Journal of Neuroscience Methods 186 (2010) 171–178
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Journal of Neuroscience Methods
journal homepage: www.elsevier.com
Sensor soc
in an in
Christop örgy
School of Life S
articl
Article history:
Received 24 O
Received in re
15 November
Accepted 17 N
Keywords:
Multielectrode
Central patter
Rhythmic motor output
Classical conditioning
Associative learning
Plasticity
Lymnaea
ding b
llula
bers
e fee
ther
) tec
. The
of the
ttern
of feeding was readily induced either by depolarizing an identified feeding-command neuron (the CV1a)
or by perfusing the chemosensory epitheliawith sucrose, a gustatory stimulus known to activate feeding.
Activity induced by sucrose is not restricted to the buccal ganglia but is distributed widely throughout
the CNS, notably in ganglia controlling locomotion, a behavior that must be coordinated with feeding.
The MEA also enabled us to record electrophysiological consequences of the associative conditioning
1. Introdu
Recent e
generation,
emphasize
taneously (B
2006).Whil
Lymnaea ha
neural basi
2008), elec
almost excl
electrodes.
that can be
six. As even
tens of thou
remain poo
tebrate mo
∗
Correspon
ences, Univers
fax: +44 0 127
E-mail add
0165-0270/$ –
doi:10.1016/j.of feeding behavior. The results suggest that MEA recording from an intact CNS enables distributed,
multiple-source neural activity to be analyzed in the context of biologically relevant behavior, behavioral
coordination and behavioral plasticity.
© 2009 Elsevier B.V. All rights reserved.
ction
fforts to understand sensory processing, motor pattern
behavioral selection and learning in neuronal networks
the need to analyze the activity ofmany neurons simul-
uzsaki, 2004; Briggman et al., 2005;Mann and Paulsen,
eworkonmolluscanmodel systems, suchasAplysiaand
s contributed substantially to our understanding of the
s of behavior (Kandel, 2001; Benjamin and Kemenes,
trophysiological studies in these systems have relied
usively on recordings made with intracellular micro-
This techniquenecessarily limits thenumber of neurons
monitored simultaneously to a maximum of about
the relatively simple brains of these molluscs contain
sands of neurons, their population coding properties
rly understood. Lymnaea is a well-established inver-
del of the organization of motor networks and their
ding author at: Sussex Centre for Neuroscience, School of Life Sci-
ity of Sussex, Brighton BN1 9QG, UK. Tel.: +44 0 1273 678508;
3 877586.
ress:m.o-shea@sussex.ac.uk (M. O’Shea).
plasticity induced by associative learning (Benjamin and Kemenes,
2008) and it is the subject of the present study to develop an effec-
tive method for multi-unit recording of the intact CNS of Lymnaea.
Activity patterns of more than 100 neurons have been moni-
tored simultaneously in the CNS of Aplysia using voltage-sensitive
dyes combined with optical imaging techniques (Zecevic´ et al.,
1989; Morton et al., 1991; Tsau et al., 1994; Antic et al., 1999).
Optical imaging of multi-neuronal activity has also been very suc-
cessfully applied in the CNS of the mollusc Tritonia (Frost et al.,
2007) and in the leech (Briggman et al., 2005). Importantly, these
studies have shown that large numbers of neurons participate
even in simple reflex behaviors, and have gone some way toward
characterizing the network properties of these neuronal assem-
blages. Optical techniques however have their limitations and not
all preparations are well suited for optical imaging of neuronal
activity. For example, our attempts to use voltage-sensitive dyes to
study multi-neuronal activity in the Lymnaea CNS have not been
successful. This is due to a combination of factors such as poor
uptake of dye, resulting in low signal-to-noise ratio and poor tem-
poral resolution.
The aim of the present study was therefore to assess the util-
ity of multielectrode arrays (MEAs) for multi-neuronal recordings
of behaviorally meaningful activity from the intact CNS of Lym-
see front matter © 2009 Elsevier B.V. All rights reserved.
jneumeth.2009.11.014y driven multi-neuronal activity and as
tact CNS on a multielectrode array
her A. Harris, Peter A. Passaro, Ildikó Kemenes, Gy
ciences, University of Sussex, Brighton BN1 9QG, UK
e info
ctober 2009
vised form
2009
ovember 2009
array (MEA)
n generator (CPG)
abstract
The neuronal network controlling fee
extensively investigated using intrace
beenpossible to record from largenum
the population coding properties of th
and neuronal networks controlling o
describe a multielectrode array (MEA
up to 60 electrodes on the intact CNS
nerves to the chemosensory epithelia
CNS containing the feeding central pa/locate/jneumeth
iative learning monitored
Kemenes, Michael O’Shea
∗
ehavior in the CNS of the mollusc Lymnaea stagnalis has been
r microelectrodes. Using microelectrodes however it has not
of neurons simultaneously and therefore little is knownabout
ding network. Neither can the relationships between feeding
behaviors be easily analyzed with microelectrodes. Here we
hnique for recording action potentials simultaneously from
preparation consists of the whole CNS connected by sensory
lip and esophagus. From the buccal ganglia, the region of the
generator (CPG), a rhythmic pattern of activity characteristic

172 C.A. Harris et al. / Journal of Neuroscience Methods 186 (2010) 171–178
Fig. 1. Semi-i
whole CNS con
long cerebral-
are shown con
shown connec
perfusion tube
outflow tube i
interneurons (
open circles in
of which there
naea. To do
of the isola
sory epithe
sensory str
behavior in
tive conditi
(Kemenes e
oped a meth
chamber su
grated into
recordings
about 20m
recording te
and stimula
manipulatin
over, the se
to be perfu
sucrose. Th
sequences of the chemosensory conditioning of behavior, to be
monitored in multiple neurons at multiple sites in the CNS. While
MEAs have been used to record from intact isolatedmolluscan gan-
ross, 1979; Novak and Wheeler, 1986), previous techniques
eparations did not involve the intact CNS and did not allow
y stim
test
nt ty
s spi
l nerv
de,
uron
aneo
S us
the f
f exp
ation
pha
MEA
lip-C
se th
by thglia (G
and pr
sensor
We
differe
taneou
centra
electro
interne
simult
the CN
rons of
type o
prepar
the eso
via the
intact
could u
ducedntact preparation of Lymnaea stagnalis. This schematic shows the
sisting of the cerebral and other associated ganglia connected by the
bucal connectives to the paired buccal ganglia. The cerebral ganglia
nected to the lips by three paired lip nerves and the buccal ganglia are
ted to the esophagus by the esophageal nerves. Lip and esophageal
s are shownwith arrows indicating the direction of flowof saline. The
s omitted. Labelled in the CNS are the cell body locations of identified
the CGC and CV1a) that are referred to in the text (black circles). The
the buccal ganglia represent the cell bodies of feedingmotor neurons
are many more than depicted.
this we have used a semi-intact preparation consisting
ted intact CNS connected by sensory nerves to sen-
lial surfaces of the lip and the esophagus (Fig. 1). These
uctures receive gustatory stimuli that activate feeding
the intact animal and are also required for associa-
oning of the feeding response to chemosensory stimuli
t al., 1986; Alexander et al., 1984). We have devel-
od for positioning this preparation in a glass perfusion
ch that the CNS lies on a sixty-electrode MEA inte-
the floor. The method produces excellent multi-site
from the CNSwith good signal-to-noise ratios and takes
in from dissection to recording. Importantly, this MEA
chnique can be combined with intracellular recording
tion methods, allowing the network consequences of
g activity in individual neurons to be assessed. More-
mi-intact preparation allows chemosensory structures
sed with behavior-activating gustatory stimuli such as
is allows feeding and related behaviors, and the con-
to these ch
et al., 1986
tionwe inve
feeding net
the CNS tha
Winlow, 19
In this p
provides an
coding prop
ent networ
memory in
feeding sys
further dev
network (V
2. Method
2.1. Anima
Adult Ly
starved for
50, KCl 1.6,
NaOH). The
before reco
and neuron
one to hold
from the su
We use
brain with
the esopha
Perfusing t
reliable wa
required to
lated with s
learning (M
nerves atta
ing it diffi
against the
removed a
ments invo
ganglia. In
bral commi
ganglia.ulation.
ed the effectiveness of this method by performing five
pes of experiment. In the firstwe simply recorded spon-
king activity in the paired buccal ganglia of the intact
ous system. In the second, using an intracellularmicro-
we stimulated a single identified feeding-command
in the cerebral ganglia (CV1a, McCrohan, 1984), while
usly recording spiking activity in the buccal ganglia of
ing the MEA. The buccal ganglia contain the motoneu-
eeding network (Benjamin and Rose, 1979). In the third
eriment we used the semi-intact lip-CNS-esophagus
and we stimulated the chemosensory epithelium of
gus with sucrose, while recording in the buccal ganglia
. In the fourth type of experiment we used the semi-
NS-esophagus preparation to investigate whether we
e MEA to monitor associative learning in the CNS pro-
e application of conditioned and unconditioned stimuli
emosensory structures involved in feeding (Kemenes
). Finally, using a semi-intact esophagus-CNS prepara-
stigatedwhether food-induced rhythmic activity in the
work is synchronized with activity in other ganglia of
t govern other behaviors such as locomotion (Syed and
91).
aper we demonstrate that the MEA recording method
extremely useful new tool to analyze the population
erties of single networks, interactions between differ-
ks and network-wide changes underlying associative
Lymnaea. Recording and analyzing the activity of many
tem neurons simultaneously will also contribute to the
elopment of a computer model of this complex neural
avoulis et al., 2007).
s
ls and dissection
mnaea stagnalis, bred at the University of Sussex, were
2–4 days and dissected in saline containing inmM:NaCl
MgCl
2
2, CaCl
2
3.5, HEPES 10 (pH adjusted to 7.9 using
outer connective tissue was removed from the ganglia
rding to reduce electrical resistance between the MEA
s. Specifically, two pairs of very fine forceps were used,
lose nerves and the other to peel the connective tissue
rface of the ganglia.
d a semi-intact preparation consisting of the intact
the lip attached by nerves to the cerebral ganglia and
gus attached by nerves to the buccal ganglia (Fig. 1).
he lumen of the esophagus with sucrose is the most
y to induce fictive feeding in the CNS, but the lip is
detect neutral taste stimuli, and both must be stimu-
ugar during CS+US presentation to induce associative
arra et al., 2007). The lip is large however and the
ching it to the cerebral ganglia are very short, mak-
cult to press ganglia other than the buccal ganglia
MEA while the lip is attached. The lip was therefore
nd only the esophagus remained attached in experi-
lving recordings from ganglia other than the buccal
preparations used for whole-CNS recordings the cere-
ssure was cut to expose the dorsal surface of the pedal