Identifiable neurons inhibited by Earth-strength magnetic stimuli in the mollusc Tritonia diomedea
1 Department of Biology, University of North Carolina, Chapel Hill, NC
27599-3280, USA
2 Friday Harbor Laboratories, University of Washington, Friday Harbor,
Washington 98250, USA
* Author for correspondence (e-mail: johnwang{at}email.unc.edu)
Accepted 31 December 2003
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Summary |
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Key words: Tritonia diomedea, magnetic, magnetoreception, orientation, navigation, mollusc
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Introduction |
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The marine mollusc Tritonia diomedea is a promising model system
for investigating the neural mechanisms underlying magnetic orientation
behavior. Behavioral studies have demonstrated that Tritonia has a
magnetic compass sense, which the animals may use during onshore and offshore
movements (Lohmann and Willows,
1987; Willows,
1999
). In addition, this animal has large, identifiable brain
cells and a central nervous system readily accessible to electrophysiology
(Chase, 2002
). Intracellular
recordings have shown that two bilaterally symmetric pairs of neurons in the
brain of Tritonia, known as left and right pedal 5 (LPd5 and RPd5)
and left and right pedal 6 (LPd6 and RPd6)
(Fig. 1), respond with
increased spiking to changes in Earth-strength magnetic fields
(Lohmann et al., 1991
;
Popescu and Willows, 1999
;
Cain, 2001
;
Wang et al., 2003
).
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In this study, we present evidence that another pair of neurons also
responds to changes in the ambient magnetic field. These cells, known as the
pedal 7 (Pd7) neurons, are adjacent to the Pd5 and Pd6 cells
(Fig. 1) and have similar
features. All three pairs of neurons have similar coloration, produce
neuropeptides known as TPeps (Willows et
al., 1997) and have electrophysiological and anatomical
characteristics of efferent neurons. In contrast to the Pd5 and Pd6 neurons,
however, the Pd7 neurons respond with decreased spiking to magnetic
stimuli.
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Materials and methods |
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Semi-intact animal preparations
Intracellular recordings from neurons were carried out in semi-intact
preparations (Willows, 1967;
Willows et al., 1973
). A small
incision was made directly above the brain on the animal's dorsal surface.
Non-magnetic tungsten hooks were used to retract the body wall, support the
animal in the seawater bath, and expose the central nervous system (CNS). A
wax-covered platform was placed beneath the brain and tungsten pins were used
to immobilize the central ganglia. Animals were allowed to recover for at
least 1 h after the brain had been immobilized. Neurons were then identified
by their size, coloration and location within the CNS and specific cells were
impaled with glass microelectrodes filled with 3 mol l1 KCl
(1530 M
). Electrical signals were amplified, monitored on an
oscilloscope, digitized using a CED 1401 A-D board, and analyzed using CED
Spike 2 software (Cambridge Electronics Design, Cambridge, UK).
Magnetic field measurements and magnetic field manipulations
The dissection chamber was located in the center of a Merritt 4-coil system
(Merritt et al., 1983). Each
of the four square coils measured 1.02 m on a side. The coil system was
powered by a BK Precision 1760 Triple output DC power supply, and a Radio
Shack TRS-80 portable computer was used to switch the coil on and off. Animals
were placed in the dissection chamber so that they faced magnetic 300°
(with 0° indicating magnetic north). When the coil was turned on, the
magnetic field was rotated 60° clockwise so that the animals were oriented
toward 240°. When the coil was off, the intensity of the ambient magnetic
field at the recording site was 53.0 µT and the inclination was 76.9°.
When the coil was turned on, alterations to the total magnetic intensity and
inclination angles were minimal (the total intensity of the magnetic field was
changed by +0.2% and the inclination angle by +0.4°).
Magnetic experiments with left pedal 7
Because the Pd7 neurons share many characteristics with the magnetically
sensitive Pd5 and Pd6 neurons, we investigated whether the Pd7 neurons also
respond to changes in the ambient magnetic field. We arbitrarily focused our
experiments on left pedal 7 (LPd7). Each LPd7 neuron was tested with two
treatments: a magnetic stimulus treatment and a control treatment. The
magnetic stimulus treatment (Popescu and
Willows, 1999; Cain,
2001
) consisted of a 15 min baseline period followed by a 30 min
magnetic stimulus period. During the stimulus period, the magnetic field was
first rotated 60° clockwise in a single step; then, after 1 min, it was
rotated 60° counterclockwise back to its original position
(Lohmann et al., 1991
;
Popescu and Willows, 1999
;
Cain, 2001
;
Wang et al., 2003
). This was
repeated so that the magnetic field was rotated once every minute
(Popescu and Willows, 1999
;
Cain, 2001
). The control
treatment consisted of a 15 min baseline and a subsequent 30 min period during
which the magnetic field was not changed
(Popescu and Willows, 1999
;
Cain, 2001
).
For each animal, the order of the two treatments was randomly determined.
After the first treatment, a recovery interval of at least 1 h elapsed before
the other treatment was applied. After the second trial had been completed,
spike frequencies (spikes min1) were calculated for each
baseline period and for the subsequent magnetic stimulus or control period
(Popescu and Willows, 1999;
Cain, 2001
). The LPd7 neurons
of nine different animals were tested using these procedures.
In addition, we tested the responses of several Pd7 neurons to a single 60° clockwise rotation of the magnetic field. In these trials, a 15 min baseline was first recorded from the Pd7 neuron before the magnetic field was rotated. Recordings continued for 15 min after the single rotation.
While recording from the Pd7 neurons, we simultaneously recorded from one or more of the following when possible: the RPd7 cell, the Pd5 neurons and the Pd6 cells. These simultaneous recordings allowed us to monitor the responses of these neurons to magnetic stimuli and also to determine whether LPd7 neurons had common synaptic inputs with LPd5 and LPd6.
Cobalt fills of the Pd7 neurons
To visualize the morphology of the Pd7 neurons, 500 mmol
l1 CoCl2 was pressure injected into the somata
(N=5 for LPd7; N=5 for RPd7) using a PV820 Picopump (World
Precision Instruments, Sarasota, FL, USA). After the CoCl2 diffused
throughout the neuron, the CNS was removed from the animal. The brain was
incubated for 15 min in 11°C filtered seawater with several drops of
concentrated ammonium sulfide (Croll,
1986). The CNS was then washed with seawater, fixed with 10%
formalin in filtered seawater for 24 h, dehydrated with an ascending ethanol
series, cleared with methyl salicylate, and mounted on a glass slide. The Pd7
somata and neurites were visualized using light microscopy.
Determining the direction of LPd7 action potentials
The direction of action potential propagation in LPd7 was determined by
recording intracellularly from this cell while simultaneously recording
extracellularly from left cerebral nerve 6 (LCeN6) and left cerebral nerve 3
(LCeN3), the two nerves found to contain Pd7 neurites (see Results).
Extracellular signals were recorded using en passant suction
electrodes in differential recording mode and amplified using a differential
AC amplifier (A-M Systems, Carlsborg, Washington, USA). LPd7 units in the
nerves were identified by determining which units corresponded one-to-one with
evoked potentials in the soma during stimulation (via current
injection through the intracellular electrode). Spontaneous LPd7 spikes were
then used to determine the direction of action potential propagation within
the left cerebral nerves.
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Results |
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Although the statistical analysis demonstrated that decreased spiking occurred during the magnetic stimulus periods, responses in individual trials were variable. Decreases in spike frequency occurred in eight of the nine individual trials. When decreases occurred, they ranged in magnitude from 0.3 to 8.7 spikes min1. Decreases in spike frequency occurred after a latency of about 3 to 10 min after the first field change (Figs 2, 4).
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For each animal, the change in spike frequency between the baseline and the subsequent magnetic stimulus period (mean change in spike frequency=3.4±1.0 spikes min1, N=9) was compared to the change between the baseline and the subsequent control period (mean change in spike frequency=0.2±0.2 spikes min1, N=9). A significantly larger change in spike frequency occurred during the magnetic stimulus treatment than during the control treatment (Wilcoxon Signed Ranks Test, P<0.01, N=9) (Fig. 3C).
Although the focus of this study was on LPd7, we were able to record simultaneously from RPd7 (the bilaterally symmetric mate of LPd7) in two animals during a magnetic stimulus treatment. In both cases, a decrease in spike frequency occurred during the magnetic stimulus period (Fig. 4A). We also recorded the responses of the Pd7 neurons to a single 60° clockwise rotation of the field in three animals, twice testing LPd7 and once testing RPd7 (Fig. 4B). In all three trials, spike frequency decreased.
Simultaneous recordings of the Pd5, Pd6 and Pd7 neurons indicated that the same magnetic stimuli that elicited decreased spiking in the Pd7 neurons elicited increased spiking in the Pd5 and Pd6 cells. In all four instances in which the activities of LPd6 and LPd7 were simultaneously recorded, LPd6 increased its firing rate during the magnetic stimulus period. Similarly, increased spiking was observed in LPd5 in four of five animals. One example of these multiple recording experiments is shown in Fig. 5A. These multi-cell recordings also allowed us to observe the postsynaptic potentials (PSPs) in the neurons. The results indicated that the postsynaptic potentials of LPd5, LPd6 and LPd7 are sometimes synchronous. However, LPd5 and LPd6 share many more synchronous PSPs with each other than with LPd7 (Fig. 5B).
|
Cobalt fills of LPd7
The somata of the Pd7 neurons are located in the dorsal, posterior region
of the pedal ganglia and often measure 200250 µm in diameter
(Fig. 6). Cobalt fills revealed
that each Pd7 cell possesses a large, primary neurite, which emerges from the
soma and enters the cerebral ganglia. At the cerebralpedal connective,
a small neurite branches and enters CeN6. The main neurite continues into the
cerebral ganglion before entering CeN3. In two of the five preparations, the
main neurite bifurcated within the cerebral ganglion with one branch entering
CeN3 while the other entered cerebral nerve 2 (CeN2).
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Direction of LPd7 action potential propagation
Simultaneous extracellular and intracellular recordings of LPd7 were
performed to determine the direction of action potential propagation. In all
preparations (N=3), spontaneous action potentials were observed in
the LPd7 soma before the corresponding extracellular units were recorded in
LCeN6 or LCeN3 (Fig. 7). This
demonstrated that action potentials in LPd7 neurons propagate from the central
ganglia toward the periphery.
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Discussion |
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The function of the Pd7 neurons is not known. Given that Tritonia
uses the Earth's magnetic field as an orientation cue
(Lohmann and Willows, 1987),
the Pd7 neurons appear likely to be components of the neural circuitry that
underlies magnetic orientation behavior. Among several possibilities, the
cells might play a role in detecting magnetic fields, in processing magnetic
field information, in generating a motor response that involves crawling along
a specific magnetic heading, or in suppressing behavior that might otherwise
impede orientation.
Characteristics of Pd7 neurons
The Pd7 neurons have neurites in nerves that innervate the anterior body
wall, oral veil and mouth. Each Pd7 neuron has neurites located in cerebral
nerve 6 (CeN6) and in cerebral nerve 3 (CeN3)
(Fig. 6). In two of five
preparations, neurites were also detected in cerebral nerve 2 (CeN2). Although
the cobalt fills did not allow us to identify the precise targets that the Pd7
neurons innervate, CeN6 innervates the ipsilateral body wall near the base of
the rhinophores (Willows et al.,
1973) as well as the eye. CeN3 and CeN2 innervate ipsilateral
areas of the oral veil and mouth (Willows
et al., 1973
). Simultaneous intracellular and extracellular
recordings of LPd7 show that spontaneous action potentials propagate away from
the central ganglia and toward the periphery through LCeN6 and LCeN3
(Fig. 7). Such an action
potential propagation pattern, from CNS to periphery, is typical of efferent
neurons (Bullock and Horridge,
1965
; Willows et al.,
1973
).
The Pd7 neurons produce a class of neuropeptides known as TPeps
(Lloyd et al., 1996;
Willows et al., 1997
;
Beck et al., 2000
). TPeps
increase the ciliary beating frequency of isolated foot epithelial cells
(Willows et al., 1997
), as
well as the ciliary transport rates of foot patches
(Willows et al., 1997
),
ciliated salivary ducts (Gaston,
1998
) and esophageal ciliated epithelial patches
(Pavlova et al., 1999
).
TPep-like immunoreactivity has been detected near ciliated epithelial cells in
the statocysts, oviduct, foot, salivary ducts, foregut and esophagus
(Willows et al., 1997
;
Gaston, 1998
;
Beck et al., 2000
). Possible
peripheral targets of the Pd7 neurons may therefore include ciliated
epithelial cells located on the anterior body wall, oral veil and the mouth.
TPeps have also been hypothesized to function as neurotransmitters or
neuromodulators within the CNS (Beck et
al., 2000
). TPep immunoreactivity has been localized in cell
bodies and neural processes throughout the CNS and in structures identified as
axosomatic synapses in the buccal ganglia
(Beck et al., 2000
). Thus, the
Pd7 neurons may interact with other neurons in the central ganglia.
An enigmatic characteristic of the Pd7 neurons, as well as the other
magnetically responsive neurons in Tritonia, is that a long latency
(about 115 min) occurs between the onset of the magnetic stimulus and
the onset of the neural response (Figs
2,
4A,
5;
Lohmann et al., 1991;
Popescu and Willows, 1999
;
Wang et al., 2003
). Similar or
longer latencies have been observed in electrophysiological responses of
honeybees (Korall and Martin,
1987
) and guinea pigs (Semm et
al., 1980
; Semm,
1983
) and in behavioral responses of spiny lobsters
(Lohmann et al., 1995
) and
bobolinks (Beason, 1989
). Some
possible reasons for these latencies are that the receptor mechanism or neural
processing of the magnetic information may require a significant period of
averaging, or that the nervous system may only periodically update magnetic
field information (Beason,
1989
; Walcott,
1996
; Wiltschko et al.,
1998
)
Comparison of Pd7 neurons with Pd5 and Pd6 neurons
To date, six neurons in Tritonia (LPd7, RPd7, LPd6, RPd6, LPd5 and
RPd5) have been shown to respond to changes in Earth-strength magnetic fields.
These cells share several similarities including the production of TPeps
(Lloyd et al., 1996;
Willows et al., 1997
),
characteristics of efferent neurons
(Popescu and Willows, 1999
;
Cain, 2001
;
Wang et al., 2003
),
responsiveness to other sensory stimuli such as rheotactic cues
(Murray et al., 1992
), and
some common synaptic inputs as indicated by synchronous postsynaptic
potentials (Fig. 5B).
The Pd7 neurons, however, differ from the Pd5 and Pd6 neurons in two ways.
Firstly, the Pd7 neurons are inhibited by magnetic stimuli whereas the Pd5 and
Pd6 neurons are excited. Secondly, the Pd7 neurons have neurites in nerves
that project to regions of the anterior body wall, oral veil and mouth. In
contrast, the Pd5 and Pd6 neurons have axons in nerves projecting to the foot
(Cain, 2001;
Wang et al., 2003
).
Given that Tritonia crawls using cilia on the ventral surface of
its foot, the Pd5 and Pd6 cells have been hypothesized to modulate cilia
involved in crawling (Willows et al.,
1997; Popescu and Willows,
1999
; Wang et al.,
2003
). Anatomical and electrophysiological characteristics of the
Pd7 neurons suggest that they too have efferent functions, but the role that
these neurons play during magnetic orientation remains poorly understood.
Although further studies will be needed to clarify the function of the Pd7
neurons, the identification of these magnetically responsive cells represents
another step toward understanding the sensory, processing, and motor
components that underlie magnetic orientation behavior in Tritonia
and other animals.
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Acknowledgments |
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