Mechanisms and significance of reduced activity and responsiveness in resting frog tadpoles
University of Bristol, School of Biological Sciences, Bristol, UK
* Author for correspondence at present address: University of Hohenheim, Institute of Physiology, Garbenstrasse 30, 70593, Stuttgart, Germany (e-mail: lambert{at}uni-hohenheim.de)
Accepted 5 January 2004
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: GABA, Xenopus, tadpole, trigeminal, immobility, cement gland, tonic inhibition
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To investigate the neuronal basis of a state-dependent reduction in
responsiveness we chose the hatchling tadpole of Xenopus laevis.
These young animals spend 99% of their time at rest, hanging from a mucus
strand secreted by a cement gland on the front of the head. While attached in
this way to objects in the water or the surface meniscus, tadpoles are less
responsive to trunk skin touch (Boothby and
Roberts, 1992a) and dimming that excites the pineal eye
(Jamieson and Roberts, 2000
).
Finally, spontaneous swimming does not occur during attachment, but is seen in
unattached tadpoles (Jamieson and Roberts,
2000
). The simplicity of both the behaviour and the nervous system
of the hatchling Xenopus tadpole has been the impetus for its use in
the study of the neuronal control of locomotion (for a review, see
Roberts et al., 1997
). In
immobilised tadpoles fictive swimming can be recorded from the motor nerves
with no requirement for anaesthesia. The sensory stimuli, which start and stop
swimming in the behaving tadpole, are also effective in the fictive
preparation, and the neuronal pathways through which they act have been
partially characterized (for a review, see
Roberts, 1997
).
Touch-sensitive neurons innervate the tail, trunk and head skin and their
activation can initiate swimming (Clarke et
al., 1984
; Roberts and Sillar,
1990
; Roberts,
1980
). Swimming can also be initiated when dimming excites pineal
ganglion cells (Jamieson and Roberts,
1999
). Significantly for our study of reduced responsiveness,
pressure on the head skin or cement gland can stop swimming. These tissues are
innervated by trigeminal sensory neurons, which fire in response to pressure
and excite GABAergic midhindbrain reticulospinal neurons (MHRs). These, in
turn, produce GABAA-mediated IPSPs (inhibitory postsynaptic
potentials) in rhythmic spinal neurons and cause swimming activity to stop
(Roberts, 1980
; Boothby and
Roberts,
1992a
,b
;
Perrins et al., 2002
;
Li et al., 2003
). Could these
same pathways be responsible for long-term reductions in responsiveness?
Our aim was to use behavioural experiments to examine the reduction in responsiveness to skin touch and to dimming, when freely behaving tadpoles hang attached by a strand of mucus. We tested whether the sensory innervation of the cement gland and GABAergic inhibition are necessary for attachment to reduce responsiveness and spontaneous activity. Since reduced responsiveness and activity during attachment may make tadpoles less obvious to predators, we looked at the effect of cement gland denervation on the predation of tadpoles by one of their most important natural predators, Odonate nymphs. To investigate the neuronal basis of reduced activity and responses, we simulated cement gland attachment in an immobilised preparation where recordings of afferent activity from the cement gland could be made over extended periods. Our aim was to define the sensory input needed to induce and maintain the state of reduced responsiveness during attachment.
Preliminary accounts of this work have been presented (Lambert and Roberts,
2000a,b
).
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Behaviour
Experiments on responsiveness were carried out in a darkroom in circular
glass dishes 7 cm in diameter and filled to a depth of 3 cm with dechlorinated
tapwater or saline. A piece of 0.3 mm diameter wire fixed to the side of the
dish was submerged below the surface of the water. Using a wire entangled in
their cement gland mucus, tadpoles could be placed on the bottom of the dish
(unattached) or attached to the wire via the mucus (attached;
Fig. 1A). Unless stated
otherwise, responses to stimuli were tested 1 min after tadpoles were placed
in position.
|
To test the effect of attachment on responsiveness to touch stimuli, individual tadpoles were stroked across the base of the tail or on the head (Fig. 1B) while viewing through a binocular microscope. Each tadpole was tested only once.
To test the effect of attachment on responsiveness to dimming, two halogen
lights (Philips PAR20, 50 W) 50 cm from the dish were used. One was set to
full brightness and the other dimmed to minimal brightness. Four tadpoles at a
time were placed in the dish in either the unattached or attached position. To
stimulate, the bright light was switched off so the light dimmed from
4800 to
180 Lux. The light was switched on again after 5 s.
Responses were considered to be initiated by the dimming if they occurred
within those 5 s. Each tadpole was tested only once.
Mandibular nerve lesioning
Under anaesthetic, skin on the side of the head overlying the distal end of
the mandibular nerve was opened, enabling the nerve to be seen. The nerve was
then severed using two tungsten needles
(Fig. 1C). The tadpole was
re-pinned on its other side and the process repeated. As a control, a sham
operation involved making a lesion of tissue just caudal to the nerve. The
tadpole was allowed to recover for 25 min in saline prior to tests on
behaviour (in saline). Testing for responsiveness began after 5 min adaptation
to the light conditions (one halogen light at 50 cm, 4600 Lux). For each
tadpole, unattached and attached tests alternated and a total of five tests
were done in each state.
For experiments testing spontaneous activity and predation, the anaesthetized tadpole was placed in a groove in the Sylgard base of a small dish and secured with a V-shaped pin around the neck region. A 1 mm wide, chisel-like blade made from a piece of razorblade was pressed down through the tadpole in a single motion until it cut into the Sylgard layer below. To cut the mandibular nerve innervating the cement gland, the blade was positioned parallel to the proximal surface of the cement gland. To perform a control cut, the blade was positioned level with the caudal edge of the eye and the most dorsal edge of the cement gland and in a dorso-ventral direction. After surgery tadpoles were placed in saline for five minutes and then into 50:50 saline:aerated tapwater for 2 h to allow the wound to heal.
Predation
Damselfly (Zygoptera) nymphs, 11.5 cm (measured from the head to the
base of the lamellae), were collected from a drainage canal near Bristol and
identified (Brooks, 1997) as
mostly Ischnura elegans and Coenagrion puella, but with some
Platycnemus pennipes and Coenagrion pulchellum. Nymphs were
kept under a 12 h:12 h light:dark cycle, fed on a diet of Daphnia,
starved for 3 days prior to trials and used only once. For testing, a nymph
was transferred to a white circular plastic dish (6.5 cm diameter) of aerated
tapwater in natural daylight. The nymph was given 30 min to acclimatise before
a tadpole was introduced to the dish. A video recorder (Sony, Tokyo, Japan)
placed 50 cm above recorded the activity in six dishes simultaneously.
Behavioural pharmacology
In order to test the effect of drugs, the skin overlying the dorsal fin and
the myotomes on the right-side was removed from the level of the 1st to the
9th post-otic myotome (see Fig.
1B). This dissection was also carried out on control animals.
Following dissection, animals were given 10 min to recover in saline and a
further 5 min in the experimental dish and lighting conditions. Drugs were
dissolved in the saline in the dish. Responsiveness to dimming or touch was
tested as above.
Drugs used were bicuculline methochloride, CGP-35348
({3-aminopropyl}{diethoxymethyl}phosphinic acid), SR-95531
(6-imino-3-{4-methoxyphenyl}-1{6H}-pyridazinebutanoic acid hydrobromibe) (all
from Tocris, Avonmouth, UK), L-NAME
(N-Nitro-L-arginine methyl ester hydrochloride) and
D-NAME (N
-nitro-D-arginine methyl ester
hydrochloride) (both from Sigma).
Electrophysiology
Anaesthetised tadpoles were pinned onto a rotatable Sylgard block in a bath
perfused with saline, slit along the dorsal fin, and then transferred to
-bungarotoxin (10 µmol l1 in saline; Sigma) for up
to 20 min. After immobilisation, tadpoles were returned to the bath and
re-pinned with their right side up and their rostrocaudal axis
perpendicular to the axis of rotation of the Sylgard block
(Fig. 4D). The skin overlying
the dorsal fin and the right myotomes was removed from the level of the 4th to
the 9th post-otic myotome (when testing responsiveness) or 1st to the 9th
post-otic myotome (when recording trigeminal activity). The preparation was
illuminated by a halogen light source and light pipe, or by a green LED
(Farnell, Wetherby, UK) with peak emission of 525nm, which is close to the
wavelength of maximum sensitivity of the pineal eye (520 nm;
Foster and Roberts, 1982
). A
minimum period of 15 min after dissection was allowed for recovery prior to
recording. To record ventral root activity a glass suction electrode (60 µm
diameter tip) was placed over an intermyotomal cleft. To record trigeminal
activity, the skin, eye and meninges overlying the right trigeminal ganglion
were removed. A 60 or 30 µm diameter glass suction electrode was placed on
the ventral lobe of the exposed trigeminal ganglion to record multiple- or
single-unit activity respectively (Fig.
4A).
|
To simulate cement gland attachment, the block was rotated so the
rostrocaudal axis of the tadpole pointed downwards at an angle of about
70° from vertical. A length of silver wire (0.1 mg mass) equivalent to
that of a typical tadpole in water (0.09 mg; volume 2.32 mm3,
density 1.04 g cm3;
Roberts et al., 2000) could be
attached to the cement gland mucus and then allowed to hang freely
(Fig. 1D).
Responsiveness was tested by giving stimuli at intervals of 2 min. Any
fictive swimming was stopped within 20 s bypressing on the head skin with a
hand-held mounted hair. Three stimuli with nothing attached to the cement
gland mucus were followed by three stimuli with the weight attached, and this
was repeated. The stimulus used was dimming of an LED (500 ms, from 8000
to either
5000 or
150 Lux).
Chemicals used were 2 mmol l1 kynurenic acid, 10 µmol
l1 -bungarotoxin, 0.1% 3-aminobenzoic acid ethyl
ester (MS-222) and 100 µmol l1 cadmium chloride (all
Sigma). Chemicals were either bath applied or delivered through a
multi-barrelled microperfusion system. Fine polyethylene tubes led to a single
nozzle with a tip diameter of approximately 120 µm (for kynurenate) or 60
µm (for MS-222). One barrel contained the chemical dissolved in saline,
another contained a solution of Fast Green in saline and another barrel
contained control saline, which was applied between drug applications to
prevent perfusion artefacts. During bath application of 100 µmol
l1 CdCl2, the skin and meninges overlying the
hindbrain and midbrain were removed to improve access.
Electrical activity was amplified and then sent via a CED1401plus digital interface (Cambridge Electronic Design, CED, Cambridge, UK) to a personal computer where it was monitored, stored and analysed off-line using Spike2 or Signal software (CED), which could also be used to control the LED. A controlled pull on the cement gland mucus was produced using a loop of tungsten wire mounted via a lever to a loudspeaker cone driven by the CED 1401plus digital interface.
Individual trigeminal units, identified on the basis of spike shape, were discriminated off-line, using the Spike2 template matching function. For analysis of single unit activity, 5 min of unattached and attached activity were used. Firing rate was calculated as a single value: total number of spikes/total time period. Interspike interval (ISI) histograms were constructed with a bin width of 100 ms. The coefficients of variation (CV) of the ISIs were calculated (standard deviation ISI/mean ISI) to describe the regularity of firing. Autocorrelograms were constructed from 5 min of activity with 200 or more spikes (bin width 100 ms).
Light levels were measured using an ISO-TECH ILM 350 (RS Components, Corby, UK).
Statistical tests used are stated in the Results and were carried out using Minitab (version 10.51) and Excel. All values are means ± standard deviation (S.D.). Non-parametric statistics were used when data failed to meet criteria of normality.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
We also tested the responses of tadpoles to head skin touch. 13 out of 22
tadpoles swam or flexed the trunk in response to a stroke to the head skin
when unattached, whereas 1 out of 16 responded after hanging, attached by
their cement glands, for 1 min (P<0.001, 2=11.09,
d.f.=1). Attachment therefore significantly reduced responses to head skin
stimulation.
Does reduced responsiveness depend on innervation of the cement gland?
Cutting through the trigeminal mandibular nerves on each side of the body
removed the effect of cement gland attachment on responsiveness to tail
strokes. In tadpoles with both mandibular nerves cut, responsiveness was no
longer reduced by attachment (unattached, 84±21% responded; attached,
82±26%; P=0.787, W=9.0, N=10, Wilcoxon
matched-pairs signed-ranks test). Sham-operated tadpoles were still
significantly less responsive to tail strokes when attached (unattached,
72±17% responded; attached, 20±13%; P=0.006,
W=55, N=10, Wilcoxon matched-pairs signed-ranks test).
Since cutting the trigeminal innervation of the cement gland abolishes the reduced responsiveness during attachment, we conclude that trigeminal sensory innervation is necessary for attachment to influence responsiveness.
Does reduced responsiveness depend on GABAergic inhibition?
If the reduced responsiveness of attached tadpoles is due to the activity
of the stopping pathway, then GABAA-mediated, tonic or long-lasting
inhibition of rhythmic spinal neurons will be necessary for attachment to
reduce responsiveness (see Introduction). The effects of GABA antagonists on
responsiveness during cement gland attachment were therefore tested in freely
behaving tadpoles. Each tadpole received five stimuli while unattached and
five while attached by its cement gland, with unattached and attached tests
alternating. Removal of a section of skin over the trunk muscles allowed
bath-applied drug access to the nervous system.
Dimming was used as a stimulus in most tests because it can be controlled precisely. Tadpoles from the same batch of eggs were tested in control saline (N=12) or saline containing an antagonist (N=12). The effect of antagonists was measured as the difference between the reduction in responsiveness produced by attachment in control medium (con) and the reduction produced in the presence of an antagonist (ant; difference=reductionconreductionant). This difference was significant in the presence of GABAA antagonists (Table 1): bicuculline (20 µmol l1; P=0.0072), SR-95531 (20 µmol l1; P=0), and bicuculline (20 µmol l1) together with the GABAB antagonist CGP-35348 (200 µmol l1; P=0.0002). In contrast, the GABAB antagonist CGP-35348 (200 µmol l1) alone produced no significant difference (P=0.4079). Swimming can also be initiated by a stroke across the base of the tail with a hand-held whisker. Attachment normally reduced responsiveness to this touch stimulus from 90±10% to 32±18% (P=0.003; not shown), but in the presence of GABAA antagonist SR-95531 (20 µmol l1) the reduction was insignificant, from 88±18% to 85±21% (P=0.371; N=12, Wilcoxon matched-pairs signed-ranks test). These results suggest that the reduced responsiveness during attachment depends on tonic inhibition acting primarily through GABAA receptors.
|
Nitric oxide (NO) can facilitate GABAA inhibition in
Xenopus tadpoles (McLean and
Sillar, 2000b). McLean and Sillar
(2000a
) suggested that the
GABAergic neurons of the stopping pathway (MHRs) also release NO in older
tadpoles. If NO is released together with GABA in the stage 37/38 tadpole,
this may amplify the inhibition of spinal neurons during attachment. Using the
same protocol as with GABA antagonists (see above), we tested for an effect of
the NO-synthase inhibitor, L-NAME, on the ability of attachment to
reduce responsiveness. In these experiments, drug access was improved by
running a tungsten needle between the myotomes and the dorsal spinal cord in
the region where the skin had been removed. An inactive form of NAME,
D-NAME, was used as a control. L-NAME (10 mmol
l1) had no significant effect on the responsiveness reducing
effect of attachment (P=0.956, see
Table 1).
Does activity increase when the cement gland is dennervated?
If GABAA-mediated inhibition is activated while tadpoles hang
from their mucus strand, this might be expected to reduce spontaneous
movements. At stage 37/38, Xenopus tadpoles hang attached for 99% of
their time and do not move while attached
(Jamieson and Roberts, 2000).
We therefore compared the activity of sham-operated tadpoles with others where
the cement gland was denervated by cutting the maxillary nerve. Tadpoles were
placed in a dish of dechlorinated tapwater and allowed 5 min to acclimatise.
Activity was then observed and all flexions and swimming movements timed to
the nearest second for a total of 30 min.
Movement of control tadpoles occurred only when they were not attached, either lying on the bottom of the dish or when the cement gland mucus strand had broken. They then swam briefly, stopping as soon as they contacted the side of the dish or the water surface. In contrast, denervated tadpoles could start and continue swimming while attached. When swimming freely, they often did not stop on contact with the side of the dish but swam along it for prolonged periods. Over a 30 min period control tadpoles made, on average, no movements (median=0, interquartile range=2, N=44) whereas denervated tadpoles made 2 movements (median=2, interquartile range=1, N=44). This increase in the number of movements, from 0 to 4 tadpole1 h1, was significant (W=2409.5, P=0.0002; Wilcoxon rank sum test). The duration of activity also increased significantly in denervated tadpoles, from 0 to 8 s tadpole1 h1 (W=2424.0, P=0.001; data not shown).
Does predation increase when the cement gland is denervated?
What is the significance of the normal inhibition of tadpole responses and
activity while they hang attached? One possibility is that tadpoles that do
not move will be less likely to be detected and eaten by predators. Odonate
nymphs are important predators of many larval anuran species
(Lawler, 1989;
Skelly and Werner, 1990
;
Chovanec, 1992
) and have been
observed in the same ponds as Xenopus tadpoles in South Africa (Alan
Roberts, personal observation). We therefore used damselfly nymphs (Zygoptera)
to compare the predation of control tadpoles to those with denervated cement
glands. Although the species used in this study are native to Europe and
therefore not a natural predator of Xenopus laevis, preliminary
studies showed that they catch and eat Xenopus tadpoles, especially
if the tadpoles move.
We placed individual control or denervated tadpoles with a single nymph and
made video recordings to monitor attacks. During a 2 h observation period, 28
out of 44 control tadpoles were eaten and this number increased significantly
to 36 out of 44 in denervated tadpoles (2=3.77, d.f.=1,
P<0.05). This suggests that one selective advantage of the
reduction in responsiveness and spontaneous activity that occurs when tadpoles
are attached might be to reduce predation.
Does simulated attachment reduce responses in immobilised tadpoles?
To investigate the neuronal pathways and mechanisms for reduced
responsiveness during attachment we devised a new tadpole preparation.
Immobilised tadpoles were pinned head down and a weight was attached to the
cement gland mucus strand to simulate the attached state
(Fig. 1D). Responsiveness was
tested using dimming stimuli, which are more controllable and repeatable than
touch. Fictive swimming responses were monitored by recording activity in the
motor nerves going to the trunk muscles
(Fig. 3A).
|
Before testing, the threshold dim level was first determined for one tadpole in each of the two batches of tadpoles tested. The lower level of the dimming was reduced until 4 or 5 out of 6 dims initiated fictive swimming. This threshold level was assumed to be similar for all tadpoles in the batch being tested. Weight attachment significantly reduced the percentage of fictive swimming responses to a threshold dim from 78±25% of dims in the unattached state to 23±21% in the weight-attached state (Fig. 3B; P<0.001, W=136.0, N=16, Wilcoxon matched-pairs signed-ranks test).
Does weight attachment reduce spontaneous swimming in immobilised tadpoles?
As in the freely behaving tadpoles
(Jamieson and Roberts, 2000)
spontaneous fictive swimming activity was present in immobilized tadpoles. The
occurrence of spontaneous fictive swimming was recorded over 5 min with no
weight attached, followed by 5 min with a weight attached to the cement gland.
This was repeated, giving a total of 10 min for each state. Each 5 min
recording began 1 min after either attaching or removing the weight. All
spontaneous swims were stopped within 20 s by pressing the head skin with a
hand-held mounted hair. The frequency of spontaneous swimming episodes was
0.23±0.21 swims min1 when no weight attached. This
frequency was significantly reduced to 0.06±0.09 swims
min1 with a weight was attached to the cement gland
(Fig. 3C;P=0.009, W=45.0, N=12, Wilcoxon matched-pairs signed-ranks test).
Does simulated attachment result in a sustained increase in trigeminal activity?
Previous studies showed that cement gland mechanosensory neurons are
spontaneously active and fire a brief burst of impulses in response to brief
distortion of the cement gland (Roberts
and Blight, 1975; Boothby and
Roberts, 1992a
). This activity was only recorded for a few seconds
and so could not be used to predict what role cement gland sensory neurons may
play over the longer periods of time for which we have demonstrated the
effects of attachment. To determine whether cement gland mechanosensory
neurons in the trigeminal ganglion could sustain firing during prolonged
attachment we hung a weight from the cement gland mucus of immobilised
tadpoles in a head-down position and recorded trigeminal activity
(Fig. 4A).
Multi-unit activity was recorded from cement gland mechanosensory neurons for 30 s periods at intervals of 5 min. In the unattached state, spontaneous activity was recorded (Fig. 4B). After 25 min, a weight was attached to the cement gland mucus where it hung freely for 1 h before being removed. When the weight was first attached there was a transient peak in activity. Increased activity was then sustained for the whole 60 min period of attachment before returning to the previous unattached level after the weight was removed. The number of spikes in each 30 s recording was counted and activity in 7 animals normalised, by dividing each count by the count at 25 min (immediately before weight attachment) in the same animal (Fig. 4C). Linear regression over the 60 minperiod of attachment showed no significant relationship between activity and time (P=0.642, F=0.218, r2=0.002). However, there was a significant increase in activity during simulated attachment (P=0.000, F=22.54, d.f.=2,20, repeated-measures ANOVA after grouping the activity from 0 to 25 min (unattached) and from 30 to 85 min (attached)).
What is the activity of single trigeminal units during attachment?
Multi-unit recording has the advantage that recordings can be maintained
for long periods of time. However, it did not allow us to determine whether
the same neurons are responsible for spontaneous activity in the unattached
state and increased activity in the attached state, or what the firing rates
were for individual neurons. When smaller electrodes were used, all individual
units (N=20 in 15 tadpoles) showed spontaneous activity, a transient
increase in firing frequency when the weight was attached, and a long-term
increase in discharge during weight attachment
(Fig. 5).
|
To study the irregular pattern of discharge in the trigeminal sensory
units, we recorded 5 min of unattached (spontaneous) activity followed by 5
min of weight-attached activity, excluding the first 30 s after weight
attachment to eliminate the transient response
(Fig. 6). Linear regression of
the summed total number of spikes every 30 s during the 5 min of the attached
state showed no significant effect of time on activity (P>0.05)
for 16 of the 20 units. We carried out further analysis of these 16 units on
the assumption that their activity arose from a stationary process
(Perkel et al., 1967). For
both unattached and attached activity, ISI histograms had a high variance and
were skewed (Fig. 6). When
attached, the firing rate was significantly higher (1.17±0.78 Hz) than
when unattached (0.28±0.31 Hz; P=0.000, t=5.28,
N=16, paired t-test). The high coefficients of variation
(CV) for the ISIs show that firing was irregular in both cases. There was no
significant difference between the two (CV unattached=1.37±0.40; CV
attached=1.15±0.51; P=0.093, W=35.0, N=16,
Wilcoxon matched-pairs signed-ranks test). Using units that fired at least 200
spikes during 5 min in the unattached (N=2) or attached
(N=11) states, autocorrelograms were constructed. These did not
reveal any pattern in the firing other than a decreased probability of firing
during a short period (
200 ms) after each spike.
|
Mechanisms underlying sustained sensory activity
Previous work has shown that chemical synaptic transmission is not
necessary for the transient response of trigeminal mechanosensory neurons to
brief stimuli, since it was not abolished either by a high Mg2+
concentration in the bathing medium, which blocked all muscular responses
(therefore presumably synaptic transmission;
Roberts and Blight, 1975), or
by kynurenate, an antagonist of glutamate receptors
(Boothby and Roberts, 1992b
).
We found that excitatory glutamatergic synaptic interactions are also not
required for sustained firing in the attached state. When 2 mmol
l1 kynurenic acid, was microperfused onto the trigeminal
ganglion, there was no significant change in either firing rate
(control=1.05±0.39 Hz, kynurenic acid= 0.99±0.46 Hz,
P=0.752, t=0.33) or CV ISI (control=1.06±0.24,
kynurenic acid=1.31±0.51, P=0.176, t=1.57;
N=6, paired t-tests), measured over 5 min.
It is unlikely that sustained firing in the attached state was due to repeated transient stimulation by the hanging weight moving in the flow of perfused saline, since stopping the flow produced no significant change in either firing rate (control=1.70±0.88 Hz, no flow=1.35±0.68 Hz, P=0.109, t=2.26) or CV ISI measured over 5 min (control=1.02±0.20, no flow=1.10±0.34, P=0.48, t=0.80; N=4 units from 3 recordings, paired t-tests).
Is sensory activity affected by efferent input?
We tested whether the activity of cement gland mechanosensory neurons was
affected by synaptic input from the CNS during fictive swimming activity. A
ventral root electrode monitored fictive swimming activity while another
extracellular electrode monitored single trigeminal units responding to cement
gland stimulation. Dimming was used to initiate episodes of fictive swimming.
The trigeminal units did not respond to the swim-initiating dim itself
(N=7 units from 5 recordings; Fig.
7). Activity in the unattached state (spontaneous activity) was
also not affected by fictive swimming. The firing rates of single units over a
period of 30 s before an episode of fictive swimming (0.57±0.46 Hz)
were not significantly different from the number in a 30 s period during
fictive swimming (0.67±0.50 Hz; P=0.41, t=0.88,
N=7 units from five recordings, paired t-test).
|
Increased trigeminal mechanosensory neuron firing during attachment therefore appears to be independent of peripheral synaptic interactions, effects of saline perfusion and efferent inputs during swimming.
What is the origin of spontaneous sensory activity?
Spontaneous firing activity occurred even in the unattached state in all
units recorded from the ventral lobe of the trigeminal ganglia, which were
excited by attaching a weight to the cement gland mucus. To rule out the
possibility that this spontaneous activity is an artefact of suction electrode
recording, we used the same technique to make single unit recordings from
neurons in the anterior lobe of the trigeminal ganglion, which innervate the
head skin and do not show spontaneous activity
(Roberts and Blight, 1975).
These head skin mechanosensory neurons were excited by broad pressure to the
head skin (N=5) but showed no spontaneous activity. This indicates
that the spontaneous activity of cement gland mechanosensory neurons is
unlikely to be an artefact of suction electrode recording.
Spontaneous activity in cement gland mechanosensory neurons does not depend
on chemical synaptic input from the CNS as it persisted when 100 µmol
l1 CdCl2 was bath-applied to the whole animal.
100 µmol l1 CdCl2 can be presumed to block
chemically mediated neurotransmission (cf.
Perrins and Roberts, 1995;
Zhao et al., 1998
) and, after
application, fictive swimming failed to occur in response to dimming. The
firing rate of individual units over 5 min of unattached activity in control
saline (0.53±0.43 Hz) was not significantly different from that over 5
min, beginning 5 min after bath application of 100 µmol
l1 CdCl2 (0.64±0.37 Hz; P=0.14,
t=1.69, N=7 units from 5 recordings, paired
t-test).
Does spontaneous activity arise in the peripheral sensory endings or within
the somata lying in the trigeminal ganglion? The mandibular nerve is
approximately 400 µm long and connects the trigeminal ganglion to the
cement gland. This separation allowed local perfusion of anaesthetic to block
any impulses generated in the receptor endings in the cement gland without
affecting the ability of the somata to fire impulses
(Fig. 8A). Solutions were
microperfused onto the distal mandibular nerve and cement gland. The flow of
the bath saline perfusion washed the microperfusate away and, by using the dye
Fast Green, it could be seen that the flow was restricted to the distal end of
the mandibular nerve and did not contact the trigeminal ganglion. During
microperfusion of control saline, cement gland mechanosensory neurons
responded to pulling on the cement gland mucus by firing a burst of impulses
(Fig. 8B). The neurons were
also spontaneously active (Fig.
8C). Current pulses (100 or 300 µs) delivered to the hindbrain
at the level of the 5th/6th rhombomere using a glass suction electrode (100
µm diameter tip) were able to backfire the trigeminal neurons
(Fig. 8D) as their axons
descend to the caudal hindbrain (Hayes and
Roberts, 1983). Switching to microperfusion of 0.1% MS-222
abolished both the response to pulls on the cement gland mucus and spontaneous
activity (Fig. 8B,C), but
current pulses to the hindbrain were still able to backfire the neurons
(Fig. 8D). After returning to
control saline, spontaneous activity returned. These results suggest that
spontaneous activity arises in the peripheral receptor endings of the sensory
neurons.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
What sensory information comes from the cement gland of the
Xenopus tadpole while it hangs at rest from mucus secreted by the
gland? We have demonstrated that trigeminal cement gland mechanosensory
neurons show sustained impulse activity at a frequency near 1 Hz during
simulated attachment. Although it was known that these sensory neurons fire in
response to an increase in cement gland mucus tension
(Roberts and Blight, 1975), it
was not known whether they could sustain firing over long periods. Multi-unit
recordings show increased activity during simulated attachment, and this is
sustained for at least 60 min. We are therefore confident that cement gland
sensory afferents can sustain low levels of increased activity throughout most
periods of attachment in the freely behaving tadpole. One of the surprises of
our study is that during the first day of the tadpole's life these
mechanoreceptor afferents are probably active nearly continuously.
Although there are many examples of behavioural states of reduced activity
and responsiveness that are induced or maintained by an external stimulus
(Gray et al., 1938;
Kramer and Markl, 1978
;
Krasne and Wine, 1975
;
Nishino and Sakai, 1996
;
Faisal and Matheson, 2001
), the
current study is, we believe, the first example where the sensory neurons
detecting the external stimulus and thus evoking the reduction in
responsiveness, have been identified. This has allowed us to define the
pattern of sensory activity that results in reduced responsiveness. The simple
behaviour and nervous system of the hatchling Xenopus tadpole has
been the key to this. Responsiveness is reduced while tadpoles are attached by
their cement glands and this behavioural state can be simulated in our
immobilised preparation.
The mechanoreceptors that innervate the cement gland may serve two
functions. Firstly, by firing a short burst of activity, they inform the
tadpole that it has contacted support and should stop swimming
(Boothby and Roberts, 1992a).
Secondly, by firing continuously at low levels (
1 Hz) during attachment,
we have shown that they give the tadpole continuous confirmation that it is
hanging attached to its mucus strand. These separate functions could be served
by two populations of trigeminal sensory neurons. However, our evidence does
not support this proposal. In contrast, the activity of single units suggests
that trigeminal cement gland mechanosensory neurons form a homogenous
population. They all have irregular spontaneous activity; fire a transient
burst of impulses when a weight is initially attached so cement gland mucus
tension is increased, and show a sustained increase in firing rate while the
weight remains attached. Our experiments indicate that the receptor terminals
of trigeminal sensory neurons lying in the cement gland
(Roberts and Blight, 1975
) are
excited directly by tension in the mucus strand secreted by the gland.
The responsiveness reducing effect of attachment was abolished by the
potent GABAA antagonist SR-95531
(Heaulme et al., 1986), and
significantly reduced by the GABAA antagonist bicuculline.
Attachment still had a significant effect on responsiveness in 20 µmol
l1 bicuculline, probably because it was not blocking all
GABAA receptors. The effectiveness of SR-95531 makes it unlikely
that the effects of bicuculline are due to the known
non-GABA-receptor-mediated effect of bicuculline, which can also block
calcium-activated potassium channels
(Seutin and Johnson, 1999
). We
conclude that GABAA inhibition is necessary for the reduction in
responsiveness during attachment. It is also necessary for the stopping
response, where the cement gland is transiently stimulated when the freely
swimming tadpole contacts an obstruction
(Boothby and Roberts, 1992b
).
In response to pulling on the cement gland mucus or pressing the cement gland,
trigeminal mechanosensory neurons fire a burst of impulses
(Roberts and Blight, 1975
;
Boothby and Roberts 1992a
).
This leads to the termination of swimming activity: the stopping response.
Present evidence suggests that the axons of these trigeminal sensory neurons
release glutamate to excite inhibitory reticulospinal neurons that, in turn,
inhibit rhythmic spinal neurons via GABAA-receptors
(Boothby and Roberts, 1992b
;
Perrins et al., 2002
;
Li et al., 2003
). Our
conclusion that GABAA-mediated inhibition is necessary for
attachment to reduce responsiveness supports the proposal that the same
GABAergic reticulospinal neurons are responsible both for stopping swimming
activity when the head first contacts support
(Perrins et al., 2002
), and
for reducing responsiveness during attachment.
Tonic inhibition mediated by GABAB receptors occurs in other
systems (Hao et al., 1994;
Lin et al., 1996
). Since
Xenopus tadpole spinal neurons also have GABAB receptors
(Wall and Dale, 1993
,
1994
), they could contribute
to the long-term inhibitory effects of attachment by raising the firing
threshold of spinal neurons. However, we found that blocking
GABAB-receptors with 200 µmol l1 CGP-35348 (an
antagonist known to be effective in Xenopus tadpoles;
Wall and Dale, 1993
) had no
significant effect on the responsiveness-reducing effect of attachment.
Our evidence suggests that GABAA inhibition plays a major role
in reducing responsiveness when tadpoles are attached, but the possibility
that other neurotransmitters are also involved cannot be excluded. Nitric
oxide (NO) is present in the tadpole hindbrain
(McLean and Sillar, 2001;
Lopez and Gonzalez, 2002
) and
may facilitate GABAergic IPSPs (McLean and Sillar,
2000b
,
2002
). However, at stage
37/38, a role for NO in facilitating tonic inhibition was not supported by our
observation that blocking NO production with L-NAME failed to have
an effect on the ability of attachment to reduce responsiveness. Neurons
containing 5-HT are present in the raphe nucleus of stage 37/38 tadpoles
(Van Mier et al., 1986
) and
5-HT has been shown to block initiation of swimming by both skin stimulation
and dimming (Sillar and Simmers,
1994
; Jamieson,
1997
). 5-HT inhibition of responsiveness to skin stimulation
involves presynaptic inhibition of primary afferent neurons
(Sillar and Simmers, 1994
) and
an increase in their firing threshold (Sun
and Dale, 1997
). This presynaptic action of 5-HT contrasts with
the postsynaptic action of GABAergic inhibition on spinal CPG neurons during
the stopping response (Li et al.,
2003
) and, as we now propose, during attachment. 5-HT is known to
have effects on CPG neurons but these are excitatory, increasing the duration
and intensity of motor bursts in swimming episodes through presynaptic
inhibition of glycine release from interneurons within the CPG
(McDearmid et al., 1997
). The
possible involvement of 5-HT in the inhibitory effects of attachment remains
to be investigated.
Significance of tonic inhibition during attachment
When Xenopus tadpoles are placed in a small dish with a naïve
damselfly nymph, the tadpoles that move appear to be the ones that are
attacked and eaten. The advantages of keeping still were made clear by Skelly
(1994), who showed that
anaesthetized tadpoles of Rana sylvatica were less likely than active
tadpoles to be attacked by dragonfly nymphs (Anax junius). We have
confirmed that during the first day after hatching Xenopus tadpoles
are very immobile, spending more than 99% of their time attached and hanging
from mucus (Jamieson and Roberts,
2000
). At this stage of development (stage 37/38) there is no
fitness cost in keeping still as the tadpoles have no mouth and do not start
to feed until about 2 days later
(Nieuwkoop and Faber, 1956
).
This contrasts with older tadpoles that need to move in order to feed but then
face increased risk of predation, for example by dragonfly nymphs
(Lawler, 1989
;
Chovanec, 1992
). Such
investigations on other tadpoles suggested that tonic inhibition during
attachment in the hatchling Xenopus tadpole could be significant in
reducing activity and as a consequence, reducing predation. In preliminary
experiments, we found that when the cement gland is removed so the tadpoles
cannot attach with mucus, they move significantly more, and significantly more
are eaten when they are placed with damselfly nymphs (A. Roberts, E. Pariser
and P. Lemon, unpublished observations). The experiment we report here shows
that denervation of the cement gland has similar effects even though the
tadpoles can still attach and hang from mucus secreted by the cement gland.
This denervation specifically interrupts the pathway that normally activates
GABAergic inhibition when the cement gland is stimulated (Boothby and Roberts,
1992a
,b
;
Perrins et al., 2002
).
Our interpretation of our behavioural experiments is that tension in the
cement gland mucus during attachment leads to tonic GABAA
inhibition. This inhibition can reduce predation by reducing the activity of
the tadpoles. Reduced activity may also help the tadpole to conserve its
energy reserves. It was quite unexpected that reduced activity appears to
result from continuous, tonic, sensory stimulation and continuous, tonic
inhibition during attachment. This means that for its first day out of the egg
the tadpole is tonically inhibited for 99% of the time. Is this long-term
tonic inhibition an unusual feature of the hatchling tadpole, or could it be
more widespread? Unfortunately, few cases have been studied in sufficient
detail, so data is lacking, but in the crayfish, tonic inhibition that leads
to reduced responsiveness has only been reported under particular behavioural
conditions such as during feeding or physical restraint
(Vu and Krasne, 1993).
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bässler, U. (1983). Studies of Brain Function, vol. 10. Berlin: Springer-Verlag.
Boothby, K. M. and Roberts, A. (1992a). The stopping response of Xenopus laevis embryos, behaviour, development and physiology. J. Comp. Physiol. A 170,171 -180.[Medline]
Boothby, K. M. and Roberts, A. (1992b). The stopping response of Xenopus laevis embryos, pharmacology and intracellular physiology of rhythmic spinal neurons and hindbrain neurons. J. Exp. Biol. 169,65 -86.[Abstract]
Brooks, S. (1997). Field Guide to the Dragonflies and Damselflies of Great Britain and Ireland. Hook: British Wildlife Publishing.
Chovanec, A. (1992). The influence of tadpole swimming behavior on predation by dragonfly nymphs. Amphibia-Reptilia 13,341 -349.
Clarke, J. D., Hayes, B. P., Hunt, S. P. and Roberts, A. (1984). Sensory physiology, anatomy and immunohistochemistry of RohonBeard neurons in embryos of Xenopus laevis. J. Physiol. 348,511 -525.[Abstract]
Faisal, A. A. and Matheson, T. (2001).
Coordinated righting behaviour in locusts. J. Exp.
Biol. 204,637
-648.
Foster, R. G. and Roberts, A. (1982). The pineal eye in Xenopus laevis embryos and larvae a photoreceptor with a direct excitatory effect on behavior. J. Comp. Physiol. A 145,413 -419.
Gallup, G. G. (1974). Animal hypnosis, factual status of a fictional concept. Psychol. Bull. 81,836 -853.[Medline]
Gargaglioni, L. H., Pereira, A. S. and Hoffmann, A. (2001). Basal midbrain modulation of tonic immobility in the toad Bufo paracnemis. Physiol. Behav. 72,297 -303.[CrossRef][Medline]
Glenn, L. L. and Dement, W. C. (1981). Membrane
potential, synaptic activity, and excitability of hindlimb motoneurons during
wakefulness and sleep. J. Neurophysiol.
46,839
-854.
Gray, J., Lissmann, H. W. and Pumphrey, R. J. (1938). The mechanism of locomotion in the leech (Hirudo medicinalis Ray). J. Exp. Biol. 15,408 -430.
Hao, J. X., Xu, X. J. and Weisenfeld-Hallin, Z.
(1994). Intrathecal -aminobutyric acidB
(GABAB) receptor antagonist CGP 35348 induces hypersensitivity to
mechanical stimuli in the rat. Neurosci. Lett.
182,299
-302.[CrossRef][Medline]
Hayes, B. P. and Roberts, A. (1983). The anatomy of two functional types of mechanoreceptive `free' nerve-ending in the head skin of Xenopus embryos. Proc. R. Soc. Lond. B 218,61 -76.[Medline]
Heaulme, M., Chambon, J. P., Leyris, R., Molimard, J. C., Wermuth, C. G. and Biziere, K. (1986). Biochemical characterization of the interaction of three pyridazinyl-GABA derivatives with the GABAA receptor site. Brain Res. 384,224 -231.[CrossRef][Medline]
Jamieson, D. (1997). The pineal eye of Xenopus laevis tadpoles. A behavioural, anatomical and physiological study of an extraretinal photosensory system. PhD thesis, University of Bristol, Bristol, UK.
Jamieson, D. and Roberts, A. (1999). A possible pathway connecting the photosensitive pineal eye to the swimming central pattern generator in young Xenopus laevis tadpoles. Brain Behav. Evol. 54,323 -337.[CrossRef][Medline]
Jamieson, D. and Roberts, A. (2000). Responses
of young Xenopus laevis tadpoles to light dimming, possible roles for
the pineal eye. J. Exp. Biol.
203,1857
-1867.
Kaiser, W. and Steiner-Kaiser, J. (1983). Neuronal correlates of sleep, wakefulness and arousal in a diurnal insect. Nature 301,707 -709.[Medline]
Krämer, K. and Markl, H. (1978). Flight-inhibition on ground contact in the American cockroach, Periplaneta americana I. Contact receptors and a model for their central connections. J. Insect Physiol. 24,577 -586.[CrossRef]
Krasne, F. B. and Wine, J. J. (1975). Extrinsic modulation of crayfish escape behaviour. J. Exp. Biol. 63,433 -450.[Abstract]
Lambert, T. D. and Roberts, A. (2000a). Tonic activity of trigeminal sensory neurons and reduced responsiveness during cement gland attachment in hatchling Xenopus laevis tadpoles. J. Physiol. 523P,272P .
Lambert, T. D. and Roberts, A. (2000b). Tonic inhibitory effects of trigeminal mechanoreceptor activity during attachment by cement gland mucus in hatchling Xenopus laevis tadpoles. Eur. J. Neurosci. S12,91 .
Lawler, S. P. (1989). Behavioural responses to predators and predation risk in four species of larval anurans. Anim. Behav. 38,1039 -1047.
Li, W-C., Perrins, R., Walford, A. and Roberts, A. (2003). The neuronal targets for GABAergic reticulospinal inhibition that stops swimming in hatchling frog tadpoles. J. Comp. Physiol. 189,29 -37.
Lin, Q., Peng, Y. B. and Willis, W. D. (1996).
Role of GABA receptor subtypes in inhibition of primate spinothalamic tract
neurons, difference between spinal and periaqueductal gray inhibition.
J. Neurophys. 75,109
-123.
Lopez, J. M. and Gonzalez, A. (2002). Ontogeny of NADPH diaphorase/nitric oxide synthase reactivity in the brain of Xenopus laevis. J. Comp. Neurol. 445, 59-77.[CrossRef][Medline]
McDearmid, J. R., Scrymgeour-Wedderburn, J. F. and Sillar, K. T. (1997). Aminergic modulation of glycine release in a spinal network controlling swimming in Xenopus laevis. J. Physiol. 503,111 -117.[Abstract]
McLean, D. L. and Sillar, K. T. (2000a). The
distribution of NADPH-diaphorase-labelled interneurons and the role of nitric
oxide in the swimming system of Xenopus laevis larvae. J.
Exp. Biol. 203,705
-713.
McLean, D. L. and Sillar, K. T. (2000b). Facilitation of GABAergic inhibition by nitric oxide in the spinal cord of Xenopus laevis embryos. Soc. Neurosci. Abstr. 26, 1996.
McLean, D. L. and Sillar, K. T. (2001). Spatiotemporal pattern of nicotinamide adenine dinucleotide phosphate-diaphorase reactivity in the developing central nervous system of premetamorphic Xenopus laevis tadpoles. J. Comp. Neurol. 437,350 -362.[CrossRef][Medline]
McLean, D. L. and Sillar, K. T. (2002). Nitric
oxide selectively tunes inhibitory synapses to modulate vertebrate locomotion.
J. Neurosci. 22,4175
-4184.
Monassi, C. R., Hoffmann, A. and Menescal, D. (1997). Involvement of the cholinergic system and periaqueductal gray matter in the modulation of tonic immobility in the guinea pig. Physiol. Behav. 62,53 -59.[CrossRef][Medline]
Nieuwkoop, P. D. and Faber, J. (1956).Normal tables of Xenopus laevis (Daudin). Amsterdam: North Holland Publishing Co.
Nishino, H. and Sakai, M. (1996). Behaviorally significant immobile state of so called thanatosis in the cricket Gryllus bimaculatus DeGeer: Its characterization, sensory mechanism and function. J. Comp. Physiol. A 179,613 -624.
Perkel, D. H., Gerstein, G. L. and Moore, G. P. (1967). Neuronal spike trains and stochastic point processes. I. The single spike train. Biophys. J. 7, 391-418.[Medline]
Perrins, R. and Roberts, A. (1995). Cholinergic
contribution to excitation in a spinal locomotor central pattern generator in
Xenopus embryos. J. Neurophysiol.
73,1013
-1019.
Perrins, R., Walford, A. and Roberts, A.
(2002). Sensory activation and role of inhibitory reticulospinal
neurons that stop swimming in hatchling frog tadpoles. J.
Neurosci. 22,4229
-4240.
Pringle, J. W. S. (1974). Locomotion, Flight. In The Physiology of the Insecta, vol.III (ed. M. Rockstein), pp.433 -476. London: Academic Press.
Roberts, A. (1980). The function and role of two types of mechanoreceptive `free' nerve endings in the head skin of amphibian embryos. J. Comp. Physiol. A 135,341 -348.
Roberts, A. (1997). Skin sensory systems of amphibian embryos and young larvae. In Amphibian Biology. Sensory Perception (ed. H. Heatwole), pp.923 -935. Chipping Norton NSW Australia: Surrey Beatty and Sons.
Roberts, A. and Blight, A. R. (1975). Anatomy, physiology and behavioural role of sensory nerve endings in the cement gland of embryonic Xenopus. Proc. R. Soc. Lond. B 192,111 -127.[Medline]
Roberts, A., Hill, N. A. and Hicks, R. (2000).
Simple mechanisms organise orientation of escape swimming in embryos and
hatchling tadpoles of Xenopus laevis. J. Exp. Biol.
203,1869
-1885.
Roberts, A. and Sillar, K. T. (1990). Characterization and function of spinal excitatory interneurons with commissural projections in Xenopus-laevis embryos. Eur. J. Neurosci. 2,1051 -1062.[Medline]
Roberts, A., Soffe, S. R. and Perrins, R. (1997). Spinal networks controlling swimming in hatchling Xenopus tadpoles. In Neurons, Networks and Motor Behaviour (ed. P. S. G. Stein, S. Grillner, A. I. Selverston and D. G. Stuart), pp. 83-89. Boston: MIT Press.
Seutin, V. and Johnson, S. W. (1999). Recent advances in the pharmacology of quaternary salts of bicuculline. Trends Pharmacol. Sci. 20,268 -270.[CrossRef][Medline]
Sillar, K. T. and Simmers, A. J. (1994). Presynaptic inhibition of primary afferent transmitter release by 5-hydroxytryptamine at a mechanosensory synapse in the vertebrate spinal cord. J. Neurosci. 14,2636 -2647.[Abstract]
Skelly, D. K. (1994). Activity level and susceptibility of anuran larvae to predation. Anim. Behav. 47,465 -468.[CrossRef]
Skelly, D. K. and Werner, E. E. (1990). Behavioral and life historical responses of larval American toads to an odonate predator. Ecology 71,2313 -2322.
Sun, Q. Q. and Dale, N. (1997). Serotonergic
inhibition of the T-type and high voltage-activated Ca2+ currents
in the primary sensory neurons of Xenopus larvae. J.
Neurosci. 17,6839
-6849.
Tobler, I. and Neuner-Jehle, M. (1992). 24-h variation of vigilance in the cockroach Blaberus giganteus. J. Sleep Res. 1,231 -239.[Medline]
Van Mier, P., Joosten, H. W., van Rheden, R. and ten Donkelaar, H. J. (1986). The development of serotonergic raphespinal projections in Xenopus laevis. Int. J. Dev. Neurosci. 4, 465-475.[CrossRef][Medline]
Vu, E. T. and Krasne, F. B. (1993). Crayfish tonic inhibition, prolonged modulation of behavioral excitability by classical GABAergic inhibition. J. Neurosci. 13,4394 -4402.[Abstract]
Wall, M. J. and Dale, N. (1993). GABAB receptors modulate glycinergic inhibition and spike threshold in Xenopus embryo spinal neurons. J. Physiol. 469,275 -290.[Abstract]
Wall, M. J. and Dale, N. (1994).
GABAB receptors modulate an -conotoxin-sensitive calcium
current that is required for synaptic transmission in the Xenopus
embryo spinal cord. J. Neurosci.
14,6248
-6255.[Abstract]
Zhao, F. Y., Wolf, E. and Roberts, A. (1998).
Longitudinal distribution of components of excitatory synaptic input to
motoneurons during swimming in young Xenopus tadpoles: experiments
with antagonists. J. Physiol.
511,887
-901.
Zhdanova, I. V., Wang, S. Y., Leclair, O. U. and Danilova, N. P. (2001). Melatonin promotes sleep-like state in zebrafish. Brain Res. 903,263 -268.[CrossRef][Medline]