Download An implantable electrode design for both chronic in vivo

Journal of Neuroscience Methods 118 (2002) 33 /40
www.elsevier.com/locate/jneumeth
An implantable electrode design for both chronic in vivo nerve
recording and axon stimulation in freely behaving crayfish
Matthias Gruhn *, Werner Rathmayer
Universität Konstanz, Fachbereich Biologie, PF5560, D-78457 Konstanz, Germany
Received 22 August 2001; received in revised form 2 May 2002; accepted 2 May 2002
Abstract
A chronically implantable electrode design permitting alternate extracellular nerve recording and axon stimulation in freely
behaving crayfish was developed. The electrode consists of a double hook made from 20 mm thin platinum wire that can be fitted to
various nerve diameters, and is easily implantable. A fast curing, flexible two-component silicone was used for insulation. The
double hook was connected to plugs and fixed on the carapace of a crayfish allowing the animals to roam freely. The setup also
allows for repeated dis- and re-connection of the crayfish for alternating recording and stimulation. Two channel recordings were
used to determine directionality and to discriminate between afferent activity of the two stretch receptor neurons and efferent
activity of several motor neurons. In addition, they were also used to determine the conduction velocity of the recorded efferent
activity. Stable two-channel recordings could be obtained for up to 5 months and 15 days without apparent effects on the animal. In
vivo stimulation could be performed for at least 312 weeks. The implantable double hook is suitable for widespread use in
invertebrate neurobiology. # 2002 Elsevier Science B.V. All rights reserved.
Keywords: Hook electrode; Cuff electrode; Orconectes; Invertebrate; Crustacea technique
1. Introduction
To expand the relevance of neurobiological and
neuromuscular data acquired in reduced in vitro preparations, it is also important to examine the activity of
muscles and neurons in animals under conditions
matching natural situations and behaviors as close as
possible.
One common method to monitor in vivo activity is to
record electromyograms with extracellular electrodes
which pick up action potentials or summated synaptic
potentials of muscles. By placing the electrodes in
different muscles of the animal, it is possible to monitor
muscle activity patterns during complex motor behaviors. In studies of locomotion in invertebrates, like
walking in crabs and locusts (Clarac et al., 1987; Wolf,
1990) or locust flight (Wilson and Weis-Fogh, 1962;
* Corresponding author. Present address: Department of
Neurobiology and Behavior, Division of Biological Sciences, Cornell
University, W 159 Seeley G. Mudd Hall, Ithaca, NY 14853-2702,
USA. Tel.: /1-607-254-4357; fax: /1-607-254-4308
E-mail address: [email protected] (M. Gruhn).
Kutsch and Usherwood, 1970), for example, myogramm
recordings have lead to important results on the
coordinated use of various muscles. This technique
also allows placement of the electrodes without restricting the animal’s movements, and thus more closely
mirrors muscle activity in vivo. Using modern transmitter technology, it is now even possible to record muscle
activity remotely, e.g. in free flight of locusts (Kutsch et
al., 1993). Since the myogramm electrodes are inserted
into muscle tissue, however, long-term use of this type of
electrodes, once implanted is difficult. On the one hand,
the electrodes are bound to cause tissue damage after
repeated muscle contractions, and on the other hand the
quality of the recordings deteriorates.
For the chronic recording of nerve activity, so called
‘cuff’ electrodes have been developed in vertebrates (for
review see Loeb and Peck, 1996). Cuff electrodes are less
sensitive to movements within the animal and therefore
can be more suitable for long-term recordings as
compared with in vivo hook electrodes, where the nerve
is drawn into a vaseline filled PVC tip (Möhl and
Neumann, 1983). In addition to recording, cuff electrodes allow chronic nerve stimulation. The designs of the
0165-0270/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 5 - 0 2 7 0 ( 0 2 ) 0 0 1 2 7 - 9
34
M. Gruhn, W. Rathmayer / Journal of Neuroscience Methods 118 (2002) 33 /40
cuff vary, but they are usually prepared from PVC- or
silicone tubes that are tightly fitted around the nerve
(Loeb and Peck, 1996; Rodriguez et al., 2000; Deurloo
et al., 2000). One to three or even more electrode wires
are placed to run along the inner side of the tube to
cover most of the inner diameter. Among the most
durable cuff designs reported so far are spiral cuff
designs which have been used for example in dogs for
continuous recordings of up to 2 years (Rozman et al.,
2000).
In invertebrates, nerve diameters can often be much
smaller than in mammals for which cuff electrodes are
usually designed. Chronic recording of nerve activity
and nerve stimulation in invertebrates is therefore often
performed using blunt-end platinum wires that are
placed in close proximity to the nerve in question
(Lnenicka and Atwood, 1985a,b; Cooper et al., 1998).
In addition, as an adjustment of the vertebrate cuff
design to smaller nerve diameters, chronically implanted
recording electrodes for the use in the pond snail
Lymnea stagnalis (Parsons et al., 1983; Jansen et al.,
1997, 1999) or the crayfish Orconectes limosus (Böhm,
1996) have been developed in the past. These designs use
a fitted hook that is insulated in situ with a fast curing
silicone. This method allows a very quick electrode
assembly and relatively easy implantation, and has been
successfully used for short term-recordings (Böhm,
1996; Jansen et al., 1996, 1999).
Electrode designs that allow stable long-term nerve
recordings as well as stimulation via the same electrode
in freely behaving invertebrates have not been described
so far. We developed a double hook electrode that can
be assembled and implanted easily and that can be used
for alternate recording and chronic stimulation in
crayfish over several months. It is of high durability
and may be well suited for use in other invertebrate
preparations as well. We chose the second abdominal
nerve root (N2) in the crayfish Orconectes limosus to test
the electrode design. This nerve is well accessible under
the dorsal cuticle in the crayfish abdomen and contains
axons of motor and sensory neurons that are active
during the well-studied escape and positioning behavior.
2. Materials and methods
2.1. Animals
Adult crayfish (O. limosus , 7/12 cm from rostrum to
telson) were obtained from Lake Constance and kept in
running lake water tanks at approximately 8/12 8C in
the animal rearing facility of the university. The animals
were fed fish and vegetable pellets ad libitum. Experimental crayfish were kept in separate tanks and fed
frozen fish pellets.
2.2. Preparation of the double hook electrodes
Uncoated 20 mm thin platinum wire (Degussa-Huels
AG, Frankfurt, Germany) was first insulated with a
non-toxic two component silicone (Extrude† -low consistency, Kerr GmbH, Karlsruhe, Germany), freshly
mixed and applied by hand. The consistency of the
silicone is high enough to ensure equal distribution on
the wire and to still be fast curing, yielding insulation
within 5 min. The insulated wire was then cut into 4/5
cm long pieces, the length to cover the distance between
the third abdominal segment and the hind third of the
carapace, leaving enough space to allow the animal to
bend the abdomen. At one end, a 1 mm stretch of
insulation was removed with a scalpel, and a hook was
bent using number five dissecting forceps. At the other
end of the wire, the insulation was removed equally and
the wire was soldered to a plug that was later glued onto
the crayfish carapace.
For the preparation of the double hook electrode, two
previously prepared hook electrodes were positioned in
parallel. Using the non-toxic two component silicone
Kwik Cast† (WPI, Berlin, Germany), the two hooks
were fixed in close apposition, if necessary adjusted with
a pair of forceps and insulated up to the very beginning
of the hooks. The Kwik Cast† silicone has a lower
consistency than the Extrude† silicone and is thus better
suited for application close to the fine tip of the
electrodes.
2.3. Preparation of the implantation
First, a metal plate or glass dish was cooled on ice and
single drops of the two Kwik Cast† silicone components
were applied separately. The cooled dish prevented the
silicone from curing too quickly once mixed. For the
application and fine dosage of small drops of silicone
during the procedure, 200 ml Eppendorf pipette tips were
heated and pulled to produce fine diameter tips.
2.4. Anesthetizing the crayfish
We tested two possibilities for anesthetizing the
crayfish.
As a first method, 3 ml of a 110 mM MgCl2 solution
were injected into the abdomen. The muscles relaxed
immediately allowing surgery without hemolymph flow
from the wound. A small amount of crayfish saline (van
Harreveld, 1936) was injected into the animal after 25
min. Too large amounts of MgCl2 solution lead to
death, whereas amounts less than 2 ml only lead to
partial immobilization even in small crayfish (7 cm in
length, rostrum to telson). The amount necessary varied
from animal to animal making this method rather
unreliable.
M. Gruhn, W. Rathmayer / Journal of Neuroscience Methods 118 (2002) 33 /40
As a second method, we chilled the crayfish in a
covered dissection dish at about /15 8C for 25 min
after having dried them superficially. At the same time,
moist paper was formed into a roll and equally frozen.
Surgery was performed on ice and the frozen paper roll
was placed under the abdomen to cool the ventral nerve
cord during the procedure. The cooling also prevented
the hemolymph from being pulsed out at the site of
surgery. The crayfish were sedated for approximately
15 /20 min after anesthesia. Full activity was restored by
transferring the animals into water at room temperature.
This form of anesthesia caused much fewer fatalities
than the use of MgCl2 injection and was therefore the
method of choice for our experiments.
2.5. Implantation procedure
The dorsal cuticle of the third abdominal segment was
cut open laterally from the lateral superficial extensors
(lsem ) using a piece of razor blade in a holder. The piece
of cuticle was removed and later used to close the wound
after the surgery. After removal of the cuticle, the
hypodermis, the epidermis and connecting tissue were
carefully removed to allow access to the distal section of
the second nerve root (N2), which is easily accessible in a
dorsal preparation. The double hook was then placed at
the anterior end of the opening and lowered into the
wound. A glass hook was used to lift the nerve onto the
two hooks of the electrode. The electrode was then
pulled out of the hemolymph and residual fluid was
sucked up with a piece of paper tissue. Subsequently, the
two silicone components were mixed and immediately
applied to and around the hooks and the nerve. During
the curing process of approximately 1 min, the pipette
tip was used to keep the silicone droplet in place around
the double hook. After the curing of the silicone, the
electrode was lowered back into the body cavity.
The previously cut out piece of cuticle was shortened
at its anterior end by 1 /2 mm in order to accommodate
the exiting wires. It was then fitted back into its place in
the tergite and fixed with a non-toxic tissue glue
(Histoacryl, Braun-Melsungen, Germany). The plugs
of the electrode were glued to the posterior part of the
carapace, using super glue (PascoFix† , Pasco Handelsgesellschaft, Berlin, Germany) and corresponding filling
material (PascoFill† , Pasco, Berlin). Care was taken not
to stretch the wires too tightly to allow for possible tail
flips.
After the procedure, the crayfish were placed in water
at room temperature to recover for at least 30 min. The
two plugs allowed for flexible tethering of the crayfish
whenever necessary and permitted free roaming in tanks
when neither recording nor stimulation was in progress.
We tested electrical insulation of the two separate hooks
by connecting their respective plugs to two different
channels of an AC amplifier. The plug on the carapace
35
was insulated from the surrounding water with low
consistency two-component silicone (Kerr).
2.6. Extracellular recordings
The two electrodes were connected to an amplifier
through long insulated and flexible copper wires, which
reduced the restraint on the animal’s movement. The
two leads from the hook electrode were each connected
to the differential input of an amplifier to measure the
nerve activity at the two points along the axons, each
against ground.
Both recording channels were amplified 1000 / and
filtered at 3 kHz (low pass) and 1 Hz (high pass). The
recordings were monitored visually and acoustically,
and stored on a PC using an A/D interface (CED model
1401 plus, Cambridge Electronic Design, Cambridge,
UK) and ‘SPIKE 2’ software (CED, UK). An additional
2 / amplifier was added before the A/D interface and
compensated by a software offset of 0.5 to increase the
overall signal to noise ratio. The approximate distance
of 0.5 mm between the two hooks of the electrode
allowed the determination of the directionality and
speed of propagation of the recorded action potentials.
2.7. Long-term stimulation
The implanted double hook electrode was also used
for daily in vivo nerve stimulation for up to 312 weeks.
For this purpose, the two hooks were connected to a
square pulse stimulator (SD9, Grass, Astro-med GmbH,
Rodgau, Germany). Stimulus duration, frequency and
strength were adjusted for to match the fit of the
electrode around N2, and to ensure efficient acute and
chronic stimulation. At the same time, the quality of the
electrode fit could be monitored by recording from the
nerve during the experiment.
3. Results
In each segment, the extensor muscles of crayfish are
innervated by 12 extensor motor neurons (Wine and
Hagiwara, 1977; Drummond and Macmillan, 1998a,b)
the axons of which exit the segmental abdominal ganglia
through the second nerve root (N2). At the location
chosen for placement of the double hook, the N2 also
contains the afferent axons of the two stretch receptor
neurons and their four efferent inhibitory control
neurons (Jansen et al., 1970a,b, 1971; Wine and
Hagiwara, 1977; Drummond and Macmillan, 1998a).
Six of the extensor motor neurons innervate the deep
extensors, the other six the superficial extensors. The
deep extensors (dem’s ) form the antagonists of the fast
phasic flexors and are responsible for the fast extension
of the tail during escape swimming (tail flip). The
36
M. Gruhn, W. Rathmayer / Journal of Neuroscience Methods 118 (2002) 33 /40
superficial extensors (sem’s ) are considered to be
responsible for the positioning of the abdomen (Kennedy and Takeda, 1965a,b; Parnas and Atwood, 1966).
The design of the implanted electrode presented here
allowed us to monitor neuronal activity in the distal part
of the N2 of the crayfish while leaving the animal with
maximum freedom of movement. Fig. 1 shows a typical
two-channel extracellular nerve recording in vivo. In this
case, a phase of no apparent behavioral activity with few
tonic discharges is followed by a phase of pre-escape
activity, followed by giant fiber-mediated escape swimming. An escape response was elicited through tapping
onto the rostrum. During this behavior the repeated
activity of the phasic deep extensor neurons (arrows)
and the activity of the adapting stretch receptor neuron
(broken line) can be observed. The pauses in discharge
of the efferent extensor neurons contained in N2
(Drummond and Macmillan, 1998b; Edwards et al.,
1999) to be expected to occur during flexion are evident
in this recording. The signals recorded represent extracellular recordings of neuronal action potentials of the
extensor neurons because of the absence of cross talk
from flexor neuron activity or artifacts by extensor or
flexor muscles. The difference in the arrival time of the
signals at the two electrodes reflects different conduction
velocity. This recording is in accord with extracellular
nerve recordings on crayfish nerves in vitro and in vivo
(Böhm et al., 1997; Drummond and Macmillan,
1998a,b) as well as EMG recordings in the abdominal
extensors (Cooper et al., 1998). Moving artifacts could
be observed in very few preparations where the double
hook was lowered too deeply into the body cavity and
into close proximity of the deep flexor muscles. The
artifacts could be identified by the slower time course of
the signal and coincidence at the two sites of recording.
Such preparations were not further analyzed. The
crayfish could be connected to or disconnected from
Fig. 1. In vivo nerve activity, recorded at two locations along the N2
(traces 1 and 2, resp.), including pre-tail flip activity (solid bar), tail flip
(arrows) and consecutive activity of the fast adapting stretch receptor
neuron (broken line). Trace 1 recording is proximal to the ganglion.
Note the strong decrease in neuronal activity during the contraction
phase in the flexor muscles during the tail flip.
the recording or stimulation setup at any time. The
electrode design, being attached to the exoskeleton also
withstands the stress of even strong sudden movements
of the abdomen such as those occurring during tail flips.
The electrode design described permitted stable recordings on both channels for 5 months and 15 days,
only being terminated by molting of the crayfish. Fig. 2
shows in vivo recordings with the double hook electrode
at two positions along the nerve, at 1 day and at 5
months and 15 days after implanting. In the recording at
5 months and 15 days, a reduction in the amplitudes to
15 and 50%, depending on the channel, compared with
those recorded on the 1st day was observed. However, a
clear distinction between individual motor neurons in
the majority of animals was still possible due to distinct
differences in the amplitude of action potentials, their
directionality and propagation velocity. In the 120
animals under investigation, a reduction of the amplitude of action potentials down to values between 60 and
70% of the original size was observed after 3 /4 weeks of
recordings.
The electrodes can be built with different spacing
between the two hooks. We normally used a spacing of
0.5 mm, only sometimes a spacing up to 1 mm. Due to
the flexibility of the thin wires, a displacement to a
smaller inter-hook spacing can occur during the implantation. The distance between the hooks was verified
post mortem and in situ after taking out the nerve for
Fig. 2. In vivo nerve activity at two locations along the N2 at 1 day
(A), and 5 months and 15 days (B) after implanting the double hook
electrode. Note the different scale for voltage in A and B. Trace 1
recording proximal to the ganglion in A and B.
M. Gruhn, W. Rathmayer / Journal of Neuroscience Methods 118 (2002) 33 /40
37
Fig. 3. (A) In vivo nerve activity at two positions along the N2 showing the activity of five different efferent neurons (EN’s), (B) In vivo nerve activity
with one stretch receptor neuron (SN) and one efferent neuron (EN) being active. The lines show cursor positions to determine directionality of signal
propagation and conduction velocity. Note the different time resolution in A and B. Trace 1 recording proximal to the ganglion in A and B.
the calculation of the conduction velocities. Small
errors, however, cannot be excluded and can lead to
variations in the calculated conduction velocities from
preparation to preparation. Still, spacing of the hooks
allows for the discrimination among efferent and
afferent activity in freely behaving animals due to
different times of arrival of identified signals at the
two electrodes. In addition, it is possible to distinguish
between different neurons in single preparations on the
basis of the respective conduction velocities of the action
potentials and their recorded amplitudes. For example,
signals from the fast and slow adapting muscle stretch
receptors could usually be distinguished from each other
and from the activity of the motor or accessory neurons
by their directionality and amplitude. A typical recording with activity of five different efferent neurons
recorded at two locations along the nerve in vivo is
shown in Fig. 3A. All of the neurons are clearly
distinguishable due to their distinct peak amplitude
and the form of their signals. Fig. 3B shows the action
potentials of a stretch receptor neuron and of an efferent
neuron in one frame at higher time resolution. The
38
M. Gruhn, W. Rathmayer / Journal of Neuroscience Methods 118 (2002) 33 /40
different directionality and conduction velocity of the
signals in the two axons are clearly visible through the
set cursor positions.
In single preparations, we were able to monitor the
activity of at least nine efferent neurons with different
amplitudes of their action potentials. The neurons could
be grouped in two classes with approximate propagation
velocities between 1/7 and 10/15 m/s, respectively.
Simultaneous observation of animals during the recording allowed the correlation of neuronal activity recorded
with certain behaviors. When the crayfish performed
simple positioning of the abdomen, characterized by
holding it in a stretched position without visible additional activity, we found at least one to two neurons
discharging predominantly in a tonic mode. Conduction
velocities of neurons producing these signals was 2/4 m/
s. In addition, under this condition, up to three more
distinct neurons with conduction velocities between 5/
10 m/s were active sporadically. Shortly before, during
and after escape swimming, up to four more distinct
neurons were recruited (Fig. 1).
The electrode was also used in long-term in vivo
stimulation experiments of the N2, which allowed
successful stimulation of the superficial extensor muscles
for up to 25 days with specific stimulation regimes. This
was evidenced by eliciting even tail flips upon stimulation. As a final control for the successful long-term
stimulation, excitatory junction potentials were recorded intracellularly while stimulating the N2 with
the same stimulus intensity in situ via the implanted
electrode in nerve muscle preparations of the superficial
extensors after 3 weeks. This indicates that the neurons
stimulated were indeed motor neurons supplying the
sem’s .
4. Discussion
Implantable electrodes for the purpose of in vivo
recording and concomitant stimulation of nerves have
been used for almost 30 years (see Loeb and Peck, 1996;
Rodriguez et al., 2000). They prove to be extremely
valuable to assess the physiological relevance of data
gathered in vitro. The research in vertebrates has
produced a number of elaborate cuff electrode designs
(e.g. Fenik et al., 2001) as well as stable multi-channel
recording or stimulation devices for long-term studies
(e.g. Jellema and Teepan, 1995; Loeb and Peck, 1996;
Crampon et al., 1999; Grill and Mortimer, 2000;
Rodriguez et al., 2000).
In invertebrates, e.g. arthropods and mollusks, the
nerve bundles are smaller (often less than 100 mm) and
contain fewer axons than in most vertebrates. Many of
the electrode designs developed for vertebrate studies
are therefore not suitable for application in invertebrate
neurophysiology. After the introduction of cuff electro-
des adapted for mollusks (Parsons et al., 1983), new
methods have been developed for in vivo recordings in
the pond snail and crayfish (Böhm, 1996; Jansen et al.,
1997, 1999). As an extension of these methods, we
developed an easily assembled, easily implantable and
durable double hook electrode that forms a flexible
connection with the nerve in question. This enabled us
not only to record from but also to stimulate small
nerves in the abdomen of crayfish for several months.
The recordings obtained with this electrode design
represent true nerve recordings. This conclusion is based
on the different arrival time of efferent and afferent
signals at the two juxtaposed electrodes, on the fast time
course of the signals (as visible in Fig. 3A and B), and on
the resemblance of the discharge pattern when compared with extracellular nerve recordings from other
published work (Drummond and Macmillan, 1998a,b;
Jansen et al., 1996, 1999; Kutsch et al., 1999). Because of
the tight fit of the electrode design and its position on
the nerve, the signals obtained were free of cross talk
from neighboring nerve bundles and from muscular
activity. Moving artifacts were observed only in very few
preparations, and could be distinguished from the
neuronal signals by their time course and their coincidence at the two sites of recording.
In addition, the nerve recording in Fig. 1 shows clear
pauses in nerve activity which are to be expected during
the contraction phase in the flexor muscle. In the cases
where moving artifacts were observed, they occurred
during tail flip behavior when the extensors and flexors,
which are both equidistant from the recording sites,
were alternately active. In these preparations, no pauses
in recorded activity during tail flips was observed. Such
preparations were not evaluated.
Using the described double hook electrode, we could
monitor at least nine efferent neurons by the difference
in signal amplitudes. The possibility of separate entities
created by the summation of action potentials at higher
frequencies was ruled out through the observation of the
different units over prolonged periods of time during the
recording. At least five out of the nine efferent neurons
observed were active during positioning behavior, the
others were active shortly before and after escape
swimming. We were also able to monitor the activity
of both stretch receptor neurons.
Of the five neurons active while a crayfish was not
moving, two discharged tonically, the others sporadically. The identity of the neurons could not be correlated with the extensor motor neurons or the accessory
neurons described in other studies (Parnas and Atwood,
1966; Sokolove and Tatton, 1975; Drummond and
Macmillan, 1998a,b) because intracellular recordings
of the respective neurons within the ganglion were not
performed in the present study. It is, however, known
that the axons of motor neurons innervating the deep
extensor muscles in crayfish have larger diameters and
M. Gruhn, W. Rathmayer / Journal of Neuroscience Methods 118 (2002) 33 /40
thus higher conduction velocities than the ones innervating the superficial extensors (Sokolove and Tatton,
1975; Atwood, 1976; Drummond and Macmillan,
1998a). In the majority of the 120 animals in our study,
the signals of faster conducting neurons had larger
amplitudes in the extracellular recordings than those of
the slowly conducting ones. Thus, there is indirect
evidence that the slowly conducting motor neurons,
active during positioning behavior, belong to the group
innervating the sem’s, whereas the neurons active before
and after the tail flips are involved in the fast reextension of the abdomen and thus could be deep
extensor motor neurons.
At the location of the double hook electrode, however, 16 efferent neurons are known to be present in the
N2 (Drummond and Macmillan, 1998a). It cannot be
excluded that we recorded from more than nine neurons,
but were unable to distinguish between some of them
due to similar conduction velocities and identical signal
amplitudes. It is possible that among the five neurons
active during positioning, there were in fact not only
motor neurons innervating the superficial extensors but
also at least two accessory neurons (Acc1 and Acc2).
The superficial extensor motor neurons (SEMN’s) 3 and
4 have been reported to be recruited at similar voltages
as the accessory neurons Acc1 and 2 (Drummond and
Macmillan, 1998a). Thus it is possible that some of these
four neurons have been occluded from our resolution. In
addition, the activity in the N2 during the tail flips
produces large summed signals, which are likely to
occlude the activity of single deep extensor motor
neurons.
The whole procedure of electrode assembly, anesthetizing the crayfish and implanting the double hook
electrode around the second nerve root of the third
abdominal ganglion takes no more than 1 h. Due to its
long durability and the possibility to adapt the electrode
to various nerve diameters within minutes, this twochannel electrode could become a useful tool for
invertebrate neuroscientists. It allows performing longterm in vivo extracellular nerve recordings and stimulation with one implanted electrode for experiments in
freely behaving animals under minimal restraint. In
combination with video monitoring and high speed
camera systems, this electrode could allow new insights
into in vivo nerve activity during known behavioral
patterns and thus help to close gaps between in vitro and
in vivo experimental data.
Acknowledgements
We are especially grateful to Dr Andries ter Maat and
Anton Pieneman (Institute for Developmental Neurobiology, Vrije Universiteit, Amsterdam, Netherland) for
invaluable initial help with the electrode design and
39
surgical procedure. We also thank Tobias Müller, Dr
Bruce Johnson as well as Bruce Land for valuable
discussions and comments, and help with the English.
We thank the Degussa-Huels AG, Frankfurt, Germany,
for the gift of the platinum wire. This work was
supported by the DFG: Ra 118/8-2 and SFB 156 grant
to W.R.
References
Atwood HL. Organization and synaptic physiology of crustacean
neuromuscular systems. Progr Neurobiol 1976;7:291 /391.
Böhm H. Activity of the stomatogastric system in free-moving crayfish
Orconectes limosus Raf. Zoology 1996;99:247 /57.
Böhm H, Messaı̈ E, Heinzel HG. Activity of command fibers in freeranging crayfish Orconectes limosus Raf. Naturwissenschaften
1997;84:408 /10.
Clarac F, Libersat F, Pflüger HJ, Rathmayer W. Motor pattern
analysis in the shore crab (Carcinus maenas ) walking freely in water
and on land. J Exp Biol 1987;133:395 /414.
Cooper RL, Warren WM, Ashby HE. Activity of phasic motor
neurons partially transforms the neuronal and muscle phenotype to
a tonic-like state. Muscle Nerve 1998;21:921 /31.
Crampon MA, Sawan M, Brailovski V, Trochu F. New easy to install
nerve cuff electrode using shape memory alloy armature. Artif
Organs 1999;23:392 /5.
Deurloo K, Holsheimer J, Bergveld P. Nerve stimulation with a multicontact cuff electrode: Validation of model predictions. Arch
Physiol Biochem 2000;108:349 /59.
Drummond JM, Macmillan DL. The abdominal motor system of the
crayfish, Cherax destructor . I. Morphology and physiology of the
superficial extensor motor neurons. J Comp Physiol A
1998a;183:583 /601.
Drummond JM, Macmillan DL. The abdominal motor system of the
crayfish, Cherax destructor . II Morphology and physiology of the
deep extensor motor neurons. J Comp Physiol A 1998b;183:603 /
19.
Edwards DH, Heitler WJ, Krasne FB. Fifty years of a command
neuron: the neurobiology of escape behavior in the crayfish. TINS
1999;22(4):153 /61.
Fenik V, Fenik P, Kubin L. A simple cuff electrode for nerve recording
and stimulation in acute experiments on small animals. J Neurosci
Methods 2001;106:147 /51.
Grill WM, Mortimer JT. Neural and connective tissue response to
long-term implantation of multiple contact nerve cuff electrodes. J
Biomed Mater Res 2000;50:215 /26.
Jansen JKS, Njå A, Walløe L. Inhibitory control of the abdominal
stretch receptors of the crayfish I. The existence of a double
inhibitory feedback. Acta Physiol Scand 1970a;80:420 /5.
Jansen JKS, Njå A, Walløe L. Inhibitory control of the abdominal
stretch receptors of the crayfish. II Reflex input, segmental
distribution and output relations. Acta Physiol Scand
1970b;80:443 /9.
Jansen JKS, Njå A, Ormstad K, Walløe L. On the innervation of the
slowly adapting stretch receptor of the crayfish abdomen: an
electrophysiological approach. Acta Physiol Scand 1971;81:273 /
85.
Jansen RF, Pieneman AW, ter Maat A. Spontaneous switching
between ortho- and antidromic spiking as the normal mode of
firing in the cerebral giant neurons of freely behaving Lymnea
stagnalis . J Neurophysiol 1996;76:4206 /9.
Jansen RF, Pieneman AW, ter Maat A. Behavior-dependent activities
of a central pattern generator in freely behaving Lymnea stagnalis .
J Neurophysiol 1997;78:3415 /27.
40
M. Gruhn, W. Rathmayer / Journal of Neuroscience Methods 118 (2002) 33 /40
Jansen RF, Pieneman AW, ter Maat AT. Pattern generation in the
buccal system of freely behaving Lymnea stagnalis . J Neurophysiol
1999;82:3378 /91.
Jellema T, Teepen JLJM. A miniaturized cuff electrode for electrical
stimulation of peripheral nerves in the freely moving rat. Brain Res
Bull 1995;37:551 /4.
Kennedy D, Takeda K. Reflex control of abdominal flexor muscles in
the crayfish: I. The twitch system. J Exp Biol 1965a;43:211 /27.
Kennedy D, Takeda K. Reflex control of abdominal flexor muscles in
the crayfish. II. The tonic system. J Exp Biol 1965b;42:229 /46.
Kutsch W, Schwarz G, Fischer H, Kautz H. Wireless transmission of
muscle potentials during free flight of a locust. J Exp Biol
1993;185:367 /73.
Kutsch W, Usherwood PNR. Studies of the innervation and electrical
activity of flight muscles in the locust, Schistocerca gregaria . J Exp
Biol 1970;52:299 /312.
Kutsch W, van der Wall M, Fischer H. Analysis of free forward flight
of Schistocerca gregaria employing telemetric transmission of
muscle potentials. J Exp Zool 1999;284:119 /29.
Lnenicka GA, Atwood HL. Age-dependent long-term adaptation of
crayfish phasic motor axon synapses to altered activity. J Neurosci
1985a;5:459 /67.
Lnenicka GA, Atwood HL. Long-term facilitation and long-term
adaptation at synapses of a crayfish phasic motoneuron. J
Neurobiol 1985b;16:97 /110.
Loeb GE, Peck RA. Cuff electrodes for chronic stimulation and
recording of peripheral nerve activity. J Neurosci Methods
1996;64:95 /103.
Möhl B, Neumann L. Peripheral feedback-mechanisms in the locust
flight system. BIONA-Rept 1983;2:81 /7.
Parnas I, Atwood HL. Phasic and tonic neuromuscular systems in the
abdominal extensor muscles of the crayfish and rock lobster. Comp
Biochem Physiol 1966;18:701 /23.
Parsons DW, ter Maat A, Pinsker HM. Selective recording and
stimulation of individual identified neurons in freely behaving
Aplysia . Science 1983;221:1203 /6.
Rodriguez FJ, Ceballos D, Schuttler M, Valero A, Valderrama E,
Stieglitz T, Navarro X. Polyimide cuff electrodes for peripheral
nerve stimulation. J Neurosci Methods 2000;98:105 /18.
Rozman J, Zorko B, Bunc M. Selective recording of electroneurograms from the sciatic nerve of a dog with multi-electrode spiral
cuffs. Jpn J Physiol 2000;50:509 /14.
Sokolove PG, Tatton WG. Analysis of postural motoneuron activity
in crayfish abdomen. I. Coordination by premotoneuron connections. J Neurophysiol 1975;38:313 /31.
van Harreveld A. A physiological solution for freshwater crustaceans.
Proc Soc Exp Biol Med 1936;34:428 /32.
Wilson DM, Weis-Fogh T. Patterned activity of coordinated motor
units, studies in flying locusts. J Exp Biol 1962;39:643 /67.
Wine JJ, Hagiwara G. Crayfish escape behavior; I. The structure of
efferent and afferent neurons involved in abdominal extension. J
Comp Physiol 1977;121:145 /72.
Wolf H. Activity patterns of inhibitory motoneurones and their impact
on leg movement in tethered walking locusts. J Exp Biol
1990;152:281 /304.