Cell Tissue Res (1991) 266:197-207
Cell
Neurons with histaminelike immunoreactivity
in the segmental and stomatogastric nervous systems of the crayfish
Pacifastacus leniusculus and the lobster Homarus americanus
Brian Mulloney and Wendy M. Hall
Department of Zoology and Neurobiology Graduate Group, University of California, Davis, CA 95616, USA
Accepted June 14, 1991
Summary. We used a polyclonal antiserum against hista-
mine to map histaminelike immunoreactivity (HLI) in
whole mounts of the segmental ganglia and stomatogas-
tric ganglion o f crayfish and lobster. Carbodiimide fixa-
tion permitted both HRP-conjugated and FITC-conju-
gated secondary antibodies to be used effectively to visu-
alize HLI in these whole mounts. Two interneurons that
send axons through the inferior ventricular nerve (ivn)
and the stomatogastric nerve to the stomatogastric gan-
glion had strong HLI, both in crayfish and in lobster.
These ivn interneurons were known from other evidence
to be histaminergic. The neuropil of the stomatogastric
ganglion in both crayfish and lobster contained brightly
labeled terminals of axons that entered the ganglion
from the stomatogastric nerve. No neuronal cell bodies
in this ganglion had HLI. Each segmental ganglion con-
tained at least one pair of neurons with HLI. Some neu-
rons in the subesophageal ganglion and in each thoracic
ganglion labeled very brightly. Axons of projection inter-
neurons with strong H L I occurred in the dorsal lateral
tracts of each segmental ganglion, and sent branches
to the lateral neuropils and tract neuropils of each gan-
glion. All the labeled neurons were interneurons; no H L I
was observed in peripheral nerves.
Key words: Stomatogastric ganglion - Interneuron -
Crustacea - Histamine - Immunohistochemistry Paci-
fastacus leniusculus (Crustacea) - Homarus americanus
(Crnstacea)
Histamine occurs in the eyes, brain, stomatogastric gan-
glion, and some segmental ganglia of lobsters (Claiborne
and Selverston 1984a; Orona etal. 1990), barnacles
(Stuart and Callaway 1988), and Limulus (Battelle et al.
1991). It is reputed to be the neurotransmitter used by
ocellar photoreceptors in barnacles (Stuart and Calla-
way 1988), where histaminelike immunoreactivity occurs
in the photoreceptors and in eight other neurons (Cal-
Offpr#~t requests to: B. Mulloney
and Tissue
Research
9 Springer-Verlag 1991
laway et al. 1988). Histamine receptors have been de-
scribed in the brains of barnacles (Stuart and Callaway
1988) and lobsters (McClintock and Ache 1989). Hista-
mine is also known to modulate the activities of certain
neurons in the stomatogastric ganglion (Claiborne and
Selverston 1984a). Biochemical and pharmacological ex-
periments have shown that the two identified IV inter-
neurons (Dando and Selverston 1972) that synapse with
these stomatogastric neurons in the lobster Panulirus
(Sigvardt and Mulloney 1982a) use histamine as a neu-
rotransmitter (Claiborne and Selverston 1984a). The cell
bodies of these interneurons lie at the back of the brain,
between the two circumesophageal connectives (Clai-
borne and Selverston 1984 b).
It seemed probable that histamine would be the trans-
mitter used by some other neurons in crustaceans, but
the numbers and distribution of histaminergic neurons
in each segmental ganglion of macruran crustaceans
have not previously been described. We used a well-char-
acterized polyclonal antiserum against histamine (Panu-
la et al. 1988) to label the IV interneurons in the lobster
Homarus americanus and the crayfish Pacifastacus len-
iusculus. This success in labeling the IV interneurons and
their stomatogastric projections constituted a critical test
of the performance of this antibody. We also used it
to map and to count neurons with histaminelike immu-
noreactivity (HLI) in the segmental ganglia of the cray-
fish. Whole mounts of these segmental ganglia revealed
a regular and quite simple pattern of HLI in inter-
neurons.
Materials and methods
Crayfish Pacifastacus leniuseulus and lobsters Homarus americanus
were obtained from commercial suppliers and maintained in aerat-
ed aquaria. Twelve crayfish and three lobsters were used for these
experiments.
The antiserum used in these experiments was raised in rabbits
against authentic histamine conjugated with 1-ethyl-3-(3-dimethyl-
aminopropyl)-carbodiimide (EDCDI, Sigma E-7750) to a carrier
protein (Panula et al. 1988). To achieve good localization of HLI
198
in these animals, it was essential to fix the tissue using E D C D I
perfused through the arterial system.
Animals were first chilled on ice, then exsanguinated with ice-
cold CaZ+-free saline. The crayfish saline (Van Harreveld 1936)
was modified by substituting Mg 2+ for Ca2+; the lobster saline
(Beltz et al. 1984) was modified in the same way. To fix segmental
ganglia, we perfused them through the sternal artery with a 4%
weight/volume solution of E D C D I in Ca 2 +-free saline for 20 rain,
followed by 4% formaldehyde in Dulbecco's phosphate-buffered
saline (PBS, Sigma D5773) for another 20 min. Fixative was per-
fused under pressure at a rate of about 1 ml/min. After the CNS
was removed from the animal, each ganglion was desheathed man-
ually in 4% formaldehyde and left in that fixative over night. We
used only freshly prepared E D C D I solutions.
To fix the brain and the stomatogastric ganglion, we perfused
fixative through the ophthalmic artery (Sandeman 1967), using
the same schedule.
Immunocytochemistry
To label neurons with HLI, ganglia were removed from fixative,
washed in 0.1 M glycine in PBS, dehydrated to 95% EtOH, rehy-
drated, and preincubated in a wash-buffer that contained 5% goat
serum (Gibco) and 0.3% reduced Triton X-100 (Aldrich) in PBS.
Fig. 1. A Whole mount of a crayfish Pacifastacus brain (supra-
esophageal ganglia), labeled with a histamine antiserum and viewed
from the dorsoposterior aspect. The pair of labeled neurons (arrow-
heads) lie in the position where the cell bodies of the IV interneu-
tons were reported in Panulirus (Claiborne and Selverston 1984b).
Anterior is to the top. B A frontal section through another crayfish
brain in the plane of the IV interneurons' cell bodies. One cell
body with HLI (arrowhead) in the cytoplasm surrounding the nu-
cleus is visible near the bases of the circumesophageal connectives
The tissue was then incubated overnight at 5~ C in primary antise-
rum diluted 1 : 250 in the same wash buffer. The ganglia were subse-
quently washed in several changes of wash buffer to remove un-
bound primary antiserum, then incubated overnight in the second-
ary antiserum, a goat-anti-rabbit FITC conjugate (Kirkegaard and
Perry) or a goat-anti-rabbit HRP-conjugated F(ab')z fragment
(Tago), diluted 1 : 25 in the same wash buffer.
Histamine conjugate
Keyhole limpet hemocyanin (KLH, Sigma H-2133) was succinylat-
ed as described in Panula et al. (1988). A synthetic antigen was
prepared by conjugating authentic histamine dihydrochloride (Sig-
ma H-7250) to succinylated KLH with EDCDI, following the pro-
tocol described in Panula et al. (1984). NaBH4 was added to the
reaction mixture, to a final concentration of 2.5 mM, for 25 rain
before final dialysis of the antigen. Histamine-KLH conjugate was
dialyzed against distilled water using Centriprep 30 concentrators
(Amicon) at room temperature.
Fluorescence microscopy
To visualize HLI, each FITC-labeled ganglion was rinsed in wash
buffer, pinned out in PBS, dehydrated in an ethanol series, and
(asterisks). The histaminelike immunoreactivity (HLI) was visual-
ized with a horseradish peroxidase (HRP)-conjugated secondary
antibody and diaminobenzidine (DAB). Thickness: 10 lain. C In
this Pacifastacus whole mount, the pair of labeled neurons (arrow-
heads) was displaced into the inferior ventricular nerve. Anterior
is to the left. D A whole mount of a lobster inferior ventricular
nerve 0vn) near its junction with the esophageal ganglion. The
brain lies to the left; the esophageal ganglion lies to the right

cleared in methyl salicylate. Ganglia were studied as cleared whole
mounts, using Nikon epifluorescence optics and planapochromatic
lenses, and photographed with a Nikon Ultraphot camera using
Kodak Ektachrome film.
To compare the intensity of labeling in the immunocytochemi-
cal control experiments, the exposure required to photograph the
normal control was determined, then the same exposure was used
to photograph the three illustrated ganglia. Similarly, the correct
exposure for printing the negative of the normal control was deter-
mined, then the same exposure was used to print the other two
images.
Fig. 2A-C. Whole mounts of stomatogastric ganglia and nerves
labeled with a histamine antiserum_ A A ganglion from Paeifasta-
cus, with heavily labeled axon terminals. B A ganglion from Ho-
marus, with heavily labeled axon terminals. C A stomatogastric
nerve from Homarus, in which two labeled axons are apparent.
Bars: 200 gm
199
Peroxidase microscopy
After incubation with the horseradish peroxidase (HRP)-conjugat-
ed secondary antibody, ganglia were rinsed in wash buffer and
several changes of PBS. The ganglia were then soaked in 0.05%
diaminobenzidine (DAB; Organon Technica 36315) in 0.05 M
TRIS-buffer, at pH 7.6 for 3 h at room temperature. H z O 2 w a s
aded to this solution to make a 0.003% solution, and the progress
of the deposition of the reaction product was monitored visually.
The DAB reaction was halted by washing the ganglia in PBS.
Ganglia that were to be sectioned were then washed in 0.1 M
sodium cacodylate buffer and postfixed in 2% OsO4 in the same
buffer for 3 h at 5~ C. The ganglia were then dehydrated, embedded
in Spurr's resin, and sectioned at 10 gm using the methods de-
scribed in Leise et al. (1986, 1987). Sections were photographed
with Nikon planapochromatic lenses using Kodak Techpan 120
film.
Results
The I V interneurons have histaminelike immunoreactivity
(HLI)
E x a m i n a t i o n o f w h o l e m o u n t s o f c r a y f i s h b r a i n s re-
v e a l e d t w o n e u r o n s at t h e b a s e o f t h e i n f e r i o r v e n t r i c u l a r
n e r v e (ivn) t h a t l a b e l e d b r i g h t l y w i t h h i s t a m i n e a n t i s e -
r u m (Fig. 1A). I n m o s t w h o l e m o u n t s , w e w e r e u n a b l e
to r e s o l v e t h e a x o n s e m e r g i n g f r o m t h e s e n e u r o n s , a n d
t h e y w e r e n o t t h e o n l y h i s t a m i n e r g i c n e u r o n s in the
Fig. 3. Whole mounts of a pair of commissural ganglia labeled
with a histamine antiserum. One cell body in each ganglion labeled
brightly (arrowheads). The neuropil contained many heavily labeled
processes. Anterior is to the right. Bar: 200 gm
200
brain. However, these cell bodies lie precisely where Clai-
borne and Selverston (1984b) described the somata of
the two IV interneurons that project to the stomatogas-
tric ganglion in Panulirus (Dando and Selverston 1972;
Sigvardt and Mulloney 1982a). In sections whose H L I
was visualized with DAB (Fig. 1 B), the cytoplasm of
these cell bodies appeared to be filled with intensely la-
beled vesicles that distinguished them from their unla-
beled neighbors. The nucleus of the immunopositive
neurons lacked H L I (Fig. 1 B).
In one whole m o u n t from Pacifastacus, the two cells
were displaced along the ivn away from the brain toward
the esophageal ganglion (Fig. ~ C, arrowheads). In Ho-
marus, the IV interneurons are normally displaced fur-
ther toward the esophageal ganglion (Cazalets, personal
communication). In our whole mounts of Homarus
brains, treated identically to our preparations of Pacifas-
tacus, we saw two brightly labeled cell bodies in the
ivn close to the esophageal ganglion (Fig. 1 D, arrow-
heads). These neurons projected axons toward the
esophageal ganglion, the route followed by the axons
of the IV interneurons in other Crustacea (Dando and
Selverston 1972).
Axonal terminals in the stomatogastric neuropil have H L I
Whole-mount preparations of the stomatogastric gangli-
on of crayfish and lobsters consistently demonstrated
9 Gd
intense labeling of neuronal processes in the neuropil
(Fig. 2A, B). These processes were continuous with ax-
ons that entered the ganglion from the stomatogastric
nerve, the route followed by IV interneurons, and did
not project beyond the stomatogastric ganglion toward
more peripheral targets. In the best preparations we
could resolve two axons of similar diameter that labeled
well above background in the stomatogastric nerve
(Fig. 2C). In contrast to these processes and to the re-
sults from other ganglia, no whole mounts of the stoma-
togastric ganglion revealed any labeled neuronal cell
bodies.
Each commissural ganglion had H L I in the neuropil
and in one cell body
Whole mounts of commissural ganglia consistently re-
vealed bright H L I within the neuropil, labeled axons
that projected through the commissures to the ganglion,
and one small neuronal cell body with H L I (Fig. 3). We
were unable to trace the axons of this neuron or any
others through the nerves that connect the commissural
ganglion to the esophageal ganglion and stomatogastric
ganglion, although intermittent labeling of axons in
these nerves did occur in some preparations, particularly
in Homarus.
Fig. 4. A A whole mount of a Pacifasta-
cus subesophageal ganglion labeled with
a histamine antiserum and viewed from
the ventral aspect. Several pairs of
brightly labeled neurons are evident
)
(large arrowheads). Two of the dorsal
midline columns of neurons had several
neurons with HLI (small arrowheads).
Anterior is at the top. Bar: 200 p.m.
B Camera Iucida drawing of the small,
dorsolaterally located neurons with HLI
that occur at the level of the first superi-
or nerves, which are shown in the fig-
ure. Same scale as A
Fig. 5 A - E . Frontal sections through a Paeifastacus subesophageal
ganglion that show details of the neurons with HLI, visualized
with an HRP-conjugated secondary antibody and DAB. Anterior
is at the top. Ai Four labeled neurons (arrowheads) are visible
in this section, one from the most anterior pair on the upper left,
one from the bright lateral pair on the right, and two midline
neurons near the bottom. Ai~ A higher magnification of the outlined
area in At, but from the next ventral section, that shows the labeled
neuron near the base of the circumesophageal connective (arrow-
head), and numerous labeled processes in the neuropil posterior
to the cluster of unlabeled cell bodies. B A more posterior view
of a more dorsal section that shows the two large lateral neurons
labeled with HLI (arrowheads). C A ventral midline neuron. HLI
is apparent as granular structures surrounding the nucleus and
continuing anteriorly into the neurite. Note the unlabeled neighbor-
ing neurons, and the change of scale. D A posterior part of the
ganglion showing one (arrowhead) of the two labeled posterior
midline cells. Numerous processes with HLI are apparent in the
neuropil. E A midline view of the ganglion somewhat dorsal to
the plane of B that shows two labeled cell bodies (arrowheads)
and extensive HLI in the neuropil. Thickness: 10 gm. Bar: 200 gm
202
The subesophageal ganglion has H L I in four ven tralpairs
of celts and in clusters of midline cells
Whole-mount preparations of the subesophageal gangli-
on consistently showed a regular, fairly simple pattern
of HLI. Most anteriorly, near the entrance of the circu-
mesophageal commissures, a relatively small pair of bi-
lateral cell bodies occurred (Fig. 4A, large arrowheads;
Fig. 5A). A pair of larger, very brightly labeled neurons
occurred more posteriorly and laterally (Fig. 4A, large
arrowheads; Fig. 5Ai, B). These brightly labeled neurons
had relatively thin neurites that projected dorsally into
the neuropil. These neurites were difficult to trace, but
in some whole-mount preparations they appeared to be
continuous with the bright axons that enter the sub-
esophageal ganglion from the connectives (Fig. 4A, open
arrows). These axons projected anteriorly into the com-
missural ganglion (Fig. 3). Two more bilateral pairs of
brightly labeled neurons occurred in these ganglia in
more posterior locations, closer to the midline (Fig. 4A,
large arrowheads; Fig. 5 D, E).
The soma of each the labeled neurons first identified
in whole mounts using an FITC-conjugated secondary
antibody was Subsequently located in a ganglion labeled
with an HRP-conjugated secondary antibody and sec-
tioned in plastic (Fig. 5). Some of these bilateral cell
bodies were visible in the same section, e.g., the second
pair of very brightly labeled neurons in Fig. 4A are visi-
ble in Fig. 5B. In each case where only one member
of a pair is illustrated in Fig. 5, the other member was
also located in a nearby section. Because the HLI in
the cell body and neurite of these neurons was restricted
to vesicular cytoplasmic compartments (Fig. 5C), we
could not reconstruct the complete structure of any of
them because the HLI in the neurite faded before it
reached any heavily labeled axons or branches.
At the dorsolateral margins of the ganglion, near the
bases of the first superior nerves (Chaudonneret 1956),
a set of small cell bodies with HLI occurred that could
~ 4
I0
Fig. 6. Frontal section through a Pacifastacus subesophageal gan-
glion in a dorsal plane that shows three small labeled neurons
in the dorsal midline column. Thickness: 10 gm
not be resolved in whole mounts. These cell bodies were
15-20 I,tm in diameter, and most of the volume of each
soma was taken up by the nucleus. The HLI in these
cells was also vesicular (compare Figs. 1 B, 5 C); these
vesicles seemed to form a single layer between the nucle-
us and the cell membrane. From a count done with cam-
era lucida, there are about ten pairs of these cells
(Fig. 4B) in the ganglion. We were unable to trace the
neurites of any of these neurons in sections.
On the ventral midline, two unpaired clusters of la-
beled neurons occurred (Fig. 4A, small arrowheads; Fig.
5Ai, C, E). In addition, two of the midline columns of
neurons that extend dorsally through the core of the
subesophageal ganglion (Mulloney and Hall 1990) also
contained a few rather lightly labeled cell bodies (Fig. 6).
These small neurons were located quite dorsally in the
ganglia, at the level of the giant axons. None of these
midline cells ever labeled as brightly as did the bilateral
pairs of large ventral neurons.
Each thoracic ganglion has one or two pairs
of neurons with H L I
The labeling of neurons in the thoracic ganglia resem-
bled the labeling of single segments of the subesophageal
ganglion. Near the ventral midline, a brightly labeled
pair of cells always appeared (Fig. 7, large arrowheads).
More dorsally and nearer to the posterior edge of ganglia
T3 and T4, a second smaller and more faintly labeled
pair of neurons appeared in the better preparations. This
variability in labeling these more dorsal cell bodies ap-
peared to reflect differences in the quality of fixation
achieved in different preparations; if one thoracic gangli-
on had a labeled pair of these smaller neurons, the other
was quite likely to do so, and the subesophageal labeling
would be spectacular.
Each thoracic ganglion also showed a consistent pat-
tern of HLI in axons and processes (open arrows in
Figs. 7, 8 Bil, 10A). In particular, two bilateral groups
of axons that projected the length of the thorax labeled
heavily. They appeared to be axons of long projection
interneurons. In sections labeled with HRP, axons in
the dorsal lateral tract (DLT) stained intensely (Figs.
8 Bii, 10A); in each ganglion, these axons sent processes
into the tract neuropil (Skinner 1985). The lateral group
of five axons (Fig. 8 Bii) terminated in T5 (Fig. 7, T 5v
open arrows). The more medial group of three axons
continued posteriorly to the last abdominal ganglion.
Each abdominal ganglion had one pair of neurons
with HLI," A2, A3, A4 and A5 each had two pairs
of labeled neurons
The labeling of neurons and processes in the abdominal
ganglia was both fainter and less reliable than that in
the more anterior ganglia. To assess the variability of
this HLI in the abdominal ganglia, we first drew the
location of each labeled cell by projecting the original
Ektachrome slides onto graph paper, and then collected
 203

Fig. 7. Whole mounts of Pacifastacus thoracic ganglia la-

beled with a histamine antiserum, viewed from the ven-
tral aspect. Tlv-T5v Thoracic ganglion 1 to thoracic gan-
glion 5, photographed in a ventral plane. T2e-T4d The
same ganglia photographed in a more dorsal plane of fo-
cus to show the cell bodies with HLI (arrowheads). These
more dorsal photos also show the pattern of the pro-
cesses with HLI (open arrows) in the longitudinal tracts,
some of which ended in T5 (open arrows). Anterior is at
the top. Bar: 200 ~tm
204
Fig. 8A, B. Cross sections of a Pacifastacus T3 labeled with a hista-
mine antiserum and an HRP-conjugated secondary antibody. Ai
A section that shows one of the two dorsal neurons with HLI
(arrowhead). The axons of the median giant neurons lie lateral
to this neuron. Ai~ A higher magnification of the outlined region
of Ai that shows four cell bodies in this dorsal group, only one
these drawings in a s u m m a r y diagram that showed b o t h
the location of each pair in one preparation and the
ranges o f these locations in each ganglion o f all prepara-
tions (Fig. 9). In each abdominal ganglion, one pair of
small neurons occurred close to the ventral midline
(Fig. 9). In each of the intermediate abdominal ganglia,
A2 through A5, we observed a second pair of cells locat-
ed more laterally, near the base of each o f the nerves
that innervate the swimmerets. These were also small
neurons, at most twice the diameter of the midline pair
(Fig. 9). These two pairs of neurons were also visible
in sections of abdominal ganglia labeled with H R P ;
Fig. 10 B and C show examples o f these cell bodies from
an A4. The H L I in cell bodies of the HRP-labeled sec-
tions appeared to be granular (Fig. 10 B).
Each abdominal ganglion also had fine neuronal pro-
cesses with H L I in both the tract neuropil and the lateral
of which has HLI (arrowhead). B i A more anterior section from
the same ganglion that shows one of the ventral neurons with
HLI (arrowhead). Bil A higher magnification of the outlined region
of Bi that shows HLI in the neuropil of the ganglion, and two
groups of labeled axons (open arrows). Thickness: 10 gm
neuropils (Fig. 10 B), and axons in the D L T s that labeled
heavily. These D L T axons resembled those illustrated
in Fig. 10A.
Immunocytochemical controls
The original description o f the primary antibody (Panula
et al. 1988) contained a careful demonstration of its
specificity, but we repeated two tests to confirm its prop-
erties in crustacean neural tissue. Three pairs of thoracic
ganglia f r o m one ventral nerve cord were labeled in par-
allel: for one pair, the normal control, our standard
protocol was followed (Fig. 11A); for a second pair, the
primary antibody was preincubated with 1.5 mg of syn-
thetic HA-antigen per milliliter o f diluted antibody
(Fig. 11 B); for the third pair, no primary antibody was

A2
A4 A5
t nerve. A1 and A6 had only the
200 pm
used (Fig. 11 C). Each of these ganglia came from the
same animal; except for the specified differences, they
were fixed and processed identically. The ganglia were
photographed at the same exposure, and subsequent
prints were also made at the same exposure to permit
readers to assess accurately the H L I in each ganglion.
Preincubating the primary antibody with the synthet-
ic HA-antigen drastically reduced the H L I relative to
that observed in the normal controls. Omitting the pri-
mary antibody eliminated all evidence of localized im-
munoreactivity. Both members of each pair gave the
same result. These results confirm Panula et al. (1988)
that the H A antiserum is highly specific for the epitope
created by EDCDI-fixation of authentic HA.
Discussion
Histamine has recently been identified as the transmitter
used by photoreceptors in insects (Simmons and Hardie
1988; N/issel et al. 1988) and barnacles (Callaway et al.
1988 ; Stuart and Callaway 1988), where there is evidence
of synthesis, synaptic localization, and postsynaptic his-
tamine receptors on the predicted target neurons. In
other crustaceans, a mix of biochemical, pharmacologi-
cal, and immunohistochemical evidence suggests that
histamine is a transmitter in the olfactory lobes of the
lobster Homarus (Orona et al. 1990) and the stomato-
gastric system of the lobster Panulirus (Claiborne and
Selverston 1984a). The Panulirus work is a particularly
important point of reference for our results because it
showed that the two IV neurons projected to the stoma-
togastric ganglion through a particular pathway (Dando
and Selverston 1972; Sigvardt and Mulloney 1982b),
had cell bodies in a particular place (Claiborne and Sel-
205
A3
~ 4~ ~
Fig. 9. Summary drawings of
whole mounts of Pacifastacus ab-
dominal ganglia labeled with a
histamine antiserum and viewed
from the ventral aspect. A1-A6
Abdominal ganglion 1 through
abdominal ganglion 6. Labeled
cell bodies are drawn in black.
A6 A2, A3, A4 and A5 had two pairs
of neurons with HLI, one pair
near the midline and one pair lo-
cated more laterally, toward the
base of N1, the first segmental
midline pair of labeled neurons.
The ranges of the locations in
which labeled neurons occurred
in all the preparations we exam-
ined are shown in gray. Anterior
is at the top. Bar: 200 gm
verston 1984b), and contained high levels of histamine.
The histamine content of these two cell bodies in Panulir-
us averaged 0.05 pmol/cell, leading to an estimate of
3 m M histamine in the cytoplasm of these neurons.
These earlier results provide both a demonstration that
authentic histamine occurs in these animals and a predic-
tion of where we might expect to find histaminergic neu-
rons in other crustaceans.
A pair of cell bodies with strong H L I occurred both
in crayfish and in Homarus at the place where the homo-
logues of the Panulirus IV neurons would be predicted
to lie (Fig. 1). This HLI would be predicted from the
known concentrations of histamine in these neurons in
Panulirus. The animals in which the pair of neurons were
displaced into the ivn provide particularly good evidence
that these cell bodies belong to crayfish and lobster ho-
mologues of the Panulirus neurons, and contain high
cytoplasmic levels of histamine. This result confirms that
this antibody and fixation can label histaminergic neu-
rons. Since the antiserum labels known histaminergic
neurons, the previously unknown neurons it labels are
probably also histaminergic.
The bright labeling of stomatogastric ganglia and
commissural ganglia in crayfish and Homarus is also
predicted from previous measurements of their hista-
mine content in Panulirus. The histamine content of the
stomatogastric ganglion and the commissural ganglia
was between 80 and 130 nmol/gm protein (Claiborne
and Selverston 1984a). The intense !abeling of the neu-
ropils of both ganglia suggests that many histaminergic
synapses occur in these ganglia, and the synaps'es made
by the IV interneurons in the stomatogastric ganglion
of Panulirus predict particular target neurons for these
interneurons in crayftsh and Homarus (Sigvardt and
Mulloney 1982a; Mulloney 1987).
206
Fig. 10A-C. Frontal sections of Pacifastacus ganglia labeled with
a histamine antiserum and an HRP-conjugated secondary anti-
body. A A frontal section of a T2 at the level of the dorsal lateral
tracts, showing three labeled axons (arrowhead) and fine processes
with HLI in the tract neuropil. Anterior is to the left; the midline
is toward the top. B A frontal section of a fourth abdominal gangli-
on showing a labeled cell body (arrowhead) in a lateral cluster
of neurons, and fine processes with HLI in the lateral neuropil.
Anterior is at the top; the midline is to the right. C A more ventral
section of the same fourth abdominal ganglion showing a labeled
cell body near the ventral midline (arrowhead). Anterior is at the
top. Thickness: 10 gin. Bar: 200 gm
T h e absence o f labeled cell bodies in the s t o m a t o g a s -
tric g a n g l i o n is c o n s i s t e n t with the k n o w n p h a r m a c o l o g y
o f these n e u r o n s . O f the 30 s t o m a t o g a s t r i c n e u r o n s , 23
are either g l u t a m a t e r g i c or cholinergic ( M a r d e r i987).
T h e t r a n s m i t t e r s used b y the others are u n k n o w n , a n d
a p p a r e n t l y n o n e o f t h e m use histamine.
Fig. llA-C. Controls demonstrating the specificity of this hista-
mine antiserum. A Normal control: a fourth thoracic ganglion,
T4, labeled with the normal protocol described in Materials and
methods. Two cells with intense HLI are apparent; compare with
Fig. 5. B Preabsorption control: a second thoracic ganglion, T2,
labeled with the same protocol, except that the primary antiserum
was first absorbed with a synthetic conjugate of histamine and
a carrier protein. C No-primary control: a first thoracic ganglion,
T1, labeled with the same protocol, except that the primary antise-
rum was omitted. Bar: 200 ~m
T h e p a t t e r n o f n e u r o n s t h a t this a n t i s e r u m labeled
was similar in the v a r i o u s segmental ganglia, a n d
r e m a r k a b l y sparse. We observed n o labeled a x o n s in pe-
ripheral nerves except the nerves o f the s t o m a t o g a s t r i c
system (Figs. 1, 2), a n d c o n c l u d e that there are n o hista-
minergic m o t o r n e u r o n s in these a n i m a l s . E v e n in the

s u b e s o p h a g e a l g a n g l i o n , w h i c h d e v e l o p s f r o m the fusion
o f six e m b r y o n i c ganglia, o n l y a small n u m b e r o f neu-
rons s h o w e d H L I . E v e r y s e g m e n t a l g a n g l i o n h a d at least
one p a i r o f n e u r o n s w i t h H L I (Figs. 4, 7, 9). T h o r a c i c
g a n g l i a T3, T4, a b d o m i n a l g a n g l i a A 2 t h r o u g h A5, a n d
s o m e s e g m e n t s o f the s u b e s o p h a g e a l g a n g l i o n h a d a sec-
o n d pair. A l t h o u g h the t h o r a c i c a n d a b d o m i n a l g a n g l i a
differ in size a n d s h a p e a n d a l t h o u g h the m i d l i n e cells
in T3, T4 a n d the s u b e s o p h a g e a l g a n g l i o n were m o r e
d o r s a l t h a n the m i d l i n e cells in the a b d o m i n a l ganglia,
we i n t e r p r e t this p a t t e r n as a serial r e p e t i t i o n o f two
p a i r s o f h o m o l o g o u s i n t e r n e u r o n s p e r segment, with ei-
ther the l a t e r a l p a i r o r the m e d i a l p a i r a b s e n t in s o m e
segments. T h e n e u r o n s in c r a y f i s h s e g m e n t a l g a n g l i a de-
v e l o p f r o m a set o f n e u r o b l a s t s like t h o s e k n o w n in in-
sects ( T h o m a s et al. 1984), a n d p r o b a b l y follow a simi-
larly d e t e r m i n a t e d e v e l o p m e n t a l plan. T h e p a t t e r n o f
H L I w i t h i n each s e g m e n t suggests t h a t either the b r i g h t -
ly l a b e l e d n e u r o n s are the p r o g e n y o f a p a i r o f n e u r o -
blasts w h o s e d a u g h t e r s d i f f e r e n t i a t e o n l y two n e u r o n s
o f this p h e n o t y p e , o r t h a t they a r e the survivors o f clones
o f d a u g h t e r cells, the rest o f w h i c h die earlier in d e v e l o p -
ment.
Acknowledgements. The antiserum used was a gift from Dr. Panula
(Helsinki). We thank him for his generosity and for his advice.
We also thank Carolyn Sherff for reading the manuscript critically.
This work was supported by NIH grant NS21194 and by NSF
grant BNS 87-19397 to B.M.
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