Anat Embryo1 (1990) 181:533-545 Anatomy and Embryology �9 Springer-Verlag 1990

Postnatal development of nonpyramidal neurons in the rat hippocampus (areas CA1 and CA3): a combined Golgi/electron microscope study Uwe Lang 1. and Michael Frotscher 2

1 Institute of Anatomy, University of Frankfurt/Main, Theodor-Stern-Kai 7, D-6000 Frankfurt/Main, Federal Republic of Germany 2 Institute of Anatomy, University of Freiburg, Albertstrasse 17, D-7800 Freiburg, Federal Republic of Germany

Accepted February 12, 1990

Summary. This study describes the morphological differ- entiation of nonpyramidal neurons in areas CAI and CA3 of the rat hippocampus as seen after Golgi-impreg- nation. Representative neurons were gold-toned and processed for an electron microscopic study of identified cells. We analyzed the postnatal stages P0 (day of birth), PS, P10 and P20. The results can be summarized as fol- lows: 1. On the day of birth nonpyramidal neurons dis- play relatively large cell bodies with short, clumsy den- drites. Great variability of the shape of the cell body and of the orientation of dendrites was observed when compared with the more stereotyped pyramidal neurons. Electron microscopy of identified nonpyramidal neurons revealed small infoldings of the nuclear membrane and immature synapses on the short dendritic shafts of these cells. 2. Developing nonpyramidal neurons from P0 and P5 display growth cones, filopodia, preterminal growth buds, and irregular varicose swellings along the den- drites. 3. Further postnatal development of nonpyrami- dal neurons is mainly characterized by an increase in dendritic length, paralled by a decrease in growth cones and preterminal growth buds. By means of the electron microscope an increase in the number of mature input synapses on the gold-toned dendritic shafts of identified nonpyramidal neurons was observed. 4. There is a signif- icant developmental difference between nonpyramidal neurons in CAI and CA3 that was most obvious on P5. Nonpyramidal neurons in CA3 appear more mature, displaying longer dendrites that sometimes traverse through several hippocampal layers. In contrast, the dendrites of nonpyramidal neurons in CA1 are still re- stricted to the layer of the parent cell body. The earlier differentiation of nonpyramidal neurons in CA3 may result from the earlier formation of neurons in CA3 than in CA1. Longer dendrites of nonpyramidal neurons in CA3, together with an earlier arrival of afferent fibers in this region, suggest that nonpyramidal neurons in

* In partial fulfilment of the requirements for the degree of Dr. med. at the University of Frankfurt/Main

Offprint requests to: M. Frotscher

CA3 are integrated into inhibitory hippocampal circuits earlier than their counterparts in CA]. 5. On P20, hippo- campal nonpyramidal neurons showed all structural characteristics as observed in adult animals both at light and electron microscopic levels. It is concluded that the structural maturation of hippocampal nonpyramidal cells is completed by that postnatal age.

Key words: Neuronal development - Ammon's horn - Nonpyramidal neurons - Inhibition

Introduction

The neurons of the hippocampus proper can roughly be divided into two groups, i.e., the pyramidal neurons and the various types of nonpyramidal cells. Several stu- dies have described their development (Schwartz et al. 1968; Purpura and Pappas 1968; Frotscher et al. 1975, 1978; Minkwitz and Holz 1975; Minkwitz 1976a-c; Pokorny and Yamamoto 1981; Schwartzkroin etal. 1982; Schwartzkroin 1982; Harris and Teyler 1983; Swann et al. 1989; Seress et al. 1989). The majority of these developmental studies have focused on the CA1 region, with the exception of those by Minkwitz (1976a, c) who analyzed the differentiation of pyramidal cells both in CA1 and CA3.

In recent years a number of studies have dealt with the development of the inibitory GABAergic system (Kunkel etal. 1986; Seress and Ribak 1988; Lfibbers and Frotscher 1988; Swann etal. 1989; Seress etal. 1989). These studies have contributed much to our un- derstanding of the differentiation of nonpyramidal cells, because the inhibitory amino acid GABA is known to be the neurotransmitter of most, if not all, nonpyramidal neurons in the hippocampus. The results of these studies may be briefly summarized as follows: 1. GABAergic nonpyramidal cells are generated prenatally, almost in parallel with the pyramidal neurons (Bayer 1980; Amar- al and Kurz 1985; Liibbers et al. 1985; Soriano et al.

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1989). 2. In the early pos tnata l period, i.e., a round post- natal day (P) 5, GABAerg ic nonpyramida l cells fo rm synaptic contacts on principal neurons, thereby provid- ing a morpholog ica l basis for inhibit ion in early develop- ment (Seress et al. 1989).

One disadvantage o f immunocy tochemica l studies using antibodies against g lutamate decarboxylase (GAD) , the GABA-synthes iz ing enzyme, or against G A B A itself, is that only cell bodies, initial dendritic segments and axon terminals are stained. There is, how- ever, reason to believe that dendritic g rowth dur ing de- ve lopment mainly occurs at dendritic tips. A systematic s tudy of the postnata l development o f nonpyramida l neurons in areas CA1 and CA3 with the Golgi method, which stains single neurons with their distal dendrit ic processes, is lacking.

W h e n compared with the pyramida l cells, nonpyra - midal neurons are less uniform. They are not a r ranged in a densely packed cell layer, but are distributed t h roughou t all layers o f the h ippocampus . They are more variable in fo rm but are less numerous , compris ing approximate ly 12% o f the neurons o f the h ippocampus proper (Dietz et al. 1987). Moreover , there is evidence for chemical differences between the various types o f nonpyramida l cells, for instance in their content o f neu- ropeptides such as somatos ta t in (SS), vasoactive intesti- nal polypept ide (VIP), cholecystokinin (CCK) and the calcium-binding protein parva lbumin (Leranth et al. 1984; Somogyi et al. 1984; Leran th and Frotscher 1986; Kosaka e ta l . 1985, 1987; Sloviter and Nilaver 1987; Sloviter 1989). This diversity within a relatively small g roup o f neurons makes a systematic s tudy o f the post- natal development o f nonpyramida l neurons more diffi- cult when compared with the more numerous and stereo- typed pyramida l neurons.

In this s tudy we have used the combined Golgi/elec- t ron mic roscopy technique for an analysis o f the postna- tal ma tu ra t ion o f identified nonpyramida l neurons in areas CA1 and CA3 o f the rat h ippocampus . By using a similar approach , the c o m p a n i o n paper by Seress and Ribak (1990) describes the postnata l differentiation o f basket ceils in the rat fascia dentata. In bo th articles the results are discussed in relation to the postnata l de- ve lopment o f inhibi tory processes because the nonpyra - midal cells are the neurons that mediate bo th feed-for- ward and feed-back inhibit ion in the h ippocampus and fascia denta ta (Andersen et al. 1964a, b; Frotscher and Zimmer 1983; Leran th and Frotscher 1983; Buzs{tki 1984; Frotscher et al. 1984; Seress and Ribak 1984; Frotscher 1985, 1989; Seress e ta l . 1989; Zipp e ta l . 1989).

Materials and methods

Male Sprague-Dawley rats (n = 38) of the postnatal stages P0 (day of birth, n=12), P5 (n=10), P10 (n=10) and P20 (n=6) were used for the present studies. The animals were sacrified by transcar- dial perfusion under anesthesia with ether or pentobarbital (Nem- butal 30 mg/kg) with a fixative containing 1% glutaraldehyde and 1% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). After

5-20 min of perfusion (duration depending on the size of the ani- mal) the bodies were stored in sealed plastic bags for at least 2 h in the refrigerator. Then, the brains were removed from the skulls and either further processed in toto (very young animals); or the hippocampi were dissected out and cut into 3-mm-thick blocks perpendicular to their long axis. After another 3 h postfixation the material was washed in phosphate buffer. For Golgi-impregna- tion, gold-toning and deimpregnation the original method (Fair6n et al. 1977) was adopted with minor modifications (Frotscher et al. 1981). After rinsing in phosphate buffer, the tissue blocks were immersed in an osmium dichromate solution (1 g osmium tetroxide and 12 g potassium dichromate in 500 ml distilled water) for 4 days. Thereafter, the blocks were briefly washed in 0.75% silver nitrate and stored in fresh silver nitrate for 2 days. The tissues were then transferred to an ascending series of glycerol (20, 40, 60, and 80%) and stored in 100% glycerol for varying periods of time. The glycerol-soaked material was either sectioned on a sliding microtome (Leitz) or on a Vibratome (Shandon) in trans- verse orientation of the hippocampus. In the first case the tissue was superficially embedded in paraffin, while in the latter the speci- mens were cut flat on one side and glued to the specimen holder. Sections were taken at a thickness of 100 gm and were examined with the light microscope and documented (photographed and drawn using a microscope equipped with a drawing tube) while floating in a drop of glycerol. Sections containing single well-im- pregnated neurons in the hippocampus proper were collected for gold-toning and deimpregnation in sodium thiosulphate (Fair6n et al. 1977). For optimal deimpregnations, samples immersed in thiosulphate were monitored under the light microscope. The pro- cedure was stopped when the neurons showed a finely granulated staining of grayish or brownish tinge.

Microtome or Vibratome sections that after the first Golgi impregnation did not contain well-impregnated cells, were collected and re-impregnated, employing a modification of the section Golgi procedure (Freund and Somogyi 1983) described in detail elsewhere (Frotscher and Leranth 1986; Frotscher and Zimmer 1986).

After poststaining in 1% osmium tetroxide the sections were dehydrated in ethanol (blockstained with uranyl acetate in 70% ethanol) and flat-embedded in Araldite between foils of aluminium and transparent plastic. Following removal of the aluminium foil, the cells were again examined under the light microscope, photo- graphed and drawn, using a Zeiss drawing tube. More than 150 neurons, at least 10 cells per age group and region (CAt, CA3), were included in the light microscopic analysis. Selected neurons were then re-embedded in plastic capsules and closely trimmed. Ultrathin serial sections were cut on a LKB Ultrotome 3 or Rei- chert OmU3, mounted on single slot grids coated with formvar film, and studied in a Siemens Elmiskop 101 or Zeiss EM 109. For each cell a sectioning protocol was made to document lost sections in the series. During sectioning, drawings of the remaining non-sectioned parts of the cells were repeatedly made on transpar- ent paper. These drawings could then be superimposed on the origi- nal drawing of the cell. In this way, parts of the cells which were no longer visible could be traced in successive ultrathin sections. With these procedures single processes of the cells could be identi- fied in the electron microscope.

Results

Light microscopic studies

The results o f the present s tudy were based on both Golgi - impregnated neurons, studied directly after silver impregnat ion while the sections were immersed in a drop of glycerol, and on gold- toned cells f la t -embedded in Araldite. We came to the conclusion that the gold- toning procedure did not give such reliable results as were

PO ( CAI/CA3 ) fh . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Fig. 1. Camera lucida drawings of nonpyramidal neurons in areas CA1 and CA3, postnatal stage 0. For comparison pyramidal neurons in CA1 (cell No. 1) and CA3 (cell No. 11) are also shown. Note the variability in the shape of the cell body and in the orientation of dendritic processes of the nonpyramidal neurons distributed over all hippocampal layers. Ceils No. 3 8 were from CAI, cells No. 2, 9, 10 from CA3. A micrograph of cell No. 2 is shown in Fig. 6a. x 170. al, alveus;fh, hippocampal fissure; sl, stratum lacunosum- moleculare; so, stratum oriens; sp, stratum pyramidale; sr, stratum radiatum

P5 ( CA1 ) fh . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

~ i !________ ~ _ _ , ,-~ . . . . . . . . . . . . . . / _ - ~ . . . . . . . . . . . . . . . . Fig. 2. Camera lucida drawings of nonpyramidal neurons in CA1 from P5. With the exception of the horizontal

~O_ _ _ i _ _ _ ~ - - _ _ ' - - - ' - - - - - - - - - - - - - - - - - - - - - - - n e u r o n s in stratum oriens (cells No. 7-9) the neurons appear rather immature and have relatively short dendrites when compared with nonpyramidal neurons in CA3 from the same postnatal stage (Fig. 3). A micrograph of cell No. 8 is

al shown in Fig. 7d. Cell No. 1 is a pyramidal neuron, x 170

known from studies of adult animals. Thus, some cells from early postnatal stages were lost during the gold- toning procedure. Nevertheless, the present data are based on the study of a large number of hippocampal sections from the various postnatal stages. For compari- son with cells in the adult hippocampus, a large collec- tion of Golgi-impregnated and gold-toned cells from previous and current studies was available (cf. Frotscher 1988).

During the study of all postnatal stages we were faced with the problem that the impregnated nonpyrami- dal cells show much larger variability when compared with the relatively uniform pyramidal neurons, which always displayed the typical bipolar vertical orientation. In contrast, nonpyramidal cells exhibited large varia- tions with regard to the orientation and length of their dendrites (Figs. 1 5). Moreover, a characteristic feature of dendritic growth, varicose swellings, persists in non-

pyramidal neurons of adult animals (Schlander and Frotscher 1986). However, with some practice the regu- lar varicosities of adult nonpyramidal neurons can be differentiated from the sometimes bizarre swellings of growing dendrites. Typical features of growing dendrites were terminal swellings, i.e., growth cones, sometimes with filopodia, and preterminal growth buds (Morest 1969) which were observed at branching points (e.g. Fig. 6 a, b). Also, a characteristic feature of pyramidal cell differentiation, the appearance of numerous dendrit- ic spines, is not present in most nonpyramidal neurons. If present at all, spines on nonpyramidal cells are rare and cannot be used for the sort of quantitative evalua- tion of developmental process that has been used for pyramidal neurons (Frotscher etal. 1975; Minkwitz 1976 c). These difficulties, together with the unpredictible results of the Golgi impregnation, have restricted our study to a merely qualitative description of the various

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fh P5 (CA3) . . . . . . . . . . . . • . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Fig. 3. Camera lucida drawings of nonpyramidal neurons in CA3 from P5. Most cells have slimmer and longer dendrites than non- pyramidal neurons in CAI of the same postnatal age (Fig. 2). Thus, dendrites are no longer restricted to the layer of the parent cell body. Also, characteristic growth cones and preterminal growth

buds are less often seen. A micrograph of cell No. 2 is shown in Fig. 7a. Cell No. 1 is a CA3 pyramidal neuron. The dendritic arbor appears more advanced when compared with the CA1 pyra- midal cell (cell No. I in Fig. 2). Note the long axon collateral which can be followed on its way through stratum oriens. • 170

P10 (CA1/CA3) fh . . . . . . . - - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Fig. 4. Camera lucida drawings of nonpyramidal neurons in CA1 and CA3 from P10. Particularly cell No. 2 which is the only one from CA3 displays thin, long dendrites. Cell No. 1 which has spine-laden apical dendrites resembles an ectopic granule cell or the inferior region interneuron described by Amaral and Woodward (1977). However, this cell was found in the inner stratum radiatum of CA1. Micrographs of this neuron are shown in Fig. Tb, c. x170

types of nonpyramidal neurons found impregnated at the postnatal stages analyzed. We have accordingly pre- sented our data largely in the form of camera lucida drawings of representative nonpyramidal neurons at the different postnatal stages. All drawings are presented at identical magnification, which allows a direct compar-

ison between the stages (Figs. 1-5). To allow for a com- parison with the principal neurons, some drawings of pyramidal cells are included.

Representative nonpyramidal neurons in the various layers of area CA1 and CA3 at P0 are shown in Fig. 1. When compared with the pyramidal neurons, especially

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fh P20 (CA1/CA3) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Q~- . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . s, , (

sr ~11 ; ~-~~~)7 :~; . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . .

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Fig. 5. Camera lucida drawings of nonpyramidal neurons in CA1 and CA3 from P20. For comparison a CA1 pyramidal neuron (cell No. 1) and a CA3 pyramidal cell (cell No. 11) are shown. There is mainly an increase in the length of nonpyramidal cell dendrites when compared with P10. Occasionally a single dendrite

is as long as the whole distance from the alveus to the hippocampal fissure (cell No. 6). The cells show all characteristics as observed in adult animals. Cells No. 3 6, 9 from CA1; cell No. 2, 7, 8, 10, 12 from CA3. A micrograph of cell No. 12 is shown in Fig. 7e. • 170

with those in CAI, the cell bodies of nonpyramidal cells are sometimes rather large (cf. cell No. 2, 5, 6, 9 and 13). The shape of the cell body is irregular, ranging from round or ovoid to rather elongated forms. In contrast, pyramidal neurons already display the characteristic ovoid or triangular perikarya (cells No. 1 and 11). The cell bodies of nonpyramidal neurons give rise to several relatively shorts dendritic processes which vary in diame- ter. Varicose swellings along the length of dendrites, pre- terminal growth buds and characteristic growth cones at dendritic tips (cell No. 8) are regularly observed. Den- drites of pyramidal neurons also showed varicose swell- ings, mainly at branching points (cell No. 1). Pyramidal cell dendrites already showed the preferred vertical ori- entation, whereas the dendrites of nonpyramidal neu- rons extended in all directions. However, some nonpyra- midal neurons were preferentially orientated horizontal- ly (cell No. 3, 4, 12, 13) whereas other cells gave rise to ascending dendrites paralleling the course of apical dendrites of pyramidal neurons (cells No. 6, 7). At this age, spines were very rare on dendrites of both pyrami- dal neurons and nonpyramidal cells. Axonal processes which were rarely impregnated could only be followed over very short distances. Figure 6 a demonstrates a pho- tomicrograph of cell No. 2 in Fig. 1. The short axon arises from the basal pole of the cell body and branches in the underlying pyramidal layer. It ends in a growth cone (arrow). A dendritic growth cone on one of the

apical dendrites of this nonpyramidal cell is also labeled by an arrow.

Changes at P5 are mainly characterized by an in- crease in the length of nonpyramidal cell dendrites. There was an obvious difference between the nonpyra- midal neurons in CA1 and CA3. We regard it as an important observation of the present study that nonpyr- amidal cells in CA3 generally appeared more advanced at P5 when compared with the same types of cells in CA1. To demonstrate this clearly, we have shown non- pyramidal cells in the two areas in separate drawings (Figs. 2, 3). Thus, the more mature neurons of P5 dis- played longer dendrites with regular varicosities that re- sembled those observed in nonpyramidal cells of adult animals (cf. cells No. 5, 7 in Fig. 3). Relatively long, horizontally or vertically orientated dendrites traversed large parts of the hippocampal layers (cf. cells No. 5, 6, 8, 10). In contrast, nonpyramidal neurons in CA1 at the same postnatal stage still displayed rather clumsy and short dendritic processes. Characteristic features of dendritic growth such as growth cones and preterminal dendritic buds are obvious (cf. cell No. 5 in Fig. 2). In general, the short dendrites of nonpyramidal cells in CA1 at P5 were still restricted to the layer of the parent cell body. Horizontal neurons in the stratum oriens ap- peared most advanced with relatively long dendrites run- ning parallel to the alveus (cells No. 7, 8, 9 and Fig. 7 d). The difference in maturity between nonpyramidal neu-

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rons in CA1 and CA3 at P5 is also obvious in the micro- graphs of Figs. 6b, c. The cell in b which represents a nonpyramidal neuron in the outer pyramidal layer of CA1 displays short dendrites ending in growth cones, and a large swelling at a branching point (arrow). The nonpyramidal neuron in CA3 (left cell in Fig. 6c) exhib- its longer and slender dendrites. The cell on the right is a CA3 pyramidal neuron with dendritic processes dis- playing both growth-related varicose swellings and a low number of dendritic spines (arrows). Another nonpyra- midal neuron with slender dendrites in stratum radiatum of CA3 is shown in Fig. 7a. Its axon descends to the pyramidal layer (see also cell No. 2 in Fig. 3).

At postnatal stages P10 and P20 we mainly noted an increase in the length of the nonpyramidal cell den- drites (Figs. 4, 5). Differences between nonpyramidal neurons in CA1 and CA3 became less obvious. At P20 several nonpyramidal cell dendrites were found to tra- verse all hippocampal layers (e.g., cell No. 6 in Fig. 5). Pyramidal neurons display an increasing number of den- dritic spines, and on CA3 pyramidal cells the large spines or excrescences for synaptic contact with the mossy fibers have formed (cell No. 11, Fig. 5). At P10 we found a nonpyramidal neuron densely covered with dendritic spines in the inner stratum radiatum of CA1 (cell No. 1 in Fig. 4; Fig. 7 b, c). This cell resembled very much the "inferior region interneuron" described by Amaral and Woodward (1977). Axons, if they were impregnated at all, could be followed for very short distances only.

The difficulties that appear in a description of the postnatal developmental changes in hippocampal non- pyramidal cells are demonstrated by comparing similar cells at different ages (Figs. 7 d and e). Both cells shown are horizontal nonpyramidal cells of the stratum oriens. However, the cell in d is from P5 whereas that in e is from a 20-day-old animal (cf. also cell No. 8 in Fig. 2 and cell No. 12 in Fig. 5). Despite the difference in the postnatal stage there are great similarities between the two neurons with regard to the orientation and length of their slender dendrites. It appears that the complex group of hippocampal nonpyramidal cells does not fol- low simple and uniform routes of dendritic development.

Fine structure of identified nonpyramidal cells

From the 12 identified nonpyramidal cells at different postnatal stages examined in the present electron micro-

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Fig. 6a-e. Differentiating nonpyramidal neurons in stratum radia- tum of CA1 and CA3. Cells photographed after goldtoning and flat-embedding in Araldite. a Neuron directly above pyramidal layer in CA1 (P0). The axon (a) and short dendritic processes end with growth cones (arrows). This neuron is also shown in Fig. 1 (cell No. 2). x 750. b Nonpyramidal neuron located at the border between stratum radiatum and stratum pyramidale of CA1 (P5, cell No. 3 in Fig. 2). Note varicose swellings at branching points (arrow) and growth cones, x 700. e Nonpyramidal neuron and pyramidal cell in CA3 at P5. When compared with the neuron in stratum radiatum of CA1 b the dendrites of the nonpyramidal neuron in stratum radiatum of CA3 (cell on the left side) are longer and have smooth contours. The dendrites of the pyramidal cell exhibit varicosities and occasionally spines (arrows). x 600

scopic study two were selected for Figures (Figs. 8, 9). The neuron shown in Fig. 8 a-c is from a newborn rat. The cell body was located in stratum radiatum of CA3. The dendrites are still rather short and end in typical growth cones (arrow in Fig. 8a). The location of this cell in stratum radiatum, and the orientation of the den- dritic processes, characterize it as a nonpyramidal neu- ron. This is supported by the electron micrograph of the cell body region (Fig. 8b) that displays a nucleus with a small indentation which has been described as a characteristic feature of nonpyramidal cells (Ribak and Anderson 1980; Seress and Ribak 1985; Schlander and Frotscher 1986). The perinuclear cytoplasm is only poor- ly developed and shows few organelles. At this postnatal stage synaptic contacts were observed only rarely on the identified neurons. The identification of synaptic contacts was generally difficult due to the immature ap- pearance of the synaptic membrane specializations. In the few examples that have been observed accumulations of synaptic vesicles and symmetric membrane apposi- tions suggested a synaptic contact. Figure 8c demon- strates an axonal varicosity with vesicle accumulations (black arrow) close to a symmetric membrane apposition on a gold-toned identified dendrite of the nonpyramidal neuron shown in a and b.

A nonpyramidal neuron from P10 located in the in- ner part of the pyramidal layer in CA3 is demonstrated in Fig. 9 a-c (see also cell No. 2 in Fig. 4). The cell has an almost mature appearance, i.e., it shows long and slender spine-free dendritic processes. This is confirmed by the electron micrograph (Fig. 9b) which demon- strates large amounts of perinuclear cytoplasm contain- ing numerous organelles. Particularly evident are the rows of endoplasmic reticulum (arrow). Large amounts of perinuclear cytoplasm have been described as a char- acteristic feature of nonpyramidal neurons in the hippo- campus and fascia dentata (Ribak and Anderson 1980; Seress and Ribak 1985; Frotscher 1985; Schlander and Frotscher 1986; Lfibbers and Frotscher 1987). At this stage, mature asymmetric synaptic contacts were ob- served on the identified nonpyramidal cell dendrites. In Fig. 9a an arrow points to a portion of the thick basal dendrite. The corresponding area is framed in the elec- tron micrograph (Fig. 9b). A higher magnification of this area displays a terminal-forming asymmetric synap- tic contact on the identified dendrite (Fig. 9 c).

On postnatal day 20, gold-toned nonpyramidal neu- rons both in CA3 and CA1 displayed all the fine struc- tural characteristics that have been described for these cells in adult animals (Ribak and Anderson 1980; Seress and Ribak 1985; Schlander and Frotscher 1986). Thus, the cell body contained large amounts of perinuclear cytoplasm packed with organelles. The cell nuclei were deeply indented and contained rods or sheets. These ob- servations in the present study are very similar to those made in developing basket cells of the rat fascia dentata which are described in great detail in the companion paper by Seress and Ribak (1990). Moreover, these au- thors have also analyzed the fine structure of developing nonpyramidal neurons in the hippocampus proper (P5, P16; Seress et al. 1989 and Seress and Ribak, personal

Fig. 7. a Nonpyramidal neuron in stratum radiatum of CA3 (P5, cell No. 2 in Fig. 3). The axon (a) descends to the pyramidal layer in which two differentiating pyramidal neurons can be seen. x 400. b Nonpyramidal neuron with spiny dendrites located directly above pyramidal layer (sp) in CA/ (P10, cell No. 1 in Fig. 4). The axon (a) traverses the pyramidal layer. Arrow pointing to dendritic seg- ment shown at higher magnification in e The cell has a similar appearance as the regio inferior interneuron described by Amaral

and Woodward (1977). x 480. e Dendritic segment (arrow in b) densely covered with spines, x 1200. d, e Horizontal nonpyramidal neurons in stratum oriens of CA1 d and CA3 e. The cell in d is from P5 (cell No. 8 in Fig. 2) whereas the neuron in e is from P20 (cell No. 12 in Fig. 5). Note the lack of major differences between the two cells with regard to dendritic differentiation, d x 4 0 0 ; e x480

Fig. 8a-e. a Nonpyramidal neuron from P0 in stratum radiatum of CA3. The cell was photographed after gold-toning, deimpregna- tion and embedding in Araldite. Note short and relatively thick varicose dendrites. Arrow pointing to growth cone at the tip of a dendritic process, x 1000. b Electron micrograph of the cell body region of the neuron shown in a When compared to more mature nonpyramidal neurons (Fig. 9) the perikaryal cytoplasm contains only few organelles. In particular, large accumulations of endoplas- mic reticulum characteristic of adult nonpyramidal neurons, are

lacking. However, the nucleus (Nc) already exhibits infoldings of its membrane (arrows) which are a characteristic feature of nonpyr- amidal neurons. Note excentric chromatin accumulations. x 13200. c Varicosity in the course of a fine nerve fiber. It is filled

with large and small, characteristic synaptic vesicles (arrow). These vesicles are concentrated at that part of the membrane which forms what appears to be an immature symmetric contact (open arrow) with a gold-toned dendritic process of the neuron shown in a, b • 30,000

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Fig. 9a-e. a Nonpyramidal neuron from P10 located within the pyramidal layer in CA3 (cell No. 2 in Fig. 4). Thin, partly varicose dendrites originate from a round or ovoid cell body. Dendrite la- beled by arrow is seen in the electron micrographs, x 400. b Elec- tron micrograph of the neuron shown in a A relatively small nucle- us (Nc) with homogeneously distributed chromatin particles is seen.

When compared to the nonpyramidal neuron at P0 (Fig. 8) the nucleus is surrounded by large accumulations of rough endoplas- mic reticulum (arrow). The boxed area is shown at higher magnifi- cation in e x 7,500. e Axon terminal (arrow) forming mature asym- metric synaptic contact on proximal dendrite labeled by arrow in a (indicated area in b). x 30,000

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communication). They found that all the fine structural characteristics mentioned above are already well-devel- oped in nonpyramidal neurons of the hippocampus proper of 16-day-old rats.

Discussion

In the discussion of our results we will mainly focus on three points: 1. Developing nonpyramidal neurons in the hippocampus display similar growth-related struc- tural characteristics as other types of neurons 2. The morphological maturation of hippocampal nonpyrami- dal neurons is mainly recognizable by an increase in dendritic length and 3. At postnatal stage P5 we have observed remarkable differences between developing nonpyramidal neurons in CA1 and CA3. These latter observations were discussed in relation to recent electro- physiological findings on the postnatal maturation of inhibitory processes in both regions (Schwartzkroin 1982; Harris and Teyler 1983; Swarm et al. 1989).

It is well established that the formation of GABAerg- ic nonpyramidal cells takes place during the prenatal period (Bayer 1980; Amaral and Kurz 1985; L/_ibbers et al. 1985; Soriano et al. 1989). Our results have shown that the maturation of nonpyramidal neurons extends into the postnatal period, and may be regarded as com- plete after the third postnatal week. Our observations are thus in agreement with other studies on the develop- ment of nonpyramidal and pyramidal neurons in the hippocampus (Minkwitz 1976a, b, c; Purpura and Pap- pas 1968; Schwartz etal. 1968; Schwartzkroin etal. 1982; Seress and Ribak 1988). It also appears that devel- oping nonpyramidal neurons in the rodent hippocampus display all characteristic features of growing neurons in other regions, such as growth cones at the growing tips of dendritic processes, filopodia, and irregular varicose swellings along the dendrites, in particular at branching points (e.g., Morest 1969). However, the postnatal differ- entiation of hippocampal nonpyramidal neurons is mainly recognizable by an increase in dendritic length. In the case of pyramidal neurons there is a clear-cut sequence in the maturation of dendritic processes. First the main apical dendrite differentiates, followed by the basal dendrites originating directly from the cell body. Only then are the side branches of the apical dendrite formed (Minkwitz 1976c; Pokorny and Yamamoto 1981). The large variability of nonpyramidal neurons due to different dendritic orientation makes the analysis of a sequential maturation of nonpyramidal cell den- drites impossible. Moreover, the disappearance of vari- cose swellings that indicates maturation for the dendrites of pyramidal cells, cannot as easily be studied in nonpyr- amidal cells. Varicosities along the dendritic processes are present in most types of nonpyramidal neurons in the adult hippocampus and fascia dentata (Cajal 1911; Lorente de N6 1934; Frotscher and Zimmer 1983; Ribak and Seress 1983, 1985; Schlander and Frotscher 1986; Liibbers and Frotscher 1987). Finally, a characteristic feature of pyramidal cell development is the increase in spine number on all parts of the dendritic tree (Mink-

witz 1976c; Frotscher et al. 1975, 1978). The increase in spine numbers cannot be used as a parameter to de- scribe the development of nonpyramidal neurons be- cause most of them lack dendritic spines. We have found that the increase in dendritic length of nonpyramidal neurons paralleled by a decrease in characteristic growth-related structures such as growth cones, clumsy swellings, and preterminal growth buds, are the most useful parameters to describe the maturation of nonpyr- amidal neurons in the hippocampus. Even with regard to dendritic length there were considerable variations. As demonstrated in Fig. 7 d there are nonpyramidal neu- rons which already have rather long and slender den- drites on P5 when compared with a very similar neuron of a 20-day-old rat (Fig. 7e).

Using the length of dendrites and characteristic fea- tures of dendritic growth as parameters, we have ob- served remarkable regional differences in the present study. Particularly at P5, nonpyramidal neurons in CA3 appeared much more advanced when compared with similar neurons in CA1. In earlier and later stages these differences were less apparent. The early maturation of nonpyramidal neurons in CA3 may result from the ear- lier formation of neurons in this region when compared with CA1 (Bayer 1980). It is interesting to note that afferent fibers from the entorhinal cortex and the contra- lateral hippocampus also arrive earlier in CA3 than in CA1 (Loy 1980). This suggests that GABAergic inhibito- ry nonpyramidal neurons are integrated earlier in hippo- campal circuits in CA3 than in CA1. In fact, recent phys- iological studies on the postnatal development of GABA-mediated synaptic inhibition in the rat hippo- campus have revealed dramatic differences in the onto- genesis of functional GABAergic inhibitory synaptic transmission between CAI and CA3 of the rat hippo- campus. Neither antidromic nor orthodromic stimula- tion could elicit identifiable inhibitory postsynaptic po- tentials in CA1 neurons from 5-day-old rats, whereas large inhibitory postsynaptic potentials could be re- corded from CA3 pyramidal cells (Swann et al. 1989).

The late development of inhibition in CA1 has al- ready been emphasized in earlier studies (Schwartzkroin 1982; Schwartzkroin etal. 1982; Harris and Teyler 1983). Preliminary studies from this laboratory have not revealed major differences between the pyramidal layer in CA3 and CA1, considering the number of GAD-posi- tive terminal-like puncta (Kfippers et al., in prepara- tion). Similarly, Swannet al. (1989) did not find a corre- lation between changes in levels of glutamate decarboxy- lase activity with age and functional GABAergic synap- tic inhibition. They concluded that glutamate decarbox- ylase activity may be inappropriate as a biochemical marker for the ontogeny of functional GABA synapses. In a recent study we have shown that GAD-positive boutons establish symmetric synaptic contacts on pyra- midal neurons in CA1 as early as on P5, thereby forming a morphological basis for inhibition in early develop- ment (Seress et al. 1989). Our present data have demon- strated that input synapses on identified nonpyramidal neurons in CA3 are already present on P0. Longer den- drites of nonpyramidal neurons in CA3, and an earlier

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ar r iva l o f afferent fibers in this region, suggest tha t inpu t synapses on n o n p y r a m i d a l neu rons are much more nu- merous in CA3 than in CA1 at ear ly p o s t n a t a l stages. Also , we had the impress ion tha t p y r a m i d a l neurons in CA3 were m o r e a d v a n c e d when c o m p a r e d with CA1 p y r a m i d a l cells o f the same p o s t n a t a l age (cf. cell No. 1 in Fig. 2 and cell No . 1 in Fig. 3). A n axon co l la te ra l o f the CA3 p y r a m i d a l neu ron could be fo l lowed for a r a the r long d is tance on its way t h rough the s t r a t um or- iens. This suggests t ha t p y r a m i d a l cell co l la te ra ls serving recur ren t inh ib i t ion are wel l -d i f fe rent ia ted in CA3 a t this pa r t i cu l a r p o s t n a t a l s tage (P5). A re la t ively large n u m b e r o f inpu t synapses , f o r m e d by ear ly a r r iv ing extr insic af- ferents a n d by recur ren t col la tera ls , increases the p r o b a - bi l i ty tha t the n o n p y r a m i d a l neurons become ac t iva ted and in tu rn inhib i t the p y r a m i d a l neurons in this region. Our p resen t m o r p h o l o g i c a l obse rva t ions thus p rov ide an exp lana t ion for the ear l ier m a t u r a t i o n o f func t iona l G A B A e r g i c inh ib i to ry synapt ic t r ansmiss ion in CA3 than in CA1 (Swann et al. 1989).

Acknowledgments. The authors wish to thank Drs. C.E. Ribak and L. Seress for their helpful comments in the preparation of the manuscript, and B. Krebs and E. Thielen for technical assistance. This study was supported by grants from the Deutsche Forschungs- gemeinschaft (Fr 620/1-4, SFB 45).

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