Calcium responses of chicken trigeminal ganglion neurons to methyl anthranilate and capsaicin
1 Monell Chemical Senses Center, 3500 Market Street, Philadelphia, PA
19104-3308, USA
2 United States Department of Agriculture, Animal and Plant Health
Inspection Service, Wildlife Services, National Wildlife Research Center, 4101
La Porte Avenue, Fort Collins, CO 80521-2154, USA
* Author for correspondence (e-mail: bryant{at}monell.org)
Accepted 19 November 2003
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Summary |
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Key words: calcium imaging, capsaicin, chick, digital fluorescence imaging, Fura Red, irritation, methyl anthranilate, pain, primary cell culture, trigeminal neuron, repellent
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Introduction |
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Despite the lack of a behavioral response to CAP in birds, the avian
nervous system is not completely insensitive to this compound. Sann et al.
(1987) showed that injections
of 1% CAP into the sciatic nerve of pigeons increased the concentration of
substance P in dorsal horn neurons. Harti et al.
(1989
) demonstrated that a
topical application of 1% CAP to the cornea of pigeons caused a decrease in
substance P in 50% of the innervating neurons. In both cases, the
physiological response to >30 µmol l1 CAP did not
appear to be associated with any overt behavioral response indicative of
irritation. These observations are consistent with behavioral studies on
starlings (Sturnus vulgaris). Mason and Clark
(1995
) showed that although
starlings did not demonstrate a congenital behavioral avoidance towards CAP,
they could be trained to avoid food treated with CAP in conditioned avoidance
paradigms, and avoidance was contingent upon an intact ophthalmic branch of
the trigeminal nerve. Together, these studies indicate that birds can perceive
and physiologically respond to CAP via pathways and mechanisms
typically associated with nociception. However, birds do not perceive CAP as
irritating.
The interest in elucidating the mechanisms underlying the mediation and
perception of avian irritants is twofold. First, taxonomic differences in the
ability to perceive plant chemical defenses such as irritants has profound
implications in understanding the evolution of plantanimal interactions
that focus on the foraging ecology of animals and seed survivorship/dispersal
in plants (Norman et al.,
1992; Clark, 1998b
;
Tewksbury and Nabhan, 2001
).
Second, understanding the neural coding of irritants will aid in the
identification and development of environmentally safe repellents. This is
important in resolving conflicts between wildlife and humans, e.g. crop
depredations and property damage, while minimizing human impact on wildlife
resources (Clark,
1998a
,b
).
There is considerable information on the effects of exogenous compounds on
avian avoidance behavior, both empirically and from a structureactivity
perspective (Clark and Shah,
1991,
1994
;
Clark, 1998a
). Yet, we know
very little about how these compounds are coded by the nervous system. In the
present paper, we characterize the concentrationresponse relationships
for MA and CAP in populations of cultured chick trigeminal neurons using
digital fluorescence imaging of intracellular calcium
([Ca2+]i). In addition, we compare mechanisms of neural
activation by MA and CAP by determining the relative dependence on
extracellular Na+ and Ca2+.
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Materials and methods |
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Calcium imaging
Changes in intracellular calcium levels ([Ca2+]i)
were measured using ratiometric digital fluorescence calcium imaging of chick
trigeminal neurons (Grynkiewicz et al.,
1985; Restrepo et al.,
1995
). Cultured neurons were loaded with Fura Red (Molecular
Probes, Eugene, OR, USA) by incubation in 10 µmol l1 Fura
Red/AM and 60 µg ml1 Pluronic-127 in Ringer's solution at
24°C for 1 h. FuraRed was used because the excitation wavelengths, 485 nm
and 440 nm, do not cause interfering fluorescence from MA as do the
wavelengths used to excite Fura 2, the more commonly used Ca2+
indicator. Images of fluorescing neurons were acquired with a cooled
charge-coupled device (CCD) camera. Autofluorescence was negligible and, with
illumination times of 100200 ms, we did not find appreciable photo
bleaching.
Responses of neurons to chemical stimulation
Coverslips with attached neurons were placed in a flow chamber through
which Ringer's solution flowed. Chemical stimuli in Ringer's solution were
applied to the flow, and pairs of ratio images were acquired every 10 s.
Chemical stimuli were applied to the neurons for 15 s and then the chambers
were rinsed for 23 min between each stimulus. The average fluorescence
ratio, F440/F485, an index of
[Ca2+]i, was calculated for each neuron using `regions
of interest' drawn automatically for each neuron using Metafluor (Universal
Imaging Corp., Downingtown, PA, USA). Responses, expressed as the change in
fluorescence ratio, were normalized to the response elicited by the positive
control, 40 mmol l1 KCl, which depolarizes neurons, causing
a concomitant influx of Ca2+ via voltage-gated calcium
channels.
Ringer's solution (pH 7.4) used for these experiments contained 138.3 mmol l1 NaCl, 5.8 mmol l1 KCl, 1.0 mmol l1 CaCl2, 1.0 mmol l1 MgCl2, 5.0 mmol l1 Hepes and 10.0 mmol l1 glucose. Calcium-free Ringer's solution contained 138.3 mmol l1 NaCl, 5.8 mmol l1 KCl, 6.0 mmol l1 MgCl2, 5.0 mmol l1 Hepes, 10.0 mmol l1 glucose and 1 mmol l1 EGTA. Sodium-free Ringer's solution contained 138.3 mmol l1 N-methyl-D-glucamine, 5.8 mmol l1 KCl, 1.0 mmol l1 CaCl2, 1.0 mmol l1 MgCl2, 5.0 mmol l1 Hepes and 10.0 mmol l1 glucose.
Experiment 1. Neuronal population response to MA and CAP
The object of this experiment was to quantify the neuronal population
response to MA and CAP stimulation. Two measures of responsiveness were used
as an index of cellular activity: (1) the magnitude of MA- and CAP-induced
changes in [Ca2+]i normalized to that of a positive
control, 40 mmol l1 KCl, and (2) the proportion of cells in
an experiment with a response greater than 5% of the response to KCl. To
determine the concentrationresponse function of chick trigeminal
ganglion (TG) neurons to MA and CAP, neurons were exposed to an ascending
concentration series of stimulus, followed by exposure to 40 mmol
l1 KCl. In seven experiments, responses to MA were obtained
from 12 neurons, and in four experiments, responses to CAP were obtained from
26 neurons.
Experiment 2. Distribution of neuronal sensitivity to MA and CAP
In a second series of experiments, we determined the colocalization of
sensitivity to MA and CAP. Fields of neurons were exposed to 100 µmol
l1 MA, rinsed for 2 min and then the neurons were exposed to
100 µmol l1 CAP. An equal number of tests had the reverse
order of presentation of MA and CAP. After a final rinse, neurons were exposed
to 40 mmol l1 KCl. We classified neurons as responsive if
the increase in [Ca2+]i was greater than 10% of the KCl
response. This criterion level was used because it represented the mean
baseline [Ca2+]i ± 2.5 S.D. recorded
during the interstimulus interval. Responses of 467 neurons stimulated with
both MA and CAP were measured in six experiments.
Experiment 3. Ion dependence of neuronal sensitivity
In a third series of experiments, we determined the ion dependence of
neuronal sensitivity to MA and CAP. Following an initial stimulation with 100
µmol l1 MA or 100 µmol l1 CAP, the
background flow of Ringer's solution was changed to either Na+-free
or Ca2+-free Ringer's solution, and the response to a second
stimulation of the appropriate compound was measured. Following replacement of
the ion-free solution with normal Ringer's solution and a period of rinsing,
the degree of recovery of sensitivity to MA or CAP was determined with a third
stimulation. In four experiments for each ion, 12 MA-sensitive neurons were
tested for calcium dependence and 11 MA-sensitive neurons were tested for
sodium dependence. In three experiments, 17 CAP-sensitive neurons were tested
for calcium dependence. In two experiments, 13 CAP-sensitive neurons were
tested for sodium dependence.
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Results |
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Response and recovery of TG neurons
Cultured chick TG neurons responded to MA and CAP stimulation with
reversible increases in [Ca2+]i. Initial neuronal
responses occurred within 510 s of exposure to the stimulus, which was
the limit of time resolution of the bath application. For both stimuli,
responses peaked within 1015 s of the stimulus flow being initiated.
Recovery to baseline [Ca2+]i typically occurred within
100 s after MA was rinsed from the chamber. Recovery from CAP stimulation took
slightly longer, approximately 120 s. Based upon these observations, we set
the minimum interstimulus interval to be 120 s for all subsequent
experiments.
Experiment 1. Neuronal population response to MA and CAP
Neurons exposed to MA and CAP exhibited positive criterion responses of
increasing magnitude as a function of increasing stimulus concentration
(Fig. 1). The proportion of
chick TG neurons that responded to stimuli (r) was defined by the
relationship r=a(1ebx)c,
where x is the concentration of stimulus, a relates to the
maximum number of neurons recruited by increasing concentration of stimulus,
b relates to the slope of this recruitment function, and c
relates to both the threshold and the slope of the function. In general, the
fit to the mean population response values for MA and CAP was good (MA
r2=0.987, d.f.=2, 4, F=157.946,
P<0.001; CAP r2=0.999, d.f.=2, 3,
F=2799.9, P<0.001;
Table 1). At the concentration
that produced a saturated response level (300 µmol l1),
the number of neurons responding to MA relative to CAP was 2.8 times greater
(47.9% vs 16.6%, respectively;
Fig. 2). MA-sensitive cells
were recruited to the criterion response levels more gradually as MA
concentrations increased. By contrast, CAP-sensitive cells were recruited to
the criterion response levels over a narrower range of concentration (c.f.
parameter c for MA and CAP; Table
1). The concentration of stimulus at which half the population of
responsive neurons was activated was higher for MA (92.3 µmol
l1) than for CAP (31.3 µmol l1).
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Differential responsiveness of neurons was apparent when the normalized
response amplitude was modeled as well. The asymptotic response amplitude was
almost twofold higher for MA-sensitive neurons relative to CAP-sensitive cells
(Fig. 3; c.f. parameter
a, Table 1). Moreover,
the first suprathreshold response concentration was lower for MA (10 µmol
l1) than for CAP (30 µmol l1). By these
two measures, chick neurons were more sensitive to MA stimulation. The
recruitment of neural response was more graded over the concentration range
for MA-sensitive cells relative to CAP-sensitive cells, with CAP-sensitive
cells expressing near asymptotic amplitude responses at the suprathreshold
stimulus concentration (30 µmol l1). By contrast,
the amplitude of responsive neurons at near threshold stimulus concentration
(
10 µmol l1) was 17% of the asymptotic response
level for MA-sensitive neurons (Fig.
3). The concentration of stimulus at which the mean response was
half maximal was 83.0 µmol l1 for MA and 24.0 µmol
l1 for CAP.
|
Experiment 2. Distribution of neuronal sensitivity to MA and CAP
Most (67.9%) of the KCl-sensitive chick TG neurons did not respond to
either stimulus. Slightly more chick TG neurons responded to only MA (16.9%)
relative to those responding to only CAP (10.3%). A smaller proportion of
KCl-sensitive chick TG neurons was sensitive to both MA and CAP (4.9%). Within
this group of neurons, there appears to be no relationship between the
magnitude of responses to MA and CAP. As noted above, KCl-sensitive chick TG
neurons had a lower threshold for response and higher amplitude of response
for MA than for CAP. Fig. 4 is
an example of the differential responsiveness to MA and CAP in three neurons
that were imaged simultaneously.
|
Experiment 3. Ion dependence of neuronal sensitivity
The responses of chick TG neurons to both MA and CAP were dependent upon
the presence of extracellular calcium. In the presence of 1 mmol
l1 extracellular calcium, MA (100 µmol
l1) induced [Ca2+]i increases to
53.0±40.4% (mean ± S.E.M., N=13) of the KCl
standard (Fig. 5A). When the
extracellular calcium was removed from the medium, cells failed to respond to
100 µmol l1 MA. When extracellular calcium was
re-introduced, the response to 100 µmol l1 MA exposure
returned to pretreatment levels (48.1±35.8% of the KCl standard;
P>0.05, Wilcoxon test).
|
A similar dependence on extracellular calcium was observed for CAP stimulation. In the presence of 1 mmol l1 extracellular calcium, [Ca2+]i increased to 46.1±30.9% of the KCl standard (N=17) after stimulation with 100 µmol l1 CAP (Fig. 5B). When extracellular calcium was removed from the medium, cells failed to respond to 100 µmol l1 CAP. When extracellular calcium was reintroduced the response to 100 µmol l1 CAP returned to pretreatment levels (43.8±27.4% of the KCl standard; P>0.05, Wilcoxon test).
The responses of chick TG neurons to MA were dependent upon extracellular sodium. For example, in the presence of 138.3 mmol l1 extracellular sodium, [Ca2+]i increased to 41.6±20.0% of the KCl standard (N=11) in response to 100 µmol l1 MA exposure. Following removal of extracellular sodium, the level of [Ca2+]i failed to increase upon exposure to 100 µmol l1 MA. Reintroduction of extracellular sodium into the medium resulted in complete recovery of responsiveness to 100 µmol l1 MA. The response level (43.8±27.4%) was similar to that seen in the pretreatment period (pre- vs post-sodium removal, P>0.05, Wilcoxon Test; Fig. 6A). By contrast, chick TG neuronal responses to CAP were not dependent upon the presence of extracellular sodium. In the presence of 138.3 mmol l1 extracellular sodium, the [Ca2+]i level in TG neurons increased to 45.6±29.4% of the KCl standard (N=15) in response to 100 µmol l1 CAP exposure. Following removal of extracellular sodium, calcium responses to 100 µmol l1 CAP exposure were unaffected (51.1±32.1% of KCl response; Fig. 6B). However, responsiveness to CAP was diminished following the replacement of sodium. The response level was 28.4±28.3% of the KCl response, suggesting cellular fatigue.
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Discussion |
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The somatosensory systems of birds and mammals are superficially similar
but differ in their response to some chemical irritants. First, many of the
same endogenous compounds (e.g. bradykinin, histamine and serotonin) elicit
pain and/or excite nociceptors in birds
(Koltzenburg and Lewin, 1997;
Gentle and Hill, 1987
) and
mammals (Kessler et al.,
1992
). Pain and irritation are modulated similarly in both taxa.
Aspirin-like analgesics reduce oral irritation in starlings to the chemical
o-acetophenone (OAP), a bird irritant similar in structure to MA.
Moreover, starlings become sensitized to chemical irritants when treated with
prostaglandin E1 (Clark, 1995
).
Second, thermonociceptors of mammals and birds respond to noxious heat in the
same temperature range (Sann et al.,
1987
; Necker and Reiner,
1980
; Nagy and Rang,
2000
). Moreover, the responses of thermonociceptive neurons of
both taxa were antagonized by capsazepine, an antagonist of the vanilloid
receptor, VR1 (Marin-Burgin et al.,
2000
; Liu and Simon,
2000
). Finally, polymodal nociceptors, an important class of
neurons that respond to both noxious thermal and mechanical stimuli, are found
in both taxa (Necker and Reiner,
1980
; Gallar et al.,
1993
). However, although mammalian polymodal nociceptors are
responsive to sub-micromolar concentrations of CAP
(Liu et al., 1996
), neural
sensitivity to similar concentrations of CAP has not been observed in birds
(Wood et al., 1988
).
Despite these similarities of the somatosensory systems of birds and
mammals, major differences also exist. First, birds lack the tetrodotoxin
(TTX)-insensitive sodium channels of mammals
(Petersen et al., 1987).
Second, at concentrations that induce depolarizing currents in rat dorsal root
ganglion or trigeminal neurons, neither CAP nor resiniferatoxin, a capsaicin
receptor agonist, induce inward currents or 45Ca uptake in chick
dorsal root ganglion (DRG) neurons (Winter
et al., 1990
). With one exception
(Petersen et al., 1987
), all
studies to date that have examined the sensitivity of avian neurons to
capsaicin have used concentrations of CAP below 30 µmol
l1, the threshold of CAP response in our study. Petersen
reported that 30 µmol l1 CAP reduced TTX-sensitive sodium
currents in chick DRG. Thus, while it is true that chick neurons are much less
sensitive to CAP than are mammalian neurons, chick neurons are not totally
insensitive. Third, neither corneal (1% CAP=3300 µmol l1)
nor prenatal intraperitoneal (600 mg kg1) application of CAP
in birds caused depletion of substance P- or calcitonin gene-related peptide
(CGRP)-containing neurons (Harti et al.,
1989
) or desensitization to algesic compounds such as histamine,
serotonin or bradykinin (Szolcsanyi et
al., 1986
), which it does in mammals
(Holzer, 1991
). Fourth, Mason
and Clark (1995
) showed that
starlings could detect CAP via the trigeminal nerve but that
responses were not nocifensive.
Consistent with the behavioral studies that show lack of nociceptive behavior towards CAP and the mechanistic studies that demonstrate the failure of CAP to deplete pain neurons of neuropeptides in birds, it is likely that the neurons responding to CAP in our study are not nociceptors. Lacking other functional characterization of these neurons, it is difficult to assign them specific physiological significance. Conversely, because MA is aversive to birds, at least some of the population of neurons that we found in chicken to be responsive solely to MA and not to CAP must be nociceptors. Whether they are sensitive to chemical stimulation only or are polymodal, analogous to mammalian polymodal nociceptors, remains to be determined.
From the experimental data, we infer that the sensitivity of chick TG
neurons to MA results from an activation of a ligand-activated transduction
mechanism. An alternative explanation is that MA and CAP cause membrane
perturbation (Feigin et al.,
1995). We do not believe this to be the mechanism for several
reasons. First, if an increase in intracellular calcium response was due to
membrane perturbation, we would expect the calcium response to be independent
of sodium. This was not the case for MA, the responses to which were dependent
on extracellular sodium. Second, if membrane perturbation was the mechanism
driving neuronal response, we would expect an increasing number of neurons to
respond as the concentration of irritant increased until at some point all
neurons responded. Rather, we observed an asymptote in the numbers of neurons
responding to increasing concentrations of both MA and CAP. A fraction of the
population of neurons remains insensitive to MA and CAP. Third, we established
that different populations of neurons responded to either MA, CAP or both
compounds. If membrane perturbation was driving the response, we would not
expect differential responses among the neurons. This segregation of the
sensitivity to MA and CAP to different subpopulations of neurons suggests that
there are at least two different transduction processes, and these are likely
to be mediated by ligandreceptor interactions. One possible mechanism
mediating the neuronal response to MA that is consistent with the observed
dependence on extracellular sodium is the opening of voltage gated calcium
channels secondary to a ligand-mediated sodium-dependent membrane
depolarization (Kostyuk et al.,
1981
). This mechanism would explain the complete loss of neuronal
sensitivity to MA when sodium is removed from the extracellular medium. It is
unlikely that the initial depolarization of sensory neurons by MA is due to
one of the transient receptor potential (TRP) ion channel family, of which
mammalian and chicken VR1 are members, because these channels are non-specific
cation channels.
Responsiveness to CAP may be similar to that described for mammals, albeit
with decreased sensitivity. In mammals, CAP activates a non-specific ion
channel (VR1), which depolarizes peripheral sensory neurons
(Caterina et al., 1997). This
channel allows the influx of calcium without dependence on extracellular
sodium. Recently, an avian ortholog to the mammalian vanilloid receptor, VR1,
has been cloned from chicken dorsal root ganglion
(Jordt and Julius, 2002
) and
characterized. While similar to mammalian VR1 in terms of sensitivity to
noxious heat (Petersen et al.,
1987
; Marin-Burgin et al.,
2000
) and antagonism of responses to protons by capsazepine
(McIntyre et al., 2001
), the
chicken vanilloid receptor, cVR1, is insensitive to 100 µmol
l1 CAP when expressed in Xenopus oocytes. Because
we observed robust responses to 100 µmol l1 CAP, it is
likely that either (1) a different ion channel mediates the neuronal responses
we observed to CAP or (2) heterologously expressed channels are less sensitive
than the native form. Jordt and Julius also reported that the heterologously
expressed channels were insensitive to MA. However, they tested MA at a
concentration below the threshold we observed.
A question arising from this research is whether the lack of sensitivity to
CAP in birds is evolutionarily primitive or derived? Few data are available to
comprehensively address this question. However, there is a suggestion that
avian insensitivity is an evolutionarily conserved trait. Facial nerves (N
VII) of catfish (Ictalurus punctatus) are insensitive to 0.1% CAP (B.
Bryant, unpublished data). Similarly, dorsal root nerve fibers in toads
(Bufo bufo) were unresponsive to CAP up to concentrations of 430
µmol l1, which is a concentration highly stimulatory to
mammals (Hawkins et al.,
1991). Brown treesnakes (Boiga irregularis) were
behaviorally insensitive to ocular application of 1% oleoresin of
Capsicum (estimated 100 p.p.m. CAP;
Clark and Shivik, 2002
). All
of these taxa, including birds, share a common trait of insensitivity to CAP.
The common ancestor of reptiles and birds post-dates the phylogenetic
divergence of mammals while the phylogenetic origin of amphibia and teleosts
pre-dates that of mammals. The implication of this pattern is that avian
insensitivity to CAP is the primitive trait and that mammalian sensitivity is
a derived trait.
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Acknowledgments |
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