THE JOURNAL OF COMPARATIVE NEUROLOGY 368285-294 (1996)

Immunohistochemical Investigation of y-Aminobutyric Acid Ontogeny

and Transient Expression in the Central Nervous System

of Xenopus laevis Tadpoles

E. BARALE, A. FASOLO, E. GIRARDI, C. ARTERO, AND M.F. FRANZONI Laboratorio di Anatomia Comparata, Dipartimento di Biologia Animale, Universita di Torino,

10123 Torino, Italy

ABSTRACT The ontogeny of the y-aminobutyric acid (GABAI-positive neurons in the brain ofXenopus

laevis tadpoles was investigated by means of immunohistochemistry, using specific antibodies both against GABA and its biosynthetic enzyme, glutamate decarboxylase (GAD). The results obtained with the two antisera were comparable. The GABA system differentiates very early during development. At stages 35 136, numerous GABA-positive neurons were seen throughout the prosencephalon and formed two main bilateral clusters within the lateral walls of the forebrain that ran caudally toward the hindbrain. Other GABA-immunolabeled cell bodies, together with a conspicuous network of GABAergic fibers, were seen in the posterior hypothalamus. In the spinal cord, the lateral marginal zone was GABA-positive, as were Rohon-Beard neurons, interneurons, and Kolmer-Agdhur cells. A very rich GABA innervation was observed in the pars intermedia of the pituitary. At stage 50, plentiful immunopositive neurons and fibers were found in the telencephalic hemispheres, the diencephalon, and the mesencephalon (optic tectum and tegmentum). By stage 54, the number of GABA- immunoreactive neurons in the posterior hypothalamus had decreased, so that, at stage 58, there were very few GABA-labeled cell bodies in the dorsolateral walls of the infundibulum, despite a strong GABAergic innervation within the median eminence and the pars intermedia. From stage 58 to stage 66, the distribution pattern was very similar to that described in the adult X. laeuis and in other amphibian species. These results point to transient GABA expression within the hypothalamus, possibly related to either 1) a naturally occurring cell death or 2) a phenotypic switch.

Indexing terms: development, amphibian CNS, neurotransmitter expression, GABAergic neurons,

o 1996 Wiley-Liss, Inc.

glutamate decarboxylase

The distribution of y-aminobutyric acid (GABA), a wide- spread neurotransmitter in the vertebrate central nervous system (CNS), has been mapped in the adult of several nonmammalian vertebrates (e.g., goldfish: Martinoli et al., 1990; silver eel: Medina et al., 1994; green frog and crested newt: Franzoni and Morino, 1989; salamander: Naujoks- Manteuffel et al., 1994; chameleon: Bennis et al., 1991; pigeon: Domenici et al., 1988). The Xenopus laeuis brain has long been exploited for developmental studies. The only work on GABA, however, is that of Roberts et al. (19871, who observed that GABA-positive neurons appeared in the hindbrain and the midhindbrain by stage 25 and who proposed a morphological classification of these GABAergic systems.

The present paper describes an immunohistochemical investigation of the ontogeny of GABA distribution through- out the CNS ofX. laeuis tadpoles from stage 35 to metamor- phic climax. Variations in GABA immunoreactivity (-IR) and in that of its biosynthetic enzyme, glutamate decarbox- ylase (GAD), were evaluated during development, and the pattern of GABA expression was compared to that in the adult.

Accepted November 17,1995. Address reprint requests to Dr. M.F. Franzoni, Laboratorio di Anatomia

Comparata, Dipartimento di Biologia Animale, Universita di Torino, Via Accademia Albertina, 17, 10123 Torino, Italy.

O 1996 WILEY-LISS, INC.

286 E. BARALE ET AL.

from Dr. M. Tappaz, Centre Hospitalier, Lyon Sud, Lyon, France) diluted 1:500.

IR was revealed by 45-minute incubation in a biotinyl- ated rabbit anti-sheep serum diluted 1:250 followed by a third 45-minute incubation in avidin-Texas red diluted 1500. Sections mounted in glycerol were observed and photographed with a Leitz microscope equipped for both standard transmitted light and fluorescence epiillumina- tion. Distribution maps of labeled structures were built up by comparing immunostained cryostat (or Vibratome) sec- tions to corresponding paraffin sections from tadpoles and brains fixed in Bouin fluid and stained with cresyl violet.

MATERIALS AND METHODS Animals

Eggs were obtained by induced breeding with chorionic gonadotrophin (Serono; Roma, Italy). Embryos were devel- oped at 20°C (k3"C) in aerated tap water and were fed with nettle powder before staging according to Nieuwkoop and Faber (1967).

Thirty tadpoles from stage 35 to stage 50 were anaesthe- tized in 0.1% tricaine methanesulphonate (MS 222; Sandoz, Basel, Switzerland), fixed for 1 hour in 0.1 M phosphate buffer (PB; pH 7.4) containing 4% paraformaldehyde (PFA) plus 0.5% glutaraldehyde or containing 4% PFA alone, and then washed in 0.1 M PB, pH 7.4.

Twenty tadpoles from stage 51 to stage 66 were anaesthe- tized as above. Their brains were rapidly dissected and immersed in the same fixatives for 1 hour.

Three adult X . laeuis were also deeply anaesthetized in 0.1% MS 222 and were transcardially perfused with 0.1 M PB, pH 7.4, containing 4% PFA plus 0.5% glutaraldehyde. Brains were rapidly dissected out and were immersed in the same fixative for 1 hour. Tissues were then cryoprotected in 7.5, 15, and 30% sucrose solutions, embedded in TISSUE- TEK O.C.T. compound (Miles, USA), and frozen in isopen- tane cooled by liquid nitrogen or in powdered dry ice. Serial 12 pm coronal sections, cut with a cryostat, were placed on gelatin-coated slides. Some tadpoles were embedded in 20% agar-agar, cut into 50-70 pn transverse sections with a Vibratome, and treated for immunohistochemistry as float- ing sections.

Immunohistochemistry Auidin-biotin-peroxidase complex (ABC) andperoxidase-

antiperoxidase (PAP) techniques. Sections were first placed in a solution of methanol PB (1:l) containing 0.3% hydrogen peroxide for 10 minutes to inactivate endogenous peroxidase activity. After washing in 0.01 M PB saline (PBS), pH 7.4, they were incubated for 20 minutes in normal goat serum diluted 1:lOO. This was followed by overnight incubation in the specific polyclonal antibody raised in rabbit against GABA (Immunotech, Luminy, Marseille, France) di- luted 1:6,000/1:12,000 or in a specific antibody raised in sheep against GAD diluted 1:2,000 in 0.01 M PBS plus 0.1% Triton X-100. IR was revealed either by ABC or by PAP complex using 3-3' diaminobenzidine (Sigma) as the chromogen.

Indirect immunofluorescence technique (IFL). After washing in 0.1 M PBS for 10 minutes, sections were incubated overnight in normal rabbit serum followed by a polyclonal antiserum raised in sheep against GAD (kind gift

Ch dp FS hab Hy

mp Oh OT P PR R St T Th

Abbreviations

cerebellum dorsal pallium fasciculus solitarii hahenulae hypothalamus infundihulum medial pallium olfactory bulb Optic tectum pituitary preoptic recess raphe septum tegmentum mesencephali thalamus

Specificity tests The specificity of immunoreactions was tested by omit-

ting one of the steps of the immunohistochemical procedure or by replacing the primary antibody with PBS.

RESULTS In the present research, we compared the distributions of

GABA and GAD, being aware that the antibody against GAD possibly identified axon terminals better than cell bodies (Oertel et al., 1981). In our hands, the results obtained with anti-GABA and anti-GAD antisera were generally comparable, except for some discrepancies regard- ing the cerebrospinal fluid (CSF)-contacting neurons in both the hypothalamus and the spinal cord (see below).

For clarity, the results have been grouped at specific critical periods of the embryonic life of X. laeuis (early embryonic stages 35/36, later embryonic stages 42146, premeta- morphic stages 50153, and prometamorphic and metamorphic stages 54/66) according to Nieuwkoop and Faber (1967).

Developing brain of X. laevis The staining of brain

sections was partly masked by abundant yolk platelets. Nevertheless, a number of neurons containing both GABA- and GAD-IR were seen within the lateral walls of the prosencephalic vesicle. Their processes formed a dense immunopositive network in the lateroventral marginal zone. More caudally, two clusters of positive neurons were also observed in the ventral thalamus and the supraehias- matic hypothalamus. After incubation in anti-GABA anti- body, a number of the labeled neurons were of the CSF- contacting type (Fig. la), whereas, after anti-GAD incubation, positive cell bodies were less numerous than GABA-positive cell bodies and did not show any stained CSF-contacting process (Fig. lb). The epiphysis was immunopositive.

In the hindbrain, neurons resembling the reticular neu- rons described by Roberts et al. (1987) formed a cluster of strongly stained elements in the lateral region. The acousti- colateral area was characterized by a dorsal group of immunolabeled neurons.

In the spinal cord, the marginal zone was intensely stained. Some Rohon-Beard neurons (RBI along with CSF- contacting cell bodies (namely, Kolmer-Agdhur cells) were immunopositive. A schematic drawing of stage 35 GABA distribution is shown in Figure 6.

Later embryonic stages (42146). A paired column of GABA-positive cell bodies was found within the lateral walls of the prosencephalic vesicle. In the caudal prosen- cephalon, each column split into two clusters of labeled neurons, one located dorsally (dorsal thalamus) and the other located ventrally (ventral thalamus). A number of GABA-immunopositive neurons were also recognizable

Early embryonic stages (35136).

ONTOGENY OF GABA NEURONS IN AMPHIBIAN BRAIN 287

Fig. 1. Photomontages of the prosencephalon at stage 35 ( ~ 4 0 0 ) . A A number of y-aminobutyric acid (GABAj-immunoreactive fibers and neurons in the suprachiasmatic hypothalamus. Some of the neurons are of cerebrospinal fluid (CSFj-contacting type (arrowheads). B:

Glutamate decarboxylase (GAD)-positive fibers and neurons in a sec- tion adjacent to a. The labeled neurons (arrowheads) do not show any intraventricular processes [avidin-biotin-peroxidase (ABC) method]. hc, Habenular commissure; 3v, third ventricle. Scale bars = 23 pm.

within the lateral walls of the infundibular recess of the hypothalamus. The median eminence and pituitary pars intermedia showed plentiful immunopositive nerve fibers and terminals (Fig. 3a).

Immunoreactive cell bodies and nerve processes were found in the dorsolateral mesencephalic vesicle. More cau- dally, immunopositive reticular neurons were seen in the medulla oblongata, and there was strong labeling in the acousticolateral area.

In the spinal cord, the majority of RB neurons were both GABA and GAD immunopositive (Figs. 5a,b). Nevertheless, in some cases, some of these cells were both GABA (Fig. 5c) and GAD negative. GABA-immunoreactive neurons send- ing dendrites toward the lateral neuropil were seen in the gray dorsal field (Fig. 5c). The small Kolmer-Agdhur cells were intensely labeled (Fig. 5b,c) after anti-GABA staining but were immunonegative after anti-GAD incubation (Fig. 5a). Strong GABA- and GAD-IR was observed within the peripheral white matter (Fig. 5a,c).

In the telencephalic hemispheres, immunopositive neurons and fibers were observed throughout both the dorsal and medial pallium and the basal areas. In the diencephalon, several GABA- positive cell bodies were seen around the preoptic recess and within the habenular nuclei. The stria medullaris in the dorsolateral diencephalic wall was strongly immunoposi- tive (Fig. 2a). The thalamus was characterized by the

Premetamorphic stages (50153).

presence of sparse immunolabeled neurons throughout the dorsal and ventral regions (Fig. 2a).

Plentiful labeled neurons and nerve fibers were detected in the lateral and ventral infundibular walls of the posterior hypothalamus (Fig. 2b). The pars intermedia was inner- vated by GABA fibers and terminals (Fig. 3b).

The mesencephalon was the most richly GABA- and GAD-immunolabeled area. Strongly immunoreactive cell bodies and nerve fibers were found in all layers of the optic tectum (Fig. 4a,b) and tegmentum.

In the spinal cord, the peripheral white matter was characterized by strong immunostaining. GABA-positive cell bodies were generally fewer, except for Kolmer-Agdhur cells, which were always recognizable (Fig. 5d). RB neurons were never seen. A schematic drawing of stage 52 GABA distribution is shown in Figure 7.

Prometamorphic and metamorphic stages (54166). From stage 54, the overall organization of GABA-immuno- reactive neurons was similar to that of the adult. In the olfactory bulbs, a number of small immunopositive cell bodies were observed within the granular layer, and, in the telencephalic hemispheres, a rich population of GABA- immunopositive neurons occupied both the dorsal and the medial pallium. Abundant GABA neurons were also ob- served in the amygdaloid nucleus.

In the diencephalon, some positive cell bodies were found in the pineal gland and in the habenular nuclei. In the

288 E. BARALE ET AL.

Fig. 2. The diencephalon at stage 50 (a,b), stage 56 (c,d), and stage 58 (e). a: The dorsolateral diencephalon. GABA immunoreactivity (-IR) in the stria medullaris (arrowheads) and several cell bodies and fibers within the thalamus (ABC method; x350). b: GABA-immunolabeled neurons and fibers in the walls of the infundibular recess (i; posterior

infundibular recess (posterior hypothalamus). (ABC method; ~ 3 5 0 ) . d GABA-immunopositive fibers and terminals in the median eminence (me; posterior hypothalamus; ABC method; ~ 3 5 0 ) . e: The infundibular wall of the posterior hypothalamus shows a strong reduction in GABA-uositivitv. (ABC method: ~ 5 0 0 ) . PD. uars distalis. Scale bars = I I

hypothalamus; ABC method; ~ 4 0 0 ) . c: Some positive neurons and CSF-contacting processes are seen (arrows) at the caudal end of the

28 pm in a, 23 irn in b, 29 I*rn i, c,d, 20 in e,

infundibular walls of the posterior hypothalamus, GABA- positive neurons began to decrease in number, and, by stage 58, they became very few both in the suprachiasmatic hypothalamus and in the infundibular walls (Fig. Be). At the posterior end of the infundibular recess, GABA-positive CSF-contacting neurons were still present (Fig. 2c). A rich immunopositive innervation was observed in both the median eminence (Fig. 2d) and the pars intermedia.

In the mesencephalon, a strong immunopositivity was found throughout the layers of the optic tectum and in the nucleus mesencephalicus lateralis profundus of the lateral tegmentum. An abundant innervation was observed in both

the acousticolateral area and around the raphe of the medulla oblongata.

From stage 54, the distribution pattern of immunostain- ing in the spinal cord was gradually changed: An increasing number of GABA-immunostained interneurons was observed, and the positivity of the lateral gray fields and Kolmer-Agdhur cells was still observable (Fig. 5e). A schematic drawing of stage 63-GABA distribution is shown in Figure 8.

Adult X. Zaevis pattern Our attention was focused mainly on the hypothalamus.

The preoptic area and suprachiasmatic hypothalamus dis-

ONTOGENY OF GABA NEURONS I N AMPHIBIAN BRAIN 289

Fig. 3. The pituitary gland at stage 42 (a), stage 53 (b), and stage 60 (c). a,b: The intense GABA-positivity within the pituitary pars intermedia (PI; ABC method; a, ~ 3 5 0 ; b, ~ 5 0 0 ) . c: GAD-like- immunoreactive innervation of the PI [indirect immunofluorescence (IFL) method; x3501. Scale bars = 29 ym in a,c, 20 ym in b.

played a few GABA-immunopositive neurons. Immunoposi- tive cell bodies were rare or absent within the posterior hypothalamus. By contrast, plentiful GABA fibers and terminals were observed in the median eminence and the pars intermedia (data not shown).

DISCUSSION This paper describes the ontogeny of GABA-immunoposi-

tive neurons in the CNS ofX. laevis, as demonstrated by the use of anti-GABA and anti-GAD antibodies.

Comparison between anti-GABA and anti-GAD immunostainings

Two forms of GAD, each encoded by a different gene, have been identified recently (Erlander et al., 1991) in the mamma- lian CNS. GADB5-IR was located predominantly in axon t e e - nals, whereas GADB7 was found mainly in the cell bodies and proximal dendrites (Eslapez et al., 1994). The antibody against GAD used in the present work (Oertel et al., 1981) primarily recognizes GADB5. In our hands, however, the antibodies against GAl3A and GAD gave comparable results. Moreover,

290 E. BARALE ET AL.

Fig. 4. The mesencephalon at stage 52 (a,b). GABA-like (a) and GADlike (b) immunopositivity within the optic tectum ofthe mesencephalon. (a: ABC method; x500; b: IFL method ~ 5 0 0 ) . OL, optic layer; SGF, superficial gray and fiber layer; CG, central gray layer; PG, periventricular gray layer. Scale bars = 20 pm.

immunopositivities for both were present by stage 35. The main differences concerned some CSF-contacting neurons in the hypothalamus and the Kolmer-Agdhur cells in the spinal cord. Because both types were immunopositive for GABA, but not for GAD, they may be not able to synthesize GABA, and they may have to take it from the CSF. In vivo and in vitro studies of spinal interneurons of X. laevis at stage 24, in fact, have demonstrated the presence of a high-affinity GABA- uptake system in these cells (Lamborghini and Iles, 1985).

GABA pattern in ontogeny At stage 35, the GABA-positive system was well devel-

oped, with a conspicuous number of strongly immuno- stained neurons in a bilateral column running through the lateral wall of the forebrain towards the hindbrain. A very rich innervation plus GABA-immunopositive neurons, a number of which were of the CSF-contacting type, were observed in the ventral thalamus and hypothalamus. By stages 4246, GABAergic nerve fibers and terminals occurred in both the median eminence and the pars intermedia.

In the spinal cord, in addition to a strongly positive lateral marginal zone, the RB cells were both GABA and GAD immunostained. However, because some were nega- tive after both stains, two subsets can be postulated. GABA-positive interneurons, with dendrites ventrolater- ally oriented, were seen in the lateral fields of the gray matter. The small CSF-contacting neurons, which have been described by Dale et al. (1987) as GABAergic elements and named Kolmer-Agdhur cells, did not show any GAD immunostaining.

By stage 50, the GABA system was extended within the mesencephalon, which was the richest GABA-IR area of the brain. From stage 54, the number of GABA-positive neu- rons in the posterior hypothalamus decreased, and, from stage 58, this region usually displayed very few GABA- immunoreactive neurons (with the exception of some CSF- contacting neurons confined within the posterior end of the infundibular recess), whereas a rich GABAergic innerva- tion was exhibited by both the median eminence and the

pars intermedia. From this stage to the metamorphic climax, the topographical distribution of GABA-containing neurons and fibers, in general, is similar to that observed in adult X. Zaeuis (present observations) and in other amphib- ian species, such as the green frog and the crested newt (Franzoni and Morino, 1989).

Transient expression of GABA-IR Our observations have shown that the distribution of

GABAergic neurons in some cerebral areas (e.g., the hypo- thalamus) underwent substantial variations during the embryonal development. In the posterior hypothalamus, GABA-IR decreased and eventually disappeared near the metamorphic climax. Because GABA-immunopositive neu- rons were rare or absent in this region in the adult, expression of GABA could be transient. On the other hand, a transient GABA-IR (but not GAD-IR) has been found in retinal horizontal cells of rat during postnatal development (Versaux-Botteri et al., 1989). Ma et al. (1992) have ob- served that the GABA expressed during the last week of embryogenesis by most rat spinal neurons (including motor neurons, relay neurons, and interneurons) disappeared during the first 2 postnatal weeks. The transient presence of GABA in motor neurons and motor nerves has also been reported in the developing chick nervous system (Von Bartheld and Rubel, 1989).

Mechanisms underlying transient GABA expression

The reduction of GABA-IR in some CNS regions may be related to 1) a programmed cell death or 2) a phenotypic switch.

Programmed cell death or apoptosis is a major mecha- nism in the regulation of neuron number and tailoring of neuronal organization during development (for review, see Oppenheim, 1991). In monkey cerebral cortex, however, the transient GABA-positive cells belong to the subplate zone, which, itself, is a transient zone (Kostovic and Rakic,

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Fig. 6. a-f: Schematic distribution of GABAiGAD immunolabeling in some representative transverse sections of the stage 35 brain ofXenopus Zaeuis. Triangles, neurons; dots, nerve fibers and terminals. Scale bar = 100 Fm.

Fig. 7. a-i: Schematic distribution of GABAiGAD immunolabeling in some representative transverse sections of the stage 52 brain ofX. Zaeuis. Triangles, neurons; dots, nerve fibers and terminals. Scale bar = 300 pm.

ONTOGENY OF GABA NEURONS IN AMPHIBIAN BRAIN 293

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Fig. 8. a-i: Schematic distribution of GABAiGAD immunolabeling in some representative transverse sections of the stage 63 brain of X. laeuis. Triangles, neurons; dots, nerve fibers and terminals. Scale bar = 380 Fm.

1990; Meinecke and Rakic, 1992). RB neurons (mostly GABA-immunopositive in the spinal cord of X . laeuis; present work) declined by about 50% from stage 48 on- wards and eventually disappeared, as in other vertebrate species (Hughes, 1957; Trevisan, 1972; Eichler and Porter, 1981; Forehand and Farel, 19821, through cell death, their function as primary sensory neurons being assumed by dorsal root ganglion cells (Lamborghini, 1987). On the other hand, pyknotic nuclei, possible markers of cell death, were observed in the hypothalamus of X. laeuis from stage 49 (unpublished personal observations).

Mechanisms other than cell death could also be respon- sible for transient GABA expression. Ma et al. (1992) have observed that the death of rat spinal motor neurons is preceded by regression of their GABA-IR, possibly due to their decreasing ability to synthesize GABA. In the retinal horizontal cells, the disappearance of GABA-IR is concomi- tant with full maturation, i.e., the acquisition of specific neuronal properties, and occurs no later than postnatal day 15, when pyknotic nuclei have not yet appeared (Versaux- Botteri et al., 1989). Phenotypic plasticity, i.e., a switch of the neurotransmitter expression, as observed in the central and peripheral nervous systems (see for example, Landis, 19901, could partly account for reduction of GABA in the hypothalamus.

Functional meaning of GABA transience Observations on mammalian brain have indicated that

the role of some neurotransmitters during ontogeny is completely different from that in the mature brain (Mangoura and Vernadakis, 1988; De Vitry et al., 1991; Ma et al., 1992). In addition to acting as neuromodulators, in

fact, they serve as trophic factors regulating the develop- ment, maintenance, and plasticity of neuronal morphology (Lipton and Kater, 1989). GABA has attracted considerable attention in this respect: In vitro experiments have shown that it facilitates synaptogenesis (Wolff, 1978; Wolff et al., 1993), stimulates (like the nerve growth factor) the migra- tion of neurons to their final position (Behar et al., 19941, and promotes neurite outgrowth and differentiation (Spoerri and Wolff, 1981). In particular, its trophic effect on neuro- nal development was suggested by Lauder et al. (19861, who showed that the trajectories taken by growing rat brain GABAergic fibers by embryonic day 13 are located within regions of both monoaminergic and peptidergic neuronal differentiation and correspond to the slightly later patterns of benzodiazepine receptors (Schlumpf et al., 1983). Ma et al. (1992) have demonstrated that, in rat spinal cord, the early presence of GABA coincides with the period of axon outgrowth, and Michler (1990) observed that axon growth was stimulated by exogenous GABA in chick tectal neurons and rat cortical neurons.

A possible developmental role played by GABA itself is also supported by the findings that GABA-IR is detectable in rat cerebral cortex before the appearance of GAD-IR (Lauder et al., 1986) and that the retinal horizontal cells are transiently GABA, but not GAD, immunoreactive (Versaux- Botteri et al., 1989; Lake, 1994).

The mechanisms by which GABA (like other transmit- ters) exerts neurotrophic effects remain to be defined. The finding (Thoenen, 1991) that synthesis of both the ciliary and the brain-derived neurotrophic factors in specific CNS neuron populations is up-regulated by glutamate and down- regulated by GABA may be highly relevant in this respect.

294 E. BARALE ET AL.

ACKNOWLEDGMENTS The authors thank Claudia Andreone and Claudio Gen-

dusa for their technical assistance and Franco Scaranari for the photographic work. This study was supported by MURST grant (60%) to M.F.F.

LITERATURE CITED Behar, T., A.E. Schaffner, C.A. Colton, R. Somogyi, Z. Olah, C. Lehel, and

J.L. Barker (1994) GABA-induced chemokinesis and NGF-induced che- motaxis of embryonic spinal cord neurons. J. Neurosci. 14:29-38.

Bennis, M., A. Calas, M. Geffard, and H. Gamrani (1991) Distribution of GABA immunoreactive systems in the forebrain and midbrain of the chameleon. Brain. Res. Bull. 26t891-898.

Dale, N., A. Roberts, O.P. Ottersen, and J. Storm-Mathisen (1987) The morphology and distribution of “Kolmer-Agdhur cells,” a class of cerebrospinal-fluid-contacting neurons revealed in the frog embryo spinal cord by GABA immunocytochemistry. Proc. R. SOC. London B 232: 193-203.

De Vitry, F., J. Hillion, J. Catelon, J. Thibault, J.J. Benoliel, and M. Hamon (1991) Dopamine increases the expression of tyrosine hydroxylase and aromatic amino acid decarboxylase in primary cultures of fetal neurons. Dev. Brain. Res. 59:123-131.

Domenici, L., H.J. Waldvogel, C. Matute, and P. Streit (1988) Distribution of GABA immunoreactivity in the pigeon brain. Neuroscience 25:931-950.

Eichler, V.B., and R.A. Porter (1981) Rohon-Beard cells in frog development: A study of temporal and spatial changes in a transient cell population. J. Comp. Nenrol. 203:121-130.

Erlander, M.G., N.K. Tillakaratne, S. Feldblum, N. Patel, and A.J. Tobin (1991) Two genes encode distinct glutamate decarboxylases. Neuron 7:91-100.

Eslapez, M., N.K. Tillakaratne, D.L. Kaufman, and A.J. Tohin (1994) Comparative localization of two forms of glutamic acid decarboxylase and their mRNAs in rat brain supports the concept of functional differences between the forms. J. Neurosci. 14:1834-1855.

Forehand, C.J., and P.B. Fare1 (1982) Spinal cord development in anuran larvae: 11. Ascending and descending pathways. J. Comp. Neurol. 209:395408.

Franzoni, M.F., and P. Morino (1989) The distribution of GABA-like- immunoreactive neurons in the brain of the newt, Triturus cristatus carnzfex, and the green frog, Kana esculenla. Cell Tissue Res. 255: 155-166.

Hughes, A.F.W. (1957) The development of the primary sensory neurons in Xenopus laeuis (Daudin). J. Anat. 91:323-338.

Kostovic, I., and P. Rakic (1990) Developmental history of the transient subplate zone in the visual and somatosensory cortex of the Macaque monkey and human brain. J. Comp. Neurol. 297,441-470.

Lake, N. (1994) Taurine and GABA in the rat retina during postnatal development. Vis. Neurosci. 11,253-260.

Lamborghini, J.E., and A. Iles (1985) Development of a high-affinity GABA uptake system in embryonic amphibian spinal neurons. Dev. Biol. 112:167-176.

Lamborghini, J.E. (1987) Disappearance of Rohon-Beard neurons from the spinal cord of 1arvalXenopus laeuis. J. Comp. Neurol. 264:47-55.

Landis, S.C. (1990) Target regulation of neurotransmitter phenotype. Trends Neurosci. 13,344-350.

Lauder, J.M., V.K.M. Han, P. Henderson, T. Verdoorn, and A.C. Towle (1986) Prenatal ontogeoy of the GABAergic system in the rat brain: An immunocytochemical study. Neuroscience 19.465493.

Lipton, S.A., and S.B. Kater (1989) Neurotransmitter regulation of neuronal outgrowth, plasticity and survival. Trends Neurosci. 12265-270.

Ma, W., T. Behar, and J.L. Barker (1992) Transient expression of GABA immunoreactivity in the developing rat spinal cord. J. Comp. Neurol. 325271-290.

Mangoura, D., and A. Vernadakis (1988) GABAergic neurons in cultures derived from three-, six- or eight-day-old embryo: A biochemical and immunocytvchemical study. Dev. Biol. Res. 4Ot25-35.

Martinoli, M.G., P. Duhourg, M. Geffard, A. Calas, and 0. Kah (1990) Distribution of GABA-immunoreactive neurons in the forebrain of the goldfish Carassius auratus. Cell Tissue Res. 260:77-84.

Medina, M., J. Reperant, S. Dufour, R. Ward, N. Lebelle, and D. Miceli (1994) The distribution of GABA-immunoreactive neurons in the brain of the silver eel (Anguilla anguilla I,.). Anat. Embryol. 189:25-39.

Meinecke, D.L., and P. Rakic (1992) Expression of GABA and GABA A receptors by neurons of the subplate zone in developingprimate occipital cortex: Evidence for transient local circuits. J. Comp. Neurol. 31 7: 91-101.

Michler, A. (1990) Involvement of GABA receptors in the regulation of neurite growth in cultured embryonic chick tectum. Int. J. Dev. Neuro- sci. 8:463-472.

Naujoks-Manteuffel, C., W. Himstedt, and G. Glasener-Cipollone (1994) Distribution of GABA-immunoreactive neurons in the brain of adult and developing salamanders (Pleurodeles Waltlii, Triturus alpestris). Cell Tissue Res. 276:485-501.

Nieuwkoop, P.D., and J. Faber (1967) Normal Tables of Xenopus laeuis (Daudin), 2nd Edition. Amsterdam: North-Holland Publishing Company.

Oertel, W.H., D.E. Schmechel, M.L. Tappaz, and I.J. Kopin (1981) Produc- tion of a specific antiserum to rat brain glutamic acid decarboxylase by injection of an antigen-antibody complex. Neuroscience 6:2689-2700.

Oppenheim, R.W. (1991) Cell death during development of the nervous system. Annu. Rev. Neurosci. 14:454-501.

Roberts, A,, N. Dale, O.P. Ottersen, and J. Storm-Mathisen (1987) Theearly development of neurons with GABA immunoreactivity in the CNS of Xenopus laeuis embryos. J. Comp. Neurol. 261,435-449,

Schlumpf, M., J.G. Richards, W. Lichtensteiger, and H. Mohler (1983) An autoradiographic study of the prenatal development of benzodiazepine- binding sites in rat brain. J. Neurosci. 3:1478-1487.

Spoerri, P.E., and J.R. WolK (1981) Effect of GABA-administration on murine neurohlastoma cells in culture. Cell Tissue Res. 21 8567-579.

Thoenen, H. (1991) The changing scene of neurotrophic factors. Trends Neurosci. 14: 165-1 70.

Trevisan, P.L. (1972) Osservazioni sulle cellule di Rohon-Beard in un Anfihio urodelo in sviluppo. Rend. Ace. Naz. Lincei 52965-969.

Versaux-Botteri, C., R. Pochet, and J. Nguyen-Legros (1989) Immunohisto- chemical localization of GABA-containing neurons during postnatal development of the rat retina. Invest. Ophthalmol. Vis. Sci. 30:652-659.

Von Bartheld, C.S., and E.W. Ruhel(1989) Transient GABA immunoreactiv- ity in cranial nerves of the chick embryo. J. Comp. Neurol. 286:456471.

Wolff, J.R. (1978) Plasticity in dendrites shown by continuous GABA administration in superior cervical ganglion of adult rat. Nature 274: 72-74.

Wolff, J.R., F. Job, and P. KAsa (1993) Modulation hy GABA of neuroplastic- ity in the central and peripheral nervous system. Neurochem. Res. 18:453-461.