). Stimulation by capsaicin at 0.5 lM

concentration caused a sevenfold increase in CGRP release

(490 ± 10.9 pg/ml) (Fig. 7a). When expressed as the per-

cent of intracellular CGRP total content from the beginning

to the end of the experiment, these values correspond to

3.6 % in the basal condition and 25.2 % in the capsaicin-

stimulated condition. Addition of non-specific nicotinic

agonist epibatidine (10-5 M) and a7-selective agonist PNU

282987 (10-6 M) or competitive antagonist CTX (10-6 M)

affected neither basal nor capsaicin-provoked CGRP

release (Fig. 7a). The experiment was repeated with some

modifications on freshly isolated DRGs to exclude any

influence of neuronal cell culture preparation procedures,

e.g. enzymatic treatment and mechanical trituration. After

dissection DRGs were incubated with oxygenated Locke’s

buffer, pH 7.4, for 1 h. Basal CGRP release was then

97 ± 11 pg/ml for 15 min (Fig. 7b, n = 4). Application of

capsaicin (0.5 lM) for the same period elevated CGRP

release to 316 ± 23 pg/ml. Nicotinic agonists such as

nicotine (10-4 M), epibatidine (10-5 M) and PNU282987

(10-6 M) did not provoke CGRP release from DRGs and

did not modulate capsaicin-stimulated release. The positive

allosteric modulator PNU120596 (10-6 M) did not mani-

fest a nicotinic agonist action in this test.

Glutamate

As a7 nAChR is known to modulate glutamatergic trans-

mission in different parts of CNS (McGehee et al. 1995;

Gotti et al. 2006; Gray et al. 1996; Jiang and Role 2008;

Wonnacott et al. 2006; Genzen and McGehee 2003), we

checked if nicotinic agonists act on basal or evoked release

of glutamate from DRG neuron terminals, and if an excess

of CGRP could modulate this process. Application of

50 mM KCl onto dorsal part of spinal cord slices led to a

2.1-fold increase of glutamate release as compared to the

basal level (Fig. 8, n = 4). Within the next 15 min wash-

out period we observed a significant decline in glutamate

release. However, we did not reveal statistically significant

changes of basal or KCl-provoked glutamate release under

application of nicotinic agonists epibatidine (10-5 M) and

PNU282987 (10-6 M). In addition we tested the reaction to

these drugs in the presence of a7 nAChR-specific positive

allosteric modulator PNU120596 (10-6 M) and competi-

tive antagonist CTX (10-6 M) as well as under excess of

CGRP (10-6 M). Neither positive nor negative modulation

of glutamate release was observed (Fig. 8).

Discussion

Here we showed that functionally active a7 nAChRs are

expressed and axonally transported by a subpopulation of

DRG neurons, mostly composed of C-, Ad- and Aa/b-fiber

* * * * *

A

B

Fig. 7 CGRP release from (a) rat DRG neuronal cell culture and

(b) whole ganglia. a Cells were exposed to four consecutive

incubation periods: (1) with buffer (basal release), (2) with nicotinic

ligands [agonists epibatidine (10-5 M) and PNU282987 (10-6 M)

and antagonist CTX (10-6 M)], (3) with capsaicin (0.5 lM) in the

presence or absence of nicotinic drugs and (4) with 2 N acetic acid to

measure the remaining intracellular CGRP content (asterisk—histo-

gram represents 10 % of the remaining CGRP content), n = 5

(mean ± SD). b Whole ganglia underwent the same procedure

without exposure to acid and with additional nicotinic ligands

[agonist nicotine (10-4 M) and positive allosteric modulator (PAM)

PNU120596 (10-6 M)], n = 4 (mean ± SD). Paired t test revealed a

significant (p \ 0.05) difference between basal and capsaicin-stimu-

lated releases in all groups, whereas nicotinic ligands modulate

neither basal nor capsaicin-stimulated CGRP release significantly

(one-way ANOVA)

Brain Struct Funct

123

peptidergic (CGRPergic) nociceptors. A part of these

neurons responded to noxious stimulus, i.e., capsaicin, and

expressed its receptor TRPV1. a7 nAChRs on DRG neu-

rons are calcium-permeable and can be activated and

positively allosterically modulated by their selective

ligands. Whereas a7 nAChRs localize almost exclusively

in CGRPergic cells, nicotinic ligands do not affect the

neuropeptide CGRP release from DRG neurons in vitro.

All our findings deal with neurons, since these are single

type of cells which, as we found, express a7 nAChRs in

DRGs, although there is an increasing number of studies

showing expression and functional role of these receptors

in CNS astrocytes (Shen and Yakel 2012; Liu et al. 2012)

and microglia (Shytle et al. 2004; Parada et al. 2013;

Thomsen and Mikkelsen 2012). Here in DRG, we dem-

onstrated plasmatic membrane localization of neuronal a7

nAChRs, in addition to their intracellular pool, that could

include both endoplasmatic and mitochondrial receptors

(Gergalova et al. 2012; Drisdel et al. 2004).

a7 nAChR-expressing cells compose an individual

population comprising one-fifth of total medium and large

DRG neurons and\5 % of small ones. This non-ubiquitous

localization might suggest a special functional role for a7

receptors. By applying a combination of well-known neu-

rochemical markers (Lawson et al. 2002; Woolf and Ma

2007; Fang et al. 2006; Djouhri and Lawson 2004), we

found that great majority of a7 nAChR-expressing DRG

neurons were nociceptive: 83.2 % peptidergic (CGRPergic)

and 15.2 % non-peptidergic. Based on their cell size

distribution and neurofilament content, we suggested that

a7 nAChR-bearing peptidergic nociceptors might include,

in addition to common small to medium rather slowly

conducting C- and Ad-fiber cells, rare medium to large

thickly myelinated fast-conducting Aa/b-fiber type (Law-

son 2002; Lawson et al. 2002).

a7 nAChRs localized on DRG neuronal cell body might

play a functional role, but in CNS they are preferentially

situated at preterminal or presynaptic sites (Gotti et al.

2009; Berg and Conroy 2002; Marchi and Grilli 2010).

This requires axonal transport from the site of protein

synthesis, i.e., the soma, towards terminals. Our crush

experiments on explanted ganglia with dorsal and ventral

roots and nerves indeed revealed an anterograde and a

weak retrograde transport of a7 nAChRs in the central and

peripheral branches of DRG neuron axons. It is consistent

with previous reports on axonal transport of aBgt-binding

sites in rat (Millington et al. 1985; Ninkovic and Hunt

1983) and chicken (Roth et al. 2000) sciatic nerve. Since in

our experiments a significant number of a7 nAChR-con-

taining axons were CGRP-immunoreactive, and a7 nAChR

transport was missing from ventral horn motoneurons, we

conclude that the majority of a7 nAChR transporting

neurons belong to the class of peptidergic nociceptors.

Being localized on nociceptive neurons, a7 nAChRs

along with other receptors, such as transient receptor poten-

tial (TRP) channels, acid-sensing sodium ion channels

(ASIC1-3), the ATP-gated P2X3 receptor, etc. (Woolf and

Ma 2007; Julius and Basbaum 2001), might make up an

Fig. 8 Glutamate release from dorsal part of rat spinal cord slices

(data normalized to basal release, mean ± SD, n = 4). Slices were

exposed to four consecutive incubation periods: (1) with buffer (basal

release), (2) with nicotinic agonists epibatidine (10-5 M) or

PNU282987 (10-6 M) and their mixture with a7 nAChR-specific

positive allosteric modulator (PAM) PNU120596 (10-6 M) or

antagonist CTX (10-6 M) or CGRP (10-6 M), (3) with 50 mM KCl

in the presence or absence of nicotinic drugs and (4) with buffer to re-

establish basal release. Paired t test revealed a significant (p \ 0.05)

difference between basal and KCl-stimulated glutamate releases in all

groups, whereas nicotinic ligands modulate neither basal nor capsa-

icin-stimulated CGRP release significantly (one-way ANOVA)

Brain Struct Funct

123

ensemble capable of detecting a broad spectrum of external

and internal stimuli. This idea is supported by findings that

non-selective nicotinic agonists excite DRG neurons in vitro

and in vivo (Steen and Reeh 1993; Bernardini et al. 2001;

Carr and Proske 1996; Rau et al. 2005; Genzen et al. 2001).

We revealed that specific agonist of a7 nAChR (PNU

282987) indeed evoked a small and fast-decaying calcium

rise in DRG neurons, which could be enhanced by a7

nAChR-selective positive allosteric modulator PNU120596.

Most of activated cells were capsaicin-sensitive C- and Ad-

fiber nociceptors and their number corresponded to per-

centage of a7 nAChR-expressing DRG neurons in histo-

chemical experiments, i.e., 12 versus 8.5 %, respectively. In

comparison with PNU 282987, a non-selective nicotinic

agonist epibatidine provoked a stronger [Ca2?]i increase in

greater number of cells (33 %), only a few of which could be

blocked by a7 nAChR-specific antagonist CTX. This finding

indicates the presence of other nAChR subtypes on the same

DRG neuron. Assortment of nAChRs varied, since the

amplitude and form of epibatidine-evoked calcium rises were

different, in accord with previous reports on non-uniform

electrophysiological responses of DRG neurons to nicotinic

drugs (Genzen et al. 2001; Rau et al. 2005).

High calcium permeability of a7 nAChR is the cause of

its efficacy as a modulator of neurotransmitter release in

CNS (Gray et al. 1996; Jiang and Role 2008; Gotti et al.

2006; Wonnacott et al. 2006; McGehee et al. 1995). It

might play the same role on DRG neurons, since cholin-

ergic interneurons presynaptically contact sensory termi-

nals in the spinal cord (Ribeiro-da-Silva and Cuello 1990)

and activation of nAChRs enhances both excitatory (Gen-

zen and McGehee 2003) and inhibitory (Liu et al. 2011)

synaptic transmission in dorsal horn. The release of glu-

tamate, the main excitatory neurotransmitter of both noci-

ceptive and non-nociceptive DRG neurons (Woolf and Ma

2007), was also analyzed in the present work. We did not

observe a significant change of basal or evoked glutamate

release from dorsal part of adult rat spinal cord in response

to nicotinic agonists. CGRP excess also did not influence

this process, although its inhibitory effect on nAChR is

well known (Di Angelantonio et al. 2003). The discrepancy

between our finding and the previously demonstrated

increase in excitatory transmission by a7 nAChR in neo-

natal rat dorsal horn (Genzen and McGehee 2003) might be

explained by significantly higher level of its expression

during earlier stages of ontogenesis, as documented for

numerous organs (Gotti and Clementi 2004; Putz et al.

2008; Zoli et al. 1995).

Peptidergic nociceptors, the main class of a7 nAChR-

expressing neurons, use neuropeptide CGRP as both a pain

signal neurotransmitter releasing in the spinal cord (Woolf

and Ma 2007) and as a pro-inflammatory agent on the

periphery (Richardson and Vasko 2002). Since a7 nAChR

is involved in release of every brain neurotransmitter (Gotti

et al. 2006), as well as in peripheral anti-inflammatory

reactions (Marrero et al. 2011; Olofsson et al. 2012; Huston

2012), and because nicotine could stimulate a CGRP

release from different organs (Franco-Cereceda et al. 1989;

Hua et al. 1994; Lou et al. 1992), we supposed that a7

receptor might modulate this process. However, in our

study nicotinic ligands including a7 nAChR-specific one

influenced neither basal nor capsaicin-evoked CGRP

release from DRG neurons. Although earlier nicotine-pro-

voked CGRP release from various tissues was explained by

its direct action through DRG neurons (Franco-Cereceda

et al. 1992), later publications have shown that nicotine

requires sympathetic efferent terminals activation to

mediate its effect upon primary afferent CGRP release

(Hua et al. 1995; Kawasaki et al. 2011). Our observations

also support the hypothesis about indirect stimulation of

CGRP release from sensory neurons by nicotinic drugs.

Elucidation of a7 nAChR functional role is in the focus

of intensive studies and appears to involve a cross talk with

other receptors (Marchi and Grilli 2010). In DRGs, we

demonstrated co-localization of a7 nAChR and TRPV1,

the capsaicin-sensitive transducer of heat, chemical (H?)

and mechanical noxious stimuli (Brenneis et al. 2013;

Wetsel 2011). All C-fiber non-peptidergic and a third of C-

and Ad-fiber peptidergic a7 nAChR-bearing nociceptors

expressed TRPV1, as follows from histochemical labeling.

In primary DRG cell culture, most neurons activated by a7-

specific agonist were capsaicin-sensitive, probably, due to

higher portion of small size cells surviving after culturing.

It is likely that a7 nAChRs co-expressed with TRPV1 in

DRG neurons might modulate its functioning, particularly,

contribute to nicotine modification of capsaicin-induced

TRPV1-mediated currents into those with faster desensiti-

zation and smaller amplitude (Fucile et al. 2005), although

application of nicotinic ligands did not alter capsaicin-

evoked CGRP release according to our study.

In summary, functional a7 nAChRs are expressed and

axonally transported to the spinal cord and periphery by

nociceptive DRG neurons, mainly by medium-sized

CGRPergic. These receptors do not modulate neurotrans-

mitter CGRP and glutamate release directly since nicotinic

ligands affected neither their basal nor provoked levels,

showing the necessity of further studies of the a7 nAChR

function in DRG neurons.

Acknowledgments This work was supported by the LOEWE Pro-

gram of the State of Hessen, Research Focus Non-neuronal cholinergic

system, by grants of Russian Foundation for Basic Research No. 13-04-

40377-N KOMFI and MCB RAS program. We thank Martin Boden-

benner, Tamara Papadakis, Silke Wiegand and Anna Goldenberg for

technical help, Dr. Christina Nassenstein for helpful discussion and

Prof. E. Weihe (Philipps-University Marburg) for providing CGRP

knockout mice. The authors declare no conflict of interests.

Brain Struct Funct

123

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