A cephalic projection neuron involved in locomotion is dye coupled to the dopaminergic neural network in the medicinal leech
1 Graduate Program in Neuroscience, University of Minnesota, 219 Hodson
Hall, 1980 Folwell Avenue, St Paul, MN 55108, USA
2 Departments of Neuroscience and Entomology, University of Minnesota, 219
Hodson Hall, 1980 Folwell Avenue, St Paul, MN 55108, USA
* Author for correspondence (e-mail: mesce001{at}umn.edu)
Accepted 28 September 2004
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Summary |
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Key words: central pattern generator, swimming, crawling, neuromodulation, serotonin, fictive locomotion, Hirudo medicinalis, leech
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Introduction |
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Dopamine often modulates the expression of locomotion and other rhythmic
motor patterns, including feeding-related programs, by inducing both short-
and long-lasting changes in the activity of neural circuits known as central
pattern generators (CPGs) (Barriere et al.,
2004; Harris-Warrick et al.,
1998
; Kabotyanski et al.,
2000
). In the leech, disruption of DA signaling induced simply by
bathing intact leeches in the DA receptor antagonist haloperidol
(Sakharov et al., 1994
)
disrupts crawling behavior, thus supporting the possibility that DA is
important for the modulation of locomotion in this preparation. The leech (in
particular, Hirudo medicinalis) has long served as a key invertebrate
model of the neural bases underlying rhythmic motor pattern generation
(Marder and Calabrese, 1996
).
In the present study, we present anatomical and physiological data documenting
the role of DA in the control of locomotion in the leech.
Locomotion in the leech is modulated by a number of cephalic projection
neurons that descend from the subesophageal ganglion (SEG) in the head. For
example, cell Tr1, a command-like interneuron, can activate the swimming motor
rhythm (Brodfuehrer and Friesen,
1986). Another descending brain interneuron, swim-inhibiting
neuron SIN-1, terminates swim episodes
(Brodfuehrer and Burns, 1995
).
Descending brain interneuron, R3b1, is a state-dependent neuron
(Esch et al., 2002
) shown to
activate swimming (when the leech is in deep water) or activate crawling
activity (when the leech is in shallow water). These examples suggest that
descending information from the head brain of the leech plays important roles
in behavioral choice, and the selection of which form of locomotion is
expressed.
Although swimming behavior has previously been shown to be modulated by the
biogenic amines in preparations lacking their head brain
(Hashemzadeh-Gargari and Friesen,
1989; Willard,
1981
), more recent studies have demonstrated the importance of the
brain and its chemical modulation (Crisp
and Mesce, 2003
; Mesce et al.,
2001
). Furthermore, descending brain interneurons, which are
modulated by the biogenic amines (Crisp and
Mesce, 2003
), have the potential to influence the activity of
down-stream aminergic cells. For example, stimulation of Tr1 excites
serotonergic Retzius cells in all segments of the central nervous system (CNS)
that have been examined to date
(Brodfuehrer and Friesen,
1986
). Together, these data suggest a tight coupling between the
activity of descending brain interneurons and the serotonergic system, which
is associated with swimming behavior.
In this study, we determined whether the catecholaminergic system is
coupled to cephalic descending interneurons. Here, we describe coupling
between the DA-synthesizing neurons of the leech head brain
(Crisp et al., 2002) and the
descending brain interneuron, Tr2, shown previously to terminate
(O'Gara and Friesen, 1995
;
Taylor et al., 2003
) or
trigger (Brodfuehrer and Friesen,
1986
) swimming. Our report is significant to the field of
locomotion, because no other studies have previously documented the
physiological actions of DA on the swim or crawl neural networks of the leech.
Data are consistent with DA biasing the nervous system to produce crawling. In
addition, we show that Tr2 receives rhythmic feedback from the crawl CPG. We
also demonstrate that DA inhibits the fictive motor rhythm for swimming, but
does not inhibit the crawling motor pattern, indicating that Tr2 is most
likely involved in both swimming and crawling.
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Materials and methods |
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Intracellular impalements, for iontophoretic injection of Neurobiotin or
physiological recordings, were performed using glass micropipettes with a
resistance of 4060 M; pipettes were tip filled with 5%
Neurobiotin (Vector Laboratories, Burlingame, CA, USA) dissolved in 2 mol
l1 potassium acetate and back-filled with 2 mol
l1 potassium acetate. Cells were filled with Neurobiotin by
iontophoretic injection using 500 ms pulses of 12 nA positive current
delivered at a rate of 1 Hz for a minimum of 15 min. Cephalic ganglia were
then fixed in 4% paraformaldehyde for 1 h at room temperature and rinsed in
iso-osmotic Millonig's buffer (13 mmol l1
NaH2HPO4, 86 mmol l1
Na2HPO4, 75 mmol l1 NaCl, pH 7.8).
Tissues were incubated for 3060 min in type IV collagenase (Sigma, St
Louis, MO, USA; 0.5 mg ml1 in phosphate-buffered saline with
1 mmol l1 CaCl2, pH 7.4) and then placed in a
blocking solution (containing 10% normal goat serum and 1% Triton X-100) for a
minimum of 2 h. Tissues were incubated overnight at 4°C in a 1:100
dilution of streptavidin [conjugated to the cyanine fluorophore Cy3 (Jackson
ImmunoResearch Laboratories, Inc., West Grove, PA, USA)] in hypo-osmotic
Millonig's buffer (13 mmol l1
NaH2HPO4, 86 mmol l1
Na2HPO4, pH 7.8).
Labeling DA-synthesizing neurons in the leech CNS was conducted according
to the methods of Crisp et al.
(2002). Briefly, ganglia (in
which neurons had been filled with Neurobiotin) were incubated for 48 h in a
mouse monoclonal antiserum raised against TH, the rate-limiting enzyme in the
synthesis of DA, diluted to 1:100 in hypo-osmotic Millonig's buffer
(containing 3% normal goat serum and 0.3% Triton X-100). This antibody was
shown previously to stain selectively only leech neurons that expressed
immunoreactivity to DA (Crisp et al.,
2002
). Tissues were then incubated for 48 h in a donkey anti-mouse
antiserum conjugated to the cyanine fluorophore Cy5 (Jackson ImmunoResearch)
diluted at 1:100 in hypo-osmotic Millonig's buffer (containing 3% normal goat
serum and 0.3% Triton X-100).
Physiological methods and analysis
Fictive crawling was monitored by recording simultaneously from the dorsal
posterior (DP) nerve, with extracellular electrodes
(Crisp and Mesce, 2003), and
from the segmentally repeated circular muscle excitor motor neuron CV, using
intracellular electrodes. Fictive crawling activity was diagnosed using
established criteria whereby the largest unit in the DP nerve, motor neuron
DE-3, fired in alteration with CV with a cycle period between 722 s
over multiple cycles within the same ganglion
(Eisenhart et al., 2000
).
Although crawling can be evoked by electrically stimulating the nerves of the
tail brain (Eisenhart et al.,
2000
), this technique was not used to elicit any of the crawling
reported here. In our study, all fictive crawling activity occurred
spontaneously (i.e., without deliberate intervention).
In only one set of experiments was electrical stimulation used to evoke
locomotion, which was in the form of fictive swimming. During these
experiments, the DP nerve (ganglion 16) was electrically stimulated once every
5 min, with a 1 s train of 20 pulses (10 ms, 5 V), to induce fictive swimming
(Mesce et al., 2001). Nerve
cords had their head brains removed, and were treated for 30 min in saline, 30
min in 50 µmol l1 DA, and 30 min in saline wash. No
electrical stimuli were presented for the first 10 min of a given treatment
period and a total of five stimuli were presented for each condition. DP
stimulation was deemed to cause swimming if swim bursts occurred within 10 s
of the shock. A paired Student's t-test was used to test the null
hypothesis that DA had no influence on the probability of shock-induced
swimming. Reported are the means ± the standard error of the means. For
all other statistical analyses, contingency tables were tested using the
Fisher Exact Test for Independence (Rees,
1985
).
Extracellular recordings were obtained using a Grass P15 amplifier (Grass Instruments, Quincy, MA, USA), and displayed and recorded digitally (at a sampling rate of 2 kHz) on a Macintosh Performa 5200 using the PowerLab data acquisition system (ADInstruments, NSW, Australia) and associated PowerLab Chart v3.6.3/s software. Intracellular signals were obtained using a Cornerstone IX2-700 electrometer (Dagan Corporation, Minneapolis, MN, USA) and recorded digitally in the same way as the extracellular signals. To maintain a fairly accurate measure of a cell's membrane potential, care was taken to balance the bridge circuit inside the cell while injecting current.
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Results |
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Dye transfer to the dopaminergic network was specific to Tr2. For example, intracellular Neurobiotin fills of other descending cephalic interneurons, including swim-trigger neuron Tr1 (N=9) and swim-inhibiting neuron SIN-1 (N=5), revealed very few or no dye-coupled neurons. Importantly, none of these dye-filled Tr1 or SIN-1 cells resulted in the transfer of Neurobiotin to any of the dopaminergic neurons.
Our observation that the command-like cell Tr2 was dye coupled to the DA
system, suggested to us that DA plays a role in the modulation of locomotion
in the leech. Thus, the following experiments were conducted to determine
whether DA could activate fictive swimming. We found that a 30 min bath
application of 50 µmol l1 DA to the leech head brain and
nerve cord did not induce any swim episodes (N=11). This result
sharply contrasted with what we had found previously in response to the bath
application of serotonin (5-HT) or octopamine (OA;
Mesce et al., 2001). Our new
data thus suggested that DA might inhibit rather than induce swimming
activity.
To study the potential inhibitory effects of DA, we first needed to
activate the swim motor pattern. We used the application of a mixture of 50
µmol l1 5-HT and 50 µmol l1 OA,
followed by a saline wash, as this was shown to be a robust and reliable
method for inducing swim episodes (Crisp
and Mesce, 2003; Mesce et al.,
2001
). This 5-HT/OA mixture was applied to the entire CNS for 30
min, followed by a 30 min washout. Then, 50 µmol l1 DA
was bath applied to the preparation (both head brain and nerve cord) to
determine if DA could inhibit swimming.
We observed that swim episodes abruptly ceased within 1 min of DA application in 100% of preparations examined (Fig. 2; N=5). By contrast, swimming persisted in four of the six control preparations in which the mixture washout was followed by saline without DA. The interaction between perfused salines (with or without DA) and the number of preparations that stopped swimming was statistically significant (P<0.05).
|
To demonstrate that DA can decrease the probability of swimming in other
contexts, we induced swimming by electrically shocking the DP nerve of
preparations perfused in saline (30 min) and in 50 µmol
l1 DA (30 min) (see Materials and methods). Because this
protocol works especially well in nerve cords lacking the head brain
(Hashemzadeh-Gargari and Friesen,
1989), we removed the head brain and ganglion 1 to maximize the
amount of swimming induced by DP nerve shock. By maximizing swim production,
we increased the rigorousness of our experiments testing whether DA can limit
fictive swimming. Among five preparations tested, we observed that 18 of 25 DP
nerve shocks initiated fictive swimming in saline (baseline control), whereas
only 6 of 25 shocks caused swimming in nerve cords perfused with DA. The mean
number of shock-evoked swims was 3.60±0.18 (saline) vs
1.20±0.43 (saline with DA). Using a paired Student's t-test
this difference was deemed statistically significant P=0.024.
The inhibitory effects of DA on swimming were not reversed within the 30 min washout period following DA application. For example, DP nerve-evoked swims occurred in only 3 of 25 stimuli (N=5), even though DP nerve activity persisted and preparations appeared viable. Although longer washes may result in greater reversibility, it is our perception that the viability of the preparation then comes into question by about 2 h, limiting us from accurately assessing the effects of extensive washings. In preparations with head brains intact, during washout periods (3060 min), fictive crawling or crawl-like patterns (see below) were expressed to the exclusion of spontaneous swimming.
The fictive crawling motor rhythm, which shares some motor neurons and
interneurons with the swimming neural network
(Baader, 1997;
Eisenhart et al., 2000
;
Esch et al., 2002
;
Kristan et al., 1988
), was
expressed in 10 of 11 preparations to which 50 µmol l1 DA
was bath applied (Fig. 3A). We
found no evidence that DA inhibited crawling. Whether DA induced crawling was
more difficult to determine, as four out of five control animals expressed
fictive crawling in saline that did not contain DA
(Fig. 3B).
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Although Tr2 has not previously been shown to be involved in crawling, it
has been shown to be excited by stimuli leading to another behavior,
shortening (Shaw and Kristan,
1997), suggesting that Tr2 has the potential of functioning in
more than one behavior. Here, we observed that Tr2 receives inhibitory
post-synaptic potentials (IPSPs) that are correlated in time with the fictive
crawling rhythm. Fig. 4 shows
simultaneous recordings from Tr2 in the SEG, the CV motor neuron, and DE-3
(largest unit in the DP nerve) in ganglion 9.
Fig. 4A demonstrates that
hyperpolarizing potentials in Tr2 were visible during the elongation phase of
each fictive crawling cycle, and correspond approximately to the peak of each
CV motor neuron burst. When the membrane potential of Tr2 was at 68 mV,
these IPSPs appeared as negative deflections.
Fig. 4B shows that when Tr2 was
hyperpolarized to 98 mV, the IPSPs appeared as positive deflections,
indicating the reversal of putative synaptic activity. Intracellular
stimulation of cell Tr2 did not cause any notable changes in the membrane
potential of CV, nor did stimulation of CV visibly alter the membrane
potential of cell Tr2 (data not shown). In addition, Tr2 stimulation did not
initiate crawling in any preparations examined (N=11), nor was it
observed to reset or alter any parameters of the crawl rhythm (e.g., cycle
period, data not shown). Recalling that Tr2 can inhibit or trigger swimming,
this finding underscores that Tr2 is linked to swim-based circuits. When the
crawl CPG was not active (Fig.
4C), depolarizing potentials in CV were correlated with IPSPs in
Tr2. (Note: Tr2 is hyperpolarized to 98 mV in
Fig. 4C, causing the IPSPs to
resemble excitatory PSPs.) The correlation of post-synaptic activity in CV and
Tr2 while the crawl CPG is inactive suggests that the rhythmic activity Tr2
received during fictive crawling is due to a pre-synaptic input shared by CV
and Tr2 (N=3).
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Discussion |
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Of the cephalic projection neurons examined, we found that Tr2 was the only
command-like neuron that transferred dye to the dopaminergic neural network of
the CNS. These DA cells in the SEG were labeled using an antiserum raised
against TH, the rate-limiting enzyme in the synthesis of DA. Crisp et al.
(2002) previously showed that
these same TH-immunoreactive (TH-ir) neurons also immunostained with an
antiserum raised against DA. Because DA, but not noradrenaline, was shown to
be the final synthetic end product of the TH-ir cells
(Crisp et al., 2002
), we can
conclude that the TH-ir cells are indeed dopaminergic.
The cessation of swimming by DA demonstrated here is quite remarkable,
because the swim-inducing paradigm we used (amine mixture removal) has been
shown to cause robust and persistent fictive swimming for up to hours
(Mesce et al., 2001;
Crisp and Mesce, 2003
). This
paradigm based on amine mixtures also has a physiological basis, which stems
from the observation that sets of OA and 5-HT-containing neurons are
co-activated by sensory neurons known to promote swimming
(Gilchrist and Mesce, 1997
).
Without such manipulations, nerve cords with intact brains rarely exhibit
spontaneous swimming (Mesce et al.,
2001
), a phenomenon likely due to the presence of descending
swim-inhibitory inputs (Brodfuehrer and
Burns, 1995
).
To demonstrate that DA can terminate fictive swimming induced by multiple
methods, we initiated swimming by electrically shocking the DP nerve in
preparations in which the brain was detached and the descending inhibition of
swimming was removed. In the presence of DA, such shock-induced swimming was
significantly inhibited. Aside from showing that DA inhibits swimming within
multiple contexts, such experiments indicate that the swim-related targets of
DA modulation may, in part, be located in the segmental nerve cord.
Segmentally repeated monosynaptic follower neurons of Tr2 have recently been
identified that inhibit swimming; these cells likely constitute an anti
swim-gating network (Taylor et al.,
2003). Future studies are warranted to determine whether DA and
its release, by way of Tr2 coupling, can influence these newly identified
targets. Dopamine's inhibitory actions, however, are likely to involve more
than segmental targets because our preliminary experiments indicate that DA
(100500 µmol l1) applied to the brain alone can
limit swimming activity.
In the leech, consistent dye transfer between neurons is associated with
electrical coupling (Davis,
1989; Wolszon et al.,
1995
). Although electrical coupling between neurons can be
accompanied by the absence of dye coupling if the neuronal tracer is too large
(e.g., Lucifer Yellow does not transfer between electrically coupled Retzius
neurons, K.M.C. and K.A.M., personal observation), to our knowledge, the
reverse has not been documented. Thus, there is no reason to doubt that Tr2
and the DA neurons are electrically coupled. Cell coupling appears to be an
important element for locomotor activity in the leech. For example, the
swim-gating neuron 204, a segmental command-like interneuron
(Shaw and Kristan, 1997
), is
weakly electrically coupled to the serotonergic swim-initiating interneuron 61
(Nusbaum and Kristan, 1986
).
Cells 204 and 61 contribute in parallel to the activation of the swim CPG, and
may coordinate their efforts through electrical coupling. Tr2 may likewise
contribute to the inhibition of swimming through coordinated efforts with the
DA system, which clearly has a strong inhibitory influence
(Fig. 2). The potential
activation of the DA system by Tr2 may, in turn, help to coordinate locomotor
activities with other behaviors. For example, dopaminergic innervation of the
stomatogastric nervous system in the medicinal leech
(Crisp et al., 2002
) and other
behavioral studies (O'Gara et al.,
1991
) suggest that DA may also regulate feeding-related behaviors.
Possibly, DA suppresses swimming while coordinating the consumption of a blood
meal, just as DA inhibits locomotion in the nematode Caenorhabditis
elegans during encounters with food substrates, thus prolonging feeding
(Horvitz et al., 1982
;
Sawin et al., 2000
). Because
we obtained no evidence that DA inhibits crawling, DA could promote crawling
behavior while the leech explores the surface of its prey looking for a point
from which to feed (Lent and Dickinson,
1984
).
Even though we demonstrated here that the swim-based Tr2 cell does not
trigger crawling under our conditions, we have shown that Tr2 is linked to the
crawling motor rhythm. This is because Tr2 shows rhythmic neural activity that
is matched to the fictive crawling rhythm. Additionally, Tr2 shares synaptic
input with the CV motor neuron, a cell responsible for the elongation phase of
crawling (Eisenhart et al.,
2000). This implies that Tr2 shares circuitry with networks
involved in the production of swimming, as well as crawling. The swim-gating
cell 204 also shares circuitry with crawling because its activity is often
time-locked to the elongation phase of crawling
(Baader, 1997
;
Kristan et al., 1988
). Because
of Tr2's coupling with the DA network, and its synaptic links to the crawl
pattern generator, both Tr2 and DA have the potential to bias the locomotor
system in favor of crawling as opposed to swimming.
It remains to be established whether bath application of DA is sufficient
to induce fictive crawling. Although crawling is clearly promoted in the
presence of DA, control preparations also expressed fictive crawling when the
applied bath solution contained no DA. One explanation is that we may have
inadvertently activated the segmental DA neurons. With the exception of the
head and tail brains, the somata of all DA-synthesizing neurons in the leech
nerve cord reside in nerve roots (specifically, within the anterior root
ganglia; Lent et al., 1983).
Each monopolar DA cell projects a single axon that projects centrally and
ramifies throughout multiple ganglia within the CNS
(Crisp et al., 2002
). Thus, by
freeing the nerve cord from the body, the axons from these cells become
transected. Such lesions may have led to a trauma-induced release of DA
throughout the CNS. In a preliminary study, we pretreated several leeches with
reserpine to eliminate the influence of endogenous DA (and other amines;
O'Gara et al., 1991
). In
normal saline, we did indeed observe that the level of spontaneous fictive
crawling was greatly reduced, further supporting a role for the amines.
Because of the intimate association between Tr2 and the dopaminergic neural
network, these neural elements, in particular, provide a fruitful area in
which to examine the cellular mechanisms of motor pattern selection. Perhaps,
studies in vertebrate systems may soon reveal a similar coupling between
command-like locomotor projection neurons and aminergic networks. Such studies
are certainly ripe for investigation, now that DA projection neurons have been
identified in the mammalian diencephalon and spinal cord
(Bjorklund and Skagerberg,
1979; Ridet et al.,
1992
), and electrical coupling among mammalian spinal neurons has
been shown to be of significance for locomotor control
(Kiehn and Tresch, 2002
).
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Acknowledgments |
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