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First published online August 9, 2007
Journal of Experimental Biology 210, 2912-2922 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.006874
Sensory-induced modification of two motor patterns in the crab, Cancer pagurus
Institute of Neurobiology, Ulm University, D-89069 Ulm, Germany
* Author for correspondence (e-mail: wstein{at}neurobiologie.de)
Accepted 16 May 2007
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Summary |
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 TOP Summary IntroductionMaterials and methodsResultsDiscussionReferences |
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AGR has a bipolar cell body in the stomatogastric ganglion; it was activated by tension increase in gastric mill powerstroke muscles. While two spike initiation zones accounted for its spontaneous activity, active membrane properties (sag potentials, spike frequency adaptation) contributed to the AGR response to current injections.
When activated, AGR diminished spike activities in two pyloric motor neurons and prolonged the pyloric cycle period. Furthermore, AGR excited gastric mill protractor neurons, inhibited the retractor neuron and evoked phase-independent resetting of the gastric mill rhythm. Repetitive spike trains entrained the rhythm to both longer and shorter cycle periods. All AGR actions seemed to be mediated via at least two premotor projection neurons in the spatially distant commissural ganglia. The response of the gastric mill neurons was independent of AGR firing frequency.
Our results suggest that homologous proprioceptors can elicit similar effects on motor patterns while utilizing different mechanisms. This work thus provides an initial framework for future studies to determine underlying common principles.
Key words: Cancer pagurus, stomatogastric ganglion, projection neuron, central pattern generation, sensory regulation
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Introduction |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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;
Büschges, 2005;
Cropper et al., 2004;
Perrins et al., 2002).
While the effects of sensory regulation on the nervous systems are well
known in many organisms, a comparison of the effects of homologous sensory
systems in different, but related, species has rarely been investigated (e.g.
Wolf et al., 2001). For
example, are the effects of a sensory organ on motor output species-specific
or a more general aspect of sensory feedback? There are few systems for which
detailed information on the functional characteristics of circuit neurons and
the motor output is available to support such investigation. One such system
is the stomatogastric nervous system (STNS) of decapod crustaceans, which has
been characterized in several different crustaceans
(Marder and Bucher, 2001;
Nusbaum and Beenhakker, 2002;
Selverston and Moulins, 1987).
The STNS is an extension of the central nervous system, which contains two
CPGs that control the movement of food throughout the foregut. While the
gastric mill circuit generates the motor output responsible for the chewing
movement of three internal teeth (two lateral and one medial), the pyloric
pattern generator drives the rhythmic movements of the pyloric filter
apparatus (Hartline and Maynard,
1975; Maynard and Dando,
1974). The circuitries of the gastric mill and pyloric CPGs are
located in the stomatogastric ganglion (STG) and have extensively been
characterized in several crustacean species.
A proprioceptor that is particularly suited for investigating the effects
of sensory feedback on the STG circuits is the anterior gastric receptor
(AGR), which was initially described in the crayfish
(Larimer and Kennedy, 1966)
and then characterized in detail in the European lobster Homarus
gammarus (Combes et al.,
1995; Combes et al.,
1997; Combes et al.,
1999; Simmers and Moulins,
1988a; Simmers and Moulins,
1988b) and the spiny lobster Panulirus interruptus
(Elson et al., 1994). In both
lobster species AGR occurs as a single bipolar cell body in the STG and
measures the tension of the muscles responsible for protraction of the medial
tooth. Its function is thus comparable to that of vertebrate Golgi tendon
organs.
AGR spikes are initiated in its dendrites close to the location where AGR
innervates the bilaterally symmetric powerstroke muscles gm1
(Combes et al., 1993). The
response of AGR to a tonic tension increase of the gm1 muscles shows no
adaptation (Combes et al.,
1995). Interestingly, receptor activity can also be oscillatory,
depending on neuromodulatory influences on its dendritic compartment
(Combes et al., 1997).
AGR participates in a long-loop reflex pathway; that is, without direct
effects on the gastric mill cells in the STG. Rather, it excites premotor
descending projection neurons in the commissural ganglia, which, in turn,
affect the STG motor neurons (Combes et
al., 1999; Elson et al.,
1994; Simmers and Moulins,
1988a; Simmers and Moulins,
1988b). As a result, AGR activity can reset and entrain the
gastric mill rhythm (Elson et al.,
1994). Additionally, the AGR pathway is involved in a complex
control of forces exerted during the muscle powerstroke. While the gastric
mill neurons receive excitation during moderate AGR firing frequencies, this
excitation is either absent or is superimposed by inhibition
(Elson et al., 1994) at higher
firing frequencies (Simmers and Moulins,
1988b). Consequently, the gastric mill rhythm is reconfigured
depending on AGR firing frequency such that low frequencies synchronize the
movements of medial and lateral teeth while strong AGR activity causes
alternating teeth movements (Combes et
al., 1999).
Here, we study AGR in the isolated nervous system of the crab Cancer pagurus. For the first time, we characterize its effects on the pyloric rhythm, in addition to its influence on the gastric mill rhythm. While AGR effects on the gastric mill rhythm in C. pagurus are similar to those obtained in the lobster – it entrains and resets the gastric mill rhythm – intracellular recordings from AGR reveal that in C. pagurus, by contrast to the lobster, AGR possesses active membrane properties such as sag potentials and spike frequency adaptation. Besides, the sign of the response (excitation/inhibition) of the gastric mill neurons is independent of AGR firing frequency. Our results suggest that homologous proprioceptors in different, but related, species regulate motor pattern via different mechanisms.
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Materials and methods |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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4°C as described
previously (e.g. Blitz and Nusbaum,
1997). Experiments were performed on the isolated STNS
(Fig. 1A). The neurons in the
stomatogastric nervous system of C. pagurus and their connectivity
and properties are similar to those in Cancer borealis
(Heinzel et al., 1993;
Stein et al., 2005;
Stein et al., 2006).
Experiments were carried out in accordance with the European Communities
Council Directive of 24 November 1986 (86/609/EEC) and with the Guidelines
laid down by the NIH in the USA regarding the care and use of animals for
experimental procedures.
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Electrophysiology
Dissections were carried out as described previously
(Blitz and Nusbaum, 1997). The
STNS was pinned down in a silicone elastomer-lined (ELASTOSIL RT-601; Wacker,
Munich, Germany) Petri dish and superfused continuously (7–12 ml
min–1) with chilled physiological saline (10–13°C).
Standard intracellular and extracellular recording techniques were used in
this study (Stein et al.,
2005; Stein et al.,
2006). Extracellular recordings of neuronal activity were obtained
by electrically isolating individual sections of STNS nerves from the bath by
building a petroleum-jelly-based cylindrical compartment around a nerve
section. The action potentials propagating through the nerve were recorded by
placing one of two stainless steel electrode wires within this compartment.
The second wire was placed in the bath as a reference electrode. The
differential signal was recorded, filtered and amplified through an amplifier
(Model 1700; AM Systems, Carlsborg, WA, USA).
In several experiments, the insertions of the gm1 muscles were left attached to the stomach wall and transferred along with the innervating nerves to the Petri dish. The anterior apodemes of the muscles were then pinned down on Sylgard®, and the posterior ossicles into which the muscles insert were either fixed to a clamp for imposing passive stretch or pinned down on Sylgard® for monitoring isometric contractions.
The gastric mill rhythm was monitored by the activity of the lateral gastric (LG; one cell), dorsal gastric (DG; one cell) and gastric mill (GM; four cells) neurons. The gastric mill rhythm was considered spontaneously active when the LG neuron (a member of the gastric mill CPG) produced bursts of spike activity. The gastric mill cycle period was defined as the duration between the onset of an impulse burst in LG and the onset of the subsequent LG burst. LG was recorded extracellularly from the lateral gastric nerve (lgn); the DG and GM neurons were recorded extracellularly from the dorsal gastric nerve (dgn). AGR activity was assessed with extracellular recordings of the dgn, stn (stomatogastric nerve) and/or son (superior oesophageal nerve) or with intracellular recordings from the AGR cell body. All activities were measured either as the number of action potentials per burst or as the instantaneous firing frequency as determined by interspike interval. Mean values for all gastric mill-related parameters were determined from measurements of 10 consecutive cycles of gastric mill activity.
To facilitate intracellular recordings and access for applied solutions,
the STG was desheathed and visualized with white light transmitted through a
darkfield condenser (Nikon, Tokyo, Japan). Microelectrodes (15–25
M
) were filled with a solution containing 0.6 mol l–1
K2SO4 and 0.02 mol l–1 KCl.
Intracellular current injections were accomplished using NPI NEC 10L (NPI,
Tamm, Germany) and Axoclamp 2B amplifiers (Molecular Devices, Sunnyvale, CA,
USA) in bridge or single electrode discontinuous current clamp mode. Sample
rates in discontinuous current clamp mode ranged from 2 to 4 kHz.
Identification of STG neurons was done by assessing their activity patterns,
synaptic interactions and axonal projection pathways in combination with
current injections, as described previously
(Bartos and Nusbaum, 1997;
Blitz and Nusbaum, 1997;
Weimann et al., 1991).
Data analysis
Data were recorded onto computer hard disk using Spike2 (ver.
5.03–5.14; CED, Cambridge, UK) and a micro 1401 AD board (CED). Data
were analyzed with Spike2 script language. Individual scripts are available at
http://www.neurobiologie.de/spike2.
Final figures were prepared with CorelDraw (version 12.0 for Windows).
Graphics and statistics were generated using Excel (Microsoft) or Plotit
(version 3.2; Scientific Programming Enterprises, Haslett, MI, USA).
Statistical tests for data analysis were Student's t-test and paired
samples t-test. Data are presented as means ± s.d. N
refers to the number of animals, while n gives the number of trials.
For all statistical tests, significance with respect to the control was
indicated on the figures using the following symbols:
*P<0.05 and **P<0.01.
Nerve backfills and intracellular stainings
To determine the projection pattern of AGR, Lucifer Yellow–CH (LY;
Sigma-Aldrich, Munich, Germany) and NiCl2 backfills of the
dgn were made using standard techniques (e.g.
Coleman et al., 1992;
Blitz and Nusbaum, 1999). In
these experiments, a VaselineTM well was built around the dgn,
and the saline within the well was replaced with distilled water. After
several minutes, the distilled water was removed and replaced with a solution
of 10% LY or 10% NiCl2 in distilled water; then, the nerve was
transected within the well. The preparation was then incubated at 4°C for
18–72 h; the dye was then removed from the well and the preparation
fixed and mounted for viewing.
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). Dye was injected into the AGR cell body by
applying 5 nA hyperpolarizing current pulses for 5 s at a rate of 0.15 pulses
s–1 for about 1 h. |
Results |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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;
Simmers and Moulins, 1988a;
Simmers and Moulins, 1988b),
AGR possessed a large bipolar cell body (length 82.0±12.6 µm, width
35.2±5.8 µm; N=6) located at the posterior end of the STG
(Fig. 1B). In C.
pagurus, one of its axons projected posteriorly to the bilaterally
symmetric gastric mill muscles 1 (gm1) via the dorsal gastric nerve
dgn and the anterior gastric nerve agn
(Fig. 1A). The ascending axon
of AGR projected to the commissural ganglia (CoGs), via the
stomatogastric nerve stn and the superior oesophageal nerves
(sons; in contrast to the lobster, in which the axon is found in the
inferior oesophageal nerve ion). Backfills and stainings revealed no
obvious arborization within the STG. In the isolated nervous system, AGR was
usually tonically active with low firing frequencies (2.26±0.98 Hz;
N=12). In contrast to the lobsters H. gammarus
(Combes et al., 1997) and
P. interruptus (Elson et al.,
1994), AGR in C. pagurus never showed spontaneous
oscillations (N>50 recordings). However, rhythmic bursts could be
elicited with focal application of neuropeptide F1 (10–6 mol
l–1) to the dendritic region of AGR (N=4; data not
shown). Application of F1 to the soma, by contrast, never elicited
oscillations (N=6).
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To assess the functional role of AGR in C. pagurus, we used a neuromuscular preparation with the gm1 muscles and their innervation left intact while extracellularly recording AGR on the dgn and/or the stn. The anterior apodemes of the gm1 muscles were fixed to the Sylgard®. We then tested the AGR response with two different approaches. (1) We applied gentle stretch to the gm1 muscles by mechanically moving the posterior ossicles to which the gm1 muscles are attached (N=5). AGR activity increased with passive stretch (Fig. 2A). Stronger stretch resulted in stronger AGR responses. (2) In preparations with spontaneous gastric mill rhythms, we monitored AGR during active contractions of the gm1 muscles (N=6). Here, the posterior ossicle was pinned down to the Sylgard® so that activity of the GM motor neurons elicited isometric muscle contractions. We found that AGR firing frequency increased when GM was active (Fig. 2B). During both isometric contraction and passive stretch of the gm1 muscles, AGR spikes originated in the dendritic region close to the gm1 muscles and were relayed towards the CoGs. Together with the fact that AGR responded to active as well as passive forces developed in the muscles, this finding suggests that AGR functions as a tension receptor of the gm1 muscles.
Intracellular recordings from the AGR soma revealed a resting potential of –60.42±5.20 mV (N=11). No synaptic inputs of any kind were obvious. Action potentials showed a mean amplitude of 57.36±28.10 mV (N=11). AGR showed strong spike frequency adaptation, when depolarized with tonic current steps into the soma (2 s duration; Fig. 3A), which contrasts with the situation in the lobsters. In steady state, AGR firing rates settled at about 20% of the maximum firing frequencies reached at the beginning of the current injection. Initial firing frequencies could reach up to 100 Hz. When negative constant-current pulses were applied, the membrane potential attained an early hyperpolarized peak and then decayed (sag) to a steady level (Fig. 3B). On termination of the pulse, the membrane response transiently overshot the resting potential such that AGR firing frequencies exceeded those obtained prior to the pulse. Both peak potential and rebound depended on the amount of the injected current (Fig. 3A,B).
Response of the pyloric circuit to AGR stimulation
As a first step to examine AGR actions on the CPGs in the STG, we
characterized its influence on the pyloric motor neurons. We measured the
spike activity of pyloric neurons and pyloric cycle frequency while we applied
current pulses of two or more seconds into the AGR soma. Since AGR showed
spike frequency adaptation, we were unable to maintain AGR firing frequencies
over the time period required for measuring the different pyloric parameters
in some experiments. We thus used a sequence of current pulses (250 ms
duration followed by a pause of 250 ms) to prevent adaptation in these
experiments.
As is obvious from the original recording in
Fig. 4A, the activities of two
pyloric neurons were affected. On average, the number of spikes per burst of
the VD and IC neurons diminished significantly (VD, from 2.7±1.2 spikes
burst–1 to 0.9±0.9 spikes burst–1
during AGR firing; IC, from 6.1± 2.7 spikes burst–1 to
1.3±1.5 spikes burst–1 when AGR was activated;
N=6, P<0.01; Fig.
4B). However, not only pyloric neurons were affected by AGR
stimulation, but also the gastric mill motor neuron LG
(Fig. 4A, top recording; see
also below). Due to its gastropyloric interactions
(Bartos and Nusbaum, 1997), LG
has a strong impact on pyloric activity. To assess whether VD and IC were
directly affected by AGR or indirectly via LG actions on the pyloric
circuit, we eliminated the LG effect by hyperpolarizing it with current
injections into its soma to prevent it from spiking
(Fig. 4C). As a result, we
found that IC and VD still showed a significant change in spike activity in
these conditions (IC, from 4.08±1.5 s without AGR stimulation to
1.59±1.1 s during AGR, P<0.01, N=11; VD, from
2.68±0.9 s without AGR to 2.11±0.9 s with AGR,
P<0.01, N=11; Fig.
4D). Additionally, the pyloric period increased (from
0.9±0.2 s without AGR stimulation to 1.0±0.2 s during AGR
stimulation; P<0.05, N=11).
The AGR actions on IC and VD appear to be entirely mediated by the actions of descending projection neurons in the CoGs, because the pyloric circuit was no longer affected (N=6; data not shown) when we transected the sons and thus also the AGR axons to the CoGs.
Response of the gastric mill circuit to AGR stimulation
As mentioned above, gastric mill neurons were affected by AGR stimulation.
We characterized the response of several gastric mill neurons with
intracellular recordings (N>6 recordings for each type of neuron).
As shown in Fig. 5A, AGR
excited the medial tooth protractor motor neurons, the GMs (four cells) and
the protractor of the lateral teeth LG (a member of the gastric mill CPG),
while it inhibited the retractor motor neuron DG. No postsynaptic potentials
time-locked to the AGR spikes were observed in either motor neuron [although
discrete post-synaptic potentials (PSPs) were obvious, see below].
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In contrast to GM, which received uniform electrical PSPs throughout the duration of the AGR stimulation in low-Ca2+ saline (intact AGR axons), LG received two different types of PSPs (Fig. 5B). Before AGR stimulation, LG received rather large PSPs (Fig. 5Bi). At the beginning of the stimulation, these PSPs disappeared and a barrage of smaller PSPs commenced, which continued throughout the stimulus (Fig. 5Bii). Towards the end of the stimulus, the large PSPs reappeared and occasionally elicited spikes (arrow in Fig. 5B). After the end of the AGR stimulus, the small PSPs disappeared and the larger ones resumed their initial frequency. While we did not further investigate the origin of these PSPs, it is reasonable to assume that they were elicited by descending projection neurons. Thus, the AGR effect on LG was mediated via the actions of at least two different projection neurons.
In the lobster, AGR actions on the gastric mill circuit depended on AGR
firing frequency (Combes et al.,
1999; Elson et al.,
1994; Simmers and Moulins,
1988a). While low AGR firing frequencies excited the protractor
motor neurons, high firing frequencies failed to excite them. By contrast, we
found that in C. pagurus different AGR firing frequencies had similar
effects on the gastric mill motor neurons, at least qualitatively
(Fig. 6A). We verified this
finding with 2 s current injections into the AGR soma (N=7). Even
when the initial firing frequency at the beginning of the AGR depolarization
was close to 100 Hz, LG and GMs were still excited by the stimulus and DG
received inhibition. This phenomenon is exemplified for a single animal in
Fig. 6B, in which we plotted
the response of the LG motor neurons over the mean AGR firing frequency during
the stimulus. Apparently, there was no switch from the active state to the
inactive state of the LG neuron. However, LG firing frequency did not increase
with stronger AGR activity.
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;
Simmers and Moulins, 1988b).
Tension increases with the activity of the gastric mill motor neurons driving
the muscles. AGR will thus be activated according to the prevailing gastric
mill rhythm, which usually shows periods of a few seconds to more than 10 s
(Stein et al., 2006). To test
the effects of AGR on the gastric mill rhythm, we thus used current injections
into the AGR soma to rhythmically activate it with a burst duration of 2 s and
a period of 10 s. To start with, we applied current injections in preparations
without spontaneous gastric mill rhythms (N=28). In all experiments,
a gastric mill rhythm was elicited that included bursting of both the pro- and
retractor motor neurons (Fig.
6C). In these experiments, all protractor motor neurons (GMs and
LG) were active in time with the AGR stimulus train. In preparations with spontaneously active gastric mill rhythm (N=12), and thus in a situation in which AGR would likely be active in intact animals, rhythmic AGR stimulation entrained the rhythm. In the experiment shown in Fig. 7A, the period of the spontaneous gastric mill rhythm was 6.4±0.4 s (n=10). When AGR stimulus trains with a period of 5 s were applied, the period of the gastric mill rhythm sped up to a period of 5.0±0.3 s (n=10). By contrast, when the stimulation period was slower (8 s) than the control period of the gastric mill rhythm, the rhythm slowed down and now showed a period of 7.9±0.7 s (n=10). Stable phase-locking was attained after three or fewer transient cycles, and after the end of AGR stimulation the cycle period immediately reverted to its free-running value. Fig. 7B summarizes the results of N=11 experiments. Here, we plotted the period of the gastric mill rhythm over stimulation period. Both parameters were normalized to the period of the spontaneous gastric mill rhythm in order to compare animals with varying control periods. The plot shows that the rhythm could be entrained between 0.4 and 1.6 normalized stimulation periods, which indicates that rhythmic AGR stimulation could decrease or increase the period of rhythm up to 60% of the original period (slope 0.99, N=11, n=55, R2=0.99, P<0.01). With shorter or longer stimulus periods, we either observed no entrainment or two gastric mill cycles per stimulus cycle (Fig. 7B, right-hand grey box), respectively.
As is obvious from the original recording in
Fig. 7A (bottom), the onset of
the LG bursts could be prior to the onset of the AGR activity during
entrainment. This resulted from the rhythmic nature of the AGR stimulation. To
further characterize the impact of AGR on the gastric mill CPG, we thus also
tested the response of the gastric mill rhythm to single AGR bursts, applied
at different phases of the rhythm (Fig.
7C). The resulting phase–response curve
(Fig. 7D) revealed a linear
relationship between stimulus phase and the phase response of the rhythm with
a slope of 0.92 (N=13, n=45, R2=0.95,
P<0.01). A slope of 1.0 would indicate a strong reset of the
rhythm (type 0 reset) (Winfree,
2001; Izhikevich,
2006). Our results thus show that AGR stimulation was capable of
resetting the gastric mill rhythm at any phase of the rhythm.
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Discussion |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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;
Combes et al., 1995;
Combes et al., 1997;
Simmers and Moulins, 1988a;
Simmers and Moulins, 1988b)
and the spiny lobster Panulirus interruptus
(Elson et al., 1994). We now
aim at expanding our knowledge about this sensory neuron by examining it in a
third crustacean species, Cancer pagurus. Two main goals motivated
this study. (1) So far, only AGR actions on the gastric mill circuit have been
investigated. For a functional processing of food, however, the movement of
the teeth needs to be coordinated with the pyloric filter apparatus. We thus
examined the AGR influence on the pyloric rhythm with gastric mill rhythm
present and with inhibited gastric mill activity. (2) Some physiological
properties of AGR and some of its effects on the gastric mill rhythm appear to
differ between both lobster species, yet its function is similar (to measure
tension of the protractor muscles gm1). We wanted to compare AGR properties
and function in the crab with those in the lobster to identify features
important for all species investigated. This work also provides an initial
framework for future studies to determine how AGR affects the STG circuits in
the crab. All AGR effects appear to be mediated via CoG projection
neurons. Since these neurons are well known in the crab (reviewed in
Nusbaum and Beenhakker, 2002),
this system is ideally suited for studying the mechanisms underlying AGR
actions. AGR in the crab shows several similarities to AGR in the lobsters. It occurs as a single bipolar soma at the posterior end of the STG (Fig. 1) and responds to an increase in tension of the gm1 muscles (Fig. 2). It thus acts as a muscle tendon organ. As in the lobsters, AGR innervates the gm1 muscles and the CoGs; however, via different routes. In C. pagurus (Fig. 1A), and also in a close relative, C. borealis (M. P. Nusbaum and C.R.S., unpublished observation), the AGR axon projects through the dgn and agn towards the gm1 muscle (instead of the dvn and agn as in the lobster). Also, as in C. borealis, AGR innervates the CoGs via the sons (Fig. 1A).
One striking feature of AGR in the crab is the strong spike frequency
adaptation when depolarizing current is applied into the soma
(Fig. 3A). This tendency of the
membrane potential to move towards the resting potential was either not
present in lobsters or only weakly affected AGR firing rate
(Elson et al., 1994;
Simmers and Moulins, 1988b).
By contrast, in our experiments, we were unable to maintain firing frequencies
for an extended time period. In fact, it proved to be necessary in some
experiments to use repetitive current pulses to prevent the attenuation of AGR
firing frequencies. The success of this procedure in turn suggests that spike
frequency adaptation may be mediated by a voltage-dependent effect because
adaptation was less effective when the membrane potential returned to values
close to the resting potential in between two current pulses. After the end of
current injection, the membrane potential temporarily dropped below resting
potential and spiking usually stopped for a few seconds.
When AGR was hyperpolarized by tonic current injection, the membrane
potential attained an early peak and then decayed to a steady level
(Fig. 3B). This `sag' is common
in many STG neurons and is often elicited by a hyperpolarization-activated and
voltage-dependent current (inward rectifier, Ih)
(Buchholtz et al., 1992). In
sensory neurons, however, such a response to hyperpolarization appears rather
unusual, especially in the sense that AGR does not receive any obvious
synaptic input. In addition, we observed a post-inhibitory rebound after the
release from inhibition such that the membrane response transiently overshot
the resting potential (Fig.
3B). Consequently, AGR firing frequencies exceeded those obtained
prior to current injection.
Spike frequency adaptation, sag and post-inhibitory rebound may serve
different functions in the perception of muscle tension. Since AGR appears to
be a conditional oscillator, one obvious interpretation is that these
properties support the membrane oscillations obtained when neuropeptide F1 is
present at the dendritic region. However, such membrane properties are often
under neuromodulatory control and are either activated or amplified in the
presence of suitable modulators (Saideman
et al., 2007). It thus remains unclear why the observed membrane
properties are so potently present in non-rhythmic preparations (in normal
saline). It is conceivable, however, that voltage-dependent membrane
properties also support the normal functioning of the receptor in non-rhythmic
conditions. If the initial high firing frequencies and their quick adaptation
are functionally relevant (see below), sag and post-inhibitory rebound may
support a quick return to the resting potential (and thus help to prepare AGR
for the detection of the next muscle tension increase) by compensating the
hyperpolarization after the end of strong AGR activity.
Effects on the pyloric and gastric circuit
The impact of AGR on the pyloric rhythm has not been previously described.
Our results show that AGR diminished the spike activities of two pyloric
neurons, IC and VD. Both are follower neurons and do not directly interfere
with pattern generation in the pyloric circuit. Nevertheless, the pyloric
cycle period increased during AGR stimulation. All observed effects apparently
were not based on gastropyloric interactions since we obtained similar results
when blocking spike activity in the gastric mill neuron LG by applying tonic
hyperpolarizing current. LG strongly affects pyloric pattern generation
via the presynaptic inhibition it exerts on the axon terminals of an
excitatory descending projection neuron
(Bartos and Nusbaum, 1997).
Since all effects on the pyloric rhythm disappeared when AGR axons to the CoGs
were severed, we conclude that the inhibitory effect on the pyloric neurons
was mediated via descending projection neurons.
The actions of AGR on the gastric mill circuit were much more pronounced
than those on the pyloric circuit. The phase–response curve
(Fig. 7D) shows that AGR was
capable of resetting the gastric mill rhythm independently of stimulus phase.
Phase–response curves show the change in oscillator period elicited by
inputs occurring at different phases in the rhythm
(Prinz et al., 2003;
Wolf and Pearson, 1988) and
thus are a solid way of confirming the functional significance of a discrete
input to an oscillatory system
(Abramovich-Sivan and Akselrod,
1998). As a result of AGR resetting capabilities, rhythmic AGR
stimulation entrained the rhythm (Fig.
7A). Entrainment of the gastric mill rhythm has also been shown in
P. interruptus (Elson et al.,
1994) but has never been studied systematically. We here show that
AGR entrained the rhythm over a broad range of cycle periods and thus has a
profound impact on gastric mill pattern generation. The period of ongoing
rhythms could be decreased or increased up to 60%
(Fig. 7B).
Functional aspects of AGR response
In H. gammarus, AGR participates in a complex control of forces
exerted during the gm1 muscle powerstroke. This is exemplified by the fact
that the gastric mill neurons receive excitation during moderate AGR firing
frequencies, while this excitation is either absent
(Simmers and Moulins, 1988b)
or superimposed by inhibition (Elson et
al., 1994) during higher firing frequencies. This is due to the
nonlinear intrinsic membrane properties of an interneuron involved in
mediating AGR effects on the gastric mill neurons
(Simmers and Moulins, 1988b).
As a consequence, low AGR firing frequencies synchronize the movements of
medial and lateral teeth, while strong AGR firing elicits alternating
movements of the teeth (Combes et al.,
1999). However, our results suggest that the sign of the response
(excitation/inhibition) of gastric mill and pyloric neurons is independent of
AGR firing frequency (Fig. 6B).
This finding stresses the relevance of the phase–response curve, which
is then not only valid for moderate but also for strong AGR firing, and it
indicates that C. pagurus may not need a whole range of influences
from AGR to the gastric mill network. It is conceivable, therefore, that AGR
has built-in buffering mechanisms preventing it from going to high activity
rates (i.e. spike-rate adaptation) or from being shut down (i.e. sag).
Interestingly, however, LG received two types of excitatory (electrical)
synaptic inputs (Fig. 5B), one
of which was initially weakened and then reappeared
(Fig. 5B), probably due to the
spike frequency adaptation of AGR. Such delayed recurrence may lead to a
delayed onset of the LG burst and thus to a different phasing of the gastric
mill neurons. In this way the involvement of interneuronal pathways in
combination with intrinsic properties of AGR could functionally replace the
nonlinear membrane properties of the interneuronal pathway involved in H.
gammarus.
In general, this study shows that homologous proprioceptors in different, but related, species regulate motor pattern via different mechanisms. It provides an initial framework for future studies to examine the mechanisms of sensory processing and common principles of sensorimotor integration in the stomatogastric nervous system.
List of abbreviations
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