Organization of the stomatogastric neuropil of the crab,Cancer borealis,as revealed by modulator immunocytochemistryAndrew E. Christie1* , David H. Baldwin2, Eve Marder1, Katherine Graubard2
1 Department of Biology and Volen Center for Complex Systems, Brandeis University, Waltham, MA 02254, USA2 Department of Zoology, University of Washington, Box 351800, Seattle, WA 98195–1800, USA
&misc:Received: 16 March 1996 / Accepted: 17 September 1996
&p.1:Abstract. We used antibodies to a number of neuromod-ulatory substances, including serotonin, FLRF Amide,red pigment-concentrating hormone, substance P, proct-olin and cholecystokinin, to investigate the distributionof molecules similar to these substances in the stomato-gastric ganglion of the crab, Cancer borealis. No immu-noreactivity was seen in the region of the cell bodies thatsurrounds the neuropil and little was found in the core ofthe neuropil (where the primary neurites of the intrinsicneurons occupy most of the space). Instead, modulatorimmunolabel was densely packed in the more peripheralportion of the neuropil that surrounded the core. Withinthis peripheral neuropil, profiles appeared quite uniform-ly distributed. Double-labeling showed that there werelimited differences in distribution between the labels ex-amined in our study. The only immunolabeled structuresthat showed a distinct differential distribution within thestomatogastric neuropil were a population of ≥10 µmvaricosities that arose from a pair of input fibers that wetermed the large varicosity fibers. These varicositieswere immunolabelled by antisera for three different pep-tides. Taken collectively, these data shows that there is astereotyped distribution of modulator immunoreactivitywithin the crab stomatogastric neuropil. However, thissegregation is more rudimentary than that reported forthe intrinsic stomatogastric neurons.
The stomatogastric ganglion (STG) of decapod crusta-ceans is one of the leading systems for studying the gen-
eration and modulation of rhythmic behaviors. This gan-glion, in concert with the three other ganglia that com-prise the stomatogastric nervous system (STNS), is re-sponsible for the coordinated movements that allow fooditems to be ingested, stored, chewed and filtered fromthe foregut (Harris-Warrick et al. 1992). The neural cir-cuits responsible for the coordination of two of these ac-tions, namely chewing and filtering, are contained withinthe STG and are commonly referred to as the gastric milland pyloric circuits, respectively (Selverston and Mou-lins 1987; Harris-Warrick et al. 1992).
In the crab, Cancer borealis, previous studies haveidentified the constituent neurons of both the gastric milland pyloric circuits, as well as their patterns of connec-tivity (Selverston and Moulins 1987; Harris-Warrick etal. 1992; Weimann 1992; Weimann and Marder 1994;Norris et al. 1995). Rather than being “hard wired”, bothnetworks are modulated by a variety of neuroactive sub-stances that alter the motor patterns of the STG (Marder1987; Marder and Weimann 1992; Marder et al. 1994;Harris-Warrick et al. 1992). Modulatory substances arereleased into the STG neuropil from input fibers thatproject to the ganglion primarily from the paired com-missural (CGs) and single oesophageal (OG) ganglia(Fig. 1; Coleman et al. 1992). Additionally, neuromodu-latory substances may also reach the STG from sensoryaxons and as circulating neurohormones via the hemo-lymph (Katz et al. 1989; Turrigiano and Selverston1990; Christie et al. 1995a). At the present time, over adozen neuroactive molecules, including both small mol-ecule transmitters and neuropeptides, have been identi-fied as modulators of the STG neural networks in C. bo-realis (Harris-Warrick et al. 1992; Marder et al. 1994;Christie et al. 1995a).
While classical small molecule transmitters are usual-ly released at conventional synapses, neuropeptides canact at a distance from their release sites (Jan and Jan1982; Kupfermann 1991). Previous immunocytochemi-cal studies of the STG have described neuropeptide-con-taining projections as “ramifying throughout the neuro-pil” (e.g., Marder et al. 1986, 1987; Goldberg et al.
* Present address:Department of Neuroscience, 215 StemmlerHall, University of Pennsylvania School of Medicine, Philadel-phia, PA 19104, USA
1988). Thus in the absence of experimental data it hasbeen conjectured that modulation by many of these mol-ecules occurs in a local hormone-like fashion. Baldwinand Graubard (1995) showed that the large neurites ofthe intrinsic STG neurons are primarily found in a coreregion within the STG neuropil. In contrast, the fine neu-rites of the intrinsic STG neurons are found in the moreperipheral region of the neuropil that surrounds the core.There has been, however, no detailed study of the distri-bution of the modulatory input axons that ramify withinthe STG neuropil to ask whether specific classes of im-munoreactive profiles are found preferentially in someneuropilar regions.
In this paper we examine the distribution of sevenneuromodulator immunoreactivities within the STG neu-ropil. We show that modulator immunoreactivity is notpresent in the region of the cell bodies, nor is it distribut-ed throughout the entire stomatogastric neuropil. Insteadit is segregated to the peripheral portion of the plexus,with the central core of the neuropil relatively devoid ofmodulator-containing profiles. These immunoreactiveprofiles fall roughly into two size groupings, namely,varicosities <10µm in major cross-sectional diameterand varicosities ≥10 µm in major cross-sectional diame-ter. Unlike the distribution of the fine processes of theintrinsic STG neurons (Baldwin and Graubard 1995),there is little evidence of large-scale segregation of mod-ulator immunoreactivity in the peripheral portion of theSTG neuropil. In fact, the only profiles that were clearlyfound to exhibit a marked segregation in the STG neuro-pil were the set of ≥10 µm varicosities (although thisstudy is limited in that each of the antibodies used labelsprofiles that arise from at least two neurons). Thus, ourdata show that there is a rudimentary organization ofmodulator-containing profiles within the stomatogastricneuropil. However, this organization is not as dramaticas that reported by Baldwin and Graubard (1995) for thefine processes of the intrinsic stomatogastric neurons.Some of these data have appeared previously in abstractform (Baldwin et al. 1992; Christie et al. 1992).
Materials and methods
Animals
Rock crabs, Cancer borealis, (n=91) were obtained from NeptuneLobster and Seafood, Boston, Mass. Animals were maintainedwithout food in artificial sea-water aquaria at 10–12°C until usedand were cold anesthetized by packing them in ice for 15–20 minprior to dissection.
Antibodies
For the localization of proctolin immunoreactivity, a rabbit anti-proctolin polyclonal antibody (Davis et al. 1989) was used at a fi-nal dilution of 1:300. FLRF Amide-like immunoreactivity was ex-amined using either of two rabbit anti-FMRF Amide polyclonalantibodies: 671C (Marder et al. 1987) and 231 (O’Donohue et al.1984) at final dilutions of 1:200 and 1:300, respectively. Both ofthe antiFMRF Amide antibodies showed equal or stronger recog-nition of extended FLRF Amide-like peptides (Marder et al.
1987), which have been shown to be the species of FMRF Amide-like molecules present in C. borealis(Weimann et al. 1993). Thus,in the remainder of this paper, we refer to these anti-FMRF Amideantisera as anti-FLRF Amide antibodies. Red pigment concentrat-ing hormone (RPCH)-like immunoreactivity was characterizedwith the rabbit polyclonal antibody of Madsen et al. (1985) at a fi-nal dilution of 1:200. Substance P-like immunoreactivity was ex-amined using a rat anti-substance P monoclonal antibody (Accu-rate Chemical and Scientific, Westbury, N.Y.) at a final dilution of1:300 (Goldberg et al. 1988; Blitz et al. 1995). Serotonin immuno-reactivity was studied using a rat anti-serotonin monoclonal anti-body (Accurate Chemical and Scientific Corp.) at a final dilutionof 1:200 (Beltz et al. 1984; Katz et al. 1989). CCK-like immuno-reactivity was studied using two mouse monoclonal antibodies tomammalian CCK8 (Christie et al. 1995b; Sithigorngul et al.1996). Both of the CCK antibodies were used at final dilutions of1:300.
The secondary antisera were goat anti-mouse, goat anti-rabbitand goat anti-rat affinity purified IgGs labeled with fluorescein,rhodamine or CY3. Fluorescein- and rhodamine-conjugated antise-ra were purchased from Boehringer-Mannheim (Indianapolis, Ind.).CY3-conjugated secondary antisera were purchased from JacksonImmunoResearch Laboratories (West Grove, Pa.). Fluorescein andrhodamine secondary antibodies were used at final dilutions of1:50. CY3 secondaries were used at final dilutions of 1:100.
Whole-mount immunocytochemistry
Prior to fixation and staining, the entire STNS was dissected fromeach animal (Selverston and Moulins 1987). The STNS is com-posed of the STG, the OG, and the paired CGs. There are 25–26neurons in the STG (Kilman and Marder 1996), ~18 in the OG (B.Claiborne and E. Marder, unpublished results), and approximately530 in each CG (V. Kilman, unpublished results). A network ofnerves connects the four ganglia to the musculature of the foregut(Fig. 1).
Tissues were processed for immunocytochemistry as wholemounts using the indirect immunofluorescence methods of Beltz
Fig. 1. A schematic representation of the stomatogastric nervoussystem. The stomatogastric nerve (stn) is the only input from thecrustacean CNS to the stomatogastric ganglion (STG). The pairedsuperior oesophageal nerves (sons) and the single oesophagealnerve (on) connect with the stn linking the commissural ganglia(CGs) and the oesophageal ganglion (OG) with the STG. Thepaired inferior oesophageal nerves (ions) connect the OG to bothCGs; the inferior ventricular nerve (ivn) links the OG to the su-praoesophageal ganglion, commonly referred to as the brain. Thedorsal ventricular nerve (dvn) and the paired lateral ventricularnerves (lvns) provide connection from the STG motor neurons tothe muscles of the foregut. The projection pattern of the large var-icosity fibers (LVFs) is also illustrated in this figure&/fig.c:
and Kravitz (1983). Dissection, fixation and all subsequent im-munoprocessing were done at a temperature of approximately4°C. The STNS was dissected in physiological saline (440 mMNaCl; 11 mM KCl; 26 mM MgCl2; 13 mM CaCl2; 11 mM Trizmabase [Tris(hydroxymethyl) amino methane]; 5 mM maleic acid;pH 7.4–7.6), fixed overnight with 4% paraformaldehyde in 0.1 Mphosphate buffer (pH 7.3–7.4), and rinsed six times over approxi-mately 6 h in a solution of 0.1 M Na phosphate (pH 7.2), 0.3%Triton X-100, 0.1% Na azide (PTA). The incubation in primaryantibody (or antibodies in the case of double-labels) was done in
PTA for 18–24 h (10% goat normal serum was added to all reac-tions containing a polyclonal serum to reduce nonspecific bind-ing). Tissues were again rinsed six times in PTA over approxi-mately 6 h. Secondary antibody incubation was also done in PTA.In double-label experiments, a cocktail of either goat anti-mouseand goat anti-rabbit or goat anti-mouse and goat anti-rat IgG wasemployed. After incubation with secondary antibody (12–24 h),each preparation was rinsed six times in 0.1 M phosphate buffer(pH 7.2) over approximately 6 h. Following the final rinse, somepreparations were mounted on glass coverslips using a solution of
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Fig. 2a–h. Global distribution of modulator immunolabeling inthe stomatogastric ganglion. This series of laser scanning confocalmicrographs shows that the majority of modulator immunolabel-ing in STG is confined to the peripheral neuropil, with the centralcore of the neuropil relatively devoid of immunoreactivity. Whilethe images in this figure are of a CCKC36-9H labeled ganglion, allantibodies used in this study show the same global organization.
a Maximum projection of 40 optical sections collected at 2µm in-tervals through the entirety of the neuropil. b–h Individual opticalsections collected at approximately 10µm intervals from b, thedorsal surface of the ganglion, to h, the ventral surface of the gan-glion. All images are shown at the same scale. stn, Stomatogastricnerve; dvn, dorsal ventricular nerve&/fig.c:
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80% glycerin, 20% 20 mM sodium carbonate, pH 9.5. Fragmentsof glass coverslips were placed next to each ganglion before cov-ering to prevent compression of the tissue. With other prepara-tions, following the final rinse, the tissue was passed though anethanol dehydration series (50%, 70%, 80%, 90%, 95%, 100%)then cleared and mounted with methyl benzoate in glass depres-sion slides. No qualitative or quantitative differences were seenbetween data obtained using the two mounting methods.
Several lines of evidence demonstrate that the conditions weused provided adequate antibody penetration into the center of thewhole-mount preparations: (1) increasing the Triton X-100 con-centrations and staining times do not further intensify staining orshow additional staining in the ganglion core; (2) occasionalstained fibers that ran through the center of the neuropil were asintensely stained as other, more peripheral ones (cf. Fig. 2, section20); (3) staining of individual plastic sections show distributionsof GABA and FLRF Amide-like immunoreactivities similar tothose seen in whole-mounts (Kilman and Marder 1996).
Confocal microscopy
All preparations were viewed with a Bio-Rad MRC 600 laserscanning confocal microscope equipped with a krypton/argonmixed gas laser and the standard YHS [for rhodamine and CY3(excitor filter, 568 nm DF10; dichroic reflector, 585 nm DRLP;emission filter, 585 nm EFLP)] or BHS [for fluorescein (excitorfilter, 488 nm DF10; dichroic reflector, 510 nm LP; emission fil-ter, 515 nm LP)] filter blocks provided by Bio-Rad. For double-la-beled preparations, the manufacturer supplied K1 (488 and568 nm dual excitation, dual dichroic reflector)/K2 (dichroic, DR560 nm LP; green emission filter, 522 nm DF35; red emission fil-ter, 585 nm EFLP) filter set was employed. The Comos softwarepackage provided by Bio-Rad was used for collecting all images.The Bio-Rad MRC 600 laser scanning confocal microscope at theUniversity of Washington involved the use of a Nikon Optiphot.Imaging done on this instrument involved the use of either a Ni-kon 20X 0.75NA lens or Nikon 60X 1.2NA oil immersion lens.The Bio-Rad MRC 600 laser scanning confocal microscope at theBrandeis University was equipped with a Zeiss Axioskop. Imag-ing done on this instrument involved the use of either a Zeiss 20X0.5 NA lens or a Zeiss 100X 1.25 NA oil immersion lens.
Data analysis and statistical calculations
Data analysis was done on single optical sections or by combiningthe sections into projections, including some stereo pair images.All projections were made using either the maximum projectionprogram provided in the Comos (Bio-Rad) software or the equiva-lent brightest projection procedure contained in NIH Image (pub-lic-domain software for the Macintosh available via anonymousftp from zippy.nimh.nih.gov).
Neuropil dimensions and volume. &p.2:Length and width measurementsof immunolabeled ganglia were obtained from projected imagesobtained at low magnification (0.825µm/pixel). Specifically, aganglion was imaged at 2-µm intervals through the depth of theneuropil (such imaging is here after referred to as a Z-series) andthen the individual optical sections were combined to produce theprojection. The length of the neuropil was defined by the maxi-mum extent of labeling seen in the stomatogastric nerve (stn)/dor-sal ventricular nerve (dvn) axis of the STG (ignoring occasionalfibers seen in the dvn or stn). The width of the neuropil was de-fined as the maximum lateral extent of labeling seen in the gangli-on. Depth was calculated based on the number of 2µm stepsneeded to image the ganglion completely.
Varicosity size. &p.2:For measurements of varicosity size, high magnifi-cation (for measures of “small” varicosities, see Results) and low
magnification (for measures of “large” varicosities, see Results)Z-series from labeled ganglia were examined using NIH Image.The maximum cross-sectional diameter of each varicosity, in theX-Y plane, was measured in the optical section in which the vari-cosity appeared to be both in focus and of maximum size. Vari-cosities were marked by hand and measured automatically by NIHImage using a previously defined calibration based on the magni-fication used in collecting the given Z-series (0.138 or0.165 µm/pixel for measurements of small profiles and0.825µm/pixel for measurements of large profiles).
Colocalization. &p.2:To determine if two immunoreactivities were co-localized in a given preparation, high magnification images ofeach label were collected simultaneously from the same focalplane. Both single optical sections and Z-series were collected foreach double-label. Simultaneously collected images, or projec-tions produced from simultaneously collected Z-series, weremerged in either Comos or NIH Image. By pseudocoloring theimages red for rhodamine or CY3 and green for fluorescein, re-gions of overlap (colocalized label) were revealed in both pro-grams as structures colored yellow. Pixel sizes for these imageswere either 0.138 or 0.165µm/pixel.
Mapping. &p.2:The distribution of CCKC36-9H, RPCH, and FLRF Amide-immunoreactive varicosities ≥10 µm was analyzed by normalizingand mapping the profiles using a Cartesian coordinate system.Specifically, the length and width of each neuropil was deter-mined as described above. The neuropil thickness was normalizedby dividing the total number of optical sections present in a Z-se-ries through a ganglion into 10 bins. The X-Y-Z center of the neu-ropil was then obtained from these measurements. For each of the10 bins in each of the neuropils measured, the location of individ-ual varicosities ≥10 µm, based on X-Y center of the neuropil be-ing examined, was calculated automatically using NIH Images.These measurements were then normalized and compiled intolength (anterior/posterior) versus width (left/right), length versusdepth (dorsal/ventral) and width versus depth scatter plots.
Statistical analysis and figure production. &p.2:Kaleidagraph (SynergySoftware) and StatView (Abacus Concepts) were used for statisti-cal computations. All measurements are given as the mean val-ue±one standard deviation. All figures were produced on a Macin-tosh Quadra 800 using NIH Image, Canvas (Deneba Software)and Photoshop (Adobe Systems) software and printed on a Tek-tronix Phaser IISDX dye sublimation printer.
Results
General organization of the stomatogastric neuropil
The organization of the stomatogastric nervous system isshown in Fig. 1. In C. borealisthe single STG has 25–26neurons (Kilman and Marder 1996), which lie primarilyon the lateral and posterior margins of the neuropil.Most modulatory inputs to the STG originate from som-ata in the paired CGs and the single OG (Coleman et al.1992) and project into the neuropil of the STG from thestomatogastric nerve (stn). Most of the axons of the STGmotor neurons exit the STG via the single dorsal ventric-ular nerve (dvn). In C. borealisfour sensory neurons, thegastropyloric receptor (GPR) neurons, with peripheralsomata also project into the STG.
Figure 2 shows the distribution of CCKC36-9H immu-noreactivity within the STG. None of the somata is la-beled. There is extensive labelling in the peripheral neu-ropil, but the neuropil core is largely devoid of labeling
(Fig. 2d–f). This general organization is found with allof the modulator labels used in the present study. Thepeptidergic projections we characterized all enter theSTG neuropil from stn fibers, while the serotonergicprojection (Fig. 3e) arises from the GPR neurons and en-ters the STG from the dvn.
Because there is no apparent large-scale segregationwithin the peripheral neuropil (e.g., Fig. 2), the maximumextent of staining within the STG can be used to delimitthe dimensions of the neuropilar plexus. Using modulatorimmunoreactivities as a measure (Fig. 3a), we found thedimensions of the STG neuropil in C. borealis(n=32 gan-glia) to be 411±56µm in length (range=319–562µm),204±42 µm in width (range=109–332µm), and65±12 µm in depth (range=42–88µm). Comparisons of
length versus width (Fig. 3b), width versus depth (Fig. 3c)and length versus depth (Fig. 3d) measurements takenfrom the same ganglion show that each measure varies in-dependently of the others.
Modulator immunoreactivities are containedwithin two distinct sizes of varicosities
For all of the immunolabels used in this study, the ma-jority of the labeled profiles were relatively small, ap-proaching the resolution of the low magnification imag-es (e.g., Figs. 2, 3a). To examine the profiles better,high-magnification Z-series for each immunolabel wereobtained from portions of several ganglia. We found that
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a
Fig. 3a–d.Neuropil dimensions of the stomatogastric ganglion. aLaser scanning confocal micrograph of substance P-like immuno-labeling in the STG neuropil. This image is a maximum projectionof 36 optical sections taken at 2µm intervals. The brightly labeledneuropil is located in the center of this image. The somata of theintrinsic STG neurons, visible due to high nonspecific backgroundlabeling, can be seen to form a ring around the neuropil. Thelength (L) of the neuropil was defined by the maximum extent oflabeling seen in the stn-dvnaxis of the STG. The width (W) of theneuropil was defined as the maximum lateral extent of labelingseen in the ganglion. Depth was calculated based on the step sizeof optical sections taken from a ganglion and the number of opti-cal sections in which immunoreactivity was present. b Scatter plot
of length versus width measurements taken from 32 immunola-beled ganglia. Regression analysis shows that these measures varyindependently (r<0.1). The average length and width is indicatedby the horizontal and vertical barsin this plot. c Scatter plot ofwidth versus depth measurements taken from the same 32 immu-nolabeled ganglia plotted in b. Regression analysis shows thatthese measures vary independently (r<0.15). The average widthand depth is indicated by the horizontal and vertical barsin thisplot. d Scatter plot of length versus depth measurements takenfrom the same 32 immunolabeled ganglia plotted in b. Regressionanalysis shows that these measures vary independently (r<0.1).The average length and depth are indicated by the horizontal andvertical barsin this plot&/fig.c:
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each immunolabel was composed primarily of varicoseswellings (Fig. 4) along the distal axon and its terminalbranches. Hereafter, such swellings are referred to as var-icosities. As shown in Fig. 5, the mean cross-sectional di-ameter of the varicosities is about 2µm. This large popu-lation of small varicosities represents a consistent featureof all of the immunolabels. We cannot give an estimate tothe number of small varicosities present, since no attemptwas made either to sample an entire neuropil at highmagnification or to subsample the neuropil in a systemat-ic manner. However, a complete sampling of varicosities≥10 µm was obtained for each immunolabel, becausethese varicosities were deemed large enough to examineusing low magnification images. For this reason Fig. 5shows the data only for varicosities <10µm.
Figure 6 shows that while the anti-proctolin, anti-CCKC37-4E and anti-substance P immunolabels show al-most no varicosities ≥10 µm (consisting almost exclu-sively of the small varicosities), the 5HT-like, FLRFAmide-like, CCKC36-9H-like and RPCH-like immunola-bels do exhibit a population of varicosities ≥10 µm incross-sectional diameter (see also Table 1). While themean major cross-sectional diameter of these large vari-
cosities is similar for the latter four labels, the range ofdiameters present in the anti-5HT label is significantlynarrower than those stained by anti-FLRF Amide, anti-CCKC36-9H or anti-RPCH (Fig. 6, Table 1). Unpaired t-tests of these populations of varicosities indicate thatwhile there is no statistical difference between the popu-lation of profiles labeled by the FLRF Amide, CCKC36-9Hand RPCH antibodies (FLRF vs CCK, P≥0.1; FLRF vsRPCH, P≥0.1; RPCH vs CCK, P≥0.1), each of thesepopulations is statistically different from that labeled byanti-5HT (5HT vs FLRF, P≤0.001; 5HT vs CCK,P≤0.005; 5HT vs RPCH, P≤0.005). This is not surprisingbecause the 5HT label is known to come from a differentsource, sensory neuron axons that enter the STG from thedvn (Beltz et al. 1984; Katz et al. 1989), whereas the oth-er three labels enter the STG from the stn(e.g., Fig. 2).
Large varicosities contain multiple peptides
To determine if the ≥10 µm varicosities labeled by anti-FLRF Amide, anti-CCKC36-9H, and anti-RPCH repre-sented common or unique sets of structures, we conduct-
Fig. 4a–d.Immunolabeling in the stomatogastric neuropil revealsthat modulators are concentrated in blob-like profiles (varicosi-ties) which are strung-like beads along fine neurites. The diversityin size of profiles labeled by the antibodies used in this study is il-lustrated in the laser scanning confocal micrographs shown in thisfigure. All images are single optical sections taken at high magni-fication (pixel sizes of either 0.138 or 0.165µm/pixel). a CCKC37-
4E-like immunoreactivity is composed primarily of small, <10µm,
varicosities. b serotonin-like immunoreactivity, while also com-posed primarily of <10µm profiles, contains a small number ofvaricosities ≥10 µm but <15µm in cross-sectional diameter. c,dCCKC36-9H-like immunoreactivity, contains a number of varicosi-ties ≥15 µm in major cross-sectional diameter, although the ma-jority of labeled profiles are <10µm. All images are shown at thesame scale&/fig.c:
Table 1.Diameter measurements of all varicosities with diameters ≥10 µm&/tbl.c:&tbl.b:
Label Mean Range Number of Number Varicosities/ Rangediameter (µm) (µm) varicosities of ganglia ganglion
Fig. 5a–g.All immunolabels contain a population of varicosities that average2–3µm in cross-sectional diameter. a–gare bar graphs showing the distribu-tion of varicosities <10µm in major cross-sectional diameter. For each im-munolabel, four Z-series (from at least two ganglia and each spanning atleast 15µm of depth) were measured. a Plots of the measurements takenfrom anti-serotonin labeled ganglia (mean diameter=2.8±1.9µm, 2753 vari-cosities total, 2 ganglia). b Plots of the measurements taken from anti-CCKC37-4E-labeled ganglia (mean diameter=1.6±0.9µm, 1289 varicositiestotal, 3 ganglia). c Plots of the measurements taken from anti-FLRF Amide-labeled ganglia (mean diameter=2.4±1.8µm, 1739 varicosities total, 2 gan-glia). d Plots of the measurements taken from anti-substance P-labeled gan-glia (mean diameter=2.1±1.5µm, 1302 varicosities total, 3 ganglia). ePlotsof the measurements taken from anti-CCKC36-9H-labeled ganglia (mean di-ameter=1.9±1.4µm, 720 varicosities total, 2 ganglia). f Plots of the measure-ments taken from anti-proctolin-labeled ganglia (mean diameter=1.9±1.2µm, 1014 varicosities total, 2 ganglia. g Plots of the measurements takenfrom anti-RPCH-labeled ganglia (mean diameter=2.5±1.6µm, 1351 varicosi-ties total, 2 ganglia). The arrow on each plot shows the mean diameter&/fig.c:
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Fig. 6a–g.Anti-serotonin, anti-FLRF Amide, anti-CCKC36-9Hand anti-RPCH immunolabels contain populations of varicosities ≥10 µm in majorcross-sectional diameter, while anti-CCKC37-4E, anti-substance P and anti-proctolin immunolabels contain almost no varicosities ≥10 µm in majorcross-sectional diameter. a–gBar graphs showing the distribution of vari-cosities ≥10 µm in major cross-sectional diameter. For each immunolabel,Z-series of four ganglia were measured. a Plots of the measurements takenfrom anti-serotonin labeled ganglia (94 varicosities total). b Plots of themeasurements taken from anti-CCKC37-4E-labeled ganglia (1 varicosity to-tal). c Plots of the measurements taken from anti-FLRF Amide-labeledganglia (141 varicosities total). d Plots of the measurements taken fromanti-substance P-labeled ganglia (5 varicosities total). e Plots of the mea-surements taken from anti-CCKC36-9H-labeled ganglia (102 varicosities to-tal). f Plots of the measurements taken from anti-proctolin-labeled ganglia(5 varicosities total). g Plots of the measurements taken from anti-RPCH-labeled ganglia (115 varicosities total). The arrow on each plot shows themean diameter&/fig.c:
7a
8
7b
Fig. 7a, b.Double-labeling reveals that alllarge profiles contain the same cotransmit-ter complement. In the merged pseudocolorconfocal micrographs shown in a and b, redcodes for profiles showing CCKC36-9Hsin-gle labeling, green codes for profiles exhib-iting either a RPCH-like or b FLRF Amide-like immunoreactivity and yellow codes forstructures showing immunoreactivity forboth CCKC36-9Hand either RPCH-like orFLRF Amide-like immunoreactivity. Thus,all large varicosities must contain CCKC36-
9H, RPCH-like and FLRF Amide-like im-munoreactivity. Some varicosities <10µmalso exhibit colocalization b and may bederived from the LVFs&/fig.c:
Fig. 8. Double-labeling reveals that modulator-specific microdo-mains exist within the peripheral neuropil of the stomatogastric gan-glion. While all the antibodies used in our study stain profiles thatramify throughout the peripheral portion of the STG neuropil, thedensity of labeling varies dramatically within this region. All immu-noreactivities show areas of dense labeling in close apposition to ar-eas with little or no staining. As is illustrated in this merged pseudo-color confocal micrograph collected from a ganglion labeled withboth anti-proctolin (shown in red) and anti-CCKC37-4E (shown ingreen), some areas are devoid of immunoreactivity in both labels (β),while other areas are devoid of staining in only one of the two labels
(α and γ). The areas that are devoid of both labels may representstructural occlusions, such as blood vessels or the major neurites ofthe intrinsic STG neurons. The areas in which one label is present butnot the other may represent modulator specific microdomains. Thefilled star symbolis centered on the core region of this neuropil.Note: the yellow label is produced by the overlap of the two antibod-ies within a single voxel (ca. 0.83µm×0.83µm×2.5µm). To deter-mine whether individual varicosities contain both labels, it is neces-sary to use a high-power, high NA objective lens, which was not usedin this figure (cf. Fig. 7). The arrows indicate the orientation of thestn-dvnaxis. stn, Stomatogastric nerve; dvn, dorsal ventricular nerve&/fig.c:
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ed a series of double-label experiments in which gangliawere stained with either anti-CCKC36-9H and anti-FLRFAmide (n=10) or anti-CCKC36-9Hand anti-RPCH (n=10).As can be seen in Fig. 7a, these double-labels show thatthe varicosities ≥10 µm labeled by CCKC36-9H also con-tain FLRF Amide-like and RPCH-like immunoreactivity.Because we previously demonstrated that all the largeprofiles labeled by CCKC36-9H appear to arise from twofibers that project to the ganglion from the paired CGs(Fig. 1; Christie et al. 1995b), we hereafter refer to the
input axons that give rise to these large profiles as thelarge varicosity fibers (LVFs).
In these same double-labels, it was apparent thatsubpopulations of the varicosities <10µm also exhibitcolocalization of the immunoreactivities. In anti-CCKC36-9H/anti-FLRF Amide double-labels, large num-bers, and perhaps the majority, of <10µm immunoreac-tive varicosities appear to be colabeled (Fig. 7b). In theanti-CCKC36-9H/anti-RPCH double-labels, only a mi-nority of <10 µm varicosities exhibit colocalization.
right
stn
leftanterior posterior
dvn
RPCH (bin 6 of 10) 100 µm
a
Fig. 9a–d.Mapping the locations of the ≥10-µm-large varicosityfiber (LVF)-derived profiles within the stomatogastric neuropil.a Mini-projection of three optical sections from a RPCH-labeledganglion. Overlaid upon this image are the maximum length andwidth boundaries used for normalizing the anterior/posterior andleft/right location of each large varicosity. The dorsal/ventral loca-tion of each varicosity was determined by position in depth bins.The normalized positions of all varicosities ≥10 µm were pooledfrom 10 ganglia (247 profiles total). b–d Scatter plots showing thelocation of each of the ≥10 µm LVF-derived varicosities in thenormalized coordinate system. The single gray dotshows the av-erage location of all LVF-derived profiles. Associated with each
plot are binned histograms of the observed distributions of vari-cosity locations and solid curves showing the distributions of a setof 247 randomly generated locations. b A projection onto the X-Yplane. c A projection onto the Y-Z plane. d A projection onto theX-Z plane. As can be seen from these plots, there is a marked seg-regation of the profiles to the anterior half of the neuropil (chi-squared, P<0.001). Likewise, these profiles tend to be located to-ward the ventral surface of the neuropil (chi-squared, P<0.001).No difference was apparent in the left/right distribution of thesestructures (chi-squared, P>0.5). RPCH, Red pigment-concentrat-ing hormone; stn, stomatogastric nerve; dvn, dorsal ventricularnerve
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Thus, it is possible that the LVFs give rise to some vari-cosities that are <10µm in major cross-sectional diam-eter.
We examined additional double-labels to look for co-localization of immunoreactivities within the small vari-cosities (including:anti-CCKC37-4E /anti-proctolin, anti-CCKC37-4E/anti-substance P, anti-CCKC37-4E/anti-FLRFAmide, anti-CCKC37-4E/anti-RPCH, anti-CCKC36-9H/anti-substance P and anti-CCKC36-9H/anti-proctolin). Howev-er, none of the other double-immunolabels that we testedshowed clear colocalization of immunoreactivity withinsmall varicosities.
Modulator-containing profiles are restrictedto the peripheral portion of the stomatogastric neuropil
The global distribution of immunolabeled profiles seenwithin the STG neuropil was the same regardless ofwhether it was labeled with 5HT, proctolin, substance P,FLRF Amide, RPCH or either of the two CCK antibod-ies. Each antibody labeled profiles that are segregatedprimarily to the peripheral portion of the neuropil(Fig. 2). The central core of the neuropil was consistent-ly found to be largely devoid of staining (cf. Fig. 2d–f).
Within the peripheral neuropil, no large-scale segre-gation of profiles was evident in any of the immunola-bels. Each label did, however, exhibit numerous smallareas (generally <25µm long by 25µm wide by 10µmdeep) with densely packed profiles in close apposition tosimilarly sized areas with little or no immunolabeling.This differential packing gives the peripheral neuropil adistinct, “honey comb”-like appearance (e.g., Fig. 4).Some of the regions devoid of labeling undoubtedly re-present the location of structural elements such as bloodvessels and sinuses or the primary and secondary neu-rites of STG neurons.
Double-immunolabels showed that a subpopulationof the areas avoided by one label can be filled by pro-files exhibiting immunoreactivity for a different neuro-modulatory substance. Examples of this differential la-beling are visible in the anti-proctolin/anti-CCKC37-4Edouble-label shown in Fig. 8. Although not shown, all ofthe antibody double-labels examined in our studyshowed similar antibody-specific microdomains withinthe peripheral neuropil.
LVF-derived varicosities are concentratedin the anterior ventral portion of the peripheral neuropil
Given the ease with which the ≥10 µm LVF-derived var-icosities can be identified and the relatively small num-ber of these profiles in a given ganglion, we mapped thelocations of these structures in a number of ganglia todetermine if these profiles are normally or differentiallydistributed (Fig. 9). Using a normalized Cartesian coor-dinate system, we found that the large LVF-derived vari-cosities map primarily in the anterior portion of the pe-ripheral neuropil (Fig. 9b). Likewise, these structures areskewed in depth toward the ventral surface of the neuro-
pil (Fig. 9c,d). No difference in lateral distribution wasnoted.
Discussion
Modulator location is ideal for synaptic interactionswith intrinsic neurons
Modulator immunoreactivity in the C. borealisSTG neu-ropil is restricted, almost exclusively, to the peripheralportion of the plexus, with the central core of the structureessentially devoid of labeled processes. This large-scalesegregation contradicts the long-held notion that modula-tor immunoreactivity ramifies throughout the entirety ofthe neuropil. A recent study of intrinsic STG neuronsshowed that the core of the stomatogastric neuropil isfilled with the large processes projecting from these cells(Graubard and Wilensky 1994; Baldwin and Graubard1995). As with the modulator labels, the fine neurites ofthe intrinsic STG neurons are also restricted to the periph-eral neuropil (Baldwin and Graubard 1995). Electron mi-croscopy done on the STG of C. borealis(Kilman andMarder 1996) and the lobsters Panulirus interruptus(King 1976a,b; Friend 1976) and Homarus americanus(Maynard 1971) shows that the majority of synaptic con-nections within the ganglion occur along stretches of fineneurite in the peripheral neuropil. Because both the modu-lator immunoreactivities and fine neurites of the STG neu-rons of C. borealisare localized to the peripheral neuro-pil, it would appear that the organization of modulator-containing varicosities in the STG neuropil is ideal formodifying synaptic efficacy and modulating intrinsic con-ductances of fine neurites within the ganglion. Moreover,recent work has shown that there are synaptic interactionsbetween these stomatogastric nerve axons (SNAXs) andSTG neurons (Nusbaum et al. 1992; Coleman and Nus-baum 1994; Coleman et al. 1995).
Inputs sharing a common modulator can producedistinct effects
As more and more work focuses on modulation of theSTG neural networks by SNAXs projecting from the an-teriorly located OG and CGs (Fig. 1), it is becomingclear that inputs sharing common transmitters are capa-ble of eliciting distinct responses from the STG neuralnetwork. For example, each of the three pairs of proct-olin-containing projections elicits a distinct subset of thephysiological responses seen with bath-applied proctolin(Nusbaum and Marder 1989a,b; Nusbaum et al. 1992;Coleman et al. 1994; Blitz et al. 1995). Thus, in a smallneuropil like the STG, there must be mechanisms for de-limiting the actions of an input, thereby allowing for thegeneration of multiple behavioral outputs from the re-lease of a common transmitter from distinct inputs. Onemechanism that has been suggested to play a role in thisdelimitation is through a segregation of input/target ap-positions. Baldwin and Graubard (1995) report that theintrinsic STG neurons of C. borealissegregate their fine
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neurites into cell-specific regions of the peripheral neu-ropil. While Baldwin and Graubard (1995) reportedmostly on uniquely identified cells, all of the immunore-activities used in this study arise from at least two neu-rons, although serotonin and substance P are known tobe derived from single neuron types (Katz et al. 1989;Christie et al. 1993). With this limitation in mind, eachof the immunoreactivities appears quite uniformly dis-tributed within the peripheral neuropil, with only smallmicrodomains of exclusion. Double-labeling shows thatsome of these microdomains appear to be modulator-and/or input axon-specific. The only clear segregation inthe periphery that we observed was in the large LVF-de-rived varicosities.
Little is known about the range over which modula-tors, particularly peptide modulators, can act in the STG.Neuropeptides can act in a paracrine-like fashion with abroad sphere of influence (Jan and Jan 1982). Thus,while a region of neuropil may be devoid of modulatorimmunoreactivity, it may still fall within a sphere ofmodulator responsiveness. Coleman et al. (1994) havedemonstrated that the peptide proctolin is enzymaticallydegraded by an extracellularly located aminopeptidase inthe STG, thereby rendering the molecule biologically in-active. If such peptidases are differentially distributedwithin the STG neuropil and/or are capable of rapid deg-radation of peptide, then it is possible that the sphere ofinfluence of a given peptide could be restricted to an areaimmediately adjacent to its site of release. If this is thecase, then modulator/cell-specific microdomains presentin the STG neuropil may have functional consequences.It is also possible that the modulator/cell-specific micro-domains arise from a random distribution of fine process-es within the STG neuropil with no functional conse-quences of a specific distribution pattern. Clearly, addi-tional studies will be needed to clarify these issues.
A second mechanism that has been hypothesized toplay a role in delimiting the physiological effects of inputaxons that share a common modulatory substance is thepresence of unique complements of cotransmitters (Mar-der et al. 1994, 1995). In the STNS of the crab, colocal-ization of neuromodulatory substances is well document-ed. Each of the three pairs of proctolinergic axons thatproject to the STG have been shown to exhibit a uniquecomplement of cotransmitters (Christie 1995). Likewise,each of the two types of allatostatin-like-peptide contain-ing projections to the STG contain a distinct constellationof cotransmitters (Skiebe and Schneider 1994; A.E. Chris-tie and M.P. Nusbaum, unpublished observations). In ourstudy we have shown that the LVFs also contain severalcotransmitters. As more work focuses on determining thepatterns of colocalization in the modulatory projections tothe STG, it will be interesting to see if each input fibertype possesses a unique cotransmitter phenotype.
Comparison of immunoreactive varicositiesto profiles seen with electron microscopy
The single example of large-scale segregation within theperipheral neuropil is the set of ≥10 µm varicosities that
arise from the large varicosity fibers. These profiles,which exhibit CCK-like, RPCH-like and FLRF Amide-like immunolabeling, show a marked preference for theanterior ventral region of the peripheral neuropil. TheLVFs and the 5HT-containing fibers are the only inputsto the STG that are known to give rise to varicosities ofthis size. All other inputs give rise to varicosities withmean cross-sectional diameters of <10µm in maximumcross-sectional diameter and, in fact, have mean varicos-ity sizes of approximately 2.0µm in maximum cross-sectional diameter. Obviously, measurements taken fromfluorescently labeled preparations, like those in ourstudy, are likely to be somewhat in error, even with theincreased resolution of the laser scanning confocal mi-croscope. However, electron-microscopic studies con-firm the existence of profiles in the size range we reportfor the large LVF-derived varicosities, as well as for thesmall varicosities arising from other inputs (Kilman andMarder 1996).
Comparative anatomy performed on several relatedspecies of decapod crustacea provide tantalizing clues asto the function of both the LVF-derived large varicositiesand the smaller profiles that arise from other inputs. Aswas reported by King (1976a,b), in P. interruptusdense-core vesicles (DCVs) are found in type C profiles, whichare generally <10µm in diameter. These same profilesalso contain classical transmitter vesicles and both pre-and postsynaptic specializations (King 1976a,b). Basedon their structure, King (1976a,b) termed these profiles“synaptic varicosities.” Based on their size and locationwithin the C. borealisneuropil, many of the <10µmmodulator-containing varicosities reported in our studyare likely to be synaptic varicosities. Electron microsco-py done on the C. borealisSTG shows the presence ofsynaptic varicosities in the peripheral portion of the neu-ropil that often contain DCVs (Kilman and Marder1996).
No profiles similar in size to the C. borealisLVF var-icosities were seen in the STG of either P. interruptusorH. americanus(King 1976a,b; Friend 1976; Maynard1971). Electron microscopy done on the C. borealisSTGshows a set of large profiles containing DCVs concen-trated in the anterior peripheral neuropil, which resemblethose found in neurosecretory structures and thereforehave been termed neurohemal-like profiles (Kilman andMarder 1996). These profiles are reported to be of a sim-ilar size and distribution to the large LVF-derived vari-cosities reported in our paper. Thus, it seems possiblethat the LVF-derived varicosities represent a populationof neurohemal-like release sites within the STG neuro-pil.
Colocalization of modulators in individual profiles
It is now the rule, rather than the exception, that multipleneurotransmitters and neuromodulators are found colo-calized in individual neurons. Moreover, as the genes forneuropeptides are cloned, it is becoming apparent thatsome peptide families consist of large numbers of relat-ed peptides (e.g., Miller et al. 1993a,b; Santama et al.
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1995; Kellett et al. 1994; Schinkmann and Li 1994;Schneider et al. 1993), many of which may be labeled bya single antiserum. Therefore, for any of the peptide im-munoreactivities studied here, it is not clear how manydifferent, related peptides are present in any of the la-belled structures. The LVF fibers contain at least threedifferent classes of peptides. There is no reason to ex-pect that there are not a number of neuropeptide familiesstill to be discovered in crustacean tissues. Therefore, itis difficult to estimate how many different peptidergicmodulators might ultimately be found to be present andreleased from the LVFs and other peptidergic terminals.
In summary, taken collectively, the work presented inthis paper represents a first step towards describing theorganization of modulatory inputs within the STG neu-ropil. We have shown that there is a gross-level segrega-tion of modulator to the peripheral neuropil with exclu-sion of modulator from the central core. This findingcontradicts the long-held belief that modulator immuno-reactivity ramifies throughout the entirety of the neuro-pil. While segregation of modulator containing profilesand their targets has been postulated as one mechanismfor delimiting the actions of inputs sharing a commonneuromodulatory substance, we have found little evi-dence that the modulator-containing profiles present inthe peripheral neuropil are themselves segregated. With-in the peripheral neuropil we have found clear segrega-tion only for a set of putative neurohemal varicosities,which arise from the LVFs.
&p.2:Acknowledgements.We would like to thank Ms. Valerie Kilman(Brandeis University) and Dr. Michael Nusbaum (University ofPennsylvania School of Medicine) for sharing unpublished resultswith us. We thank Mr. Ray Price and Ms. Ann Wilensky for theirassistance with data analysis. Research supported by NS17813(E.M.), NIH Shared Instrument Grant RR05615 (Brandeis Univer-sity), NS15697 (K.G.), ISIORRO4646-01 (University of Washing-ton) and a grant from the Human Frontiers Science Program Orga-nization. E.M. gratefully acknowledges the support of the W.H.Keck Foundation.
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