THE JOURNAL OF COMPARATIVE NEUROLOGY 261~435-449 (1987)

The Early Development of Neurons With GABA Immunoreactivity in the CNS of

Xenopus laevis Embryos

ALAN ROBERTS, N. DALE, O.P. OTTERSEN, AND J. STORM-MATHISEN Department of Zoology, The University of Bristol, Bristol, U.K. (A.R., N.D.);

Anatomical Institute, N-0161 Oslo 1, Norway (O.P.0, J.S.-M.)

ABSTRACT We have used an antibody to glutaraldehyde fixation complexes of y-

amino butyric acid (GABA) to stain the developing central nervous system of Xenopus laevis embryos. Neuronal somata, growth cones, axons, and dendrites were found with GABA-like immunoreactivity. Transmission elec- tron microscope (TEM) observations were made of axons and synapses. By observation of the earliest stages of differentiation of neurons, seven classes of putative GABAergic interneurons were discerned. 1) Ascending neurons are first stained in the hindbrain at stage 26 and later extend caudally in the spinal cord. They have ascending ipsilateral axons. 2) Midhindbrain reticulospinal neurons are first stained at stage 25 and develop as a compact group with descending ipsilateral and contralateral axons. 3) Vestibular complex commissural neurons are first stained at stage 29/30 in a dorsal position near the entry of the seventh and eighth cranial nerves. They have ventral commissural axons that descend contralaterally and their somata form a compact mass. 4) Rostral hindbrain commissural neurons are first stained at stage 33/34 just rostral to the entry of the trigeminal nerve. They each have a decussating projection. 5) Rostral midbrain neurons are first stained in the midbrain at stage 29/30 and are later associated with promi- nent dorsal and ventral commissures. 6) Optic tract and 7) rostral forebrain neurons are found in the forebrain associated with strongly stained axon tracts.

The direction of axonal growth from its earliest stages was distinct for each class of hindbrain and spinal cord neuron.

Key words: immunocytochemistry, propriospinal neurons, GABAergic interneurons, neuron differentiation

An essential step in understanding any nervous system is to be able to identify the different classes of neuron it contains. For the brainstem and spinal cord of vertebrates, that is proving to be a difficult task. We have therefore studied the very simple nervous system of frog embryos in the hope that, at early stages of development, the basic organization of the central nervous system may yield to careful study and provide a framework to help understand the nervous systems of more mature and complex animals. Studies using retrograde filling with horseradish peroxi- dase (HRP) are beginning to distinguish classes of neuron in the spinal cord and hindbrain of developing lower verte- brates. In the young zebrafish larva a number of reticulo- spinal neuron types with specific axonal projection patterns have been described (Kimmel, '82; Kimmel et al., '82, '85).

0 1987 ALAN R. LISS. INC.

In embryos of the clawed toad (Xenopus Zueuis), five classes of propriospinal neuron have been described (Roberts and Clarke, '82) in the late embryo at stage 37/38 (Nieuwkoop and Faber, '56), and one of these classes has been shown to extend as a column into the reticulospinal region of the hindbrain (Roberts and Alford, '86). Studies in Xenopus have also looked at the development of spinal (Nordlander, '84) and hindbrain neurons including reticulospinal neu- rons (van Mier and ten Donkelaar, '84; Nordlander et al., '85). However, the method is not suitable for seeing the

Accepted February 18,1987. N. Dale's present address: Howard Hughes Medical Institute,

Columbia University, 722 West 168th St., New York, NY 10032.

436 A. ROBERTS ET AL.

earliest stages of neuronal differentiation. To do this Jacob- son and Huang ('85) have injected HRP into blastomeres at the 32-cell stage, then allowed time for further development before fixation and processing. An alternative method is to use immunocytochemical staining for putative transmit- ters chemicals. This was first used in Xenopus embryos to show that sensory Rohon-Beard neurons had substancep- like immunoreactivity (Clarke et al., '84). Van Mier et al. ('86) have used antibodies against serotonin to study the development of raphespinal projections. We have previously used antibodies that recognize the glutaraldehyde fixation products of glycine to stain putative glycinergic neurons in the spinal cord (Dale et al., '86). We report here on our use of polyclonal antibodies, which bind to the gluteraldehyde fixation products of y-aminobutyric acid (GABA) (Storm- Mathisen et al., '83; Ottersen and Storm-Mathisen, '84), to reveal GABA-containing neurons in the central nervous system of embryos of Xenopus laevis. This has allowed us to distinguish seven broad classes of putative GABAergic neuron and to follow the development of some of these cell classes in considerable detail from their earliest stages of outgrowth of axons and dendrites. We have also used the GABA antibody to reveal and observe the development of Kolmer-Agduhr cells, a class of cerebrospinal fluid contact- ing neurons found in Xenopus embryos (Dale et al., '87a,b).

METHODS Embryos of Xenopus laeuis were produced in Bristol by

induced breeding with the aid of chorionic gonadotrophin. They were allowed to develop at 20 3°C in aerated tapwater before staging (Nieuwkoop and Faber, '56) and fixation. This was in 5% gluteraldehyde (diluted from 25% with 100 mM phosphate buffer, pH 7.4) for 1 h for light microscopy or in 2% gluteraldehyde in 50 mM cacodylate buffer with 70 mM sucrose and 3 mM CaClz at pH 7.4 for 10 h for electron microscopy. After fixation embryos were placed in 100 mM phosphate buffer with 0.1% gluteralde- hyde and 0.04% sodium azide, where the CNS was isolated by dissection before processing in Oslo by the methods of Ottersen and Storm-Mathisen ('84). Details of the purifica- tion of the antiserum, the staining protocol that uses per- oxidaze-antiperoxidae, and controls for specificity of the antibody applied in Xenopus are given in Dale et al. ('87a). However, all staining was blocked by addition of 0.1 mM GABA processed with glutaraldehyde to the serum but not by addition of other gluteraldehyde-treated amino acids, including glycine and taurine.

After staining, whole-mount CNSs were dehydrated and mounted between coverslips in Canada balsam. For sec- tions, CNSs were embedded in wax or Araldite and cut at 15 pm. All drawings were made with a camera lucida and details were checked under oil at ~ 5 0 0 or ~1 ,000 . For transmission electron microscopy (TEM) CNSs were embed- ded in Araldite, cut on an LKB Ultramicrotome, stained with lead citrate and uranyl acetate, and examined in a Phillips 200 microscope. The number of embryos examined as whole-mount CNSs at each stage were as follows: stages 22-24 (4), stage 25 (31, stage 26 (191, stage 27 (61, stage 28 (S), stage 29/30 (5), stage 31 (12), stage 33/34 (14), stage 351 36 (3), stage 37/38 (19).

RESULTS General distribution of somata and axons

The first neurons showing GABA-like immunoreactivity appeared in the hindbrain at stage 25 (Nieuwkoop and

Faber, '56). The subsequent development of marked neu- rons was followed to stage 37/38 and the positions of their somata are illustrated in Figures 1 and 2. Axons and growth cones were also strongly stained and are considered below. Between stages 25 and 28 two groups of neurons started to show GABA immunoreactivity. The first group is in the midhindbrain and forms a prominent ventral commissure (midhindbrain reticulospinal group (rnhr); unfilled somata in Fig. 1). The second group is first stained in the caudal hindbrain and develops caudally as a column extending into the spinal cord (ascending group (as); filled somata in Fig. 1). By stage 31/32 three further groups of neurons are stained. In the rostral, dorsal hindbrain a vestibular com- plex (vc) group differentiates and forms a strong, local ven- tral commissure (see stage 29/30 in Fig. 1). In the midbrain a rostral midbrain group stains in the lateral rostral region, whereas in the forebrain a group stains near the position of the optic tract (r and ot at stage 31/32 in Fig. 1). Between stages 33/34 and 37/38 the numbers of stained cells in each group increases, the extension of the column of ascending neurons caudally into the spinal cord being very clear. A new group of stained cells forming a local ventral commis- sure appears rostrally in the hindbrain between stages 35 and 38 (rh in Fig. 2A) when another group stains rostrally in the forebrain (rf in Fig. 2A).

Since more than one cell type shows GABA activity, the identity and origin of stained longitudinal axons in the marginal zone is usually unclear at later stages of embry onic development. Transverse sections (Fig. 2B) show that commissural axons are absent in the spinal cord but are produced by all the hindbrain cell groups. Though not indi- cated in Figure 2B, such sections show stained axons throughout the marginal zone of the spinal cord (see also next section and Fig. 4A) and hindbrain. The extent of stained axons a t stage 37/38 is shown in Figure 3. The commissural projections of hindbrain cell groups are clear. In addition, the rostral midbrain group is associated with commissures located dorsally just caudal to the pineal ves- icle and ventrally in the midbrain. However, the exact origin of these commissures was unclear. The optic tract group connects via stained axons to the optic chiasm, and

as f m mfb mhr oc 0s ot P rf rh rm sc vc

Abbreviations

ascending neurons forebrain midbrain median forebrain bundle midhindbrain reticulospinal neurons optic chiasm optic stalk optic tract neurons pineal body rostral forebrain neurons rostral hindbrain neurons rostral midbrain neurons spinal cord vestibular complex neurons

Fig. 1. Distribution of somata with GABA-like immunoreactivity from stages 25 to 33/34. Lateral views of whole-mount nervous systems in which all marked neuronal somata on the right side were drawn. Stages are given at left of each drawing. Ascending (as) and vestibular complex (vc) neurons have black somata; midhindbrain reticulospinal neurons (mhr) and more rostral neurons have open somata. Axons and growth cones are not shown except for three (arrows) at stage 26 that come from mhr somata on the opposite side. Midbrain to hindbrain junction (large arrowhead), obex (small arrowhead).

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GABA NEURONS IN CNS OF X . Zueuis 439

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Fig. 3. Distribution of GABA activity in axons and somata at stage 371 38. A Photomicrograph of whole-mount of the brain from right side to show main cell groups and axon tracts. Dots indicate the brain outline. B: Draw- ing of specimen in A. B , C Drawings from two specimens of whole-mount brains and rostra1 spinal cord from right side to illustrate reproducibility of

cell distributions. Somata traced and marked as in Figures 1 and 2. Axons indicated by stippling. Commissures hatched diagrammatically. Asterisks and roman numerals indicate entry points of cranial sensory nerves. Small arrowhead is obex, large arrowhead is midbrain-to-hindbrain junction.

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diameter p m Fig. 4. GABA-positive profiles in rostral trunk spinal cord at stage 371

38. A: Tracing, from a photomontage of TEM pictures a t x 8,880 and from a transverse section, showing all GABA-positive profiles. There are 68 on the left and 67 on the right. Arrow indicates a probable stained dendrite. Asterisk shows approximate location of synapse in Figure 5C. B: Histogram of profile diameters (mean of longest and shortest diameters) measured from TEM pictures.

the rostral forebrain group is associated with a commissure just rostral to the optic stalk.

Fine structure of axons and synapses Transverse thin sections were examined in the TEM to

see if GABA-immunoreactive cells could be recognized and particularly to see if GABA-stained axons made en passant synapses and if these had any diagnostic ultrastructural features. In embryos not treated with Triton X 100 but frozen briefly in liquid nitrogen, preservation for TEM was acceptable. Stained profiles were seen throughout the mar- ginal zone of the spinal cord a t stages 37/38 (Fig. 4A). These could arise from the interneurons described here or from Kolmer-Agduhr cells (see Dale et al., '87a). The size and number of profiles in rostral trunk spinal cord were mea- sured from photomontages (at X 8,800) of transverse sec- tions made at five levels separated by 30-40 pm. In nine marginal zones the mean number of profiles was 67 (SD 11.2, range 43-85) and the mean diameter was 0.4 pm (n = 91, SE 0.015) with a skewed distribution (Fig. 4B). The vast

A. ROBERTS ET AL.

majority of profiles were sectioned axons with roughly cir- cular outlines, containing microtubules and small mito- chondria (Fig. 5A,B). Some more complex profiles lacking any presynaptic features were probably dendrites (e.g, ar- row in Fig. 4A).

Synapses were clear where the staining was lighter and intracellular organelles could be resolved (Fig. 5B-F). They were recognized as close contacts between a stained presyn- aptic profile, with clustered clear vesicles adjacent to a dense region of membrane, and a postsynaptic profile. The synaptic cleft contained electron-dense material (cf., Hayes and Roberts, '73). In 10 of the 14 examples there was thick- ening of the subsynaptic membrane, in 7 there were presyn- aptic dense-cored vesicles. About half of the presynaptic profiles had mitochondria. Twelve of the 14 were axoden- dritic, sometimes located near the surface of the cord quite distally on the dendrites (Fig. 5B). One synapse was axoso- matic (Fig. 5C), most probably onto a motoneuron, since the soma was in a ventrolateral position (see asterisk in Fig 4A for approximate position). Since most presynaptic profiles were simple in outline and a number contained microtu- bules (e.g., Fig. 5B), it is likely that most are made en passant from axon varicosities. Comparison with other un- stained synapses (Fig. 5D, E) did not reveal obvious diag- nostic features of GABA-stained presynaptic structures.

Characterization of stained neurons At stage 37/38 it is difficult to resolve the shapes and

processes of different neuron classes. However, by following each group back to the stage at which it was first stained, axonal projections and, in some cases, the patterns of den- dritic branching can be resolved. With this approach one class of propriospinal neuron and three groups of hindbrain neurons have been broadly characterized and will now be considered in turn.

Ascending neurons. At stage 37/38 stained somata are present in the dorsal half of the spinal cord forming a column that appears to extend into the caudal hindbrain (Fig. 2A). The somata vary in position from superficial dorsolateral to a deep middorsoventral one (see transverse sections in Figs. 2B and 6C). Density of somata is maximal near the obex at 12-14 per 100 pm per side. In the 1 mm caudal to the obex the mean density is 6.7 per 100 pm (SD 2.8, n = 20). The somata are generally unipolar (Fig. 7C,D) but occasional more dorsal dendrites can emerge from the soma. Since there are no stained commissural axons in the spinal cord, we assume that these neurons have ipsilateral axons but whether they ascend or descend is unclear at stage 37/38.

The axonal projections and distribution of ascending neu- rons are clear at earlier stages. They first stain as a distinct group in the caudal hindbrain at stage 26 (Fig. 1). Their numbers increase to stage 31/32, and up to this stage they are distinct from other hindbrain cells by virtue of 1) paler staining, 2) absence of strong ventral initial process of axon, and 3) location in a dorsal tract of stained axons with ascending stained growth cones. This dorsal ascending tract, present at the dorsoventral level of the somata of the as- cending neurons and not present caudal to them, is partic- ularly clear at stages 28 and 29/30 (Figs. 8 and 1OC). It would partly overlap that region of the marginal zone also occupied by Rohon-Beard cell axons. This type of prepara- tion shows that the ipsilateral axons of these neurons are ascending and by stage 29/30 reach the level of the trigem- inal nerve. By stage 31/32 their somata stain more strongly

GABA NEURONS IN CNS OF X . laevis

Fig. 5. Fine structure of axons and synapses with GABA activity seen in transverse sections of rostra1 trunk spinal cord. A Three stained axons in the marginal zone close to the external surface of the cord, which has basal lamina (arrow) and collagen fibrils. Microtubules and mitochondria are clear in largest, lightly stained axon and in unstained profiles. R: Lightly stained axodendritic synapse 1.4 pm from the external surface of the cord with two, possibly three, stained axons (near top). Synaptic area, here and in C, D and E, is between arrowheads placed in the postsynaptic profile. C

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Darkly stained axosomatic synapse. For approximate location see asterisk in Figure 4A. Scale bar, 0.5 pm, applies also to A and B. D,E: Axodendritic synapses from stained (marked by arrowheads) and unstained profiles. Preservation tended to be worse in stained profiles, which here contain dense-cored vesicles (arrows). F: Stained synaptic profile curved around narrow dendritic postsynaptic profile (between arrowheads). Cleft and post- synaptic membrane density are clear. Scale bar, 0.5 pm, applies also to D and E.

442 A. ROBERTS ET AL.

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D Fig. 6. Ascending neurons. A,B: Drawings from whole mounts at stage

31/32 in approximate positions indicated on diagram of nervous system to show dendrites and axons. Oblique dorsal view with dorsal midline (dashed line) and Rohon-Beard neurons shown as faint outlines. C: Transverse sections of rostral spinal cord at stage 37/38 to show soma positions and dendrites. Outline of marginal zone is dotted. (Note absence of commissural

in the caudal hindbrain (Fig. 7B) and their axons are still distinct in a dorsal tract with ascending growth cones (Fig. 9B and C). In some neurons at this stage an ascending axon can be traced from the soma for a short distance (Fig. 6A,B). After stage 31/32 the dorsal ascending tract merges with more ventral axons (see Fig. 3) and the most rostral ascend- ing neurons cannot clearly be distinguished from midhind- brain reticulospinal neurons (see below). By stage 37/38 the ascending neurons extend from the level of the vagus cau-

stained axons ventrally). D: A selection of neurons drawn from spinal whole mounts at stage 31/32 (rostral to the left) with the most mature rostral cells on the left and early &ages of process outgrowth on the right. Growth cones are cross-hatched and dorsoventral position is related to the level of the most dorsal stained axon in the marginal zone (dotted line). Scale in A applies also to R and D.

dally for about 1.8 mm, approximately to the level of the 10th postotic somite (Fig. 1). At later stages they may con- tinue to develop more caudally.

The development of ascending neuron dendrites can be followed at progressive stages or by observing the longitu- dinal developmental gradient, for example at stage 31/32. In the caudal hindbrain these neurons are unipolar (Fig. 7B) with dorsal and ventral dendrites in the dorsal half of the marginal zone (Fig. 6). When viewed obliquely from

GABA NEURONS IN CNS OF X. Zueois 443

Fig. 7. Photomicrographs of neurons with GABA-like immunoreactivity made from whole mounts, A Midhindbrain reticulospinal and ascending neurons at stage 31/32. The same region is drawn in Figure 9B; rostral is to the right and the view is oblique ventral. The mhr neurons have large ventral processes from darkly stained somata. Their commissural axons are seen ventrally together with axons from contralateral homologues, which run obliquely to join the ventral edge of the bundle of stained fibers in the

marginal zone (at arrow). B: Ascending neurons seen in dorsal view of the rostral spinal cord (rostral to right) at stage 31/32. Depth of somata from the surface varies, deeper cells being clearly unipolar (asterisks). Refractile inclusions are yolk platelets. Midline and neurocoel are between arrow- heads. C,D: Ascending neurons seen in lateral view at stage 37/38 with other stained axons. Principal ventral processes can be seen. C: Rostral to left. D: Rostral to right. Scale in B, 30 pm, applies to A, C and D.

444 A. ROBERTS ET AL.

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Fig. 8. Drawings of whole mounts a t stages 28 and 29/30 showing all stained somata and clear growth cones, and indicating the extent of stained axons. At stage 28 ascending neurons (black somata) are overlain by stained axons forming a distinct dorsal tract in the marginal zone, which runs rostrally over the somata of midhindbrain reticulospinal neurons and ends in two ascending growth cones. Note absence of this tract caudal to stained neurons. Clear somata of midhindbrain reticulospinal neurons contribute commissural and descending axons to a ventral tract with descending growth cones. At stage 29/30 axon tracts of stage 28 are more extensive, two vestibular complex neurons are present, and rostral midbrain and optic tract groups appear in association with dorsal and descending growth cones.

below, the dendrites are well displayed (Fig. 9B, C). In the mid-spinal cord dendrites are generally unclear under stained axons but more caudally the early stages of den- dritic growth with dendritic growth cones can be seen (Fig. 6D). Little change occurs in more rostral cells by stage 371 38 (Fig. 7C, D). The dendrites generally extend through the dorsal half of the marginal zone, some extending more dorsally than most of the GABA-stained axons into the dorsolateral region occupied by Rohon-Beard cell axons (Roberts and Clarke, 1982).

Midhindbrain retieulospinal neurons. At stage 37/38 midhindbrain reticulospinal neurons form a strongly stained group extending about 200 pm caudal of the otic capsule and forming a strongly stained ventral commissure (Figs. 2 and 3). These neurons are larger and have clearer dendrites than more rostral neuron groups. Their dendrites are directed mainly ventrally. However, at stage 37138 an individual neuron's dendrites and axons are unclear and they can be resolved only at earlier stages. These neurons are seen first at stage 25 or 26 as the most rostral stained somata (Figs. 1 and 10). When first stained, the neurons have a single ventral commissural axon formed by a large flattened growth cone. On the side contralateral to the somata these large growth cones turn caudally in the mar- ginaI zone (see arrows at stage 26 in Fig. l and Fig. 10B). This growth forms a crossed descending tract of stained axons in the ventral part of the marginal zone that is clearest at stages 27 to 29/30 as it extends candally into the spinal cord (Fig. 8). The presence of many descending growth cones in the tract makes its direction unambiguous at these stages. As development proceeds the ventral commissural axons of some individual cells can be traced (Figs. 7A, 9B,C,

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and 10 C-E). After the formation of a commissural projec- tion at least some of these neurons grow an ipsilateral descending axon. These can be seen clearly as neurons differentiate at the rostral end of the group between stages 28 and 33/34 (Fig. 8). At stage 28 many midhindbrain reticulospinal neurons were seen with a main ventral pro- cess that branched in the ventral marginal zone to form a descending ipsilateral axon before running ventrally as a commissural axon. In one case a descending growth cone was clear close to the branch point (see Fig. lOC). Ipsilateral axons could be followed for short distances at later stages but were never so clear (Figs. 7A, 9C, and lOD,E). Few dendrites were present up t o stage 29/30, after which a mainly ventral and lateral dendritic arbor developed (Figs. 7A, 9B,C, and 10D,E), extending throughout the dorsoven- tral extent of the marginal zone but more concentrated in the ventral half. The number of midhindbrain reticulo- spinal neurons is about 15-20 per side at stage 37/38 but cannot be counted because of ambiguity in distinguishing more caudal cells in the group. At earlier stages when counts were possible the number of commissural axons was roughly double the number of neurons on one side in some cases (e.g., Fig. 9B), but less in others (e.g., Fig. 9C), presum- ably because of the difficulty of resolving single from paired or grouped axons.

Vestibular complex commissurat neurons. Vestibular complex commissural neurons differentiate later than the previous two groups, first staining in the rostral hindbrain at stage 29/30 as a distinct dorsal group of small unipolar somata with ventral commissural axons (Figs. 1 and 8). The number of neurons in this group increases and by stage 371 38 (Figs. 1 and 2) a short longitudinal column of somata is formed (Figs. 2 and 3) lying just caudal and dorsal to the entry of the trigeminal nerve in the region of entry of the

GABA NEURONS IN CNS OF X. laeuis 445

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Fig. 9. Neurons and processes with GABA-like activity a t stage 31/32 C : Arrowheads indicate a neuron with ipsilateral descending axon and drawn from whole mounts. A The brain. B,C: Caudal half of the hindbrain growth cones. Two rostra1 oblique axons (stars), one with a descending in oblique ventrolateral view showing ascending neurons (stippled) associ- growth cone, come from the commissure of the vestibular complex group. ated with dorsal tract and ascending growth cones (arrows), and all mi- All growth cones are cross-hatched. Dashed line indicates midventral axis dhindbrain reticulospinal neurons (clear) with associated ventral of the cord. Transverse section at top indicates orientation of B and C , which commissure and ventral tract with descending growth cones apparent in C . are viewed in direction of the big arrow.

446 A. ROBERTS ET AL.

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GABA NEURONS IN CNS OF X . laeuis 447

seventh and eighth cranial nerves. These cells are all clearly unipolar with single ventral axons forming a compact ven- tral commissure. At stage 31/32 some axons in this commis- sure can be seen to turn caudally and run obliquely into the dorsal part of the marginal zone (Fig. 9C). At later stages individual axons could not be followed, so their direc- tion, whether they branch and extend into the spinal cord, was not clear. The dendrites of these neurons were weakly stained and unclear.

Rostral hindbrain commissural neurons. The rostral hindbrain commissural neurons are the last group of hind- brain neurons to stain. They appear at stage 33/34 or 35/36 as small cells with ventral commissural axons (Figs. 1 and 2). By stage 37/38 a clear group is present at the rostral edge of the hindbrain with a prominent, local, ventral com- missure (Figs. 2 and 3). The nature of dendrites and direc- tion of projection of axons after they had crossed were unclear.

Rostral midbrain neurons. Rostral midbrain neurons appear stained at stages 29/30 or 31/32 and increase in number by stage 37/38 (Figs. 1 and 2A) when they may be separable into two subgroups both intimately associated with strong dorsal and ventral commissures (Fig. 3). The initial axon outgrowth from a t least some of these neurons is dorsal and can be seen from growth cone orientation at stage 31/32 (Figs. 8 and 9A). However, the cellular origin of the stained commissural axons cannot be determined with confidence at present. Dendrites were also not clear.

Optic tract neurons. Optic tract neurons generally ap- pear stained at stage 31/32 and increase in number to form a dense group at stage 37/38 (Figs. 1, 2A, and 3). They are associated with commissural axons in the optic chiasm, but at stages 29-32 at least some have short descending axons with growth cones (e.g, Fig. 8). Again the origin of stained commissural axons was unclear. Dendrites were not clear.

Rostral forebrain neurons. Rostral forebrain neurons appear stained at stage 35/36 and are associated with the medial forebrain bundle (Figs. 2A and 3). Their direction of growth and dendrites were unclear.

DISCUSSION Immunocytochemical staining for putative transmitters

provides a powerful technique for distinguishing and defin- ing classes of CNS neurons. This task is also easier at early stages of development since those neurons so far investi- gated all expressed transmitter activity a t an early stage of morphological differentiation. In addition to providing in-

Fig. 10. Development of midhindbrain reticulospinal neurons drawn from whole mounts. A Location of cells shown in B. View of brain at stage 26 from right side. B: Ventrolateral view (see arrow and transverse section above) showing somata (dotted on left side), axons and growth cones (cross- hatched) at stage 26. Dashed line is ventral midline. Dotted line indicates approximate ventral edge of unstained axons in marginal zone of right side. C: Three unipolar neuron somata at stage 28 with ventral processes that branch to give one ipsilateral descending axon and one ventral commissural axon. Growth cone on ipsilateral axon of most rostral cell is clear. The

formation on the chemical identity of the neurons the method therefore offers a new technique for studying neu- ronal development. In Xenopus it has been used by van Mier et al. (‘86) to study development of neurons with serotonin-like activity. We have used a GABA antibody in the present study and also to define spinal Kolmer-Agduhr cells and observe their development (Dale et al., ’87a,b). We have also used a glycine antibody to reveal spinal cord commissural neurons, which we know from physiology are the reciprocal inhibitory neurons active during swimming (Dale et al., ’86). We now consider the anatomical and developmental implications of our results.

Neuroanatom y Neurons with GABA immunoreactivity were found

throughout the CNS, but only those in hindbrain and spinal cord are considered here in detail. Fibers and many small somata were well stained in the midbrain and forebrain, but in these positions dendrites and projection patterns were not clear. There were no stained somata in the pineal vesicle; however, since both eyes and nasal epithelia were removed, we cannot be sure whether they contributed stained fibers to the tracts seen in the forebrain. Only further study will resolve these uncertainties. What is needed is staining of later stages so that the identity of tracts, commissures, and cell groups can be established in these regions by following them through development until they can clearly be identified with adult structures.

Even in the hindbrain and spinal cord, characterization of some neuron classes was possible only after examination of their early development. Again exact parallels with nu- clei defined in mature animals is not possible. The features used to characterize cell types were position, size, and time of first staining of the somata; projection pattern of axon or axons; and pattern of dendritic outgrowth. Table 1 summa- rizes the features of the cell classes with GABA activity shown in Figures 2 and 3 as well as the probably sensory Kolmer-Agduhr cells (Dale et al., ’87a,b). Since all the defining features for each group cannot be checked for each cell, soma position and presence or absence of a ventral commissural axon were the most useful features. By stage 37/38 some groups begin to overlap, and in these regions assignment of cells to classes was uncertain. Outlying cells, such as the most rostral midhindbrain reticulospinal cells or the most caudal vestibular complex cells, could belong to nearby groups. Alternatively, they could be unique cells, or the first cells to differentiate in new groups, in either case having a distinct function.

Ascending neurons. Ascending neurons were described by Roberts and Clarke (‘82) together with a second class of propriospinal neuron with ascending axons. These “dorse lateral interneurons’ were distinguished by having multi- polar somata in a more dorsal and superficial position. However, with the GABA antibody spinal neurons were stained that had features of both these cell classes but did not obviously fall into two classes (see Fig. 6). This suggests that the Drevious ascending and dorsolateral neurons may

Y

rostral growing end of the dorsal ascending track is seen at the level of the somata with six growth cones. All stained axons were drawn, D: The most rostral two neurons at stage 33/34, each with a ventral commissural axon and large dendrites. More rostral cell also has an ipsilateral descending axon (partly dotted). E: One neuron at stage 37/38 with ipsilateral and commissural axons and extensive ventrolateral dendrites in marginal zone. In D and E clear outlines are other midhindbrain reticulospinal neurons, filled outlines are vestibular complex commissural neurons, and thin longi- tudinal lines indicate dorsal and ventral extent of stained axons. Scale in B applies to C, D, and E.

be a singie class and that the superficial dorsolateral posi-

Consequently, in Table 1, ascending neurons are now de-

the features of “dorsolateral interneurons.” The present

form a longitudinal column extending along the spinal cord

tion for somata may not be a useful diagnostic feature.

fined rather more broadly so as to incorporate cells with

GABA staining of ascending neurons has shown that they

and up into the hindbrain roughly to the level of entry of

448 A. ROBERTS ET AL.

TABLE 1. Hindbrain and Spinal GABA Neurons at Stage 37138

Stage of first Axon

Type staining Soma projection Dendrites

Kolmer- 25 In spinal Ipsilateral, None (cilia and Agduhr' cord ventrolateral edge of neurocoel, ascending, in microvilli in

ventral half of neurocoel) marginal zone

unipolar

Ascending 26 In spinal cord and caudal hindbrain, Ipsilateral , In dorsal half of dorsolateral, uni- or multipolar ascending, in dorsal marginal zone,

marginal zone dorsal Ventral commissural In middle to Midhindbrain 25 In midhindbrain

reticulospinal (level of 1st postotic somite), descending and ventral middorsoventral in marginal zone, ipsilateral marginal zone, multipolar descending, in extensive

ventral half of mainly ventral marginal zone to soma

Vestibular 29130 In rostra1 half of hindbrain Ventral commissural ? complex (level of otic capsule), descending,

dorsal edge of marginal zone in dorsal half of marginal zone

Rostral 33134 Rostral to nerve V in hindbrain, Ventral ? hindbrain mid-dorsoventral, commissural

? unipolar

'Data from Dale et a1 ('87a).

the vagus. This is also the level to which other classes of spinal neurons extend rostrally - for example "descending interneurons" revealed by retrograde HRP labeling (Rob- erts and Clarke, '82; Roberts and Alford, '86) and "commis- sural interneurons" revealed by a glycine antibody (Dale et al., '86). Lamborghini and Iles ('85) report that commissural neurons have a specific GABA uptake mechanism. How- ever, it is likely that the cells they describe are ascending neurons, since they did not demonstrate that their cells had ventral commissural axons (Roberts and Clarke, '82). We find no spinal ventral commissural axons in preparations treated with GABA antibody but do see such axons and clearly stained commissural neurons in specimens treated with a glycine antibody (Dale et al., '86).

Hindbrain neurons. Moving into the hindbrain, mi- dhindbrain reticulospinal neurons have bilateral descend- ing axon projections. Similar neurons have been retrogradely filled with HRP applied to the spinal cord (van Mier, personal communication) but are not characterized in published studies of reticulospinal projections (van Mier and ten Donkelaar, '84; Nordlander et al., '85). These cells would not be confused with descending neurons, which do not extend as far rostrally into the hindbrain, have dorsal dendrites, and have only ipsilateral axons (Roberts and Alford, '86). Bilaterally projecting reticulospinal neurons have not been seen in zebrafish larvae (Kimmel et al., '82) or in lampreys (Rovainen, '78). Decussating vestibulospinal neurons with somata similar in form and position to the present vestibular complex commissural neurons were filled with HRP by Nordlander et al. ('85) but are not mentioned at this stage by van Mier and ten Donkelaar ('84).

The physiological function of putative GABAergic neu- rons in the spinal cord and hindbrain can be established only by the physiological methods we have applied to other cells (Roberts et al., '86). Rohon-Beard cells and spinal motoneurons have GABA receptors (Bixby and Spitzer, '84; Soffe, '87) and the activation of these is antagonized by 30 pM bicuculline. GABA-dependent responses could therefore

be revealed by bicuculline, coupled with local lesions to the ventral commissures formed by the three hindbrain cell groups (Fig. 3). The dorsal location of the vestibular com- plex commissural neurons suggests that they are associated with reciprocal inhibition related to vestibular-lateralis input via cranial nerves VII, VIII, and X (Herrick, '48). The more ventral location and bilateral descending projections of midhindbrain reticulospinal neurons suggest that these could be excited by trigeminal afferents responding to slow head skin and cement gland indentation, since this type of stimulation usually inhibits swimming (Roberts and Blight, '75; Roberts, '80). However, the role of these and the ascend- ing neurons will depend vitally on whether they are active or silent during swimming, and only intracellular record- ings will give this information.

Development GABA immunoreactivity appears in Xenopus neurons at

the time when they initiate outgrowth of processes. This is also true for GABA in the cerebrospinal fluid-contacting neurons, Kolmer-Agduhr cells (Dale et al., '87a,b), for gly- cine in spinal commissural neurons (Dale et al., '86), and for serotonin in raphe-spinal neurons (van Mier et al., '86). Transmitter synthesis therefore coincides with the start of morphological differentiation of processes. For GABA it is also likely that specific uptake mechanisms (Lamborghini and Iles, '85) and receptors appear at a similar stage (Bixby and Spitzer, '84).

The initial axonal outgrowth is strictly directed for each class of spinal or hindbrain neuron with GABA activity. Axonal outgrowth generally precedes the growth of den- drites. The orientation of axonal growth cones does not simply depend on orientation of the soma, since in the case of midhindbrain reticulospinal neurons a descending ipsi- lateral branch forms at a quite repeatable position (Fig. 10C,E). The specificity of the orientation of initial axon outgrowth has been measured for Xenopus spinal Kolmer- Agduhr cells (Dale et al., '87b). This specificity is also clear

GABA NEURONS IN CNS OF X . Zaeuis 449

for Rohon-Beard neurons (Jacobson and Huang, ’85), com- missural neurons (Jacobson and Huang, ’85; Dale et al., ’86), retinal ganglion cells in the eyecup (Halfter et al., ’85) and brain (Harris, ’861, and peripherally for trigeminal sensory neurites (Davies et al., ’82) and motoneuron axons (Eisen et al., ’86). For the interneurons with GABA activity this precise axon outgrowth orientation is not preceded by any random outgrowth as suggested by Jacobson and Huang (‘85). In general, the immunocytochemical method offers exciting experimental prospects for examination of what factors might influence the direction of initial outgrowth, since specific populations of neurons can be followed from their earliest stages of morphological differentiation.

ACKNOWLEDGMENTS We thank J. Ablett, A.T. Bore, T. Eliassen, I. Fridstrbm,

G.F. Lothe, M. Shannon, K. Stentoft, L.J. Teagle, and J. Line Vaaland for assistance and Drs. R. Nordlander, K.T. Sillar, S.R. Soffe, and P. van Mier for advice. This work was supported by the Science and Engineering Research Coun- cil, the Company of Biologists, The Norwegian Research Council for Science and Humanities, the Norwegian Coun- cil on Condiovascular Disease, and the Norwegian Society for Fighting Cancer.

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