Distribution of GABAergic neurons in late fetal and early postnatal rat hippocampus

Developmental Brain Research, 50 (1989) 177-187

177

Elsevier BRESD 50979

Distribution of GABAergic neurons in late fetal and early postnatal rat hippocampus F. Rozenberg, O. Robain, L. Jardin and Y. Ben-Ari INSERM U29 Maternit~ de Port-RoyaL Paris (France)

(Accepted 23 May 1989) Key words: ~,-Aminobutyric acid-immunocytochemistry; Hippocampus; Development

The ontogenesis of GABAergic neurons in the rat hippocampus was studied using an anti-GABA serum. GABA immunoreactivity appeared at the 18th day of gestation. At this stage, GABA-immunoreactive (GABA-IR) cells are grouped in two layers, one located deeply in the intermediate zone near the ventricular zone, and the other found superficially in the marginal zone near the hippocampal fissure. During the late embryonic and early postnatal life, GABA-IR neurons progressively disappeared from these two layers. The transient appearance of an abundant network of GABAergic neurons might be due to transient expression of GABA in some neurons or to cellular death. Later on, from the third postnatal day, the GABA-IR neurons appeared throughout the whole hippocampus according to a dorsoventral and lateromedial gradient. The setting of neuronal bodies preceded that of GABA-IR puncta (supposed to be mainly synaptic boutons) around the neuronal cell bodies and along the dendritic shafts. The puncta are only visible from the sixth day onwards and their number increased rapidly during the first 3 postnatal weeks. Our results indicate that GABA may have a role in neurotransmission in the hippocampus from a very early stage of development.

INTRODUCTION ~,-Aminobutyric acid ( G A B A ) is a major inhibitory neurotransmitter in the mammalian brain. The distribution of G A B A e r g i c neurons in the hippocampus of the adult rat is well documented on the adult animal. It has been studied using immunocytochemical methods with antibodies directed against glutamic acid decarboxylase ( G A D ) 21, or G A B A itself 2'1°'18'27'28"31. G A B A e r g i c neurons are found in all layers, particularly outside the pyramidal and granular layer (in the stratum oriens, stratum radiatum, the molecular layer of dentate gyrus and the hilus). In contrast, there is little information concerning the ontogenesis of G A B A e r g i c neurons. Biochemical data in G A B A and G A D activity during development have been reported a. Autoradiographic studies using [3H]thymidine labelling 4'12'24'2s indicate that the neurons of stratum oriens and stratum radiatum, known to consist mainly of G A B A e r g i c

interneurons, are generated between embryonic (E) day E l 5 and E l 7 , i.e. two days before appearance of the A m m o n ' s horn pyramidal neurons ( E l 7 and El9). The neurons in molecular layer of dentate gyrus and of hilus are also generated between E l 5 and E l 9 , i.e. before the granule cells of the dentate gyrus. More recent studies, using a combination of [3H]thymidine autoradiography and G A D immunocytochemistry have confirmed the early ontogenesis of hippocampal G A B A e r g i c neurons 1,14'29, that are generated from the 13th to the 15 th embryonic day. Nevertheless, the precise distribution of G A B A e r g i c neurons in the late embryonic and early postnatal life is still unclear. Recently a study from Seress and Ribak 26 has provided some information on the early distribution of G A B A e r g i c neurons in the rat hippocampal formation only after the fourth postnatal day. In the present study, using an a n t i - G A B A serum, we have analyzed in detail the distribution of G A B A e r g i c neurons from E17 and examined the

Correspondence: Y. Ben-Aft, INSERM U29, Maternit6 de Port-Royal, 123 Bd. Port-Royal, 75014 Paris, France.

0165-3806/89/$03.50 (~) 1989 Elsevier Science Publishers B.V. (Biomedical Division)

178 temporal relation between the genesis of GABAergic neurons and their expression of G A B A inside the neuronal cell bodies. MATERIALS AND METHODS

Animals and tissue preparation Wistar rat fetuses and pups were used. For the prenatal study, the day after mating was considered as E0 (birth occurred at E22). Fetuses were examined at E17, El8, E20, E21. For the postnatal (P) study, pups were examined at P0, P3, P6, P l l , P18 (3-5 rats were studied at each of these ages). Embryos were removed from the mother under nembutal anaesthesia. Fetuses and young pups were anesthetized with ether, then perfused intracardially with a 0.1 M cacodylate buffer (pH 7.4) containing 3% glutaraldehyde. After perfusion, brains were removed and kept overnight in the same fixative at 4 °C, then washed in buffer solution. The hippocampus was cut into 1-mm thick blocks along frontal and horizontal planes. The blocks were dehydrated in alcohol and in two acetone baths, before araldite embedding. The polymerization was performed at 56 °C for 48 h. After embedding, the blocks were cut serially (1 p m thick), and mounted on gelatinecoated slides. One slide out of each group of 5 was stained with Toluidine blue, and the 4 following ones were used for immunocytochemistry.

M Tris buffer (5 rain). Slides were subsequently incubated overnight at room temperature with a 1/200 dilution of rabbit antiserum raised against BSA-glutaraldehyde-GABA reduced (Immunotech Marseille France) then incubated in goat amirabbit serum (1/100 for 45 rain at room temperature) and later in peroxidase-antiperoxidase 1/200 for I h at room temperature. Slides were reacted with diaminobenzidine tetrahydrochloride containing 0.01% H20 2 for 20 min exposed to 0.05% osmium tetroxide for 20 min. The sections were finally dehydrated and coverslipped. The reaction specificity was checked with two procedures. In one case, the primary antibody was omitted, in the other case, it was replaced by a serum which had been incubated with G A B A : in both cases, there was no selective staining of neurons.

Quantitative analysis Coronal sections from the middle of dorsal hippocampus were used for the quantitative analysis. Five to ten sections taken from 3-5 rats were counted, and only sections that displayed good immunostaining were used. In order to normalize data and to reduce variability between sections, the results from each hippocampai area studied were expressed as a percent of the total number of GABAergic neurons found in the entire hippocampus. Such an approach was used for all developmental ages investigated.

Immunocytochemical staining In order to remove the araldite, the slides were processed as follows: they were dipped in a 6% solution of sodium methylate (30 min), then, in a sequential fashion in a 1:1 methanol-benzene solution (2 min), in pure acetone (2 min twice), in distilled water (5 min twice), in 2 mM CaC12 + 0.02 M Tris (pH 7.6) at room temperature (5 min twice), in 2 mM CaCI 2 + 0.02 M Tris plus pronase (Sigma) 0.001 mg/ml at 37 °C (15 min), and in 0.1 M Tris buffer (pH 7.6) at 37 °C (5 min). The slides were then exposed to 3% H202 for 10 min to block non-specific activity, and exposed to a solution of 0.1

RESULTS

Morphological characteristics of hippocampal development Although the principal features of the development of hippocampus have been described in recent studies 4'24'25, in order to facilitate the understanding of the main features of the present investigation, it is useful to give a brief outline. The primordium hippocampi develops from the dorsomedial wall of the telencephalon at E16-E17. At this early stage, the limits of hippocampus are

Fig. 1. E17: coronal section, x 150. A: Toluidine blue: the pyramidal layer (PL) is quite visible, but the dentate area (DA) is still immature without clearcut granular layer. Notice the widening of the marginal zone at the hippocampal boundary (arrow). B: immuno-GABA: notice the complete absence of G A B A immunoreativity in the DA compared to the adjacent cortex. Note also the strong labelling of inner part of marginal zone.

"..-,I

181 defined by thinning of the ventricular zone and widening of the marginal one x7. From E16 to El7, the dorsomedian wall of telencephalon curves towards the lateral ventricles, and this progressive infolding of primordium hippocampi delineates the hippocampal fissure (Fig. 1A). At El8, the main structures are clearly recognizable on horizontal sections (Fig. 2A). The ventricular zone, consisting of a tightly packed pseudostratified cell layer, reaches its peak volume. The intermediate zone containing large numbers of migrating neurons, stretched from the ventricular zone to the pyramidal layer. It consists of a thin lamina that develops along a subiculo-dentate gradient. The outer zone is the marginal zone which is larger in the hippocampal formation than in the rest of neocortex. The hippocampal fissure delineates the area dentata from the pyramidal layer of Ammon's horn. At this stage, the area dentata consists of a rich cellular germinal zone. From E l 8 onwards, the ventricular zone and the intermediate zone rapidly disappear, the former being reduced to one cell layer at E20, and the latter gradually restricted to the stratum oriens as soon as P0. The pyramidal layer thickens and becomes prominent from E20 onwards. As early as P3, the apical dendrites of the pyramidal neurons are sufficiently individualized to constitute a stratum radiatum. The marginal zone, initially rich in cells, is progressively replaced by the parvicellular stratum lacunosum moleculare. The dentate gyrus appears following dorsoventral and lateromedial gradients 4'24'2s. At E20, the suprapyramidal limit is individualized, and at P3, the infrapyramidal limit is visible.

GA BA-immunocytochemistry (1) Hippocampus proper At El7, GABAergic neurons were numerous in the subiculum, but not visible in the hippocampus proper. At El8, GABA-labelling was observed in the hippocampus itself: immunoreactive neurons

were principally found in two layers (Figs. 1B, 2B). The first layer was in the intermediate zone (near the ventricular zone) where GABA-immunoreactive (GABA-IR) neurons were mostly directed in a horizontal plane, parallel to the underlying ventricular surface. At some distance from the ventricle, orientation of the GABA-IR neurons was less regular. Many large neurons showed a GABAimmunoreactive thick primary dendrite with initial bifurcation. The second layer of GABA-IR cells was in the marginal zone where the density of labelling was particulary marked in its inner part. Nearly all visible cell bodies were immunoreactive. Immunoreactive processes transected horizontally or transversally formed with these cell bodies a very dense GABAergic network. In contrast, the labelling was almost nil in the superficial part of the marginal zone. A few GABA-IR neurons were observed in the pyramidal layer, between the somata of the pyramidal cells of the CA1 Superior Regio. Fig. 2B from sections taken at El8 shows only sparse GABA-IR cells near the hippocampal fissure in the area that will become the molecular layer of the fascia dentata. From El8 onwards, in the Ammon's horn, the GABA-IR neurons progressively disappeared from the intermediate and marginal zones, and gradually appeared in the whole hippocampus. As the size of the intermediate zone decreased markedly, immunoreactive cell bodies became sparser near the ventricular zone, while they were more numerous near the pyramidal layer. At P3, GABA-positive cells were not found in the intermediate zone although they were present in the well-defined stratum oriens (Fig. 3C). These cells are round or spindle-shaped with their body oriented in a horizontal plane along the pyramidal layer. During the same period, GABA-IR cells moved away progressively from the hippocampal fissure. At P3, as the space separating the pyramidal layer from the hippocampal fissure increases, GABA-IR neurons were only found in the lower half of stratum

Fig. 2. El8: horizontal section, x250. A: Toluidine blue: several mitoses are visible in the ventricular zone (VZ), bundles of fibers pass through the intermediate zone (double arrow). The pyramidal layer (PL) is clearly indicated, while the granular layer cannot be seen in the immature DA. B: immuno-GABA; immunoreactive neurons are mainly concentrated in two layers: the superficial one in the marginal zone (MZ), the deeper one outside the V Z (mainly made of horizontal neurons).

183

Fig. 4. Hippocampus at postnatal day P18. Coronal section, x 45. Adult pattern of distribution of GABAergic cells. Notice the disappearance of the fetal labelling around the hippocampal fissure. GABAergic neurons are distributed throughout the whole hippocampus.

radiatum, while the stratum lacunosum moleculare included occasional immunoreactive cell bodies, mainly found along the hippocampal fissure.

(2) Fascia dentata G A B A - i m m u n o r e a c t i v i t y appeared later in the dentate area than in the A m m o n ' s horn. As shown in Fig. 2B, the dentate area differed sharply from the hippocampus proper, since in the former at E18 there was a complete lack of G A B A labelling. The first labelled cells were observed at E20, in

the molecular layer and were opposite to the hippocampal fissure (Fig. 3A), while the hilus itself included very few G A B A - I R cells, situated mostly in the suprapyramidal blade. The labelling of the molecular layer, already clear at P0 (Fig. 3B), diminished in density at P3 (Fig. 3C), while it increased in the hilus (following the development of the fascia dentata). At P0, most of the G A B A - I R neurons in the hilus were found along the dorsal segment of the granule cell layer.

Fig. 3. E20: hippocampal fissure and dentate area. A: E20 coronal section, x 120. GABA neurons are numerous around the hippocampal fissure, inside the MZ and in the molecular layer of DA. B: P0 coronal section, x 120. Notice progressive decrease in labelling outside the hippocampal fissure and accumulation in stratum radiatum (SR). Progressive increase in labelling in the upper part of DA was present. C: P3 coronal section, x 120. Notice progressive decrease of density labelling in SR, and in presence of GABA neurons in the suprapyramidal part of DA, especially below the granular layer, forming a polymorphous layer (arrow).

4~

185 100

considerably, and at P18, an adult pattern was visible (Fig. 5B), with every pyramidal neuron surrounded by about 20 puncta.

80 60 40

Quantitative results 20

iz 0

El8 E20 PO P3 P6 PII PI8 Fig. 6. Percentage of the total number of GABAergic neurons found in the entire hippocampus in each area. SO + IZ, stratum oriens + intermediate zone; SP, stratum pyramidale; SR + LM, stratum radiatum + lacunosum molecularis; MDG, molecular of the dentate gyms; FD, fascia dentata; H, hilus.

At P3, the GABA-IR polymorph layer was conspicuous (Fig. 3C). It was present along the dorsal and ventral segment of fascia dentata at P6, when some GABAergic pyramidal basket cells were clearly identified, with their dendrites running between the granule cells. In the center of the hilus, labelled neurons appeared first in the suprapyramidal area (P0), then in the infrapyramidal area (P3).

(3) Developmental gradient Cell analyses along coronal and horizontal planes during development demonstrated two gradients in the appearance of the GABA-IR cells, a finding similar to those previously described for pyramidal and granular neurons 4'24'25. In Ammon's horn, GABA-IR neurons followed mainly a lateromedial gradient (Fig. 1B). In the area dentata (Fig. 3A, B,C), they followed instead a dorsoventral gradient (E20-P6). Adult pattern was observed from P18. At this age GABAergic cells were distributed throughout the whole hippocampus (Fig. 4).

(4) GABA-IR puncta We have also examined the changes in GABApositive puncta, presumably synaptic boutons, during development. As shown in Fig. 5A, at P6, most pyramidal or granular neurons had few if any puncta around their somata. Occasionally, a neuron was surrounded by 6-10 GABA-positive puncta. At P l l (not shown), the number of puncta had increased

Fig. 6 indicates the evolution of the distribution of GABAergic neurons in the different areas of the hippocampus. It shows the decrease of the percentage of GABAergic neurons accumulated in the intermediate zone after E18, and the decrease of the percentage of GABAergic neurons accumulated in the stratum radiatum after P3. The appearance of GABAergic neurons in the dentate area is delayed. The main events occurred before the third day of life. DISCUSSION

The first conclusion in the present study is that GABA-IR neurons were absent in the rat hippocampus at E17, but appear at E18 (two days later than in the neocortex13). This is a novel finding since previous work based on the use of G A B A or GAD antibodies did not examine tissue before P4 (ref. 26). Previous studies based on radioactive labelling of DNA have shown that neurons which will eventually express G A B A are mostly generated between E13 and E15 (refs. 1, 14, 29). There is a 2-4-day lag between the last mitosis and phenotypic expression of the neurotransmitter. This lag may be due to the lack of sensitivity of the method or to a delayed expression of G A B A in the hippocampal inhibitory intemeurons. The GABA-IR neurons were found to be first distributed in two layers, a deep one in the intermediate zone beneath the cortical plate and a superficial one in the marginal zone. This distribution is reminiscent of that seen in the whole neocortex 13 and especially in the visual cortex 6'3°. In this latter area, the cells initially present in the subplate and in the marginal zone are the first to be generated TM. Like in the visual cortex, these two populations of hippocampus GABA-IR neurons

Fig. 5. Pyramidal layer in the CA3 area. A: P6, x 600. Only a few puncta are visible around the neuronal soma of pyramidal cells. One non-immunoreactive pyramidal neuron surrounded by several immunoreactive puncta is indicated (arrow). B: P18, x 600. Many puncta are visible around all the neuronal soma and proximal dendrites.

186 progressively disappeared from E20 in the intermediate zone, and from P0 in the marginal zone. Various hypotheses may account for this regression. The simplest one is that the density of neurons was decreased by the tremendous increase in size of the cortex in the neonatal period. A n o t h e r possibility is a transient expression of G A B A in some neurons of the developing bippocampus. This phen o m e n o n has already been proposed for other neurotransmitters 19'2°. Nevertheless it is likely that at least part of these early G A B A e r g i c neurons disappeared because of death in the course of maturation. Although cell death is rarely seen in the normal developing hippocampus, we have found some evidence of necrotic cells at E20 and P0 in the molecular layer. Y a m a m o t o has shown with the Fink-Heimer method patterns of degeneration in the rat hippocampus between P0 and P10 (ref. 32). Such a regression has also been observed in the case of Cajal Retzius cells in the molecular layer of the neocortex 9'22. In the visual cortex Luskin and Shatz x5 have shown that cells appearing first are mostly transient and may provide a cellular framework for the development of the cortical plate. Interestingly an earlier study 23 has shown that there is a larger percentage of G A B A - I R cells among E l 8 dissociated hippocampal cells grafted into the adult hippocampus than in the host hippocampus. Since in this condition the graft has little connection with the host, the establishment of normal circuitry is seemingly associated with a reduction in the number of G A B A - I R cells in the hippocampus. Whatever the precise mechanism of their regression, eventually the gradient of final

location of G A B A - I R neurons followed the pattern previously shown for the pyramidal and granular neurons 4"24"25. They appeared first in A m m o n ' s horn following a lateromedial gradient, then in the area dentata following mainly a dorsoventral gradient between E20 and P6. Another important conclusion of the present study is that, in spite of the presence of a large population of G A B A - I R neurons during the early postnatal period, there were no visible puncta which are supposed to be axon endings on the neuronal somata. The presence of neuronal bodies and their processes precedes that of axosomatic contact which are demonstrable only during the second or third postnatal week 16. Recent in vitro studies on the neocortex 3'11 suggest that a non-synaptic G A B A release from growth cones may precede G A B A e r g i c synaptogenesis. Interestingly, a recent study on hippocampal slices indicates that until P4-P5, G A B A acting on G A B A A receptors has an exclusively depolarizing action s. At this stage, intracellular recordings reveal the presence of spontaneous and evoked giant synaptic depolarizing potentials which are mediated by G A B A and modulated by presynaptic N M D A receptors. Application of bicuculline or picrotoxin blocks completely spontaneous or evoked excitatory responses, indicating that, in fact, at this early developmental stage, G A B A mediates all excitatory activity. The release of G A B A with such a prominent excitatory activity during a restricted period of development may represent a significant signal for cell growth and differentiation.

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