Mechanosensory Neurons With Bend- and Osmo-sensitivity in Mouthpart Setae From the Spiny Lobster Panulirus argus

2004·5.8 MB·

by  Anders Garm

Reference: Biol. Bull. 207: I95-20S. (December 2004)
2004 Marine Biological Laboratory
Neurons With Bend- and
Mechanosensory
From
Osmo-sensitivity in Mouthpart Setae the Spiny
Lobster Panulirus argus
ANDERS GARM1'*, CHARLES D. DERBY2. AND JENS T. H0EG1
1 Zoological Institute, University ofCopenhagen. Denmark; ami ~ Department ofBiology,
Georgia State University. Atlanta, Georgia
Abstract. The mouthparts of the spiny lobster Panulirus uli. These specializations, called setae, are hollow, hairlike
argus hold primarily two types of setae simple setae and extensions of the cuticle that contain the dendrites of the
cuspidate setae. Mechanosensory neurons from these setae sensory neurons. Individual setae ofaquatic crustaceans are
were examined by electrophysiological recordings. The either mechanosensory. chemosensory. or bimodal (both
population of simple setae contained two types of mech- mechanosensory and chemosensory), the latter being the
anosensory neurons: displacement-sensitive neurons, which most common type based on morphological evidence
responded to deflection at the setal base; and bend-sensitive (Schmidt and Gnatzy, 1984; Derby, 1989; Schmidt, 1989;
neurons, which responded to bending of the setal shaft. Gate and Derby, 2001, 2002a. 2002b; Garm et al.. 2003).
Displacement-sensitive neurons, in general, responded pha- Crustaceans have been shown to respond behaviorally to
sically and only during actual displacement. Typically, their changes in the osmolarity oftheir surroundings (Jury etal.,
response changed with alteration of the direction, ampli- 1994; Dufort et al.. 2001); while the sensory structures
tude, and velocity/acceleration of the mechanical stimulus. behind this behaviorare unknown, there are indications that
Bend-sensitive neurons, in general, responded phaso-toni- the setal mechanorecptors may sense osmotic changes
cally and carried information on the direction and region of (Tazaki, 1975).
bending. This is the first experimental demonstration of Unimodal mechanosensory setae are locatedonthe dorsal
bend sensitivity forarthropod setae. Cuspidate setae contain side of the carapace and abdomen and on the antennae of
highly sensitive mechanosensory neurons; however, due to many decapod crustaceans. They are typically plumose
the rigid nature of these setae, whether they were bend (featherlike) in shape, innervated by one to four mech-
sensitive ordisplacement sensitive could not be determined, anosensory neurons, and specialized for detecting water-
and they were thus called "tactile neurons." Bend-sensitive borne vibrations (Wiese. 1976; Vedel and Clarac, 1976;
neurons, but not displacement-sensitive neurons or tactile Tautz et al.. 1981; Vedel. 1985). They are sensitive to
neurons, showed graded responsestochanges in osmolarity. displacement, being responsive to movements of the entire
The osmosensitivity ofthese neurons could mediate behav- seta around the basal membranous region, and can respond
ioral responses to changes in the osmolarity of seawater or to displacements as small as 0.01 degrees (Wiese, 1976).
food. Moreover, they respond most strongly to displacement in
onedirection, usually perpendiculartotheoutgrowthsofthe
Introduction seta. Equipped with fields of these setae, each with a dif-
Because they are completely covered by an exoskeleton. ferent directionality, animals can obtain detailed informa-
tion about the location of the source of the vibrations.
crustaceans need specialized sensors todetectexternal stim-
Bimodal setae with mechanosensitivity have been exper-
imentally demonstrated for a variety of setal types on the
Received 18 February 2004; accepted 10 September2004. pereiopods and antennae of decapods (Shelton and Laver-
and*TOorgwanhiosmmcBoirorleosgpyo.ndLeunncde Usnhiovuelrdsibtey.adHderlegsosnedavaatgDeenpa3.rt2m2e3n6t2ofLuCnedl.l ack. 1970; Halt and Bauer, 1980; Altner et al.. 1983; Gate
Sweden. E-mail: [email protected] and Derby, 2001. 2002a. 2002b; Schmidt et al.. 2003).
195
196 A. GARM ET AL.
These mechanosensory neurons are believed to be displace- krill. and squid. A SONY DXC 950P color (Y/C) 3CCD
ment-sensitive, but little is known about their sensory prop- camera equipped with a Micronikkor 105-mm macro lens
erties such as directionality or response to changes in ve- was placed outside the aquarium and enabled 5-/u,m resolu-
locity. tion. Recordings were made on PAL super VHS. Light was
Decapod crustaceans are known to have flexible feeding obtained from a 120-W bulb. Representative images of
behavior. They can change how theirmouthparts are usedin mouthpart movements were captured with a time resolution
feeding, depending on the nature especially the size of of 20 ms (50 images/s) using the frame grabber card
the prey item (Greenwood. 1972; Caine. 1975a. 1975b; DVRaptor from Canopus; the images were imported into
Gerlach eta!., 1976; Kunze and Anderson. 1979; Schembri. CorelDraw 10.0, with a resolution of 720 X 564 pixels.
1982; Hunt et <//., 1992; Johnston. 1999; Garm and H0eg,
2001; Garm et ui. 2003; Garni, 2004). Moreover, crusta-
Electrophysiology
cean mouthparts are known to have a high density of setae
with a great diversity in external morphology (Schembri. Spiny lobsters, Paiui/irns argiis, with carapace lengths of
1982; Stemhuis eta!., 1998; Garm and H0eg, 2000; Coelho of 40-80 mm, were captured in the Florida Keys and
et a/., 2000). and the sparse morphological evidence sug- maintained in two 400-1 aquaria with artificial seawater
gests that most are bimodal mechanoreceptors and chemo- (Instant Ocean. Aquarium Systems, Mentor, OH) at 23-28
receptors (Paffenhofer and Loyd. 2000; Garm et /., 2003). C, and fed shrimp and squid. Mouthparts examined in this
To modify their feeding behavior, therefore, crustaceans are study included the mandibularpalp (/; = 4), basisofmaxilla
likely to acquire tactile information about the texture and 1 (// = 2), basis of maxilliped 1 (/; = 6), endopod of
shape of food while it is being handled by the mouthparts. maxilliped 2 <;i = 10), and endopod of maxilliped 3 (n =
Most ofthis information probably comes from the setae on 2\ ). Between one and four neurons were studied per ap-
the maxillipeds, since these mouthparts perform most ofthe pendage. To gain access to the nerve bundles and artery of
manipulation and orientation ofprey items during handling. the various mouthparts, one of the appendages was ablated
In this study, we have tested the hypothesis that mech- immediately before each experiment and dissected in cold
anosensory neurons in the mouthpart setae ofdecapod crus- lobster saline (gram/liter: 28 NaCl, 0.75 KC1, 3.4
tfaeceedainnsgpbreohvaviidoert.acWtielehianvfeorumsaetdiotnheiCmaporritbabnetanfosrpcionnytrloolblsitnegr 0M.g7C2l2H-E6PHE,SO),. 2F.o5rCmaaCxli:l-l2ipHe2dO,3.3boNtah:StOhe4. d0a.c3tylgluuscoasned,
Panulirns urgiix (Latreille. 1804) because it is an estab- propodus were removed from the endopod. The cuticle was
lished model animal forcrustacean sensory biology (Derby. removed from the propodus, and after the apodeme was cut
2000; Derby eta/., 2001; Harrison etat.. 2001; Ache. 2002; at its insertion onto the dactylus, the muscle was gently
McClintock and Xu. 2002). and because its setae are large removed with forceps. This dissection left behind only the
and thereby accessible for experimental work. Our focus is principal artery flanked by the major nerve to the append-
on the mandibular palp, the medial rim of the basis of age. The artery and nerve bundle were separated from each
maxilla I and maxilliped 1. the propodus and dactylus ofthe other with a pairoffine tungsten needles, and the nerve was
endopodofmaxilliped 2. and the dactylus ofthe endopod of divided into four to six bundles. For the mandibular palp
maxilliped 3. These structures are often in direct contact and endopod of maxilliped 2. the procedure was similar,
with prey during food manipulation (Garm, 2004), and thus although the endopod of maxilla 2 was cut at the merus-
are likely to have sensory functions; moreover, they are of carpus joint, and the nerve in the carpus was used instead.
sufficient size for convenient experimental manipulation. For maxilla 1 and maxilliped 1. the basis was cut from the
We examined the sensitivity of these neurons to setal dis- limb, and the cuticle was removed from the proximal half.
placement (deflection at their insertion), setal bending (de- In these limbs, only a small amount of muscle and connec-
flection along their shaft), and osmotic changes in the sea- tive tissue had to be removed to reveal the nerve and artery.
water around them. When maxilla 1 or maxilliped 1 was used, the lobster was
anesthetized on ice before dissection. Even though involved
Materials and Methods in food handling, the basis of maxilla 2 was not examined
due to its small size.
Video recordings After the dissection, the preparation was secured in a
stimulating-recording chamber made of two petri dishes
Adult male and female spiny lobsters. Ptmitlirm urgus, separated by adental-wax barrier(Fig. 1A). The preparation
with carapace lengths of 45-90 mm. were obtained from was secured in the wax such that its two parts were bathed
Bermuda and kept in a 300-1 aquarium at Danmarks Ak- in different solutions. The distal part of the preparation,
varium in Copenhagen. The video recordings were made in which housed undissected cuticle and setae, was situated in
50-1 aquaria. Both systems had running seawater at 24 C. a stimulating dish that contained artificial seawater (gram/
Forthe recordings, the animals were fed mussels, fish meat. liter: 24.7 NaCl. 0.66 KC1, 4.7 MgCK- 6H2O? 1.9
MECHANO- AND OSMORECEPTORS IN P. ARdl'S 197
B
Manipulate
hook
Stationary
Piezo-crystal
V
M
/Recording
electrode Primary
bending point
Reference
electrode
Cut setae
Cannul
Saline
Figure 1. Experimental setup. (A)Schematicoverviewofthe stimulating-recordingchamber,showinghow
mechanical stimuli were applied with a piezoelectric-crystal-controlled probe. (B) Stimulation of a bend-
sensitive cell. Neighboring setae were removed to allow space for stimulation of the seta innervated by the
neuronofinterest.Thissetawasbentaroundtheattachmentofastationaryhook.Thebendinghook wasmoved
eitherby apiezoelectric crystal (see A) or by hand.
CmaalClpvai2lH,,wOh.ic6h.3coMntgaSinOe4d-t7hHe:Oe,xp0o.s1e8dNnaerHvCeOa,n)d:arttheeryp,rowxais- afmrpolmifi0e.r1.2-T1h.i2smprmovmiedaedsumreodvewmietnhtasmriacnrgoimnegteinrasmcpalleitbuadre.
situated in a recording dish that contained saline. After the Simply attaching the hook to a simple seta resulted in no
wax barrier was sealed, the artery was cannulated and detectable alteration of the movements. To ensure that no
perfused with pressurized, oxygenated lobster saline at a force was applied in other than the direction of displace-
rate of0.4-1.1 ml/min (see Derby, 1995, for more details). ment, the hook moved freely along the setal shaft. This had
The time from ablation of the mouthpart to perfusion was the side effect that the angular velocity ofthe displacement
10-15 min. and nerve recordings were initiated 15 min was not constant, but declined within a single displacement
later. following the equation: tan a = D/L, where a is the angle
Electrophysiological recordings were made en passant ofdisplacement, D is the distance traveled by the hook, and
with fine-tipped extracellular suction electrodes (Derby. L is the distance from the socket of the seta to the initial
1995). To determine whether the recording included mech- attachment point of the hook. This also meant that the two
anosensory neurons, a probe was used to brush the setae. It parameters, velocity and acceleration, could not be sepa-
neural responses were detected, the seta innervated by the rated in our experiments. This coupling was enhanced by
active neuron was identified by stimulating progressively the failure of the hook to attain maximum velocity instan-
smaller areas with the probe. The neighboring setae were taneously; rather there was a period of acceleration, which
then removed with a pair ofscissors to ensure space for the was most significant when square signals were used. In
micromanipulators and toensure free movements ofthe seta effect the displacements started with a short period of ac-
ofTinwtoeretsytpe(Fsigo.fImBe)c.hanical stimulation were presented celeration to maximum velocity, then slowly decelerated
displacement and bending. Displacement is defined as mov- untAil cthheandgierecitniotnherevveelrosceidt.y/acceleration and amplitude of
iicftulnstegeiclctft,liheoesnoasitetnthiaattetsh,etshwoseicettkaahenltgnlscohehafabatnep,gpteawwsrie.etenhnBtoeutbntheedanibndnayigsnedgiesotofedfcettfhtaeihbnelseeedsteadtaiasaslnpaldsahdcateefh--te ttdhhueersasettiitoemnsutlos,ufsttrhwieaasnelgeauclctacrroicmsatplilmisuislghineawdlebrfyerocumhsaetdnh.geiWwnhagevtenhegtevhnoeelrvtaeatlgooerc.iatnIydn/
mseetnatwaatsitasttbaacshee.dTtoo eaxsammailnletduinsgsptleancehmoeonkt,sewnhsiitcihvitiyn,tutrhen aatcceeiltehreart0i.o4n8smenmsit(i2vi0t0yVw)asort0e.s7t2ed,mtmhe(3a0m0plVi)t,udaendwsatsimfuilxuesd
was connected to a piezoelectric crystal via an extension duration was then varied using a stimulus frequency range
arm (Fig. 1A). Movement of the crystal was effected by of 1-10 Hz. When amplitude sensitivity was tested, the
applying 50-500 V, either in square or triangular pulses, velocity/acceleration was fixed by using halfthe stimulation
using a wave generator amplified by a high-voltage DC duration when doubling the voltage. Stimuli were presented
198 A. GARM ET AL.
continuously for 15 s. Most displacement-sensitive neurons Results
were also tested for bend sensitivity.
Toexamine bend sensitivity, one hook was situated at the Theresults are summarized inTables 1 and 2. Recordings
place ofdesired bending, and the distal part was then moved were obtained from neurons innervating two types of se-
to an angle of about 45 by either a handheld needle or a tae simple and cuspidate (Fig. 2A, B). None of the neu-
hook connected to a piezoelectric crystal (Fig. IB). The rons showed any spontaneous activity. Simple setae con-
neurons were also tested for their sensitivity to bending tained neurons that appeared to belong to either of two
proximally relative to the holding point. All bend neurons categories (Tables 1 and 2, Fig. 3). The first were displace-
were tested for displacement sensitivity by using maximum ment-sensitive neurons that responded to deflection of the
amplitude as described above. entire seta at its socket. The second were bend-sensitive
When testing directionality, both displacement and bend- neurons that responded to bending of the setal shaft; a few
ing were presented in four directions: distally (towards the ofthese neurons also gave small responses when displaced,
atipppeonfdtahgee)a,ppaennddamgeed)i,alplryoxainmdallaltyer(alalwya(ytofrthoemltefhteatnipdotfo tthhee pberiensgumdaibslplyacbeed.caTuhsee voifdesolirgehtcorcdoinncgosmisthaonwtedbetnhdatinbgenudpionng
right of the appendage). Four displacement-sensitive and does occur during food manipulation (Fig. 2C. D; supple-
four bend-sensitive neurons were tested for the persistence mentary video clips can be viewed at <http://www.mbl.
edu/BiologicalBulletin/VIDEO/BB.video.html>).
of their response after the distal half of the seta was re-
moved with a pair of scissors. The cuspidate setae contained neurons sensitive to the
rigBide,cathuesyeccouuslpdidnaottebseetmaeovaneddtbhyeirthreedcruycsetdal,sobcuktetonalrye vbeyrya rcsleoifugelhrdtreesndtottmoobaevses"mtteaincmttuilslaetonefedutrihonensas.e"ctoa,ntrboultlesdinmceanntheers,e tnheeuyroanrse
handheld needle. Thus, displacement and bending could not
be reliably separated.
Sensitivity tochanges in osmolarity was tested by placing Displacement-sensitive neurons
the appendage in a tube with full-strength artificial seawater Displacement-sensitive neurons displayed phasic re-
(i.e. 3.5%) flowing at 5 ml/min. The seawater was then sponses, spiking only during the actual setal displacement
exchanged with a 8-s pulse of either deionized water or a (Fig. 3A). None of the neurons responded to bending, and
concentration series of artificial seawater from 1.75%- all of the neurons continued to respond to displacements
5.25% (i.e., 50%-150% full-strength seawater) made by after the distal half of their seta was removed. They were
changing the concentration of NaCl. The concentration se- differentially sensitive to the direction, velocity, and ampli-
ries was presented in steps first from 100% down to 50% tude of displacement, as described below.
and then from 100% up to 150%, with intertrial time inter- Halfofthe displacement neurons (9 of 18 tested) showed
vals of 1 min. Three of each neuron type were also tested directional sensitivity; in most cases, they responded much
with deionized water for 5-15 min. more strongly (i.e. with more spikes) to one of the four
Each recording was made for 10 s with a sampling directions (Fig. 4). The "best" direction varied among the
frequency of 56 kHz using Axoscope 9.0 software (Axon nine neurons, but distal and proximal displacement was
Instruments, Inc.. Union City. CA). Spike sorting and quan- most frequently the best direction. Only one neuron gave
tification were performed using Datapac 2000 software most spikes to lateral displacement, and no neuron re-
(RUN Technologies. Mission Viejo, CA). All spikes for a sponded most strongly to medial displacement. Some re-
given stimulation were used when analyzing both the num- sponded exclusively to displacement in one direction, but 7
ber of spikes per stimulation and the interspike interval of 9 neurons responded to two or more directions (Fig. 4).
during a single stimulation. There was no significant difference between the number of
Table I
Number<'/ r<7/\ ro;ci/fareuch stimulationparameterorcombination ofparameters
Type
MECHANO- AND OSMORECEPTORS IN P. ARGUS 199
Table 2
Summary ofsensitivity ofmechanosensory neurons
Setae type
200 A. GARM ET AL
Figure2. Morphologyofthesetae: scanningelectronmicrograph(SEM)andvideoimages. (A)SEMimage
ofastoutcuspidate seta from the endopodofmaxilliped 2; the arrowhead indicates the very reduced socketof
theseta. (B) SEM imageofsimple setae from thebasisofmaxilliped 1. (C, D) Videostill imagestaken ca. 1 s
apart showing a simple seta (arrowhead) that is from the carpus ofmaxilliped 3 (M\p3 car) and bends during
food manipulation. The seta isalmost straight before preycontact (C)but bends in the distal halfwhencontact
is made (D). The broken line indicates the prey held by the mouthparts. car = carpus, Lb = labruni. Mdp =
mandibular palp. Mxp2 = maxilliped 2, Mxp3 = maxilliped 3.
phaso-tonic high-frequency response, while otherdirections tested with a set of seawater stimuli at different osmolari-
produced phasic responses and lower response rates (Fig. ties 1.75% to 5.25% salt concentration. (Full-strength sea-
8). Eight of the ten neurons tested responded unequally to water is 3.5%, so these range from 50% to 150% ofnormal
bending at different regions in that they only responded to seawater; Fig. 10B). One neuron responded most strongly to
bending of the distal half of the seta (Fig. 9), but the hyperosmotic stimuli (Fig. 10B. neuron 1); one neuron
experimental setup did not allow for precise location ofthe responded only to hyposmotic stimuli (Fig. 10, neuron 2);
most sensitive region. Two neurons responded to bending and two neurons responded in similar ways to hypo- and
anywhere along the setal shaft. None of the four tested hyperosmotic stimuli (Fig. 10. neurons 3-4). but with dif-
neurons responded tobending afterthe distal halfofthe seta ferent intensities. The response to changes in salinity
was removed. showed a response latency of several seconds, even when
Bend neurons were sensitive to changes in osmolarity the neurons were exposed to deionized water (Fig. 10A).
=
(Fig. 10). All tested neurons (n 11) responded with
high-frequency spiking to pulses of deionized water. The Tactile neurons from cuspidate xetac
response to bending did not change after stimulation with
8-s pulses of deionized water. Three neurons were kept in The 10 tactile neurons obtained from cuspidate setae
deionized water fora prolonged period, and they all stopped were highly sensitive, responding to the slightest touch by
responding to the deionized water after 1-2 min. One neu- the probe that caused no visible setal movement (Fig. I 1A).
ron spiked constantly for 1.5 min before it stopped respond- Eight of the neurons gave phaso-tonic responses to pro-
ing. These three neurons showed a profound reduction in longed displacement (Fig. 11B): these neurons tended to
responsiveness to bending before they stopped responding. adapt similarly to bend neurons (Fig. 11C). Sensitivity to
None ofthe three neurons responded to bending after 5 min direction, amplitude, or velocity of mechanical stimulation
in deionized water, and none ofthem recovered after30 min was not examined because stimulation was necessarily per-
in full-strength seawater. formed by hand and therefore was not accurate enough for
Bend neurons differed in their responses to the salinity such quantification.
treatments. Four bend neurons from maxilliped 3 were The tactile neurons were not sensitive to pulses ofdeion-
MECHANO- AND OSMORECEPTORS IN P. ARGUS 201
Displacement-sensitivecell
B
202 A. GARM ET AL.
A
12-
C
MECHANO- AND OSMORECEPTORS IN P. ARGUS 203
tively) (Lavalli and Factor. 1W2; Garm and H0eg, 2001).
Directionofbending. DDistal Bend sensitivity, perhaps in coordination with propriocep-
Proximal tors, may provide animals with information on the hardness
DLMaetdeiraall ofthe prey, affecting the decision about whetherto crush or
bite the prey item with the mandibles. The phaso-tonic
response ofbend neurons should ensure that the animals get
information on the position ofthe objecteven when the prey
is notmoving orbeing moved. The directionality and region
of sensitivity of bend neurons should give information on
shape, texture, and location of the prey item, in addition to
the stimulus direction and velocity information provided by
Figure8. Sixoften tested bend neurons were directionally sensitive, displacement neurons (discussed below).
respondingwith morespikeswhenbentinonedirectionthaninothers.The An interesting question is how the directional sensitivity
given direction ofbending is relative to the long axis of the appendage. ofthe bend neurons is enabled. It could be due either to the
Noneofthesixneuronsrespondedexclusivelytoonedirectionofbending, mechanical properties of the setal cuticle making bending
tnbhuat=tiint\sgfeeoneremarelaldlrtteohceodryidsipsnlhgaosyw.ead"waor"sbte"std"irdeicrteicotni.onN.oNeerurroornba6rswaarsedgiifvfeenresnitncien micoarlearlrikaenlgyeminenotneofornemuorroensdiirnescitdieontsh,eosrettaoltlhuemmeon.rpWhoelodgi-d
not examine the mechanical properties of the setae in any
detail; but since the recorded responses are all to the same
manipulation (Fig. 2), and this should activate the bend- degreeofbending (approximately45), stiffnessofthe setae
We
sensitive neurons. As seen in video recordings, the distal cannot account forthe observed directionality. therefore
part ofthe seta is most likely to bend during food manipu- believe that the arrangement ofthe outerdendritic segments
lation, consistent with the fact that most bend neurons are is causing the directionality of some of the bend neurons.
only sensitive to bending distally (Fig. 9). Decapods in Normally the sensory cilia are arranged in a ciliary com-
general have mandibles with separate areas for biting or partment enclosed by a dendritic sheath with no noticeable
crushing the prey (incisor and molar processes, respec- order or orientation, but this may be different for these
Bend
tip
Stimulus Bend
B
Bend distalthird f
Bend
Bend midways
Bend
Os 0.5s 11ss 1.5s 2s
Figure9. Responsesfromabendneuron(arrow)tobendingatdifferentregionsofthesetalshaft.(A)When
thesetawas bentcloseto its tip, theneuron respondedphaso-tonically throughouttheentire periodofbending.
(B)Whenthe setawasbentintheregion about athirdofthe way fromthetip.theneuron respondedphasically
withonlytwospikes. (C)Thisneuron,similartofiveoftheotherbendneuronstestedforregion-sensitivity,did
notrespondtobending at the proximal halfofthe setal shaft. Arrowheads indicate responses from unidentified
neurons.
204 A. GARM ET AL.