GABA-Like Immunoreactivity in the Nervous System of Oikopleura dioica (Appendicularia)

6 Pages·1991·2.1 MB·English

by  T Bollner

Reference: Biol. Bull 180: 114-124. (February. 1991)
GABA-Like Immunoreactivity in the Nervous System
of Oikopleura dioica (Appendicularia)
TOMAS BOLLNER' *. JON STORM-MATHISEN2. AND OLE FETTER OTTERSEN:
*Department ofZoology. Stockholm University, 5-706 91. Sweden, andAnatomical Institute.
University ofOslo, KarlJohans gt. 47. N-0162 Oslo 1. Norway
Materials and Methods
Abstract. The cellular localization of -y-aminobutyric
acid(GABA)hasbeen visualized immunocytochemically Specimens of0. dioica Fol, 1872. were collectedatthe
iannttihseernuemrvtoougslustyasrtaelmdeohfydOeifkioxpaltieounracodmipoliecxaebsyofusGiAnBgAa.n KMrairsitnieneBbieorlgogMiacrailnLeabBioorlaotgoircya,l bSotatthioonnatnhdeawtestthecTojasatrnoof
TheresultsshowGABA-likeimmunoreactivityinneurons Sweden. Forimmunocytochemistry thefollowingfixatives
ofthe brain, in cells ofthe sensory vesicle, in the caudal were used: (1) 5% glutaraldehyde, (2) 3% glutaraldehyde
ganglion, and in the nerve cord. Positive reactions were and 1% paraformaldehyde, or(3) 1% glutaraldehyde and
also found at the neuromuscular terminals in the tail. 1% paraformaldehyde, all in 0.1 A/ sodium-phosphate
buffer at pH 7.4. After 1 h fixation at room temperature,
Introduction
animals were kept and transported in cold sodium-phos-
Amino acids are considered to be important neuro- phatebufferwith0.5% glutaraldehydeadded. Free floating
transmitters in the vertebrate central nervous system (re- whole tissues were processed for immunocytochemistry
view: Ottersen and Storm-Mathisen, 1984a). Among as described by Storm-Mathisen et ai (1983) and Dale et
these, 7-aminobutyric acid (GABA) isa dominant inhib- ai, (1986); the primary anti-serum was diluted 1:300 be-
itory neurotransmitter in the brain and spinal cord, and fore processing according to the peroxidase-anti peroxi-
even occurs in the peripheral nervous system (Jessen et dase technique (Sternberger, 1979).
ai. 1986). GABA has also been reported to be a major After fixation in 3% glutaraldehyde and 1% paraMform-
inhibitory neurotransmitter in a wide variety ofthe in- aldehyde or 1% glutaraldehyde alone, both in 0.1 so-
vertebrate phyla (Gerschenfeld, 1973; Meyer et ai. 1986; dium-phosphatebufferat pH 7.4. animalswereembedded
Vitellaro-Zuccarello and De Biasi, 1988). But no infor- in Epon resin andcutwith aglassknife. One-^m sections
mation is available concerning this amino acid in the wereprocessedonglassslidespreviouslycoatedwitheither
Urochordata,agroupthat isoftenconsideredtobeaphy- chrome alum gelatin or poly-L-lysine, and processed by
logenetic link between invertebrates and vertebrates. the immunogold-silver (IGS) method, as follows.
The organization ofthe nervous system ofOikopleura The sections were etched for 45 min in sodium-etha-
dioica has been investigated by several authors (Gait and nolate, washed 3X5 min in absolute alcohol, followed
Mackie. 1971; Holmberg, 1984; Bollneretai. 1986), and by 2 X 5 min in distilled water, and rinsed briefly in 20
the presence of acetylecholinesterase in this species has mMTrisbufferatpH 7.4containing 155 mMNaCl,0.1%
been reported (Durante, 1959; Flood, 1973). The aim of BSA,and20mA/NaN3.Thesamemedium wasalso used
this investigation was to establish whether Oikopleura for subsequent rinses and for diluting sera. The sections
dioica exhibits GABA immunoreactivity in its central or were then incubated with a droplet of 5% normal goat
peripheral neurons. serum in a moist chamber for 20 min. Thereafter they
were incubated overnight in 50 n\ primary anti-serum
*RePcreeisveendta2d2drJeassn:uaDreypa1r9t9m0e;natccoefpBtieodlo6gyN.oUvnievmebrseirty1o9f90L.ondon.TRWoy2a0l d3iXlut1e0dm1i:n10i0n.bAufftfeerraatrpiHnse8.i2n,tbuhfefseer,ctfioonlslowweerdebiyncwuabsahteesd
HollowayandBedfordNewCollege.Egham Hill.Egham,Surrey for60 min with GAR G5 (goat anti-rabbit immunoglob-
OEX, UK.
119
120 T. BOLLNER ET AL
ulin adsorbed to 5 nm colloidal gold. Janssen. Belgium)
diluted 1:80 in the same buffer. Finally the sections were m
washed with the same medium as after primary serum,
rinsed in distilled waterseveral times(5 min each at least),
developed in 100 ^1 silverenhancer kit (Janssen), washed
in water, and coverslipped.
Indirect immunofluorescense aswellastheperoxidase-
antiperoxidase (PAP) method ofSternberger (1979) were
also tried; the same primary antibodies were visualized
by either a second layer ofFITC-conjugated sheep anti-
rabbit serum (SIGMA), orby unlabeled sheep anti-rabbit
serum(StatensBakteriologiska Laboratorium, Stockholm)
followed by PAP complex (Dakopatts). Figure 1. SchematicdrawingofOikopleiira dioicashowingthepo-
The antiserum against glutaraldehyde-conjugated 7- sitionofthenervoussystemwiththeimmune-positiveneuronsindicated,
aminobutyric acid (GABA antiserum 26) was raised, pu- the position ot the sensory' vesicle is indicated by the dotted circle, ab.
rified, and characterized asdescribed previously (Ottersen amnotuetrhio:rnb.ulnbe:rvber,cobrrda;inn;me.g,nceauurdoamlusgacnugllaironj:ungc.tigoonn.adBsa:ri.=in2t0e0stiMnme.: m,
and Storm-Mathisen, 1984b; Ottersen el ai. 1986). For
all methods used, the controls included absorption of
GABA
antiserum with GABA-glutaraldehyde complexes sections(Fig. 7)andinwholemounts.Transversesemithin
(GABA-G) and glutamate complexes (Glu-G) at final sections through the tail showed GABA-like immuno-
concentrationsof300^Af. orreplacement ofthe primary reactivity in the large nerve terminals(Fig. 9) innervating
antiserum with normal rabbit serum. Furthermore, the the muscles (cf. Flood, 1973, 1975) and in fibers ofthe
immunoreactivity of the antiserum used was tested ac- dorsal nerve cord. Furthermore, positive staining was of-
cording to the filter disc method described by Ottersen ten seen in the epithelial cells ofthe notochord. Because
and Storm-Mathisen (1984b). The fixation conjugates the tail of the animal is twisted 90 counter-clockwise,
spotted on thediscswere made from macromolecules ex- the dorsal nervecord isseen tothe right ofthe notochord
tracted from ratbrain homogenateandfrom homogenate in a frontal view ofa transverse section oftail.
oftheneural complex from theascidian Cionaintestinalis. The semithin sections from animals fixed with 1% glu-
taraldehyde processed using the immunogold technique
Results gaveastrongerreaction thananyothermethod. The PAP-
method showed weak staining in the anterior bulbs and
The central nervous system ofOikopleiira dioica con- in the caudal ganglion, whereas the FITC-incubated sec-
sists ofan anterior ganglion (brain) and tail ganglia. The tions showed clear label in the same regions as did the
anterior part ofthe brain is extended into paired bulbs, immunogold method. However, all methods showed
and in the mid-region it has a sensory vesicle. The brain similar patterns ofimmunoreactivity.
is connected to several ganglia in the tail by a solid nerve Theanti GABA serum, eitherabsorbedwith glutamate-
cord. The largest ofthese tail ganglia is referred to as the glutaraldehyde complex (Glu-G) or not, produced selec-
caudal ganglion (Figs. 1, 2). tive staining ofthe GABA conjugates on the filter discs,
Although the brain is hard to see in whole mountsdue butnosignificantstainingcouldbeseen afterpretreatment
to the thick oikoplast epithelium, GABA-like immuno- of the GABA antiserum with GABA-glutaraldehyde
reaction was observed in the paired anterior bulbs de- complex (GABA-G) (Fig. 8). Similarresultswereobtained
scribed by Bollner el a/., (1986). In semithin sections, a with spots ofamino acids conjugated to macromolecules
positive reaction is easily seen in some ofthe neurons in from rat and Ciona, suggesting that the previously dem-
tthhee bGuAlbBsA(Fiagn.ti3-).seIrnumthewerreestfoofunthde: birnaionn,estcaeillninlgocwaittehd wonhsotlreatmeodunsptesciafsicwietlylaissivnalsiedmiatlhsionfsoerctiuornosc,hotrhdearteeasc.tioInn
ventrally in the mid region, in one of the most caudal wasvirtually abolished when antisera treated with GABA-
cells (Figs. 2, 4), and in a dorsal cell close to the sensory G or normal rabbit serum were used instead ofGABA
vesicle (Fig. 5). Furthermore, immunopositive staining antiserum. Treatment ofthe anti serum with Glu-G did
was seen in the epithelial cells referred to as the "brain not have this effect.
vesicle cells" by Holmberg (1984) (Fig. 6).
Two immunostained cell bodies situated in the caudal
Discussion
part of the caudal ganglion were observed in semithin
sections (Fig. 7) and in the whole mount preparations Many investigations have established the presence of
(Fig. 8). Immunoreactive fiberswereseen in both semithin either GABA or glutamic acid decarboxylase (GAD),
GABA IN OIKOPLKL'RA NERVOUS SYSTEM 121
IL br
<^
Y|Ai-4Q*ij '
,''
Figure 2. Sagittal section through the whole animal showing the localization and GABA-like immu-
noreactivity (arrowheads) ofthe brain (br), one ofthe anterior bulbs (ab). the nerve cord (n), the caudal
ganglion (eg) and in a neuromuscularjunction ofthe tail (arrow). Stainingcan also be seen in thegonads
(g),at the apical surfaceofsomeofthe intestinal cells(i). and in the rectum (r). Nostainingcould beseen
in an adjacent section treated with anti-GABA/GABA-G. 1GS method. Differential interference contrast.
Bar= 100Aim.
which catalyzes the synthesis ofGABA from glutamate, be neurons with inhibitory functions, it should be re-
in lower chordates and in invertebrates (Osborne, 1972; membered thatGABA may also havedepolarizingeffects
Osborne ct at., 1979; De Biasi, 1986). More recently, the (Alger and Nicoll, 1982). In addition to the neural local-
use of specific antibodies (Storm-Mathisen ct ai. 1983) ization,GABA wasclearly present in epithelial cells. This
has led to a more precise knowledge about the cellular agrees with the situation in vertebrates, where GABA has
localization ofGABA in both vertebrates (Ottersen and been demonstrated in non-neural epithelial cells (Oren-
Storm-Mathisen 1984, a, b; Roberts et ai. 1987) and in- sanz el ai, 1986; Davanger et ai, 1989). Amino acids in
vertebrates(Bickerel ai. 1985; Meyer et ai. 1986; Hom- general are also known to modulate osmoregulation (see
berg et ai. 1987). This is, to our knowledge, the first im- Gilles, 1979, for review). Synthesis of GABA has been
munocytochemical studyon theoccurrence ofamino ac- reported to occur in fish erythrocytes where it may par-
ids in the nervous system of a protochordate. Using ticipateinthemaintenanceofaconstantcell volume(Fu-
biochemical analyses, Osborne etai (1979) foundGABA gelli et ai, 1970). O. dioica is an isosmotic animal, and
and several other putative amino acid neurotransmitters the vesicle and thechorda arethe only internal structures
in homogenates ofthe cerebral ganglion ofanother pro- not totally surrounded by hemolymph, and therefore
tochordate, the tunicate Ciona intestinalis. might use GABA for regulating the intracellular osmo-
Thepresent investigation showsthecellularlocalization larity.
of a GABA-like substance in the nervous tissue of O. The muscles in the tail are innervated both by fibers
dioica. TheGABA-positivecellsintheanteriorbulbsand branchingdirectly fromthe nervecordandfrom perikarya
in the rest ofthe brain are thought to be neuronsjudging alongthecord(Flood, 1973). These nerveshaveelaborate
from their location and their ultrastructural appearance end-arborizations on the surface ofthe muscle cells and
previously described by Bollner et ai. (1986, unpubl.). are thought to be cholinergic (Flood, 1975; Bone and
Also, most ofthe cells in the caudal ganglion are consid- Mackie. 1982). Cholinergic neuromuscular transmission
ered to be neurons. However, one ofits anterior cells, a iswidelydistributed throughout theanimal kingdom, but
large ependymal cell, produces the Reissner's fiber GABA-ergic inhibition of muscles is only known in in-
(Holmbergand Olsson, 1984). The significance ofGABA- vertebrate phyla (Gerschenfeld, 1973). GABA-like im-
like immunoreactivity in the neurons ofthe central ner- munoreactivity has been demonstrated in inhibitory
vous system is difficult to evaluate. Although they may nerves in insect muscle (Bicker et ai, 1988; Robertson
122 T. BOLLNER ET AL
m
it
7 nc k
a
anti-GABA
GABA
anti-GABA
null
\ +Glu-G
\
Gly Gin
anti-GABA
8 +GABA-G
Figure 3. Transverse section through the anterior bulbs with staining in neurons (arrowheads) and in
someofthefibersofthe neuropile(arrows), m, mouth. IGS method. Differential interferencecontrast. Bar
=
15 Aim.
Figure4. Sagittalsectionshowingpositivestaininginonecellinthemidpartandinonecellintherear
part ofthe brain (arrowheads) and also in a fiber of the nerve cord (arrow). IGS method. Differential
interferencecontrast. Bar = 15 ^m.
Figure5. Transversesection through thebrain in theregion ofthesensoryvesicle(sv).with stainingin
onecell body(arrowhead). IGS method. Differential interferencecontrast. Bar = 15 ^m.
Figure6. Section through the sensory vesicle(sv) showingpositive staining in one ofthe brain vesicle
cells(arrowhead)and in theepithelialcells formingthe vesicle wall. IGS method. Differential interference
contrast. Bar = 15 ^m.
Figure 7. Transversesection through thecaudal ganglion showingpositive staining in cell somata (ar-
rowheads) and in neuropile fibers (arrow); nc, notochord. IGS method. Differential interference contrast.
Bar = 10jjm.
Figure8. Whole-mount preparationsofthecaudalganglionshowingpositivereactionin neurons(arrows)
aftertreatment with anti-GABA (a) and anti-GABA/Glu-G (b) and no reaction after treatment with anti-
GABA/GABA-G (c), corresponding control filter discs with amino acids conjugated to macromolecules
from dona neural tissue by glutaraldehyde are shown at the right. Gin. glutamine; Gly. glycine; null,
glutaraldehyde-treated protein with no amino acid added. PAP method. Bar = 15 ^m.
GABA IN OlKOPLEUR.-\ NERVOUS SYSTEM 123
Figure9. Transversesection through thetail nearthetrunk, frontal view. GABA-like immunoreaction
can be seen in a neuromuscular terminal (arrowhead! and in the nerve cord (n). This section also shows
stainingin a part ofthe fiberbetween the nervecord and the neuromuscularterminal (arrow), m, muscle;
nc, notochord. IGS method. Differential interferencecontrast. Bar = 2(1j/m
and Wisniowski, 1988). Our rinding that neuromuscular I)eBiasi,S.,C.Frassoni,and I,.VitellaroZuccarello. 1986. Glutamic
synapsesofO. dioicashowGABA-like immunoreactivity acid decarboxylase (GAD)-like immunoreactivity in the pedal gan-
is the first indication that GABA may also act as neuro- glion ofMyiiliis galloprovincialis. Cell TissueRes 244: 591-593.
Durantc, M. 1959. Sullalocalizzazioneistochimicadellaacetilcolines-
musMcourlearwoinrhkibiistonreyedseudbsttoanecsetaibnlisshomwehecthhoerrdaGteAs.BA is a tLemrbarsiyollu.ngMoorlophsovliluEpxppo d2i:a2l3c4u-n2e43as.cidie e in appendicularie. Acta
neurotransmitter in O. dioica. Its possible role in osmo- Flood, P. R. 1973. Ultrastructural and cytochemical studies on the
regulation in the vesicle should also be subject to further muscleinnervationinAppendiculana.Tunicata.J Microsc. 18:317-
326.
investigation.
Flood, P. R. 1975. Scanningelectron microscopeobservationson the
muscleinnervationofOikopleuradioicaFol (Appendiculana,Tun-
Acknowledgments icata)withnotesonthearrangementofconnectivetissuefibers. Cell
We are grateful to A. T. Bore and Y. Lilliemark for Fol,TiIIs.su1e87R2e.s 1E6t4u:de3s57s-u3r69l.es appendiculaires du detroit de Messine.
technical assistance. This investigation was supported by Mem. Soc. Phys. Hist. Nat. Geneve21:445-449.
the Hierta-Retzius and Lars Hiertas Minne foundations. Fugelli, K., J. Storm-Mathisen, and F. Fonnum. 1970. Synthesis of
vaminobutyricacid in fish erythrocytes. Nature228: 1001.
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