Download physiological and chemical architecture of a lobster ganglion with

PHYSIOLOGICAL
OF A LOBSTER
REFERENCE
AND CHEMICAL
ARCHITECTURE
GANGLION
WITH
PARTICULAR
TO GAMMA-AMINOBUTYRATE
AND GLUTAMATE1
M. OTSUKA,2
E. A. KRAVITZ,
Department of Neurobiology, Harvard Medical
(Received
for
publication
AND
D.
D.
POTTER
School, Boston, Massachusetts
November
18, 1966)
IS LITTLE
DOUBT that gamma-aminobutyric
acid (GABA)
is the
inhibitory
transmitter
compound
at the lobster neuromuscular
junction
(see 20). Special interest, therefore, is attached to the chemistry
of this
compound. GABA is about 100 times more concentrated
in inhibitory
than
fibers
in excitatory
axons; the concentration
is about 0.1 M in inhibitory
if GABA is freely dissolved in the axoplasm (13,15). The origin of the GABA
difference appears to lie in an asymmetric
distribution
of the enzyme that
synthesizes GABA from glutamate,
glutamic
decarboxylase,
the pathway
for subsequent degradation
being about equally active in the two types of
axons (14). The mechanisms
regulating
the levels of the enzymes, on the
other hand, are unknown; it is possible that differential
enzyme synthesis or
destruction
accounts for the decarboxylase
difference. Since the neuronal
cell body is presumably
the site of enzyme synthesis, the aim of the present
work was to develop methods for finding and isolating
the cell bodies of
efferent excitatory and inhibitory
neurons within the lobster central nervous
system. As a first step toward understanding
enzyme regulation
in the
GABA pathway, the contents of the substrates, GABA and glutamate,
were
measured. During the course of this work observations
were made on the
physiological
organization
of a ganglion, and these are also presented.
The present study, like earlier ones on peripheral axons, was aided by the
favorable anatomy of the lobster. The total number of efferent neurons is
small, usually between two and six per muscle, at least one of which is inhibitory (cf. 12, 31); this eases the problem of locating any particular
cell body.
Like the axons, the cell bodies are large enough to be isolated with hand-held
instruments.
In contrast to the cell bodies of most vertebrate neurons, they
are apparently free of synapses (1); thus interpretation
of the chemical analysis is simplified.
Finally, the ganglia, like the peripheral
nerves, show a
striking constancy of organization;
many of the efferent cell bodies can be
recognized by their size and position alone.
A brief abstract of this work has been published (21).
THERE
by Public
Health
Service
Grants
5 K3 NB 7833-05,
6 K3
and NB 02253-07.
Fellow.
Present
address:
Dept.
of Pharmacology,
Faculty
and Dental
University,
Tokyo,
Japan.
Downloaded from http://jn.physiology.org/ by 10.220.33.5 on June 18, 2017
1 This work
was supported
5899-OIAI,
NB 02X3-06,
2 Rockefeller
Foundation
of Medicine,
Tokyo
Medical
HD
726
OTSUKA,
KRAVITZ,
AND
POTTER
Animals.
Lobsters
(Homarus
americanus)
were
obtained
from
local dealers
and
kept
in moist
seaweed
in a
cold room
at 3 C, sometimes
for several
days. They weighed
0.5-1.5
kg.
Preparation.
A portion
of
the abdomen
containing
the
first through
the fourth
ganglia was isolated
(Fig.
1A).
If
the extensor
muscles
(cf. Fig.
12B)
were
used,
the
main
branch
of the second
ganglionic
root was exposed
byremoving
the
overlying
shell,
skin,
and
muscle.
The
extensor
muscles
were isolated,
remaining
connected
to the
rest
of
the
abdomen
by
to permit
the extensor
muscles
to be
of the abdomen
is placed
ventral
side up
the nerve
bundle.
The
nerve
is sufficiently
long
placed with their deep face exposed
when the rest
(Fig. 1A).
If the extensor
muscles
were not needed,
they were cut away
with the shell to which
they are attached.
Further
dissection
was done in oxygenated
physiological
solution
(hereafter called saline,
see below
for composition),
with
the temperature
maintained
between
7 and 14 C by thermoelectric
cooling
units
upon which
the chamber
rested.
The ventral
surface
of the ganglionic
chain was exposed
by removing
midline
portions
of the overlying
ribs, the joint
membranes,
and the underlying
pigmented
skin (Fig. 1A). To permit
transmitted
light to reach the ganglia,
the flexor musculature
dorsal
to the ganglionic
chain was
divided
in the midline,
care being taken
to minimize
damage
to the third
roots.
The only
Downloaded from http://jn.physiology.org/ by 10.220.33.5 on June 18, 2017
FIG. 1. A: an abdominal
segment
of the lobster,
viewed
from
the ventral
side.
The
ganglionic
chain
was exposed
by removing
ribs and skin in
the midline.
Flexor
muscles
were
divided
at the midline.
At the left, the extensor
muscles are attached
to the preparation
by the nerve
supply
of
one segment
(the main branch
of the second root).
B : ventral
surface
of the
nerve
cord,
showing
the second abdominal
ganglion.
The cell bodies were
exposed
by removing
the connective
tissue
sheath
and
perineural
tissue.
The
cell
bodies
are shown
at higher
magnification
in Fig.
3. Rl,
R2, and R3; first, second,
and
third
ganglionic
roots.
GABA
AND
GLUTAMATE
IN
CELL
BODIES
The ventral
surface,
ganglion
consists
of
with
toluidine
blue.
bodies were exposed
12 and 13.
structure
joining
the two sides was now the ganglionic
chain with its roots. The two halves
were fixed with forceps
or pins so that slight
tension
was placed
on the roots,
holding
the
ganglia
taut
(see Fig. 1, A and B). Light
was reflected
obliquely
from
below
to give a
dark-field
effect.
To expose the cell bodies,
the outer
connective
tissue sheath
(Fig. 2A) was cut along
a lateral
margin,
reflected
to one side, and removed.
The mass of soft perineural
tissue
(Fig.
2A) was washed
away
with
a gentle
stream
of saline
from
a fine-tipped
pipette
(see Fig. 2B). The washing
process was stopped
as soon as the cells were clearly
recognized.
Under
the dissecting
microscope
the ganglion
now had the appearance
shown
in Figs.
1B and 3.
Cold, oxygenated
saline was usually
directed
into the neighborhood
of the ganglion
throughout
the experiment.
An outlet
attached
to a suction
line maintained
a fluid level
barely
covering
the tissues.
In some experiments
the bath was oxygenated
directly
without
perfusion.
The methods
for identifying
the cell bodies
of efferent
excitatory
and inhibitory
neurons
are described
in RESULTS
section.
Downloaded from http://jn.physiology.org/ by 10.220.33.5 on June 18, 2017
FIG. 2. A: transverse
section
of the third
abdominal
ganglion.
on which
the cell bodies
lie, is uppermost.
The main
mass of the
neuropil
in which
synapses
are found.
The ganglion
was stained
B : transverse
section
of a second abdominal
ganglion
in which
the cell
before
fixation.
The highly
vacuolated
cytoplasm
is characteristic
of
727
728
OTSUKA,
KRAVITZ,
AND
POTTER
Isolation
and transfer
procedures.
Two
procedures
for isolating
the cells were used.
In method
1, the cell bodies
were isolated
by free-hand
dissection
using
forceps
under
a
dissecting
microscope
at a magnification
of 40 X. The sharpened
points
of stainless
forceps
were polished
with rouge to reduce
the tendency
of the isolated
cells to stick to the forceps.
The cell body
was first separated
from
its neighbors
by softening
and separating
the
surrounding
tissue with
a fine stream
of saline
or with
forceps.
When
the cell body
had
been freed,
the axon was grasped
with forceps
as far from
the cell body
as possible
and
severed
(Fig. 4A). Remaining
tissue was removed
from
the isolated
cell as thoroughly
as
possible
to reduce
the chance
of contamination
by small
cell bodies
that lie beneath
the
large cells (Fig. 2). A histological
section
of such an isolated
cell body is shown
in Fig. 4C;
some contaminating
tissue remains,
but most of the sample
consists
of the nerve
cell body.
under
the
abdominal
dissecting
ganglion,
microscope.
showing
the appearance
of the living
This is the same ganglion
as Fig.
cell bodies
14B.
Resting
potentials
of isolated
cells were usually
between
15 and 40 mv, with none higher
than 45 mv; in most cases, the resting
potential
was not measured.
The isolated
cell was then drawn
into a pipette
with a constricted
neck (Fig. 4B) and
transferred
to a 0.2-ml
conical
tube with about
1 ~1 of saline.
Two microliters
of 0.2 N HCl
were added
to extract
GABA
and glutamate
and to denature
eneymes.
The sample
was
then stored
at - 20 C until
analyzed.
Isolation
and extraction
usually
took less than 4 mm.
Method
2 for isolating
the cell bodies
resulted
in more consistent
chemical
measurements.
The whole
ganglion
was quickly
removed
and placed
in isopentane
kept
near its
freezing
point
with liquid
nitrogen.
The preparation
was dried in vacua
(about
20 ,.L Hg)
for a week at -30 C. The dry ganglion
was placed
in xylene
to make it more transparent.
The desired
cells were
removed
with
forceps
and transferred
and extracted
as above.
Xylene
was chosen as a clearing
agent because
GABA
and glutamate
are virtually
insoluble
in it. In one series of experiments,
cells were physiologically
identified
before
the ganglia
were frozen;
neighboring
cells were stained
by dye injection
(see below)
so that the identified
cells could
be recognized
in the dried
ganglia.
In another
series of experiments
(reported
Downloaded from http://jn.physiology.org/ by 10.220.33.5 on June 18, 2017
FIG. 3. The third
GABA
AND
GLUTAMATE
IN
CELL
BODIES
Downloaded from http://jn.physiology.org/ by 10.220.33.5 on June 18, 2017
FIG. 4. Isolation
and transfer
of single
cell bodies.
A : the cell body at the lower right
was separated
from its neighbors
by free-hand
dissection.
Its axon
was severed,
and the
cell lies free on the ganglion.
B: the cell body
has been drawn
into a transfer
pipette.
The constricted
neck of the pipette
prevents
the cell from
being
lost,
and by slowing
fluid movement
improves
control.
C: section
of an isolated
cell body,
which
was fixed in
1 y0 OsOa, embedded
in Epon
and stained
with
toluidine
blue. Adhering
tissue is present
at the upper
right
and lower left.
730
OTSUKA,
KRAVITZ,
AND
POTTER
Downloaded from http://jn.physiology.org/ by 10.220.33.5 on June 18, 2017
in the bottom
two columns
of Table
3) an attempt
was made to minimize
disturbance
to
the ganglia
before freezing.
Instead
of isolating
a segment
of the abdomen
in the usual way,
the brain
was crushed
and the whole animal
immobilized.
The second
abdominal
ganglion
was exposed
and the connective
tissue sheath
and perineural
tissue over the ventral
surface
removed.
Circulating
blood
was cleared
from
the ganglion
during
this operation
with
a
jet of cooled,
oxygenated
saline.
The ganglion
was removed
and frozen,
the operation
taking
about
10 min in all. Less than 1 min elapsed
from cutting
the roots
and connectives
to immersing
in the isopentane.
The cell bodies
were presumably
frozen
instantly
since
they were directly
exposed
to the freezing
solution
(cf. Fig. ZB). The cells were identified
in the dried ganglion
by visual
inspection.
GABA
clnd glutamate
assays. The microtubes
containing
the cell bodies
were lyophilized.
GABA
was assayed
by a modification
of the method
of Jakoby
and Scott
(7).
The details
of this assay have already
been published
(15). With
large inhibitory
cell bodies
the sample
could be divided,
so that a control
tube containing
aminooxyacetic
acid could
be run to give tissue blank
levels of fluorescence.
Glutamate
was determined
by dividing
the sample
in half and treating
one portion
with a bacterial
decarboxylase
which
converts
glutamate
to GABA.
Both
portions
were
then assayed
for GABA;
the difference
between
them
represents
the glutamate
in the
sample.
Glutamine
will also react in this assay but independent
checks have demonstrated
that glutamine
levels are low in these cells. The details
of the glutamate
assay were as
follows:
The lyophilized
sample
was dissolved
in 5 ,~l of pyridine-hydrochloride
buffer,
with NaCl
(25). Samples
of 2 ~1 were transferred
to two
pH 4.6 adjusted
to 0.1 M chloride
O.l-ml
test tubes. Two microliters
of decarboxylase
were added
to the first tube (containing
sufficient
enzyme
to convert
all the glutamate
present
to GABA
within
15 min)
and 2 ~1
of heated
decarboxylase
to the second
tube.
The tubes
were capped
with
parafilm
and
incubated
for 15 min at room
temperature.
They
were heated
at 95 C for 1 min, 1 pl of
1 M Tris, pH 7.9 was added
and a standard
GABA
assay was run, as previously
described.
The bacterial
decarboxylase
was prepared
from dried Escherichia coli cells purchased
from
Worthington
Biochemical
Corp.
The enzyme
was extracted
by homogenization
in a French
pressure
cell, precipitation
of contaminating
proteins
at pH 4.6, fractionation
with
solid
(NH&SO*
(O-60 (y(, saturation)
and dialysis
against
1,000
volumes
of 0.01 M potassium
phosphate,
pH 6.5. This purification
procedure
is a modification
of the method
of Shukuya
and Schwert
(25).
Estimation of cell body volume. Only rough measurements
of cell volume
could be made
on the living
cell with
the dissecting
microscope.
It was found
that
the manipulation
required
to measure
the diameter
of living
cells under
the compound
microscope
(cell
transfers,
temperature
changes,
etc.) resulted
in reduced
GABA
contents.
The method
used, therefore,
was to derive
an average
volume
for each cell type by reconstruction
from
serial sections
of fixed,
plastic-embedded
ganglia.
These ganglia
were from lobsters
of t,he
same size and condition
as those used for chemical
analyses.
In two ganglia,
certain
cells
were physiologically
identified
in the usual way and their positions
in the ganglion
sketched.
The ganglia
were
then fixed
in ice-cold
acrolein-glutaraldehyde
fixative
modified
after
Sandborn
et al. (23) and postfixed
in phosphate-buffered
0~04. The ganglia
were dehydrated
in methanol,
stained
with
toluidine
blue, and embedded
in Epon
9. They
were serially
sectioned
with a steel knife
(J. Alvarez
and E. J. Furshpan,
unpublished
data)
at 4 ,u.
Shrinkage
of the ganglia
during
these procedures
was found
to be negligible.
The cells
could
be easily
identified
by comparing
a reconstructed
ventral
view
from
the serial
sections
with the sketch
of the living
ganglion.
The volume
of a cell was derived
by tracing
each section
at known
magnification
on paper,
weighing
the tracings
to determine
their
area, and multiplying
the area by the known
thickness
of the sections.
It was found
that
the large inhibitory
cells could
be confidently
identified
on histological
grounds
alone;
they contained
a higher
proportion
of large vacuoles
(5-50
,U in diameter;
see Fig. 2B)
than neighboring
excitatory
cells. Moreover,
certain
excitatory
cells could
be identified
from their positions
in the ganglion.
The second abdominal
ganglia
from five other animals
were fixed without
physiological
manipulation.
They
were
treated
as described
above,
except
that they were sectioned
at 11 or 13 ,u. The proportion
of the cell volume
occupied
by vacuoles
was estimated
in 13 cells.
PhysiologicaL solution. The solution
had
the following
composition
(millimoles)
:
NaCl,
462; KCl, 15.6; CaC12, 25.9; MgS04,
8.3; Tris(hydroxymethyl)aminomethane,
10.0;
GABA
AND
GLUTAMATE
IN
CELL
BODIES
731
FIG. 5. Electrode
used for stimulating
the peripheral
nerve
in the
middle
of its
course.
One
lead
(dotted
line)
is surrounded
by a
polyethylene
tube and is exposed
to
the bath only at the bottom
of the
groove
in which
the nerve
lies. The
other lead (solid black line) is wound
around
the polyethylene
t*ube.
was exposed
to the saline only in the groove.
the tube. Stimulating
voltages
were as small
assembly
was carried
on a ball-joint
manipulator
When
the nerve
in the groove
was stimulated,
centrifugal
directions
at the same threshold.
A second
lead was wound
on the outside
of
as those used with
suction
electrodes.
The
and could easily be bent into any position.
impulses
spread
in both
centripetal
and
RESULTS
PART
I
Physiological
architecture
of the ganglion
The abdominal ganglia were used because the cell bodies lie nearly in one
plane (Figs. 2A, 3), and are among the largest in the lobster nervous system,
several being over 200 ,U in diameter. Usually the second and third abdominal
ganglia were chosen. Cells of exposed ganglia were impaled under visual control. The resting potentials
recorded with our low-resistance
microelectrodes
were usually 40-70 mv, and with few exceptions the cell bodies showed action
potentials
of less than 25 mv, suggesting
that the membrane
of the cell body
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maleic
acid,
10.0; NaOH,
about
10 to bring
the pH to 7.5. Just before
using,
glucose
(2 mg/ml)
was added.
The saline
was cooled
and oxygenated.
Electrodes.
Glass micropipettes
were used for recording
and passing
current.
They
were filled
with 3 M KC1 or 2 M K- or Na-citrate
and had resistances
of 2-20 megohms
when measured
in 3 M KCl.
The electrodes
could be connected
via a manual
switch
either
to a high impedance
preamplifier
or to a pulse generator.
For marking
cell bodies,
organic
dyes
were
injected
electrophoretically
from
the
microelectrodes.
Electrodes
with
tips about
1 ,U in diameter
were filled with dye solutions,
about
one-quarter
saturated,
by pushing
the dye down the shank
with a fine glass filament
(Alvarez
and Furshpan,
unpublished).
The dyes used were
fluorescein
sodium,
chromotrope
2R, light
green S F yellowish,
and fast green FCF.
They
are listed
in order
of decreasing
rate of diffusion
within
the cells.
It was frequently
necessary
to stimulate
a peripheral
nerve
in the middle
of its course
without
disturbing
its central
and peripheral
connections.
A convenient
electrode
for
doing this was made by enclosing
a platinum
wire with a flattened
end in a polyethylene
tube about
4 cm long (Fig. 5). The end of the polyethylene
tube was sealed and a groove
for supporting
t,he nerve
and confining
the current
was made with a hot needle.
The wire
732
OTSUKA,
KRAVITZ,
AND
POTTER
FIG. 6. Simultaneous
intracellular
recordings
of spontaneous
activity
in 11 of the
second ganglion
and in the superficial
flexor
muscle
innervated
by this neuron.
A and B
are different
preparations.
Arrows:
EPSP’s.
This
figure
illustrates
the correspondence
between
impulses
in 11 and IJP’s
in the opposite
superficial
flexor
muscle.
In B, the first
IJP was superimposed
on the decline
of a spontaneous
EJP. The time course of the action
potential
is shorter
than in A; such variation
was frequently
seen in our preparations.
In B, 2-mv
scale refers to upper
trace;
4-mv
scale to lower
trace.
Downloaded from http://jn.physiology.org/ by 10.220.33.5 on June 18, 2017
and its slender process behaved passively
under our experimental
conditions
(cf. 26).
At the outset our problem was to find the efferent neurons among all the
cells in the ganglion (Fig. 3) and to determine
the function
of each. Efferent
excitatory
cells were found relatively
easily because a visible muscle contraction usually appeared when they were stimulated
intracellularly
with a single
shock. The possibility
that the cell was an interneuron
could then be virtually eliminated by showing that antidromic
impulses reached the cell body.
The first efferent inhibitory
cell body was located by trial and error, after a
prolonged
search. Following
the demonstration
that this cell was rich in
GABA, chemical analysis provided a rapid means for discovering
others. Our
progress was entirely dependent on the fact that each cell has a characteristic
size and position, so that once identified it could readily be found from animal to animal.
Identification
of efferent inhibitory cell bodies. The first inhibitory
cell body
identified
innervates
the superficial
flexor muscle illustrated
in Fig. 12%
muscle 1. Advantage
was taken of the fact that this muscle receives a spon
GABA
AND
GLUTAMATE
IN
CELL
BODIES
733
taneous barrage of excitatory
and inhibitory
impulses,
as can be seen in
Figs. 6 and 7 (cf. 12). The inhibitory
junctional
potentials
(IJP’s)
in the
muscle disappeared
if the ganglion of the same segment was cut away. Thereupon, in a series of such ganglia, cell bodies were impaled systematically
in
search of one whose spontaneous
activity
was related to the IJP’s in the
muscle. Eventually
such a cell was found on the contralateral
side; this cell
is shown, labeled 11, in Figs. l2A and 14. Simultaneous
recordings
in the cell
body and the muscle are shown in Fig. 6; an action potential an the cell body
invariably
preceded the IJP’s in the muscle by about 20 msec. The one-toone relation between the action potential in the cell body and an IJP in the
muscle demonstrated
that the neuron was at least associated with the inhibitory pathway
to the muscle fiber. Two further
observations
indicated that
the neuron lay directly in the inhibitory
pathway;
subthreshold
activity
in
the cell body (arrows,
Fig. 6) were not followed by IJP’s in the muscle, and
when the cell body was stimulated
with injected current, an IJP appeared in
the muscle with about the same delay as with spontaneous
firing (Fig. 7).
To test whether
the neuron was directly
connected
to the muscle, the
nerve leading to the muscle was stimulated
in midcourse
with gradually
increasing intensity.
At a sharply defined threshold
an IJP appeared in the
muscle fiber and an antidromic
action potential was observed in the cell body
(Fig. 8B); this observation
greatly reduced the chances that we were dealing
with an interneuron.
The action potential in the cell body of this neuron was
characteristically
less than 10 mv in size; nevertheless,
it could be distinguished from excitatory
postsynaptic
potentials
(EPSP’s)
by its faster time
course and conspicuous afterpositivity
(cf. Figs. 6, 9).
When the 11 cell bodies (one on either side of the ganglion) were impaled
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FIG. 7. Effect
of stimulating
II
on the membrane
potential
of the
superficial
flexor
muscle
innervated
by this
cell. Short
current
pulses
were delivered
to II of the second
ganglion
through
an intracellular
electrode.
The
muscle
received
a
high-frequency
barrage
of spontaneous EJP’s
upon
which
the evoked
IJP’s
were superimposed
at nearly
constant
latency
(dotted
line).
with microelectrodes,
synchrony
of action potentials
was often observed
(Fig. 9A; see description
of electrical
coupling, below). The procedures
discussed above would, therefore, not have distinguished
which of the two cells
was attached to a particular
muscle. However,
as the experiment
proceeded,
the action potentials
did not remain synchronous,
an impulse in one cell
being accompanied
only by an EPSP in the other (Fig. 9AZ). An IJP in the
superficial
flexor muscle was invariably
associated
with an impulse in the
FIG. 9. The upper
part of the figure
shows
simultaneous
recording
of spontaneous
activity
in both 11 cell bodies of the second abdominal
ganglion.
In Al, early in the experiment,
action
potentials
in the two cells are synchronous.
Later
in the experiment,
A2,
the synchrony
is no longer
perfect.
Arrow
indicates
an EPSP.
Part B is from
a different
preparation,
in the same condition
as A2. It shows
that
IJP’s
in the superficial
flexor
muscle
are not invariably
associated
with impulses
in the homolateral
Il.
(B3; compare
with Fig. 6.) All records
are intracellular.
Downloaded from http://jn.physiology.org/ by 10.220.33.5 on June 18, 2017
FIG. 8. Stimulation
of the nerve
to the superficial
flexor
muscle
while recording
intracellularly
from the muscle
and from the opposite
II of the third ganglion.
In A, the stimulus
intensity
was subthreshold.
In B, activity
appeared
in the muscle
fiber and neuron
at the
same stimulus
strength.
l-mv
scale refers
to upper
trace.
GABA
AND
GLUTAMATE
IN
CELL
BODIES
735
FIG. IO. Stimulating
and recording
electrodes
were inserted
into the cell body
of 13,
while recording
from the extensor
muscle
it innervates,
M. dorsalis
Prof. In A, the current
pulse was just subthreshold
for 13; in B just
suprathreshold.
An IJP
appeared
in the
muscle
at the same stimulus
strength
as the impulse
in 13. 0.4-mv
scale refers to the upper
beam.
GABA analysis was then used to locate two more pairs of inhibitory
cell
bodies, 12’s and 13’s. The characteristic
position of these high GABA cells is
shown in Figs. 12A and 14. The physiological
procedures
for demonstrating
an inhibitory
function
were similar to those just described for Il.
One-to-one
correspondence
between
the neuronal
action
potential
and the
muscle
was established
for 12 and 13 with stimulation
in the cell body and in the
The experiment
with conduction
in the orthodromic
direction
is illustrated
for 13
at a sharply
defined
threshold
an all-or-none
action
potential
was produced
body and an IJP in the muscle
about
30 msec later.
IJP in the
periphery.
in Fig. IO;
in the cell
Identification
of efferent excitatory
cell bodies. The cells that produced
visible contraction
in the periphery
are labeled Ml through Ml4 in Fig. 12A.
In the smaller cell bodies (M8’s, Mll)
trains of stimuli were occasionally
required to produce contraction;
in the others a single suprathreshold
pulse
was sufficient.
For six cells, M2, M5, M6, M9, MlO, and M14, a one-to-one
correspondence
between an antidromic
action potential in the cell body and
Downloaded from http://jn.physiology.org/ by 10.220.33.5 on June 18, 2017
contralateral
cell body, but this correspondence
did not hold for the ipsilateral cell body (Fig. 9B). From this consistent
finding we conclude that the
axons are crossed.
The presence of bidirectional
one-to-one
synapses in crustacean
ganglia
(described
below; see also 27) still leaves some doubt whether
the 11’s are
efferent cells or interneurons.
However,
if they are interneurons
there should
be other cells in the ganglion, the true efferent neurons, with the same behavior; thorough exploration
in many preparations
never revealed such cells.
Moreover,
the cells in question
consistently
have a high GABA
content
(PART
II) not seen in excitatory
neurons. From all this evidence we conclude
that the 11’s are the efferent inhibitory
neurons innervating
the superficial
flexor muscles.
736
OTSUKA,
KRAVITZ,
AND
POTTER
FIG.
11. Stimulation
of the third
root
while
recording
intracellularly
from
M9 of
the second ganglion
and the muscle
it innervates,
M. obliquus
ant. 4. (The experimental
arrangement
is thus similar
to that of Fig. 8.) The stimulus
strength
was subthreshold
in A.
Activity
appeared
in both cell body
and muscle
at the same threshold
(B). The lo-mv
scale applies
to the upper
beam.
Functional
architecture of the abdominal ganglia. There are three conspicuous features of the ganglionic
organization:
1) Each cell has a functional
partner with the same size and position on the opposite side of the ganglion;
2) each cell lies in close association
with particular
other cells, forming
a
characteristic
grouping;
and 3) the groups are quite constant in location, but
within each group relative positions of cells are not necessarily
constant.
The positions
and certain features
of the identified
cells are described
below. The description
applies to the second and third abdominal
ganglia; all
but the sixth ganglion appear to be similar, but the others have not been
studied in detail. No consistent
differences
were observed between 0.5-kg and
1.5-kg lobsters except that the cells were somewhat
smaller in the former.
The identified cells are shown in six representative
ganglia in Figs. 12, 13,
and 14, and the muscles they control are listed in Table 1. We did not attempt to find the entire field of innervation
of any neuron. Most cells were
traced to a single muscle only, but it is highly likely that the innervations
of
domen.
The numbered
muscle
4 it has been
V = ventral;
D = dorsal.
muscles
are named
in Table
cut to reveal
8 and 9 more
Gl = first abdominal
ganglion.
1. Muscle
7 corresponds
clearly.
A =anterior;
to 10; like
P =posterior;
Downloaded from http://jn.physiology.org/ by 10.220.33.5 on June 18, 2017
an excitatory
junctional
potential
(EJP) in the muscle leaves little doubt
that the cells are connected directly
to the muscles. Such an experiment
is
shown for M9 in Fig. Il. There is little doubt that M4 is the giant motoneuron described by Johnson (8) and Wiersma
(28) since its axon is crossed
and leaves through the third root of the same segment, and since it was the
only cell that regularly produced contraction
in more than one flexor muscle.
The other M cells are probably
directly
connected to muscles since a single
shock or low frequency
of stimulation
produced
EJP’s in muscle fibers.
The ganglion contains a pair of interneurons
that form segments of the
lateral giant fiber system. These cells drive the M4 axons and other efferent
axons on a one-to-one basis (32) and ought to produce a pronounced
contraction when stimulated,
but we did not succeed in finding their cell bodies.
Downloaded from http://jn.physiology.org/ by 10.220.33.5 on June 18, 2017
B
ANTERIOR
POSTERIOR
D.
FIG. 12. Identified excitatory and inhibitory
neurons of the second ganglion and the
muscles they innervate. A : a composite physiological
map derived from two ganglia from
different animals. Physiologically
identified cell bodies are traced with black lines. The cell
bodies are named as described in the text. The peripheral field of innervation
of each cell
is given in Table 1. Not all of the M8 cell bodies were identified in this experiment
(compare with the right side of Fig. 14A). B : drawing of the musculature
of the lobster ab-
738
OTSUKA,
KRAVITZ,
AND
POTTER
FIG. 13. Physiological
maps of
the anterior flexor excitatory
neurons in three ganglia. In B and C the
midline of the ganglion falls approximately at the right border of the
photograph.
This figure illustrates
the variability
of the relative positions of Ml, M2, and M3 (compare
also Fig. 12A) but it can be seen
that the relative sizes of the cells and
their
position
as a group
are
constant.
Medial flexor excitatory cells, M4 and M5. These two large cell bodies are
always adjacent and in the center of the ganglion near the midline, M4 being
usually the larger (Fig. 12A). M4 lies anterior or anteromedial
to M5. As
indicated in Fig. 14, their axons cross and leave through the third root of the
same segment. These cells are so constant in size and position that they can
be recognized by visual cues alone.
Lateral flexor excitatory cells, M6 and M7. These are two cell bodies of
about the same size, usually in contact, and, with 12, the largest in the ganglion (Fig. 12). Their axons emerge, uncrossed, through the third root of the
same segment (Fig. 14, A and Bl). As a pair they are conspicuous, lying over
the base of the first root in relation to a cluster of small cells (MS’s) and to
12; however, their relative positions are variable (Figs. 12A; 14, A and Bl).
These neurons often show a burst of action potentials,
usually overshooting,
after penetration
with a microelectrode.
It is sometimes difficult to produce
contraction
by intracellular
stimulation
of these cells.
Downloaded from http://jn.physiology.org/ by 10.220.33.5 on June 18, 2017
12,13, and M4 are broader than is indicated in Table 1. The functions of the
cells are indicated in Fig. 14 and the destinations
of the axons are shown with
arrows.
Anterior flexor excitatory cells, Ml, M2, and M3. These three cells lie at
the anterior pole of the ganglion; their relative sizes are constant, Ml being
the smallest and M3 the largest (Figs. 12,13, and 14). They innervate flexor
muscles and are labeled with an F in Fig. 14. Their axons cross the midline
and leave through the third root of the ganglion just anterior. While the
groups are in corresponding
positions on the two sides of the ganglion, the
arrangement
of the cells within the cluster appears to be random (Figs. 12,
13, and 14). Since other cell bodies of similar size are found in this region,
these three neurons must be physiologically
identified in each preparation
GABA
AND
GLUTAMATE
IN
CELL
BODIES
739
Downloaded from http://jn.physiology.org/ by 10.220.33.5 on June 18, 2017
Swimmeret
excitatory cells, M8’s. These are part of a compa .ct cluster of
relatively
small cell bodies lying at the base of the first root and usually
bordered by 12 and the lateral and posterior flexor excitatory cells. They are
labeled “s” in Fig. 14, A and Bl. Their axons leave uncrossed through the
first root. As many as five cell bodies have been found in this group (e.g.,
stimulation.
Fig. 14A). They must be identified by intracellular
Posterior flexor excitatory cells M9, MlO, and Ml 1. These are two cell
bodies (M9, MlO) of similar size and a smaller one (Mll,
labeled with an
“f” in Fig. 14) located at the posterior-lateral
part of the ganglion. All send
axons through the ipsilateral
third root of the same segment. Since we have
not distinguished
M9 and Ml0 functionally,
we arbitrarily
call the cell
adjoining the small cell cluster M9. The positions of M9 and Ml0 are variable, and at least one of the two often needs physiological
identification.
They probably innervate different parts of M. obliquus ant. The small cell,
Mll,
is usually located medial or posterior to the larger ones, but in many
preparations
it was difficult to find, possibly because it lies deeper. Ml1 can
be recognized only by stimulation.
Extensor excitatory cells M12, M13, and M14. These three neurons lie
posterior to the inhibitory
group and medial to the posterior flexor excitatory
cells (Figs. 12A and 14). Their axons run through the ipsilateral second root
of the same segment (Fig. 14). Their relative positions are variable, and usually they can be recognized only by intracellular
stimulation.
Inhibitory
cells II, 12, and 13. These cells are adjacent to each other in
spite of the fact that 11 innervates a weak tonic flexor, 12 innervates the
main twitch flexor and 13 extensor muscles. This is in contrast to the groups
of excitatory cells which always innervate synergist muscles. The inhibitory
axons decussate while the axons of neighboring
excitatory cells do not (Fig.
14). Therefore, inhibitory
cells are not associated with excitatory cells which
innervate the same muscles (Fig. 17), nor is there a consistent relation between inhibitory
and excitatory cells which innervate synergistic or antagonistic muscles (Fig. 12, Fig. 14). For these reasons, 11, 12, and 13 appear to
form a natural group.
12 is the largest cell in the ganglion. 13 lies posterior or posteromedial
to
12. 11 is usually in contact with 12, but its position is variable. 12 and 13
could be located with reasonable confidence by visual inspection,
but 11
required physiological
identification.
11 and 12 send their axons through the
third root, 13 through the second. Confirmation
of the identity of 12 and 13
was easy since no other large cells near 12 and 13 send their axons to the
contralateral
roots. When the superficial or main branch of the third root was
stimulated
with low intensity, the only large cell in the posterior and contralateral half of the ganglion responding with an antidromic
action potential
was 12. Similarly,
on stimulation
of the second root, 13 was the only large
cell responding with antidromic
action potentials.
It is likely that 12 corresponds to the common flexor inhibitor
described in the crayfish abdomen
by Kennedy and Takeda (ll), since it sends branches into all major subdivisions of the third root.
740
OTSUKA,
KRAVITZ,
14A.
POTTER
(See opposite page.)
General remarks. We have seen no exception to the observations
by
Wiersma (29) that efferent axons to the swimmeret,
extensor, and flexor
muscles run through the first, second, and third roots, respectively.
In Fig.
14, A and B, it can be seen that the excitatory cell bodies in the anterior part
of the ganglion send axons to the opposite side, while the posterior groups of
excitatory cells give rise to uncrossed axons. The three pairs of identified
inhibitory
axons on each side are crossed. About 80% of the large cells in the
ganglion have been identified:
this constitutes about 10% of the total cell
population
(30).
Electrical coupling between cell bodies. Electrical
synapses were sought
between pairs of inhibitory
cells by the methods used in earlier studies (e.g.,
5, 27); the membrane
potential
of one cell was altered by passing hyperpolarizing pulses while recording from both cells. The potential
changes in
pairs of 11’s and pairs of 12’s had the characteristics
of passive spread of current, and hyperpolarizations
spread equally well in either direction. These
results show that these cells are linked to each other by electrical synapses.
We have no convincing demonstration
of electrical coupling between 13’s.
The attenuation
of the potential spreading from one 12 cell body to the
other was usually about 60 : 1. In the case of 11’s, the attenuation
was about
50 : 1. Evidence from histology and from experiments with injections of dyes
that diffuse rapidly within the cells indicates that the axons of the inhibitory
neurons decussate near the dorsal surface of the ganglion. If this is the place
Downloaded from http://jn.physiology.org/ by 10.220.33.5 on June 18, 2017
Fig.
AND
GABA
AND
GLUTAMATE
IN
CELL
BODIES
Downloaded from http://jn.physiology.org/ by 10.220.33.5 on June 18, 2017
FIG. 14. Physiological
and chemical
maps of two ganglia.
Part A shows the second
ganglion
of a l&kg
lobster,
and B the third
ganglion
of a 0.5-kg
lobster.
In A and Bl,
physiologically
identified
inhibitors
have been marked
with white
and excitors
with black.
B2 is a chemical
map of the same ganglion
as Bl; cells containing
more than 2 X lo-l1 moles
of GABA
were marked
white;
others
containing
no detectable
GABA
were marked
black.
F =excitatory
cells innervating
flexor
muscles;
f =excitatory
cells innervating
the superficial flexor
muscles
(i.e., Mll).
E =excitatory
cells of the extensor
musculature.
s =excitatory
cells of the swimmeret
muscles
(i.e., M8).
11, 12, and 13 =inhibitory
cell bodies.
Arrows
indicate
whether
the axons
are crossed
or uncrossed;
a diagonal
arrow
indicates
that the axon leaves
through
the third
root. All but the most
anterior
cells send axons
through
roots
of the same ganglion.
Not
all cells were identified
in both
ganglia,
e.g.,
the swimmeret
neurons
on the left side of photograph
A. This figure may be compared
with Fig. 12A.
OTSUKA,
742
KRAVITZ,
AND
POTTER
of electrical coupling, it is not surprising
that the interaction
appears to be
weak when recorded far away in the cell bodies on the ventral surface. As
was mentioned above, the spontaneous
activity
of the 11’s was often synchronous (Fig. 9A).
The experiment
illustrated
in Fig. 15 suggests that this synchrony
is not
produced by common interneurons,
but is achieved directly. When one of the
Table
_____I~~-~~
1. Cells
Axons
13
Axons
M4
M5
11
12
abdowlinal
ganglion
and the muscles
they
control*
crossed,
emerging
through
3rd root of lstl ganglion
M. obliquus
posterior
2 (3), medial
part
M. obliquus
anterior
2 (2)
M. t,ransversus
abdominis
2, ventral
part (9)
rossed, emerging
through
M. do rsalis profundus,
crossed,
emerging
M. obliquus
M. obliquus
M. obliquus
M. kansversus
M. obliquus
M. ventralis
M. obliquus
2nd root of 2nd
1ateral
part ( 13)
tlhrough
3rd root of
posterior
3 (5), lateral
anterior
2, caudal
part
anterior
4 (6)
abdominis
3, dorsal
anterior
3 (4)
superficialis
abdominis
anterior
2, caudal
part
ganglion
2nd ganglion
part
(10)
part-/
2, 3 (I)
(10)
Axons
M8
uncrossed,
emerging
through
M. remotor
II pedis spuris
1st root of 2nd ganglion
(swimmeret
muscle)
Axons
Ml2
Ml3
Ml4
uncrossed,
emerging
through
2nd root of 2nd
M. dorsalis
profundus,
medial
part
(11)
M. dorsalis
profundus,
middle
part (12)
M. dorsalis
profundus,
lateral
part (13)
ganglion
Axons
M6
M7
M9
Ml0
Ml1
uncrossed,
emerging
through
3rd root
M. transversus
abdominis
3, dorsal
M. obliquus
anterior
2, caudal part
M. obliquus
anterior
4(6)
M. obliquus
anterior
4 (6)
M. ventralis
superficialis
abdominis
ganglion
of 2nd
partt
(10)
2, 3 (l),
lateral
part
* Posit,ions
of the cells and muscles
are shown in Fig. 12. Numbers
in parentheses
the labels on tlhe muscles
in Fig. 121% The nomenclature
is that, of Schmidt
(24).
corresponding
to 8 in segment
posterior
to second rib.
refer to
7 Muscle
11’s was internally
stimulated,
the muscle innervated
by the other consistently showed IJP’s. It is probable that the electrical synapse is the mechanism for coupling the activities
of the two inhibitory
neurons.
Weak electrical interaction
was also observed between neighboring
large
excitatory
cells within
the anterior,
medial, and lateral flexor groups. This
coupling persisted after the cell bodies were separated by removing the tissue
between them, demonstrating
that the site of the current spread is not at the
cell body.
Downloaded from http://jn.physiology.org/ by 10.220.33.5 on June 18, 2017
Axons
Ml
M’t
M3
of the second
GABA
AND
GLUTAMATE
IN
CELL
BODIES
743
PART
II
Chemical architecture of the ganglion
GABA content of excitatory and inhibitory cell bodies. A correlation between GABA content and physiological function was established in experiments in which many cells were removed by free-hand dissection from living
ganglia, following physiological identification.
The results of such assays are
shown in Table 2. Individual values are given to illustrate the variability
of
the results.
GABA analyses were made on 36 excitatory cell bodies. In 32, no GABA
was detected (less than lo-l1 moles per cell). Four of the cells appeared to
contain l-l.7 x lo-l1 moles, but this is below the limit of accurate measureI’abLe
2. GABA
contents
of excitatory
and
inhibitory
cell bodies
from
fresh
ganglia
-Cell Type
GABA,
lo-11 moles
Average,
10-l” moles
Excitors
Anterior
flexor
Medial
flexor
Lateral
flexor
Pas terior
flexor
Extensor
Inhibitors
11
12
13
2.1, 3.5,
2.6
2.7
7.2, 12.5, 5.1, 7.1, 10.0, 6.3, 7.1,
7.3, 7.8, 7.3, 7.9, 7.3, 8.0, 9.0
6.9, 2.6, 1.9, 6.8, 2.7, 5.7
B&h 0.5- and 1.5~kg lobst.ers
were
was 2 X lo-l1
moles. Semicolons
separate
same ganglion
or from
different
ganglia.
used. The limit
values
obtained
of accurate
from cells
7.9
4.4
measurement
on opposite
of GABA
sides of the
Downloaded from http://jn.physiology.org/ by 10.220.33.5 on June 18, 2017
FIG. 15. Effect
of stimulating
homolateral
11 of the third
ganglion
on the membrane
potential
of the
superficial
flexor
muscle
3, 4. Short
current
pulses
were delivered
to 11
cell body
through
an intracellular
electrode.
Note that latency
is variable from stimulation
to stimulation.
Arrow
indicates
the onset of the IJP.
744
OTSUKA,
KRAVITZ,
AND
POTTER
Downloaded from http://jn.physiology.org/ by 10.220.33.5 on June 18, 2017
ment (2 X lo-l1 moles). In these cases the possibility
of contamination
of
samples with small inhibitory
cell bodies must be considered.
In an attempt to determine more accurately the GABA content within
excitatory cell bodies, two samples containing
six and eight cells were analyzed. The GABA contents of these samples were less than 1.3 x lo-l1 moles,
again below the limit of accurate measurement;
the average GABA content
per cell was therefore less than 2x lo-l2 moles. The conclusion wa s that
GABA if present in excitatory
cell bodies is in amounts too small to be
reliably measured by our methods.
This stands in sharp contrast to the results with physiologically
identified
inhibitory
cells from fresh ganglia (Table 2); in all but one of the 23 cells
more than 2 X lo-l1 moles of GABA was present. The differences in average
GABA content are due mainly to differences in cell volume. 11 is small and
difficult to isolate with hand-held instruments;
therefore few analyses were
performed on this cell.
Figure J4B illustrates the correlation between inhibitory
function and a
high GABA content, and excitatory function and a low GABA content. All
the marked cells in Fig. 14Bl were physiologically
identified
and then the
ganglion was photographed.
Cells with more than 2 X lo-l1 moles of GABA
are presented in white in Fig. 1442; those without detectable
GABA in
black. Twenty-three
of the cells were assayed for GABA including many of
the identified cells. The GABA contents of eight identified cells were assumed
from the data of Table 2, and these cells have been marked black or white
accordingly.
While the procedures described above established a clear difference between excitatory
and inhibitory
cells, within each type of inhibitory
cell
there was considerable variation in the GABA content. This could be due to
1) differences in cell volume from animal to animal (see below), 2) leakage or
changed GABA metabolism
during the impalement
and stimulation
required for physiological
identification,
and 3) damage during isolation of the
living cell with forceps. The results of three experiments
designed to test
these points are shown in Table 3. In the first experiment
the inhibitory
cells
were identified by visual inspection alone and were removed quickly from the
living ganglia with forceps; there were no failures to detect GABA and the
average GABA contents were somewhat higher than those reported in Table
2. In the second experiment the cells were physiologically
identified, but they
were removed after the ganglia had been freeze-dried, as described in METHODS. Again, the GABA
contents were higher, especially in the 13’s. Finally,
the two lower columns of Table 3 show the results obtained when ganglia
were rapidly removed from the whole animal and frozen, as described in
METHODS.
These cells were visually identified.
The average GABA contents
were highest of all. These values are the most reliable because there was the
least chance for damage to the cells, and because it is unlikely
that the
GABA contents were affected significantly
by altered metabolism,
assuming
that the enzyme activities in the cell bodies are similar to the optimal ac-
GABA
Table
--__----___
3. GAHA
AND
contents
1-1-
GLUTAMATE
of excitatory
and
IN
inhibitory
cell
GABA Content,
Visually
from
12
13
bodies
BODIES
isolated
lo-11 molgnt
745
in
several
Average,
ways*
lo-11 molest
identified
inhibitors
fresh ganglia
8.3,
5.0,
Visually
from
12
13
11.4, 6.5, 8.6
5.7, 5.2, 7.8, 4.1,
8.7
5.4
4.5
identified
indried ganglia
7.6, 9.0, 8.4
6.6, 8.4, 6.0,
8.3
6.5
5.0
identified
inhibitors
dried ganglia
Visually
from
M4
M6
M9
Ml
identified
excitors
dried ganglia
and M5
and M7
and Ml0
and M2
* For the methods
of identifying
0.5-kg
lobsters
only.
7 The
11.0, 6.5, 9.7, 9.8
6.1, 7.5, 5.9, 7.8
9.2
6.8
4
0,
0,
2
pooled
0, 0, 1.3
0.5
pooled
0.5
cells
0.5,
0
cells
and
limit
isolating
of accurate
the cells, see text
of PART II. Data
measurement
was 2 X lo+
moles.
tivities measured in peripheral axons (14). (These ganglia were immersed in
circulating
blood, and the neurons were intact, until less than 1 min before
freezing. In 1 min, a volume of homogenized
axoplasm equal to the 12 cell
body (see Table 5) is able to synthesize or degrade only about 0.3y0 of the
amount of GABA in the 12 cell body.) It should be noted that the highest
GABA contents per cell found with the various isolation procedures were
similar (Tables 2 and 3); we conclude that the major causes of variation
in
the results were damage during isolation and differences in cell volume from
ganglion to ganglion (see below).
Glutamate content of excitatory
and inhibitory
cell bodies. Glutamate
analyses were performed on 12, 13, M6, and M7 cell bodies (Table 4). The
limit of accurate measurement
in all cases was 4 X UP moles. In contrast to
Table
4. Glutamate
contents
of excitatory
Cell
and inhibitory
Glutamate,
cell bodies from
lo-11 moles
fresh
Average,
ganglia”
10-u moles
-
12
13
15.0, 8.2, 5.9, 13.0, 11.0,
11.4, 5.2, 9.9, 6.5
8.6, 15.0, 7.2, 9.5, 7.8
M (6 or 7)
9.6,
* Data
are from
0.5-kg
6.5,
12.7,
lobsters
6.5,
only.
7.3
6.8,
14.0,
16.5,
10.3
9.6
8.5
Downloaded from http://jn.physiology.org/ by 10.220.33.5 on June 18, 2017
physiologically
hibitors
from
12
13
from
CELL
OTSUKA,
746
Table
KRAVITZ,
5. Volumes
AND
of cell bodies
POTTER
of efferent
neurons*
~~
Cell
Total Volume,
12
M6 and
M4
M5
M7
Cytoplasm,
10-g liter?
6.74-6.48
7.2-7.0
8.42-8.07
6.65-6.25
8.93-6.79
5.35-4.9s
3.34-3.42
3.85
3.57
3.4
4.51-4.63
3.64-3.5
4.97-4.95
5.4 -5.4
4.21
6.29-6.73
3.45-3.35
2.24
2.32
3.32
2.5
4.4-4.5
4.16-4.23-3.79-3.81
4.95-4.4
-5.13
6.05-4.14
3.81
5.32
4.47
4.38
3.32
3.75
5.33
3.05
Average,
10-g litert
Total
volume:
Cytoplasm:
6.89
3.75
Total
volume:
Cytoplasm:
4.67
2.97
Total
volume:
4.64
Total
volume:
4.49
Total
volume:
3.86
* All cells are from
second
abdominal
ganglia
of 0.5-kg
lobsters
treated
in METHODS,
Measurements
(5) were on physiologically
identified
cells; all
visually
identified.
Hyphens
separate
measurements
on the same ganglion.
sents the total volume
of the cell body less the volume
of cytoplasmic
vacuoles
$ When two values
were available
for a ganglion,
they were averaged
and the
computing
the value in this column.
as described
others
were
t This repre(cf. Fig. 2B).
mean used in
the result with GABA, excitatory
and inhibitory
cells contained
similar
amounts of glutamate.
This is similar to the findings in isolated axons (14).
It provides an internal control for the GABA analyses, demonstrating
that
the excitatory cells were not selectively damaged by the isolation procedures.
Glutamate
is a fairly uniform constituent
of tissues, and the uncertainty
introduced by contamination
with glia and connective tissue is greater than
with the GABA analyses. An isolated cell with a small amount of adhering
tissue is shown in section in Fig. 4C. This qualification
must be borne in mind
in considering the results obtained with these methods.
Concentration
of GABA and glutamate in excitatory and inhibitory
cell
bodies. Our calculations
of the concentrations
of these substances depend on
volume measurements
made from sections of fixed ganglia (see METHODS).
These volumes are given in Table 5. Values obtained from cells of the same
ganglion are separated by hyphens; such pairs of cells generally had similar
volumes. A conspicuous feature of the cell bodies was vacuoles of an uncer-
Downloaded from http://jn.physiology.org/ by 10.220.33.5 on June 18, 2017
13
10-g liter
GABA
AND
GLUTAMATE
IN
CELL
BODIES
747
Table
6. GAHA
and
glutawzate
concentrations
in excitatory
and
GABA, 10-S molesJiter
inhibitory
Glutamate,
cell bodies*
10-S moles/liter
Cell
12
I3
M (6 or 7)
M (4 or 5)
Total cell volume
Cytoplasm
Total cell volume
Cytoplasm
13.4
14.6
24.5
22.9
14.9
20.6
18.3
27.5
32.3
(1 *a
* These values
were obtained
by dividing
the average
GABA
and glutamate
contents
(Table
3, bott,om
categories
and Table
4) by the average
volumes
(Table
5). It should
be
noted that the concentration
of GABA
in M (4 and 5) is uncertain
because the GABA
content,
is below the limit
of accurate
measurement.
GABA contents shown in the lower columns
tents shown in Table 4.
of Table
3 and glutamate
con-
DISCUSSION
Architecture of the abdominal ganglia. A high degree of constancy in the
size, position, and connections of individual
neurons appears to be general in
invertebrate
nervous systems (e.g., 1). Indeed, from available physiological
and anatomical
information
there is no reason to doubt that this is also true
of chordate nervous systems. This constancy was essential to the present
study, as it was in studies on other forms (e.g., 4, 9, 10, 19).
With regard to the way cells are arranged in the ganglion, it is found that
neurons controlling
certain muscles do not lie together, or even at corresponding points in successive ganglia. For example, at least seven excitatory
cells innervate M. obliquus ant. 3 (Fig. 16); these lie in four cell groups in
three ganglia, both sides being represented.
Figure 17 illustrates
that the identified
inhibitory
cells are not found
adjacent to the excitatory cells with which their axons run in the periphery.
(It should be noted that the course of axons through the ganglion is not
known in detail; Figs. 16 and 17 are diagrammatic
in this regard.) That cells
lying at widely separate points may send axons to the same target is familiar
Downloaded from http://jn.physiology.org/ by 10.220.33.5 on June 18, 2017
tain nature (Fig. 2B). In 12 and 13 these vacuoles consistently formed a large
portion of the cell body, about 40%. Usually less than 15% of the cytoplasm
of M6, and M7, and other excitatory cells was composed of the vacuoles. This
difference between the large excitatory
and inhibitory
cells was so pronounced that it could be used to locate 12 and 13 rapidly in the sections. In
13 cells an independent
measure of the vacuoles was available; this was subtracted from the total volume to give the “cytoplasmic
volume.” The nucleus
was found to comprise about 1 y0 of the total, but the contributions
of mitochondria, endoplasmic
reticulum,
and other cellular components are not yet
known.
In Table 6 the concentrations
of GABA and glutamate
are shown, computed both for total volume and cytoplasmic
volume, using the average
748
OTSUKA,
KRAVITZ,
AND
POTTER
FIG. 16. Excitatory
of M. obliquus
ant.
tion, see text.
innervation
3. For descrip-
reasonable
to suppose
that the same
Figs. 16 and 17.
Similarly, we may suppose that the excitatory
cells are grouped according
to their reflex connections.
In some cases adjacent excitatory
cells send axons
to the same muscle (e.g., M8’s; M9 and MlO), in other cases synergistic
muscles are innervated
(e.g., Ml-M3;
M4 and M5; M6 and M7). One is
reminded that in the mammalian
spinal cord, motoneurons
innervating
the
same muscle are clustered
and may also be closely associated
with motoneurons innervating
synergistic
muscles (e.g., 22).
The invariable
clustering
of the inhibitory
cells 11, 12, and I3 is more
surprising.
These cells innervate
muscles of different
or contrary
function
prof.
lateral part
M.vent.
superficialis
922 R3
caudal
FIG. 17. Locations
of excitatory
For description,
see text. R2 and
ganglionic
roots,
respectively.
part
R3
and inhibitory
indicate
that
cell bodies
the axons
innervating
lie in the
three muscles.
second
and third
Downloaded from http://jn.physiology.org/ by 10.220.33.5 on June 18, 2017
in neuroanatomy;
an example
from
primates
is the convergence
of
spinal
interneurons
and cortical
cells on
spinal motoneurons.
In such
instances
the converging
neurons
have
different
inputs.
Kennedy
and Takeda
(12)
have
demonstrated
that
axons innervating
the superficial
flexor
muscle
of the
crayfish
abdomen
subserve
different
reflexes,
and it is
is true of the cells illustrated
in
GABA
AND
GLUTAMATE
IN
CELL
BODIES
749
Downloaded from http://jn.physiology.org/ by 10.220.33.5 on June 18, 2017
and doubtless receive quite different inputs. It is possible that they do have
certain connections
in common or that they interact in a way that we did not
detect; still another possibility
is that 11, 12, and 13 have a common embryological
origin. Perhaps cells destined to have inhibitory
chemistry
differentiate
at special points in the developing
ganglia and then acquire distinct and appropriate
incoming and outgoing connections.
In this regard it
may be noted that another pair of high-GABA
cells of unknown
function
lie in the midline in the immediate
vicinity
of this cluster.
It must be borne in mind that so far only three inhibitory
neurons have
been identified;
of course, clustering
of synergistic
inhibitory
neurons may
also occur in the lobster CNS. The present point is simply that in this instance neurons of antagonistic
function
are consistently
found together.
The grouping of neurons with common transmitter
chemistry
is familiar
in the mammalian
nervous
system. Motoneurons
are clustered,
as are cell
bodies of preganglionic
neurons of the autonomic
nervous
system.
In the
cerebellum
it has been reported
that the layers of cells are discrete with
respect to function,
e.g., granule cells being excitatory
and Purkinje
cells
inhibitory
(3,6).
Another point of interest concerns the constant
relative sizes of the cell
bodies. Alm .ost certainly
this is related to the number of muscle fibers innervated, as in the case of mammalian
motoneurons
(18) . The largest cell body
is 12 which innervates
the massive caudal part of the anterior oblique muscle,
and doubtless other flexor bundles as well. In contrast,
one of the smallest of
the identified cells, 11, innervates
only the thin superficial flexor muscle. The
smallest excitatory
neurons we identified
innervate
the diminutive
swimmeret muscles.
The possible role of electrical
coupling in coordinating
the activities
of
cells on the two sides of the ganglion was discussed
above. We find that
neurons with chemical inhibitory
synapses in muscle also form excitatory
synapses in the CNS that are electrical
Thus a cell can be ex .citatory
and
inhibitory
without
the need for a second transmit ter compou .nd or an altered
postsynaptic
chemistry.
Another case of a neuron that forms both excitatory
and inhibitory
contacts has recently been reported in the snail Aplysia
by
Kandel et al. (9). In this case, a single transmitter,
acetylcholine,
and two
different postsynaptic
chemistries
are apparently
involved.
GABA and glutamate in the cell bodies of efferent neurons. Our long-term
interest in the cell bodies is to discover the mechanism
of regulation
of the
enzymes of the GABA pathway
in excitatory
and inhibitory
neurons. As yet
we have no information
about the enzymes; the present paper provides
information
only about the substrates,
GABA and glutamate.
There is no doubt that the GABA
content of inhibitory
cell bodies is
considerably
higher than that of excitatory
cell bodies and that glutamate
is
present in about equal amounts in the two types of cells. We have confidence
in the values for GABA
contents
of inhibitory
cells because similar high
values were obtained with several methods
of identification
and isolation
750
OTSUKA,
KRAVITZ,
AND
POTTER
Downloaded from http://jn.physiology.org/ by 10.220.33.5 on June 18, 2017
(Tables 2 and 3). The glutamate
contents
are more tentative
because of
inevitable
contamination
with small amounts
of other tissues.
Our calculations
of concentration
within
the cells are less certain for
several reasons. First, we do not know how these amino acids are distributed
in the cytoplasm,
Since there is no evidence in lobsters that either the substrates or enzymes are bound to cellular constituents
in axons or cell bodies
(cf. 14), it would be reasonable
to assign GABA and glutamate
contents
to
cytoplasm
exclusive of organelles.
Light microscopy
provides
a rough estimate of the volume occupied by vacuoles and nucleus (Table 5), but the
volumes of mitochondria
and other formed elements are not Yet known.
In
view of these uncertainties,
the only safe conclusion
is that the concentrations calcula ted from total cell volumes represent
lower limits.
Another
uncertainty
regarding
GABA
and glutamate
concentrations
arises from the fact that we could not measure the volumes of the cells that
were chemically
analyzed.
By dividing
average
substrate
contents
by
average volumes we may be obscuring
differences
from animal to animal. It
would be interesting
to know why the volumes of 12 and 13 vary over about
a twofold
range (Table 5).
It is unlikely
that there are great differences
in GABA
concentration
between cell bodies and axons of inhibitory
neurons. The average concentrations of GABA in the abdominal
cell bodies (Table 6) are about one-seventh
(total volume) or one-fourth
(cytoplasmic
volume) those previously
found in
inhibitory
axons of the walking
legs (15). Since we have not measured substrate contents in two parts of the same neuron, however,
this comparison
between cell body and axon is tentative.
If the con .centrat ion of GABA in
excitatory
cell bodies is similar to that in lim b axons (lo-”
M or less), it is not
surprising
that the amounts
we observed were below the limit of accurate
measurement;
a cell body with a volume of 4.5 x lO-g liter would contain
4.5 x lo-l2 moles or less (cf. Table 3).
The rather high concentrations
of the physiologically
active compounds
glutamate and GABA throughout
the lobster neurons may be compared with
the distribution
of acetylcholine
and catecholamines
in mammalian
nerve
cells. By histochemical
means it has been demonstrated
that catecholamines
are found throughout
the cells (2), but the concentration
gradient from cell
body to nerve terminal
is reported
to be steep (1: 100 to 1: 1000). Acetylcholine is found in axons (17) at a considerable
distance from the terminals,
in axoplasm is not known.
The relatively
high concenbut the concentration
tration
of GABA
and catecholamines
in cell bodies permits
a conclusion
about transmitter
chemistry
from anal yses of this part of the cell. It is not
yet known whether
this is also true of cholinergic
neurons.
SO far, in the lobster nervous
system a high content of GABA has labeled
a cell as inhibitory.
If this is true in other nervous
systems
it could be
valuable in mapping
certain
inhibitory
pathways.
Recent
work
on the
cerebellum
has indicated
that Purkinje
cells are inhibitory.
The layer in
which they lie contains
considerably
more GABA
than other cerebellar
layers (16). Analyses
of single cells could show whether
GABA is an index
GABA
AND
GLUTAMATE
IN
CELL
BODIES
751
of inhibitory function in the mammalian CNS. Neither acetylcholine nor
noradrenaline analyses can be used in this way to indicate neuronal function,
SUMMARY
ACKNOWLEDGMENTS
We gratefully
acknowledge
the histological
assistance
of Mrs.
Florence
Miss
Karen
Fischer.
Robert
Bosler
gave
us unfailing
assistance
throughout
We received
much
helpful
advice
from
Professors
Kuffler,
Furshpan,
Hubel,
Foster
and
the work.
and Wiesel.
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