gaba-immunoreactive neurons in the nematode ascaris

THE JOURNAL OF COMPARATIVE NEUROLOGY 307:584-597 (1991)

GABA-Immunoreactive Neurons in the Nematode Ascaris

JOHN GUASTELLA, CARL D. JOHNSON, AND ANTONY O.W. STRETTON Neuroscience Training Program (J.G., A.O.W.S.) and Department of Zoology

(C.D.J., A.O.W.S.), University of Wisconsin, Madison, Wisconsin 53706

ABSTRACT y-Aminobutyric acid (GABA) immunoreactive neurons in the cephalic, somatic, and caudal

regions of the Ascaris nervous system were visualized with serial section and whole-mount GABA immunocytochemistry. In the ventral and dorsal nerve cords, GABA-like immunoreactivity (GLIR) is localized to the neurites and cell bodies of identified inhibitory motor neurons and to two fibers, one in each cord, that arise from neurons in the nerve ring. GLIR is absent from identified excitatory motor neurons and from ventral cord interneurons. In neurons containing GLIR, immunoreactivity was present throughout the cell, which argues against an exclusive localization of GABA at conventional synapses.

In whole mounts, ten GABA-immunoreactive neurons were present in the cephalic region. These include four nerve ring-associated cells (the WE-like cells), two bilaterally symmetrical pairs of lateral ganglia neurons (the amphid-GABA and deirid-GABA cells) and one bilaterally symmetrical pair of ventral ganglion cells (the VG-GABA cells). In sections, the RME-like cells and the VG-GABA cells were consistently stained through the cephalic region. However, anti-GABA staining of the lateral ganglia cells in sections was light, thus suggesting that they contain less GLIR than the other more intensely stained GABA-immunoreactive neurons. In the caudal region, a single GABA-immunoreactive neuron was present in the dorsal rectal ganglion. Our data suggest that these ten cephalic neurons, and a single dorsal rectal ganglion neuron, use GABA as a neurotransmitter.

Key words: serial section immunohistochemistry, invertebrate nervous system, neurotransmitter localization

The cellular localization of neurotransmitters is an important part of the structural-functional description of a nervous system. The nematode Ascaris is an advantageous preparation for studying neurotransmitter localization since its nervous system contains only about 298 neurons, most of which are identifiable from preparation to preparation. Morphologically, these neurons are generally much less complex than neurons in other species; most are monopolar or bipolar with unbranched neurites whose paths can be followed over long distances by light and electron microscope serial section techniques or in whole mounts (Stret- ton et al., '78; Johnson and Stretton, '87; Angstadt et al., '89; Sithigorngul et al., '90). The ventral and dorsal nerve cords are particularly suited for morphological studies, since they contain a highly ordered array of nerve fibers and cell bodies, some of which have been analyzed electrophysi- ologically (Walrond et al., '85; Davis and Stretton, '89). These morphological characteristics enable one to study the nematode nervous system at the level of single cells, and to catalog the neurotransmitter phenotypes of individual identified neurons (Johnson and Stretton, '85, '87; Sithigorngul et al., '90). In addition, the morphology of individual cells is

highly conserved between different nematodes, and so corresponding cells in different species can be compared (White et al., '76, '86).

GABA appears to be an inhibitory transmitter in the motor nervous system of Ascaris. Del Castillo et al. ('64) have shown that GABA produces a chloride-dependent hyperpolarization in Ascaris muscle, and Martin ('85) has shown that GABA opens chloride channels in Ascaris muscle. Both GABA and glutamic acid decarboxylase (GAD, the enzyme of GABA synthesis) are biochemically detectable in extracts of the ventral and dorsal nerve cords (Burden and Stretton, unpublished observations; Johnson and Stretton, unpublished observations). Recently, the localization of GABA to individual neurons has been carried

Accepted January 30,1991. John Guastella's present address is Division of Biology 156-29, California

Institute of Technology, Pasadena, CA 91125. Carl D. Johnson's present address is Cambridge Neuroscience Research,

One Kendall Square, Cambridge, MA 02139. Address reprint requests to A.O.W. Stretton, 105 Zoology Research

Building, 11 17 W. Johnson Street, University of Wisconsin, Madison, WI 53706.

O 1991 WILEY-LISS, INC.

GABA-IMMUNOREACTIVE NEURONS IN ASCARIS 585

out with immunocytochemical methods: GABA-like immunoreactivity (GLIR) is present in 19 identified inhibitory motor neurons, but absent from excitatory motor neurons (Johnson and Stretton, '87). Taken together, these results strongly support the hypothesis that GABA is a neurotransmitter in nematode inhibitory motor neurons.

In this paper we extend our analysis of the cellular localization of GLIR in the Ascaris nervous system. First, we describe GLIR in serial sections of the ventral and dorsal nerve cords and compare the results with those reported from whole mounts (Johnson and Stretton, '87). Second, we describe the GAF5A-immunoreactive cells in the cephalic region of the Ascaris nervous system from both whole mounts and sections. This region contains the majority of neuronal cell bodies in the animal and also includes a neuropil (the nerve ring), which represents a concentrated site of synaptic interactions. Finally, we describe GABA staining in whole mounts of the caudal region. Our results show that there are ten neurons in the cephalic region and a single GABA-immunoreactive neuron in the caudal region that are consistently labeled with anti-GABA antiserum.

Portions of this work have been published in abstract form (Stretton and Johnson, '85; Guastella et al., '86).

MATERIALS AND METHODS Tissue preparation

Reagents. The antiserum used in these experiments (anti-GABA #5) and the amino acid-protein conjugates utilized in the competition studies were prepared and provided by Dr. R. Wenthold, NIH, according to the methods described in Wenthold et al. ('86). Affinity-purified, horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (heavy and light chains) was from BioRad. Gabaculine was purchased from CalBiochem.

Live specimens of Ascaris suum were obtained from a local slaughterhouse and stored in 150 mM NaC1, 50 mM sodium phosphate, pH 7.4 at 40°C. Worms were prepared for histology within 2 days of obtaining them from the host.

The anterior 20 mm of adult female worms (total length 0.30-0.35 m) were fixed with 1-3% glutaraldehyde and 1-3% acrolein in 0.1 M cacodylate buffer, pH 6.8, at 4°C. Some preparations were fixed overnight, and then postfixed in 2% OsO, in the same buffer for 90 minutes at 4°C. In later experiments, preparations were made with reduced fixation times, or without postfix- ation. Since immunostaining of the cephalic neurons was more intense and more consistent after light fixation (4-5 hours in 1% aldehydes), most of the experiments reported here were carried out on such lightly fixed samples. Follow- ing fixation, tissue samples were dehydrated in graded ethanols and embedded in Epon (Stretton, '76).

Serial sections 4-6 pm thick were cut with a steel knife, mounted on slides coated with 5% gelatin and 0.5% chrome alum, and dried overnight at 80°C. The embedding medium was removed from the sections with a solution containing acetone, benzene, and 0.5% KOH in methanol (1:l:l) (Hogan and Smith, '82). The sections were then rehy- drated, treated with 1% phenylhydrazine-HC1 in phosphate- buffered saline (PBS, containing 140 mM NaC1, 27 mM KCI, 8 mM Na,HPO,, 1 mM KH,PO,, pH 7.2) and 1% ethanolamine (pH 9.0) in the same buffer at room temperature for 20-30 minutes. After washing, the anti-GABA antiserum (diluted 1:1,000 to 1:2,000 in PBS containing

Specimens.

Serial sections.

10% normal calf serum and 1 mgiml BSA) was applied overnight at 4°C; the secondary antibody (HRP-labeled goat anti-rabbit IgG diluted 1:1,000 in the same buffer) was applied for 4 hours at room temperature. The sections were washed, then stained with 0.03% 3,3-diaminobenzidine- HC1 (DAB; Sigma) and 0.006% H,O, in PBS, and cover- slipped in glycerine jelly.

Whole mounts. Whole mounts were prepared as described in Johnson and Stretton ('87). Briefly, worms were injected with 4 mdml collagenase, which dissociates muscle cells from the body wall, cut down one lateral line, and pinned out flat in Sylgard-lined dishes. The preparations were fixed in 2% glutaraldehyde and 0.5% paraformalde- hyde in PBS (1 hour at room temperature), treated with 1% ethanolamine (pH 9.0) and 1 mg/ml pronase (each for 30 minutes at room temperature), and washed prior to application of antibodies. Antibodies were applied in 100 mM Tris, pH 8.0,300 mM NaCl, 1 mg/ml BSA, containing 10% BSA and 1% Triton X-100. Anti-GABA serum was generally used at a dilution of 1:2,000 and applied for 12-36 hours; HRP-labeled goat anti-rabbit second antibody was applied overnight at a dilution of 1:1,000. Both antibody incubations were carried out at 4°C. Preparations were stained for 5 minutes in DAB and H,O, and mounted in glycerine jelly.

For sections, the specificity of the GABA antiserum was tested by preabsorption with the following amino acid-protein conjugates: GABA, glutamate, aspartate, glutamine, taurine, glycine, and p-alanine. Antise- rum was mixed with conjugate to the final desired concentrations (1:2,000 dilution of antiserum and either 10 pg/ml or 1 mg/ml of conjugate) and incubated overnight at 4°C. Just prior to use, the complex was centrifuged at 12,OOOg for 15 minutes. The supernatant was then applied to sections as described above.

For whole mounts, the anti-GABA serum was mixed with conjugate to the final desired concentrations (1:2,000 dilution of antiserum and 4 pg/ml, 20 pg/ml, or 100 pg/ml of conjugate). Incubations were carried out overnight and the mixtures used to stain whole mounts without centrifuga- tion.

The slides were viewed with brightfield and phase optics. Photographs were taken on a Zeiss Universal microscope; drawings were also made, with a Zeiss RA microscope equipped with a drawing tube. Recon- structions were performed by tracing fibers through serial sections with the photographs and drawings as references.

The ventral nerve cord contains the cell bodies and neurites of motor neurons, which innervate the body wall muscle cells, and the processes of interneurons, some of which make synapses to motor neurons (Stretton et al., '78). The dorsal nerve cord consists almost entirely of the neurites of motor neurons; these processes are joined to their cell bodies in the ventral cord by single lateral processes called commissures. Commissure position along the nerve cord is highly reproducible and can be used to identify neurons reliably. The morphological and physiological phenotypes of the motor neurons allow them to be classified into seven different types; five of these types have commissures. There are three types of dorsal excitors (DE1, DE2, and DE3), one type of dorsal inhibitor (DI), and one type of ventral inhibitor (VI). The dorsal excitors have commissures that arise directly from their cell bodies; the dorsal and ventral inhibitors have commissures that lie anterior to their cell bodies. The remaining two types of

Blocking experiments.

Microscopy.

Anatomical background. Nerue cords.

586 J. GUASTELLA ET AL.

A

B

Figure 1


motor neuron (V-1 and V-21, which are putative ventral excitors, do not possess commissures; their processes are confined to the ventral cord. The motor neurons are arranged in a pattern that repeats five times along the length of the animal; the experiments reported in this paper examine neurons in the first repeating unit.

The anterior 4 mm ofAscaris contains about 162 of the 298 neurons in the animal (Goldschmidt, '08). This region contains the following structures (Fig. 1): 1) a small ganglion in the ventral nerve cord (the retrovesicular ganglion), which contains 13 neuronal cell bodies as well as a full complement of ventral cord neurites (including motor neurons and interneurons) (Angstadt et al., '89); 2) a group of ganglia located near the circumpharyngeal nerve ring named according to their position in the coronal plane of the animal (the ventral ganglion, dorsal ganglion, left lateral ganglion, and right lateral ganglion); 3) the nerve ring; and 4) two lateroventral fiber tracts, termed the amphid and deirid commissure groups, containing the commissures of neurons located in the lateral ganglia and lateral line.

The caudal nervous system comprises 30 neuronal somata arranged in four small ganglia (Voltzen- logel, '02; Deineka, '08; Sithigorngul, '87). These ganglia include: 1) a preanal ganglion (PAG-4 neuronal cell bodies), which lies at the posterior end of the ventral nerve cord; 2) two lumbar ganglia, containing 12 neurons (left lumbar ganglion) and 11 neurons (right lumbar ganglion); and 3) a dorsal rectal ganglion (DRG-3 neuronal cell bodies) located on the dorsal surface of the rectum.

Identification of neurons. Motor neurons and their associated commissures were identified according to the criteria described by Stretton et al. ('78) and Johnson and Stretton ('85). These criteria include the relative position of the commissure and cell body along the body axis. In addition, the processes of excitatory and inhibitory motor neurons form separate bundles in well-defined positions at

Cephalic region.

Caudal region.

Fig. 1. A Diagram of the anterior ganglia of Ascaris in a preparation that has been slit near the dorsal axis and opened flat. NR, nerve ring; DC, dorsal nerve cord; VC, ventral nerve cord; LLL, left lateral line; RLL, right lateral line; VG, ventral ganglion; DG, dorsal ganglion; LG, lateral ganglion; RVG, retrovesicular ganglion. Nerve cell bodies are shown as filled profiles. Individual neuronal processes, called commissures, are embedded in the hypodermal tissue and project between the nerve cords. There is a triplet of left ventrodorsal commissures just anterior to the retrovesicular ganglion and a pair of right ventrodorsal commissures just behind the ventral ganglion. Two larger bundles of ventrolateral fibers, located immediately posterior to the nerve ring, are called the amphidial commissures (AC), and two smaller bundles of ventrolateral fibers, located at the posterior margin of the lateral ganglia, are the deirid commissures (DeC). (Taken from Sithigorngul et al., '90). B: Drawing of cephalic region illustrating GABA-immunoreactive neurons in the nerve ring, ventral ganglion, and lateral ganglia. The RME-like cells (labeled RMEV, RMED, RMEL, and RMER) are found in the nerve ring; each sends two lateral processes into the nerve ring (these processes are truncated in the diagram); in addition, the RMEV-like and WED-like neurons send processes down the ventral cord and dorsal cord, respectively. Two pairs of GABA-immunoreactive cells (the amphid and deirid pairs) are located within the lateral ganglia. A single pair of GABA-immunoreactive cells is contained within the ventral ganglion. The lateral cells possess slender processes that travel through the amphid and deirid commissural bundles and ventral cord to enter the nerve ring. The ventral cells send processes directly into the ring. The two GABA- immunoreactive somatic motor neurons present in this region, one in the left triplet and the other in the right doublet of ventrodorsal commissures, have been omitted.

the neuromuscular surface of the nerve cords (Walrond et al., '85); this feature can be used in transverse sections of the cords to identify inhibitory and excitatory motor neurons. Assignments based on this criterion are reliable; in a large number of reconstructions they have been confirmed by tracing fibers to their cell body and/or commissure (Stretton et al., '78; Walrond et al., '85; Donmoyer and Stretton, unpublished observations). Neurons in the cephalic region of the nervous system were classified as originally described by Goldschmidt ('08) in Ascaris and from the complete description of the morphologies of neurons in the anatomically related nematode Caenorhab- ditis elegans (White et al., '86).

RESULTS GLIR in the ventral cord

The distribution of GLIR in sections of the ventral nerve cord was examined in four different regions contained within the first 20 mm of the worm (see Fig. 1): 1) immediately posterior to the ventral ganglion, where bundles of fibers from each side of the nerve ring merge to form the nerve cord; 2) anterior to the retrovesicular ganglion; 3) within the retrovesicular ganglion; and 4) just posterior to the retrovesicular ganglion. The general staining pattern was similar in all four regions. The ventral cord contains a group of three to four GABA-immunoreactive medium- caliber profiles located at the neuromuscular surface of the cord (Fig. 2A). Based on the fact that they were grouped on the left side of the ventral nerve cord, these fibers were identified as inhibitory motor neurons (both VI and DI); this conclusion was verified by tracing profiles through serial sections to their cell bodies (see below). Smaller immunoreactive profiles are frequently seen in the vicinity of the motor neurons. In some instances these small profiles were identified as spinous extensions of the DI dendrite; previous work has shown that these spines are postsynaptic to V-1 and V-2 neurons, putative ventral excitatory motor neurons (Donmoyer, Desnoyers, Angs- tadt, and Stretton, unpublished observations). In other cases these small profiles were the presynaptic extensions of VI axons, which form synapses with both muscle and V-1 and V-2. The GABA-immunoreactive spines are sporadic in their location-consequently they are not present in every section. GLIR was not detectable in the processes of excitatory motor neurons, nor in the processes of large and small ventral cord interneurons.

Apart from the profiles of the inhibitory motor neurons, sections of the ventral cord contain only a single additional GABA-immunoreactive fiber. This fiber has a characteristic position deep within the cord (Fig. 2A). Both whole mounts and serial section tracing indicate that this fiber is the ventral process of a neuron closely associated with the nerve ring (the RMEV-like neuron) (see below).

A similar pattern of staining was observed in the dorsal cord (Fig. 2B). Three to four medium-caliber GABA- immunoreactive fibers were present at the neuromuscular surface and a single immunoreactive fiber was characteris- tically present at the base of the dorsal cord (Fig. 2B). From their position in the cord, the immunoreactive profiles at the neuromuscular surface were identified as the neurites of the inhibitory motor neurons (both VI and DI). Examina- tion of whole mounts and serial sections indicates that the immunoreactive fiber at the base of the dorsal cord is the


Fig. 2. Cross sections through the ventral and dorsal nerve cords. A Ventral cord showing primary neurites (asterisks) and spines (small arrows) of inhibitory motor neurons. A single GABA-immunoreactive fiber (large arrow) is present towards the base of the cord. Staining is absent in the other ventral cord fibers, the hypodermal chalice (Hy),

and the muscle. MA, muscle arms; Mu, muscle contractile fibers. B: Dorsal cord showing five GABA-immunoreactive fibers. The fibers at the neuromuscular surface are the dorsal processes of motor neurons (asterisks). A single GABA-immunoreactive fiber (large arrow) can he seen at the base of the cord. Scale bar = 10 pm.


process of the RMED-like neuron (see below). The remainder of the dorsal cord fibers lack detectable GLIR.

In order to confirm the identities of ventral cord GABA- immunoreactive fibers, stained profiles were traced to their cell bodies and commissures. The region reconstructed comprised about 5.5 mm extending from the posterior border of the retrovesicular ganglion to the anterior portion of the first repeating unit. This region contains three inhibitory motor neurons: VIl, VI2, and DI1 (nomenclature as described in Johnson and Stretton, '87). VI1 was traced from its cell body, located just posterior to the retrovesicular ganglion, to its commissure, located just posterior to the ventral ganglion. The cell bodies of V12 and D12 are closely associated as a pair in the anterior region of the first repeating unit. These neurons were also traced to their commissures, which occur at the beginning of the first repeating unit (for VI2), and just anterior to the retrovesicular ganglion (for DI2). These experiments thus serve as proof that the labeled commissures are extensions of identified inhibitory motor neurons.

Serial sections through the somata of inhibitory motor neurons showed that GLIR is present throughout the cell body region (50-75 pm); the nucleus was also stained, usually as intensely as the cytoplasm (Fig. 3). The distribution of GLIR was generally homogeneous. In particular, there was no indication of higher levels of GLIR in inhibitory motor neuron spines, which are sites of presynaptic and postsynaptic interactions. Occasionally, differences in staining intensity were observed along the longitudinal extent of these fibers. These differences could usually be ascribed to fiber damage.

GLIR was examined in two putative cholinergic motor neurons present in these series (Fig. 4). V-1 (Fig. 4A), a motor neuron with output to ventral muscle, was identified based on the location of its cell body within the ventral cord (at the base of the cord just behind the retrovesicular ganglion). The DE1 motor neuron (Fig. 4B), located near the beginning of the first repeating unit, was identified by position and by the presence of its commissure, which extends directly from the soma. As predicted, GLIR was not detected in either of these neuronal cell bodies or in their neurites.

GLIR in the retrovesicular ganglion Since four retrovesicular ganglion neurons possess a

GABA uptake system (Guastella et al., '86; Guastella and Stretton, '91), we were particularly interested in determin- ing whether any of these cells contained endogenous GABA. In sections of eight different preparations, GLIR was not detectable in any of these cells, nor in any of the other nine retrovesicular ganglion neurons. Furthermore, GLIR was not observed in the retrovesicular ganglion in any of the whole-mount preparations examined. In sections, the staining pattern of motor neuron profiles in the retrovesicular ganglion is identical to that seen in other regions of the ventral cord, although the absolute positions of these profiles in the coronal plane change as the cord shifts to accommodate the retrovesicular ganglion cell bodies.

It was possible that the absence of GLIR in the four GABA-uptake-positive cells was due to rapid metabolism of endogenous GABA by GABA-transaminase, which could potentially reduce GABA concentrations to levels below the sensitivity of the detection method. This hypothesis was tested by treating samples with the GABA-transaminase inhibitor gabaculine (Rando and Baumgarten, '77) prior to

fixation. Fig. 5 shows sections of neurons from a gabaculine- treated retrovesicular ganglion. The anti-GABA staining pattern of this and one other gabaculine-treated retrovesicular ganglion was identical to two control samples and to the staining pattern seen in eight other retrovesicular ganglion preparations stained for GABA immunocytochemistry. Gabaculine had no effect on staininglevels in hypodermis or muscle (not shown). We can conclude that the absence of GLIR in these cells is not due to metabolism of GABA by GABA-transaminase. This experiment does not, however, rule out the possibility that these cells contain levels of GABA below the limit of detectability of the immunocytochemical assay.

GLIR in the cephalic region Whole mounts. The staining pattern observed in some

whole-mount preparations was frequently incomplete, probably due to removal of only part of the cephalic muscula- ture, damage during dissection, or excessive digestion by collagenase or pronase. However, the examination of a large number of preparations (a total of 77 whole mounts) enabled us to identify a standard set of cephalic GABA- immunoreactive neurons. These neurons fall into two categories. One category consists of neurons that are consistently and intensely stained. A second class consists of neurons that are less consistently stained or that are stained less intensely.

The consistently and intensely stained neurons include ten cell bodies: four cell bodies associated with the nerve ring; two pairs located in the lateral ganglia; and one pair in the ventral ganglion. The nerve ring-associated neurons consist of two large spindle-shaped neurons, located ven- trally (Fig. 6A) and dorsally (not shown) in the nerve ring, and two smaller spindle-shaped neurons located in the left and right regions of the nerve ring. The ventral and dorsal nerve ring cells are tripolar: two lateral processes travel circumferentially within the nerve ring while the third process enters the ventral cord (for the ventral nerve ring cell) or the dorsal cord (for the dorsal nerve ring cell). GLIR in the lateral nerve ring cells was difficult to detect because these neurons are often obscured by the intense GLIR of processes in the nerve ring itself. In their position and morphology, these four nerve ring-associated neurons are very similar to the RME neurons found in C. eleguns (White et al., '86). We therefore refer collectively to these cells in Ascarzs as the RME-like neurons, and individually to the ventral, dorsal, left, and right cells as the RMEV-like, RMED-like, RMEL-like, and RMER-like cells respectively.

The consistently and intensely stained neurons also include three bilaterally symmetrical pairs of GABA- immunoreactive ganglionic neurons. One pair is present in the posterolateral portion of the ventral ganglion (Fig. 6A). These are small monopolar neurons, and their processes exit the anterior pole of the cell body, move to the midline, and travel a short distance in the ventral cord before entering the nerve ring on the same side as their cell body. We refer to these as the VG-GABA neurons.

Two pairs of consistently stained neurons have cell bodies in the lateral ganglia. One pair consists of medium-sized ( - 15 pm in diameter) monopolar neurons located in the lateral ganglia just posterior to the nerve ring (Fig. 7A). These cells possess lateroventral commissures that join the amphidial commissure bundle, travel anteriorly in the ventral cord, and enter the nerve ring ipsilateral to the cell body. These cells sometimes also possess short posterior


Figure 3

GABA-IMMUNOREACTIVE NEURONS IN ASCARZS 591

Fig. 4. Cross sections through the ventral cord showing the absence of GLIR in the cell bodies of two excitatory motor neurons (arrows). A V-l motor neuron, B DE1 motor neuron. Scale bar = 20 pm.

processes. We refer to these neurons as the amphid-GABA neurons. The second pair of lateral ganglia GABA-immunoreactive cells possesses small monopolar cell bodies ( - 5 pm in diameter) located near the posterior margin of the lateral ganglia (Fig. 7A). These cells possess lateroventral commissures that enter the deirid commissure bundle; like the amphid-GABA cells, these processes then run anteriorly in the ventral cord and enter the nerve ring ipsilateral to the cell body. In some preparations, these cells have an additional short process extending from the cell body. We refer to these neurons as the deirid-GABA cells.

Additional neurons were stained in less than 5% of the preparations. The most commonly observed cells of this type were a weakly immunoreactive pair of neurons with large (25-30 pm) fusiform somata located in the middle of the lateral ganglia. These cells have thick anteriorly di- rected processes that connect to lateroventral commissures in the amphidial commissure group. A second pair of inconsistently stained neurons were located at the level of or just anterior to the nerve ring. When these cells were observed, they were generally intensely stained. Occasion- ally, light staining of other neurons in the cephalic ganglia was seen, but in fewer than 5% of the preparations. I t is not clear whether this staining represents authentic GLIR.

Four series of sections through the nerve ring and cephalic ganglia, and two additional series through the nerve ring only, were examined for GABA-immunoreactive cells. All of the GABA-immunoreactive neurons observed in whole mounts were stained in sections; however, for some of these cells, the intensity of staining in sectioned material was lower than that observed in whole mounts.

Sections.

Fig. 3. Cross sections through the ventral cord showing GABA- immunoreactive cell bodies and commissures of three inhibitory motor neurons. A, B: Cell body (A, large arrow) and commissure (B, arrowhead) of VI1, a ventral inhibitory motor neuron. C, D: The cell body (C, large arrow) and commissure (D, small arrowheads) of DI1, a dorsal inhibitory motor neuron. The cell body of the V12 neuron is also present in C (small arrow). A small, unstained retrovesicular ganglion neuronal cell body can be seen in D (large arrowhead). E, F: The cell body (E, large arrow) and commissure (F, arrowheads) of VI2, a ventral inhibitory motor neuron. LL, Lateral line, Ph, pharynx. Scale bar = 10 Fm.

All four of the RME-like neurons and their processes were consistently and intensely stained; the RMEV-like neuron is shown in Fig. 6B. GLIR was exceptionally heavy in the nerve cord processes of the RMEV-like and RMED- like neurons, enabling us to trace the path of these fibers through the ventral cord. GLIR remained high in these neurites within the region traced, a distance of about 10 mm. The RMEV-like and RMED-like nerve cord neurites are aspinous; this is in contrast to the inhibitory motor neuron processes, which possess both presynaptic and postsynaptic spines (Donmoyer, Desnoyers, and Stretton, unpublished observations). The processes of RMEV and RMED are moderately varicose, particularly in the region posterior to the retrovesicular ganglion, and GLIR is strong in both the varicosities and intervaricose intervals.

A pair of small GABA-immunoreactive cells was observed in the posterolateral ventral ganglion in all five preparations examined; one member of the pair is shown in Fig. 6C. In some cases, staining of these neurons was quite intense; in other preparations staining was patchy. Sections through the cell bodies of these neurons revealed that they are monopolar with their neurite originating from the anterior pole of the soma. Based on this morphology, and on their size and position, we conclude that they are identical to the GABA-immunoreactive ventral ganglion cells (VG-GABA neurons) observed in whole mounts.

GABA-immunoreactive lateral ganglia neurons were seen in some of these series, but the staining intensity was low (Fig. 7B, C). A pair of lightly stained neurons was observed in the lateral ganglia at the level of the amphid bundle in 315 series; one member of this pair was observed in an additional series. Weakly GABA-immunoreactive lateral ganglia neurons were also observed at the level of the deirid bundle in 415 series. The GLIR present in the processes of these cells was barely detectable. The position and morphology of these lateral ganglia neurons suggest that they are the amphid-GABA and deirid-GABA neurons seen in whole mounts.

In 214 series, a pair of moderately to heavily stained neurons was observed in the lateral ganglia anterior to the nerve ring. Another series of sections contained a single weakly immunoreactive large neuron in the midlateral


Fig. 5. A-D: GABA immunoreactivity in the retrovesicular ganglion from a preparation treated with 100 JLM gabaculine for 4 hours at 37°C. Phase-contrast micrographs of cross sections through the cell bodies of four members of a class of retrovesicular ganglion neurons that take up 3H-GABA (asterisks). These cells do not contain detectable GLIR. Note the intensely immunoreactive fibers of the inhibitory motor neurons (large arrowheads) and the RMEV-like neuron (small arrowheads). Scale bar = 20 km.

ganglia. These cells may correspond to similar neurons occasionally observed in whole mounts.

The nerve ring contained high levels of GLIR in both whole mounts and sections (Figs. 8, 9). One primary source of GABA-immunoreactive profiles is probably the RME-like cells; in sections, thick, intensely stained neurites could often be traced back to the RME-like cell bodies. The RMEV-like and RMED-like neurons also possess GAl3A-immunoreactive somatic spines. I t is clear from the whole mounts that the other GABA-immunoreactive cephalic neurons send processes into the ring; these cells probably represent an additional source of GLIR observed in sections of this neuropil.

Nerve ring.

GLIR in the tail From studies of whole mounts, only 2 of the 30 neuronal

cell bodies in the tail ganglia contain GLIR. One of these cells, located in the preanal ganglion, is the V113 motor neuron, which has been described previously (Johnson and Stretton, '87). The preanal ganglion also contains the GABA-immunoreactive terminations of processes from more anterior inhibitory motor neurons. The GABA-immunoreactive neuron in the dorsal rectal ganglion (the DRG-GABA neuron) is a bipolar cell whose posterior process branches and forms terminal end bulbs at the base of the anal

depressor muscle, which it appears to innervate. The anterior branch of the DRG-GABA neuron passes around the rectum and enters the ventral nerve cord at the preanal ganglion. Its anterior extent has not been determined.

GLIR in nonneuronal tissue GLIR was examined in several nonneural structures in

the somatic and cephalic regions. GLIR was not detectable above background levels in the muscle and hypodermis (Fig. 2A, B) and cuticle (not shown) of the body wall. Furthermore, GLIR was not detectable in pharyngeal muscle (Fig. 9) and the excretory system (not shown). Lightly stained profiles were occasionally seen in the pharynx (Fig. 9); these probably represent neuronal fibers from two neurons within the pharyngeal nervous system that have been shown to contain GLIR in whole-mount preparations of the pharynx (Johnson, unpublished observations).

GLIR in male Ascaris The pattern of GABA-immunoreactive neurons observed

in whole-mount preparations of male worms is identical to that seen in females. In particular, six male-specific motor neurons whose cell bodies are located in the ventral nerve cord and the male-specific sensory neurons, motor neurons,


Fig. 6. GABA-immunoreactive neurons in the nerve ring and (large arrow). Note immunoreactive profiles (arrowheads) in the nerve ring. Pharyngeal muscle (Ph) and hypodermis (Hy) do not contain detectable GLIR. C: Cross section through the right half of the ventral ganglion showing one of the ventral ganglion neurons (arrow). The nerve cord process of the RMEV-like neuron (large arrowhead) and an unidentified small-caliber GABA-immunoreactive profile (small arrowhead) can also be seen. Scale bar = 10 km.

ventral ganglion. A: Whole mount showing GLIR in the RMEV-like neuron (large arrow) and in a pair of ventral ganglion neurons (small arrows). The nerve cord process of the RMEV-like neurons (large arrowheads) and one of its nerve ring processes (small arrowheads) can be seen. The ventral ganglion cell processes (open arrows) run into the nerve ring. Anterior is at the top of the micrograph. B: Cross section through the cell body of the RMEV-like neuron showing intense GLIR

and interneurons located in the tail do not contain detectable GLIR (data not shown).

Blocking experiments Blocking experiments in whole mounts were performed

with three different concentrations of GABA-BSA conjugate (4 p,g/ml, 20 pg/ml, and 100 pgirnl) and with GABA- ovalbumin conjugate (20 pg/ml). In these experiments, we specifically compared the staining intensities of the amphid-

GABA and deirid-GABA neurons and the inhibitory motor neurons. Complete blockage of anti-GABA staining in the amphid-GABA and deirid-GABA neurons was obtained in all cases. Complete blocking of staining in the inhibitory motor neurons generally required GABA-BSA at a concentration of 100 p,g/ml; at 20 p,g/ml staining was greatly diminished, and in most cases eliminated. The most anterior inhibitor motor neuron commissures (VI1 and DI1) were completely blocked by 20 pg/ml of GABA conjugate


Fig. 7. GABA-immunoreactive neurons in the right lateral ganglion. A Whole mount of the right lateral ganglion showing the cell bodies of the amphid (large arrow) and deirid (small arrow) neurons. Portions of the proximal neurites of these two cells can also be seen; the remainder of their processes are out of the plane of focus. The GABA-immunoreactive commissure of VI1 (arrowheads) crosses through this part of the lateral ganglia. Anterior is at the top of the micrograph. B: Phase-contrast micrograph of a cross section through the right lateral ganglion at the level of the amphid bundle showing a moderately immunoreactive neuron (large arrow). Two unstained profiles are nearby (arrowheads). C: Phase-contrast micrograph of a cross section through the right lateral ganglion at the level of the deirid bundle showing a lightly stained spindle-shaped neuron (large arrow). Scale bar in A = 50 pm; in B, C = 10 pm.

and diminished by 4 pgiml, although some staining was still detectable. These results suggest that staining of the amphid-GABA and deirid-GABA neurons is blocked by lower concentrations of GABA conjugates than is staining in the inhibitory motor neurons. None of the other amino acid-BSA conjugates (glycine, glutamate, aspartate, p-alanine, taurine, and glutamine, all at 20 pglml) had any effect on cephalic neuron staining.

Blocking experiments were performed on sections with conjugates at concentrations of 10 pglml and 1 mg/ml. At 10 pg/ml, only the GABA conjugate blocked staining in immunoreactive neurites and cell bodies. At 1 mg/ml all of the conjugates had some blocking effect. The strongest compet- itors at this concentration were p-alanine, glutamate, and aspartate, which reduced staining to background or near background. This result indicates either that the specificity of this antiserum for GABA-protein conjugates is high but

not absolute, or that the ligands used to make the other amino acid conjugates contain low levels of GABA as a contaminant.

DISCUSSION The present results, in combination with those reported

previously (Johnson and Stretton, '87), identify a total of 30 consistently stained GABA-immunoreactive neurons in As- caris. The 11 additional GABA-immunoreactive cells, over and above the 19 GABA-immunoreactive inhibitory motor neurons, include the 4 RME-like neurons, the bilaterally symmetric pairs of VG-GABA, amphid-GABA and deirid- GABA neurons, and the DRG-GABA neuron.

Our comparison of the anti-GABA staining patterns in whole mounts and sections has revealed an interesting difference. In sections, four of the consistently observed


Fig. 8. GABA immunoreactivity in a whole mount of the nerve ring. The cell body of the RMEV-like neuron (arrow) and its nerve ring processes (large arrowheads) are stained. Other small caliber fibers can also be seen within the ring (small arrowheads). Anterior is at the top of the micrograph. Scale bar = 50 pm.

neurons (the amphid-GABA and deirid-GABA cells) stain less intensely than the other cells of this class. This staining difference was not seen in whole mounts. Part of this discrepancy may be due to the different experimental conditions, in particular enzyme digestions and fixation methods, used in processing whole mounts and sections. In the procedures used for making sections, two of the causes of variation, damage during dissection and the enzymatic digestions, have been eliminated, but optimizing the fixation conditions still leads to a less than ideal compromise. Of the ten consistently staining neurons identified in head whole mounts, six neurons (the four RME-like cells and the two VG-GABA cells) gave strong staining in all of the serial section preparations. However, both pairs of LG cells gave low-intensity staining in sections. We believe that the staining differences observed in sections reflect a genuine difference in levels of GABA between the LG neurons and the strongly GABA-immunoreactive cells. This conclusion is supported by the results of the blocking experiments in whole mounts: staining of the LG neurons is blocked by lower levels of GABA-conjugate than is staining of the inhibitory motorneurons. Further support is provided by experiments using a different anti-GABA antibody. In 32 whole-mount preparations made using a GABA-specific monoclonal antibody (Sithigorngul et al., '891, in which, in contrast to experiments with the GABA antiserum, the conditions were not adjusted to give maximal staining the staining intensity was in the order: motorneurons > RME-like neurons > amphid-GABA and deirid-GABA neu-

rons > other immunoreactive neurons (Sithigorngul and Stretton, unpublished data). This result, together with the results from serial sections, suggests that the amphid- GABA and deirid-GABA neurons actually contain less GABA than the other consistently observed neurons.

Identification of GABA-immunoreactive neurons

The morphological criteria used to identify cephalic neurons in Ascaris include cell body location, commissure presence and position, and direction of entry into the nerve ring. Identification of C. elegans neurons employs the additional criterion of synaptic connections, which, together with the above morphological features, allows the unique identification of neurons. The synaptic connections of Ascaris cephalic neurons are not yet known, which precludes the unique identification of Ascaris GABA- immunoreactive neurons. However, based on morphological criteria, it is possible to define a subset of C. elegans neurons to which the Ascaris cells might correspond. The four Ascaris nerve ring-associated cells almost certainly correspond to the C. elegans RME neurons, which are also GABA-immunoreactive (McIntire and Horvitz, '85). These cells possess a highly distinctive shape and are the only neurons in the cephalic region whose cell bodies are located within the nerve ring. In C. elegans these neurons innervate head muscle cells; they probably also function as ring motor neurons in Ascaris. For the remaining consistently


Fig. 9. GLIR in a cross section through the nerve ring. The cell bodies of the RMEV-like (large arrow) and RMED-like (small arrow) neurons are intensely stained. Numerous GABA-immunoreactive profiles (examples labeled with arrowheads) within the nerve ring are present. GLIR is not detectable in pharyngeal muscle (Ph) or somatic muscle (Mu). A lightly GABA-immunoreactive neuron is stained in a pharyngeal nerve cord (open arrow). Scale bar = 50 pm.

stained neurons, the possible comparisons (with the nomenclature of White et al., '86) are as follows: 1) For VG-GABA: AIA, AIM, MY, RMDD, RMF, or RMH; 2) for amphid- GABA AIB, AIZ, AVB, RIB, RIC, RIM; and 3) for deirid- GABA ADA or RMG. The Ascaris DRG-GABA cell probably corresponds to the C. elegans DVB neuron, which is GABA-immunoreactive and innervates the rectal muscle (McIntire and Horvitz, personal communication). Both neurons have cell bodies in the dorsorectal ganglion and fibers that project anteriorly in the ventral nerve cord. The identities of the inconsistently or weakly stained neurons cannot, at this point, be assigned with any confidence. In summary, the nervous system of Ascaris includes 26 neurons that are strongly and consistently GABA-immunoreactive, 4 neurons that are consistently but weakly GABA- immunoreactive, 2 neurons tha t a re strongly but infrequently GABA-immunoreactive, and 2 neurons that

are weakly and infrequently GABA-immunoreactive. Two moderately immunoreactive neurons are located within the pharynx.

Functional implications The serial section approach allowed us to examine GABA-

like immunoreactivity in single cells over relatively long distances at a resolution higher than is possible with whole mounts. In general, GLIR persists at a constant level in the nerve cord processes and commissures of the inhibitors and in the nerve cord processes of the RMEV-like and RMED- like cells. This observation suggests that GABA is present throughout the neuron and argues against an exclusive localization at morphologically identified synapses. Our finding is in agreement with GABA immunocytochemical experiments in mammals (Ottersen and Storm-Mathisen, '84; Somogyi et al., '85) and with biochemical experiments in crustacea (Otsuka et al., '67; Kravitz and Potter, '65). It is, however, possible that higher levels of GLIR in synaptic regions might not be detected if immunoperoxidase staining is already at its maximal value.

GLIR is not detectable in the large or small ventral cord interneurons. These results suggest that, if there are inhibitory interneurons present in the ventral cord, they do not use GABA as a neurotransmitter. This result is relevant to the interpretations of the mode of action of avermectin in Ascaris (Kass et al., '80, '82). Avermectin has effects that mimic GABA agonists. One speculation, that avermectin causes a release of GABA from a putative GABAergic interneuron, must now be abandoned. If the action of avermectin is mediated by GABA release, the source of this GABA must be the inhibitory motor neurons or the RME- like neurons.

The nerve cord fibers of the RMEV-like and RMED-like neurons are as intensely immunoreactive as their cell bodies. Although these fibers extend for at least 20 mm in the nerve cords, their physiological role is not clear. An electron microscopic analysis indicates that the nerve cord processes of the RMEV-like and RMED-like neurons contain few, if any, synaptic vesicles (Donmoyer and Stretton, unpublished observations). A similar dearth of synaptic vesicles has been observed in the C. elegans RMEV and RMED nerve cord processes (White et al., '86). The absence of conventional morphological synapses obviously does not rule out these fibers as a functional source of GABA-it is possible that they release GABA from nonvesicular cytoplas- mic stores. A similar proposal has been made for some GABAergic horizontal cells in the teleost retina (Schwartz, '87). A subset of these cells make vesicle-poor, but appar- ently physiologically active, contacts with bipolar cells; secretion of GABA by these horizontal cells may occur by a reversal of the GABA transporter, resulting in a voltage- regulated, but calcium-independent, release of neurotransmitter (Schwartz, '87). It is interesting that all four RME- like neurons in Ascaris possess a GABA uptake system (Guastella et al., '86; Guastella and Stretton, '911, which, in the case of the RMEV- and RMED-like cells, extends into the nerve cord processes. I t is possible that GABA may be released from the nerve cord neurites of these cells by a reversal of the carrier-mediated transport mechanism. Al- ternatively, these processes could function in the clearance of extracellular GABA from the nerve cords.

GABA-associated neurons in the nematode A complete description of GABA function in an organism

requires the cellular localization of at least six markers: 1)


endogenous GABA; 2) glutamate decarboxylase; 3) GABA receptors; 4) GABA-transaminase; 5 ) GABA transporters; and 6) GABA release sites. In this paper we have localized GLIR, which, considering the specificity of the antiserum used, probably represents authentic GABA stores. Clearly, the presence of GABA in a neuron is not sufficient to identify that neuron as GABAergic (i.e., capable of releasing GABA in a regulated fashion). Independent evidence for functional release of GABA is necessary. Nonetheless, it might be expected that the cells with the highest levels of endogenous GABA would be the physiologically important sources of GABA. Based on this criterion, we predict that, besides the inhibitory motor neurons, the RME-like neurons, the VG-GABA neurons, and the DRG-GABA neurons are GABAergic. The remaining GABA-immunoreactive cells, which include the amphid-GABA and deirid-GABA neurons, as well as the less consistently immunoreactive neurons, possess less intense GLIR. We suspect that these cells contain lower levels of GABA. However, at least some of the less consistently immunoreactive neurons appear to contain GABA transporters (see companion paper), and may still play a role in GABAergic mechanisms, either as functional sources of GABA or as GABA sinks. Further- more, although no retrovesicular ganglion neurons contain detectable GLIR, four of them possess GABA transporters. We therefore refer to these Ascaris cells, and other cells containing at least one of the above described GABA markers, as “GABA-associated” neurons, since they may be involved in aspects of GABA physiology other than release.

ACKNOWLEDGMENTS The authors thank Dr. R. Wenthold for anti-GABA

antiserum and amino acid-protein conjugates and P. Brack- ley for technical assistance. This work was supported by NIH grant AI20355.

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gaba-immunoreactive neurons in the nematode ascaris

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