NERVOUS SYSTEM 0 F HYDRA VULGARIS
INTRODUCTION
Coelenterates are the first multicellular
organisms to have· evolved a nervous system.
The hydrozoan
nervous system as a result appears very important from
the evolutionary point of view.
Although, it is consi-
dered to be the most primitive form of nerv'ous system,
little is known about its structure· and rec·a.'"ltly, even
its presence has been do':_lbted (Hes·s et al. ,1957;
Slautterback and Fawcett, 1959; Hess, 1961) •. Various
'
behavioural patterns like periodic contraction which
reduce hydra, making it look like a tightly contracted
ball, basal gliding, loopir.g, ·somereaulting, response to
light by moving to-vrards it (~vilson, 1891), contracting
(Haug, 1933; Rushforth et al$, 1963; Singer et al. , 1963)
or ceasing periodic contractions (Passano and Nccullough,
'
1962), nematocyst discharge, feeding, gro-vrth and rege-
neration all appear to be intimately associated with
the nervous system.
Although various observations
sho1:r that the hydra muscular system is poorly innervated.
' This is not surprising in vievl of the relatively
uncomplicated behaviour of' this orga,..'1ism.
This does
-: 2 : ..
not mean that nervous system is not the primary factor
in controlling the behaviour of Hydra.
The study of the nervous system of Hydra
started as early as 1890.
The "continuity throughout
the nerve net" has been the subject of several early
investigators (Hadzi, 1909; Marshall, 1923; McConnell,
1932).
Their investigations are not in complete agree-
ment and it is generally believed that "Yrhile some
processes end freely, many processes unite such that
nerve cell bodies lie in a so called net worlr.
NcConnell 1 s wo:ck ( 1932) on the development of epidermal
nerve net in the buds stated as, "the formation of the
nerve net takes place by growing together of the
processes from the ganglion cells:
adv~!ce be~veen
the epitheliomuscular cells until they
meet other processes from the
they fuse.
These processes
g~VJ.glion
cells with which
Other processes from the ganglion cells
grow out in various directions and end among the
muscular cells."
More recently Spangenberg and Ham (1960)
using a modified version of the original McConnell
technique have also confirmed the existence of an
epidermal nerve net.
According to these authors "• ••••
it is easY to distinguish a \·Tell organized continuous
nerve net work both in the tentacle and in the stalk
-: 3 :-
and pedal disk region - the observable net work of the
tentacles extending for a short distance into the column
region and the neries of the pedal disk forming a circle
around the aboral pore."
All the studies performed
above were at light microscopic level and utilized one of
the two basic methods : ( 1) specific chemicals to
dissociate cells without destroying them and (2) selective
staining usually with methylene blue.
The histological study of nerve net in hydra
has been made by several investigators (Hadzi, 1909;
Marshall, 1923; McConnell, 1932).
Various investigators
have employed different techniques like dissociation
~--
(Schneider, 1890), vital staining (Hadzi, 1909; McConnin~;
1932; Spangenberg and Ham, 1960; Burnett and Diehl, 1964)
Heidenhain's Hematoxylin and PAS toludine blue staining
(Noda, 1969;) etc:~·.
All these studies are clearly based
on general stains that are not specific to nervous
system.
It is apparent that all these investigators
except Mackie ( 1961) have failed to employ specific
methods like ailYer impregnation to study the nervous
system.,
However, for reasons unknO\vn, Mackie's work has
not produced satisfactory demonstration of nervous system
of hydra.
Although the presence of acetylcholinesterase
in the nervous system of Hydra has been shown by classical
Histochemical techniques at the light microscopic level
•
(Lentz and B~rrnett, 1961), biochemical studies are
-: 4 :--
entirely lacking.
In this context study of nervous system
has been undertaken in addition to the fo~1entioned
conventional
techniques~
Specific gold and silver impre-
gnation methods also have been utilised.
Acetylcho-
linesterase has been studied using the techniques of
polyacrylamide gel electrophoresis and the biochemical
demonstration of esterase activity with fluorogenic
substrate like n-methylindoxyl acetate (NMIA).
Behaviour
On the basis of various observations it has
been possible in t..he present study to explain some of
the coordinating mechanisms, despite the apparent absence
of centralization in the basal region.
Nematocyst
·,
discharge appears to be effected by the sensory and
motor neurons only as evidenced by the absence of interneuron.
A through conducting S,Ystem is also observed.
Simple responses to the touch,by hydra demonstrated the
phenomenon of facilitation and decremental conduction.
The types of cells composing the nervous
sYste.1n have also interested several investigators.
Some
of the earlier studies indicate that there are three
types of nerve cells, g4nglionic, neurosensory and
..
intermediate types (Hadzi, 1909; Narshall, 1923;
193~;
Burnett and Diehl, 1964; Bullock and
Herridge, 1965).
The ganglienic cells located at the
McConnell,
base of epitheliomuscular cells are bipolar or
--: 5 : ..
multipolar and the processes may divide and subdivide
before they terminate into other cell processes.,
Spangenberg and Ham
.< 1960)
have indicated that nerve
cells usually contain two to seven processes.
The
origin of the various types of cells composing the
nervous system has been studied by several investigators.
Generally it is believed that all nerve cells arise from
interstitial cell~ \Marshall, 1923; McConnell, 1932;
Burnett and Diehl, 1964).
Since nerve cells do not
divide unlike some specialized cells in Hydra (Burnett,
1973) (e., g. epi theliomuscular cells, mucous cells,
digestive cells), their origin is considered to stem
exclusively from the.differentiation of interstitial cells.
HATERIALS AND METHODS
Hydra vulgaris (Annandale, 1911) was used as
tbe experimental material for this vrork..
The animals
were raised by the method of Loomis and Lenhoff ( 1956)
at a constant temperature of 23° + l°C..
The culture
medium vlas prepared as follows:
Stock Solution A
~
Calcium chloriae 7.5 grams in 500 ml of hot
tap v,rater.
Stock Solution B :
1 .. Sodium bicarbonate 10 grams.
--: 6 : ...
2-e
EDTA ( tetrasodium ethylene diamine tatraacetate) - 5 grams.~issolved in 500 ml of
'
tap water ..
Ten ml of solution A and 10 ml of B are mixed and
made up to 2000 ml wi t.."IJ. tap watere
It was nee essary to add
EDTA as the copper present in the tap vJ"ater was found to be
toxic to Hydra®·
.Animals 1vere fed daily vTith living naupili
of Artemia salina.
All artemia 11ere removed from culture
dish one hour after feeding to prevent fouling of water
and thus rendering Hydra inactive.
Demonstration of the
(a) Golgi
1
~mpregnation
nervenet' o.f Hydra
method
Prior to transferring to osmium tetraoxide and
potassium dichromate mixture, it wq.s found necessary to
fix hydra in Bouin's fixative to avoid blackening of the
material.
Paraffin sections of 10
~
thickness were cut.
The sections were brought to vTater and were immersed in
an aqueous solution containing potassium dichromate and
osmium tetraoxide
mixture~
Accordi1~
to original Golgi
technique, slides were kept in the fixative for 2 daYs and
were then transferred to 0.,75% aqueous silver nitrate for
tvJO daYs.
All slides vrere dehydrated in a graded series
of alcobol and mounted in DPX.
(b) Holme's silver stain
Like the Golgi technique it is specific for tte
nerve cells.
Bouin 1 s fixed paraffin sections of 10. )-1
-: 7 :thickness were brought to water and transferred to 20%
silver nitrate for one hour.
The. slides were ·then
processed through impregnating solution (100 ml Holme's
boric acid - borax buffer, pH 8.4 containing 1% aqueous
silver nitrate, 5.0 ml of 1% aqueous pyridine at 37°C).
They were then processed through a series of solutions.
Reducing solution - (Hydro quinone - 1. 0 g. sodium sulphate - ·
10.0 g. and distilled water to 100 ml) for 2-3 min.
The
slides were then dipped for 2-3 min in 0~2% gold chloride
for 3 min., . 2% oxalic acid for 3-10 min till the nerve
cells were differentiated, and in 5% sodium thiosulphate
for 5 min.
(c) Methylene blue s.tain
As used by other investigators (Stark et al,
1969) for staining the axons in the Ventral nerve cord
of insects vTas tried as a supplementary stain for
demonstrating the nerve net.
Solution I.,
Hethylene blue stock solution
This was prepared by dissolving 0.5 gm of
methylene blue chloride in 100 ml of distilled water.
The solution was heated, stirred and filtered.
Solution II.
Leucomethylene blue solution
lm unacidified Unna-type solution was prepared
by adding 6s0 ml of 12% w/v sodium formaldehyde sulfoxalate
~:
8
:~
(rongalit) to 30 ml. of methylene blue stocl:'.: solution.
The mixture was stirred and 1·1armed gently until it began
to turn deep dirty g.reen, tten removed from the flame and
stirred well till it turns colourless®
an unadjusted pH of
s,
The solution had
and -vras ready for immediate uses-
The leucosolu tion tended to oxidise within a few hours
when freshly exposed to air, but it could be reduced by
the addition of enough sodium dittionite to remove blue
colour (Larimer aJld Ashby, 1964).
Solution III
ft~onium
picrate
This was made by adding 3N NH 0H to a saturated
4
aqueous _solution of picric acid. pH of the solution ~>Tas
approximately 7.,
\\lhole hydras were kept in leucomethylene
blue solution for
1-lt
hours.,
Then ammonium picrate was
added and allo-vred to stand for at least 10 min.
Finally
the hydras are mounted on a slidesIdentification of acetylcholinesterase in the nervous
system of Hydra
For each experiment 30 hydras were bomog ~nized
in all glass homogenizer in ice cold TRIS buffer pH 8.,3.,
Homogenate vTas subjected to polyacrylamide gel electrophoresis at 4°C according to the standard procedure of
Davis ( 1964).
suitable.
Usually 7% polyacrylamide gel was found
The follO\ving stocl:: solutions were prep are d.
-: 9 :-
Reagent A
i.
I N HCI
24 ml
ii.
Tris (Hydroxymethyl)
Methylamine
N, N, N', N1 Tetramethyl et;hylene
diamine ( TEHED)
18.3 g
iii.
0.115 ml
Added distilled water to make 50 ml, pH was
adjusted at 8., 9.
Reagent B
12 ml
i.
IN HCI
ii.
Tris (Hydroxymethyl)
Methyl amine
1.495 g
iii.
N,N,N',N' -Tetramethyl ethylene
diamine
0.12 ml
Added distilled water to make 25,ml.
Reagent C
i.
Acrylamide
ii.
N,N'
methylene
Bisacrylamide
14 g
0.,3675 g
Added distilled water to make 50 ml.
Reagent D
i.
Acrylamide
2.5 g
ii.
N ,N'
methylene
Bisacrylamide
0.625 g
Added distilled water to make 25 ml.
Reagent E
i.
Riboflavin
4 mg
Added distilled water to make 100 ml.
Reagent F
i.
4P
Sucrose
g
Added distilled water to make 100 mle
Reagent G
i.
Ammonium persulphate
0.14 g
Added distilled water to make_lOO ml
Stock solutions were mixed in tt.e follovring
proportions, for making polyacrylamide gels:
a) Running g el
i.
Stock solution A
2 ml
ii.
Stock solution
c
4 ml
iii.
Stock solution G
iv.
Distilled water
S ml
~a.ml
b) Stacking gel
i.
Stock solution B
0.5 ml
ii.
Stock solution D
1.0 ml
iiie
Stock solution E
01!>5
iv.
Stock solution F
2e0 ml
ml
Tris-glycine buffer Oe4 M pH 8.3, was used in
both the upper and lo1rrer chamber of electrophoresis
apparatus.
Preparation of running gel
Thoroughly cleaned gel tubes were fixed
vertically in the rubber gromets for filling with gel
solution which is prepared as above.
Running gel was
-: 11 :-
poured into the gel tubes taking care to avoid air bubbles
in the gel after polymerization..
Immediately after'
dispensing the running gel solution into the gel tubes,
water was layered carefully over the
to flatten the gel sl.1rface.
runn~ng
gel solution
Runnifl..g gel solution was
polymerized with Ammonium persulphate as a catalyst.
T.he
polymerization was complete in 30 minutes.
Preparation of stacking gel
The stac}:ing gel solution which vras prepared by
mixing carefully the stock solutions as mentioned above
\vas placed over the running gel.
Again water was layered
on the stacking gel solution and photcpolymerized for 20 min.
Electrophoresis
For electrophoresis 0. 2 ml of the homogenate
was mixed with a little amount of sucrose, and was layered
on the stacking gel.
These gel tubes were inserted into
~G;~n the upper chamber of the apparatus.
Tray
buffer diluted 10 times with distilled water, was poured
in the lower and upper chambers of the apparatus.
Botb
the lower and upper chambers were assembled as a single
unit and the gel tubes were dipped in the tray buffer.
One ml.(O.OO]Jb)aa.queous solution of Bromophenol blue was
added to the upper chamber as a tracking dye.
The whole
apparatus was kept at 4°C in the refrigerator ..
A current of 2 mA per tube was passed through
FIGURE le
Diagramatic representation of monopolar
neuron w'i th the fiber directly innervating the
nematocyst.
This particular monopolar cell 1vas
present in the outer ectodermal layer which is
depicted from a section stained by Golgi method
specific for nerve cell.
M - monopolar neuron, N - nematocyst.
FIGURE 2.
Diagramatic reprEfsentation of a bipolar
neuron.
FIGURE 3e
B - bipolar neuron.·
Diagramatic representation of a multipolar
neuron.
Mn - multipolar neurqn.
N - nematocyst.
FIGURE 4
Photograph shovling the nerve net of
Hydra distributed on the entire surface®
Inter-
connections bet\veen one nerve, cell with that of
the adjacent nerve cell can be cl.early seen ..
Holme's silver metho.d was used for st.aining.
Note : Nonneuronal components are left
unstained
( x 280).
BC - body cells,
Mn- multipolar neuron.
~--~-
•
-----.------
FIGURE 5.
. Higher magnification of Fig.4.
ShovJing
clearly the interconnections of the fibers of multipolar nerve cells witb that of the other nerve cells
(
X
700) •
Mn - multipolar neuron.
•
FIGUHE 6 : Longitudinal section of the tentacular
region stained by Holme's silver technique.
It
shows a large number of monopolar nerve cells
distributed on the entire surface of the tentacle.
These cells contribute to the formation of a
common fiber tract (
x 280).
Mft,- mixed fiber tract, M - monopolar neuron.
-: 12 :the gel tubes for the first 10-15 minutes for stacking
the protein sample and increased to 3 rnA/tube for separation.
Electrophoresis was carried' out for 45 minutes.
One of the gels v-ras stained 1vith coomassie -brilliant
blue by standard techniques to localize proteins.
gel was stained for acetylcholinesterase activity
Second
usir~
the flourogenic substrate, n - methylindoxylacetate (~~I~
(Habibulla and Nev1burgh, 1973).
For this purpose gel
was incubated in a mixture containing
of NMIA in phosphate buffer pH 7.2.
sa~1rated
solution
Third gel, which v-ras
immersed ino-1% e{:t,erine for 40 minutes and then transferred to n-methylindoxyacetate served as a control.
OBSERVATIONS
--------
I:Jith these stains three types of cells could
be observed in Hydra namely monopolar· (fig. 1) , bipolar
(fig.2) and multipolar (fig.3).
An attempt was made to
study their differential localization in 3 regions,
tentacular, middle and basal regions.
nerve cell ending
direc~ly
Branches of one
into the adjacent nerve cell,
gives a net appearance of the nervous system (figs.4 & 5).
(a) Tentacular region
Large number of monopolar nerve cells
wer~
observed distributed on the entire outer surface of the
· tentacle (fig. 6).
The branches of the monopolar cells
are joined together to form a continuous fibre tract
FIGURE 7 :
Magnification of a portion of .Fig. 6,
showing the monopolar nerve cells and the contribution of their fibres to form a common tract
(
X
700) •
Mft - mixed fibre tract, 1'1 - monopolar neuron.
•
'•
•
·~
.,tB;;
r~r!!'l
.
"':#!.
.••.
-
"<
""w·,.
.
FIGURE 8: Cross section of the tentacular region
showing the presence of bipolar nerve cells at the
base of each
nematocy~t.
These have one fibre
contributing to the formation of a common fibre tract
and the other innervating the nematocyst.
I
silver staining technique was used
Holme's
( x 700).
dN - discharged nematocyst; B - bipolar
neuron~
FIGURE 9.
Longitudinal section of the tentacular
region sho1,v-ing 2-4 bipolar nerve cells present at
•
the base of each nematocyst.
. silver technique ( x 700).
B-bipolar neu.ron
Stained by Holme• s
•
"
FIGURE 10
This photomicrograph shov1s the section
of the middle region, stained by Golgi method •
.
Note : The presence of a monopolar nerve cell
~n
the
epitheliomuscular layer and its fiber innervating
the nematocyst ( x 700).
E- ectoderm, N - nematocyst,
M - monopolar neuron.
..-
FIGURE 11.
Photomicrograph of the middle region
showing a multipolar nerve cell in the outer epitheliomuscular layer with one fibre innervating
the nematocyst and the other two branches innervating the outer surface of the ectodermal laYer.
Gastrodermal elements are evident as a large number
of fibres in the gastrodermal region and these
fibres join to form a continuous fibre tract at the
base of the mesoglea.
gastrodermal cells.
These fibres innervate the
Golgi preparation.
E - epi theliomuscular laYer, Mn- multipolar
neuron, N - nematocyst, G - gastrodermal plexus,
Mft - mixed fibre tract.
FIGURE 12:
Photomicrograph of the Golgi preparation
of a section through the middle region showing
branched fibres in the gastrodermal region.
Some of
these fibres have their nerve cells located in the
ectodermal region.
region.
These fibres cross the mesogleal
Large number of branched fibres are seen
arising from the common fibre tract ( x 700).
E - epi theliomuscular layer, G - gastrodermal nerve
fibres, 11 - monopolar neuron,
Mft - mixed fibre tract.
FIGURE 13 :
Photomicrograph of the basal region
stained by Holme ' s silver technique showing bipolar
and multipolar nerve cells forming a network in the
gastrodermal layer.
All the nerve cells have long
and branched fibres which
m~~e
fibres of the other nerve cell .
cell is seen ( x 700).
Nu - neurons.
contact with the
No monopolar nerve
-: 13 :that can be distinctly seen (~igs. 6 & 7).
This ~ibre
tract has fibres originating from the nerve cells which
are present at the base of each nematocyst.
These nerve
cells have their other fibre innervating the base of each
nematocyst i.e. they are bipolar.
There are 2 to 4 of
these bipolar nezwve cells present at the base of each
nemato cyst ( Figs. 8 & 9).
(b)
Middle region
In the epitheliomuscular layer monopolar and
multipolar cells are ( fig.lO) present.
Monopolar cells
have their branch going to the cnidoblast.
The multipolar
cells have one branch going to the cnidoblast and the
other going to the outer surface of the ectodern:al layer
( fig.ll) .
Gastrodermal nerve elements were also observed.
Large number of nerve fibres were seen in the gastrodermal
region (figs. 11 & 12).
Some of these fibres have their
nerve cells located in the ectodermal region (fig.l2).
These fibres cross the mesogleal region and join a continuous fibre at its base.
Large number of fibres are
seen arising from this continuous fibre tract and innervating the gastrodermal cells ( fig.ll & 12).
These nerve
fibres form the gastrodermal plexus.
(c)
Basal region
In this region nerve net is more pronounced
than in any other region (fig.13).
Most of the cells are
FIGURE 14 :
Photomicrograph of the basal region
showing bipolar nerve cells located in the gastrodermal layer.
region nerve
Stained by Golgi technique.
cel~s
in the
be distinguished (x 700).
ectoder~al
In this
region cannot
B -bipolar neuron.
FIGURE 15 :
Photomicrograph of the basal region
showing a bipolar nerve cell in the gastrodermal
laYer (x 700) B- bipolar neuron.
I
FIGURE 16 :
Electrophoretic pattern of acetylcho-
linesterase of Hydra vulgaris.
A.
Gel stained with fluorogenic substrate for
acetylcholinesterase.
B.
Gel stained vrith coomassie - brilliant blue to
localize proteins.
c.
Gel treated with
1%
eserine, an inhibitor of
acetylcholinesterase and then stained with
fluorogenic substrate.
the text.
For details refer to
0
AChE
AChE
M
M
lJ)
2
u
z
w
....J
<(
u
lJ)
6.5
s.o
8
A
M
c
~
Stacking Gel
-
AChE
-
Protein Band
-
Marker Dye
Dia.,gramatic representation of the composite picture
of the nervous system of Hydra indicat.ing the location of
different types of neurons in the tentacular, middle and
basal regions. It sho1.vs the presence of the mixed fibre
tract traversing through all the three regions. The
enlarged picture of the tentacular region clearly sho1.vs the
presence of the sensory neurons on the surface and bipolar
neurons 2-4 in number at the base of each nematocyst.
Hiddl e reg ion shotvs monopolar and bipolar neurons
in the epi theliomuscular layer. Gastrodermal plexus can
Basal region shows large number of
also be clearly seen
multipolar neurons.
A well defined nerve
N
M
Mft
B
E
G
Mn
Tr
Mr
Br
ne~vork
is apparent.
- nematocyst
- monopolar neuron (sensory neurons)
- mixed fibre tract representing through
conducting sYstem
- Bipolar neuron
- Epi thelio muscular layer
- g strodermal plexus
-Multipolar neuron
Tentacular region (an enlarged portion
is shown in 1)
- Middle region
Basal region (an enlarged portion is
shown in 2)
-: 14 :-
bipolar and multipolar.
layer (fig.l4).
Nervenet is in the gastrodermal
In this region ectodermal cells are
closely packed and nerve cells cannot be easily made out.
All the nerve cells have long and branched fibres
~;hicb.
make contact with the fibres of the other nerve cell
(fig.l4). No monopolar cell could be seen in the basal
region.
Here also nerve fibres join to form a common tract
that runs at the base of the mesoglea ( Fig.l5).
Activity staining for AChE
After polyacrylamide gel electrophoresis the
gel was incubated in a mixture· containing saturated solution
of n-methylindo:xylacetate - a fluorogenic substrate for
AChE (Habibulla and Newburgh, 1973).
A distinct green
band of acetylcholinesterase activity appeared after 30
minutes.
RF value of the enzyme band was found to
correspond with the third band in the gel stained for
proteins.
RF value of the band was ,found to be
o. 28
(fig.l6)" (RF =mobility of enzyme band in em/mobility of
the marker dye in em).
The proteins were stained by
co6massie dye ..
Relationship between the nervous system and the simple
behavioural responses of Hydra
It is apparent from the above findings that
the nervous sYstem of Hydra is not centralized in the
anatomical sense.
Ho\vever, there is an attempt towards
-: 15 :centralization in the basal region as evidenced by the
~ccumulation
•Of a large number of neurons.
cells are bipolar as well as mul'tipolar.
nerve cell could be located.,
These nerve
No monopolar
It is also observed that a
large number of sensory cells are present in tentacular ·
region.
These monopolar sensory cells recaive the
stimulus and pass it to a common branch that can be
observed going to the basal region.
is a mixed tiber tract.
This common branch
Fro1.1 here the sensory stimulus
appears to be tra,.'1smi tted dmvn to the motor neurons
(bipolar and multipolar), in the basal region.
This
appears to be a coordinating region and probably could be
considered as a primitive brain.
Thirdly, nematocyst discharge can also be
explained by the presence of a large number of sensory
cells present on the surface of the tentacle.
sensory cells receive the stimulus and
common fibre tract.
tr~'1smit
These
it to a
From this the stimulus is trans-
mitted to the nematocyst through the mediation of. a
bipolar nerve cell present at its basee
The absence of
interneurones in Hydra appears to be in keeping with the
primitive type of organization.,
A through conducting system is present in
hydra.
This can be observed by the presence of a conti-
nuous fibre tract, traversing below the mesoglea throughout the body of Hydra.
This is a mixed fibre tract
--: 16 :-
having both sensory (monopolar) and motor fibres in it
as mentioned earlier.
Facilitation
This could be observed in Hydra easily when
tentacles are touched with a fine needle.
lightly, response is slow.
there is
If touched for the second time,
an increased response.
In the third time, if a
strong stimulus is given it will again show
response.
If touched
an increased
This stimulus is apparently relayed through
the adjoining 'nerve cells of the nerve net.
The decremental conduction
--------------------------This could be perceived in Hydra by obser·..ring
the progressive contraction starting from the tentacular
:;'egion to the basal region in response to touch.
Relaxation in the presence of light
Apical portion of Hydra is much more sensitive
to light than the basal region.
MlOreover, it is also
observed that monopolar cells are much more abundant in the
apical region.
These tvTO observations put together explain
the existance of photoreceptive nerve cells in the apical
region.