Morphological properties of intracellularly labeled layer I neurons in rat neocortex

THE JOURNAL OF COMPARATIVE NEUROLOGY 376~198-213 (1996)

Morphological Properties of Intracellularly Labeled Layer I

Neurons in Rat Neocortex

FU-MING ZHOU AND JOHN J. HABLITZ Neurobiology Research Center and Department of Physiology and Biophysics,

University of Alabama at Birmingham, Birmingham, Alabama 35294

ABSTRACT The morphology of neurons in layer I of rat neocortex, including Cajal-Retzius (CR) cells,

was studied by using intracellular biocytin staining in brain slices obtained from rats during the first 22 postnatal days. Within the first postnatal week, horizontal bipolar neurons or CR cells were prominent in layer I. Typically, CR cells had one main dendrite and one axon originating from opposite poles of the somata. Even though the main dendrites and axons could be quite long, complex dendritic or axonal arbors were not observed. Starting around postnatal day 6 (PN 61, CR cells were less frequently observed. From PN 10 to PN 21, nonpyramidal neurons with diverse morphologies became the main neuronal component in layer I. The somata of layer I nonpyramidal neurons were quite variable in size and shape. Dendrites were smooth or sparsely spiny, and the dendritic trees were mainly restricted to layer I, covering an area with a diameter of about 200 pm. Axon collaterals of these cells formed elaborate arbors with diameters of around 700 pm in layer I and extending, in many cases, to layer II/III and even layer IV. This extensive axonal plexus provides a rich anatomical base on which layer I neurons, functioning as local circuit elements, may interact with each other and with neurons in other layers. o 1996 Wiley-Liss, Inc.

Indexing terms: Cajal-Retzius cell, cerebral cortex, cortical development, interneuron

Layer I of the mammalian neocortex lies just under the pial surface, is thin (in rat about 50 pm at birth and 150 pm in adult) and so extremely low in neuronal density that it has been called a molecular or cell-free layer. About 100 years ago, Cajal (see DeFelipe and Jones, 19881, using the Golgi method, made a series of observations on layer I in several different species. Modern investigators confirmed and extended Cajal’s original anatomical observations (see Marin-Padilla, 1984, 1988 for review). Furthermore, it has been suggested that the fetal layer I, or primordial plexi- form layer, is uniquely important for cortical development (Marin-Padilla, 1984, 1988, 1992). Histochemical studies have shown that most layer I neurons are immunoreactive for y-aminobutyric acid (GABA) and its synthesizing en- zyme, glutamic acid decarboxylase, suggesting that they are inhibitory neurons (Gabbott and Somogyi, 1986; Martin et al., 1989; Winer and Larue, 1989; Hornung and Tribolet, 1994; Imamoto et al., 1994; Li and Schwark, 1994; Prieto et al., 1994a,b).

Pyramidal neurons of layers 11-VI extend their apical dendrites into layer I (Marin-Padilla, 1992) and axon collaterals of pyramidal and nonpyramidal neurons also project into layer I (Martin, 1984; Kawaguchi, 1993; Cowan and Wilson, 1994). Therefore, layer I neurons may interact

with neurons in deep layers as suggested originally by Cajal over a century ago (DeFelipe and Jones, 1988) and more recently by Martin et al. (1989). Quantitative anatomical studies indicate that considerable information processing may occur in layer I (Beaulieu and Colonnier, 1985; Beau- lieu et al., 1994).

To understand possible interactions between layer I neurons and cells in deeper cortical layers, it is necessary to have precise knowledge about the detailed morphological, physiological, and cytochemical properties of layer I neurons. Recently we have characterized the action potential and repetitive firing properties of layer I neurons (Zhou and Hablitz, 1996a,b,c). We found that layer I neurons display fast-spiking behavior typical of cortical interneurons, consistent with other physiological studies showing that cortical GABAergic interneurons are fast-spiking cells (Connors and Gutnick, 1990). However, knowledge about the morphology of layer I neurons has come mainly from work using the classic Golgi method (Bradford et al., 1978; Marin-Padilla, 1984; DeFelipe and Jones, 1988), which fails

Accepted July 17, 1996. Address reprint requests to John J. Hablitz, Neurobiology Research

Center, University of Alabama at Birmingham, Birmingham, AL 35294.

O 1996 WILEY-LISS, INC.

MORPHOLOGY OF LAYER I NEURONS 199

break into the cell and achieve the whole-cell mode. Electri- cal signals were amplified by an Axopatch 200 amplifier (Axon Instruments). Data concerning the electrophysiology of layer I neurons are reported in separate papers (Zhou and Hablitz, 1996a,b,c).

Biocytin labeling Biocytin histology was done according to the methods

described by Hirokawa and Armstrong (1988) and Kawagu- chi (1993). Biocytin (0.5%; Sigma Chemical Co., St. Louis, MO) was dissolved in the pipette solution before each experiment and allowed to passively diffuse into neurons from the recording electrode. After recording, individual slices were fixed overnight, at 4”C, in 0.1 M phosphate buffer containing 4% paraformaldehyde and 0.5% glutaral- dehyde. Following two 20-minute washes in 0.1 M phosphate buffer, slices were dehydrated in a graded ethanol series. After two 20-minute rinses in 0.1 M phosphate buffer, followed by a 10-minute wash in 0.05 M Tris buffer, slices were incubated in an avidin-biotinylated horseradish peroxidase complex (ABC Kit, Vector Laboratories, Burlingame, CA) dissolved in 0.05 M Tris buffer at room temperature for 4 hours. After excess ABC was removed by two 10-minute washes in 0.1 M phosphate buffer, individual slices were reacted with 0.01% HZOz and 0.05% 3,3’-diaminobenzidine tetrahydrochloride (DAB) in the presence of 0.025% (w/v) CoClz and/or 0.01% (w/v) NiS04(NH4)zS04 for 2 minutes in 0.05 M Tris buffer. Slices were then thoroughly washed in 0.1 M phosphate buffer, mounted on gelatin coated glass slides, cleared in xylene, and coverslipped.

Biocytin-filled neurons were photographed and/or drawn using a camera lucida drawing device under a 40 or 50 x oil objective. A 1 0 0 ~ oil objective was used occasionally. Processes were judged to be axons on the basis of their relatively smooth, thin appearance and uniform caliber. The term beaded dendrite is used to refer to dendrites with periodic enlargements, as exemplified by the cell in Figure 11. Dendrites with very few or no spines are referred to as smooth, whereas sparsely spiny dendrites are dendrites with an intermediate number of spines, as typified by the cell in Figure 9. The term “spiny” was used when the spine level was higher than those in sparsely spiny cells even though the spine density was below that typically seen in pyramidal neurons (Feldman, 1984). Spine density was not used as a criterion in grouping cells in the present study because it was observed that cells with similar dendritic and axonal profiles had different spine levels (see also Bradford et al., 1978). Tissue shrinkage was not corrected.

to reveal the full extent of axonal processes. A recent attempt by Anderson et al. (1992) using dye injection in fixed tissue also failed in this regard. The present study used a brain slice preparation and whole-cell patch recording techniques to intracellularly label individual neurons. Layer I neurons were found to have diverse morphological features including extensive axonal arbors which spread widely in layer I and, in many cases, to deep layers. Cajal-Retzius (CR) cells were prominent during the early postnatal period but became sparse after PN 13. Some preliminary results have appeared in an abstract (Zhou and Hablitz, 1995).

MATERIALS AND METHODS Brain slice preparation and identification

of layer I neurons Sprague-Dawley rats aged 0-21 days (PN 0-21, the day of

birth being designated PN 0 ) were used in this study. All animals were housed and handled according to approved local guidelines (Institutional Animal Use Committee). Brain slices were prepared according to previously described methods (Zhou and Hablitz, 1996a). After decapita- tion, brains were rapidly removed from the skull and immersed in ice cold saline for 1 minute. One hemisphere (right or left) was dissected out and 150-250 pm thick coronal slices were cut using a Vibratome. Parasagittal slices were also used in a few experiments for the purpose of comparison. In PN 0-6 animals, slices were obtained from frontal and parietal areas. Most other experiments used slices from the frontal area; parietal and cingulate slices were occasionally used for comparison. The definition of cortical areas was according to Paxinos and Watson (19861, Paxinos et al., (1991), and Zilles (1990). Slices were kept in a saline filled storage chamber bubbled with 95% 0215% COz at room temperature (21-23°C).

After at least 1 hour’s incubation, individual slices were transferred to a recording chamber mounted on the stage of a modified Zeiss standard microscope. Visualization and identification of individual layer I neurons in living brain slices was achieved by using differential interference con- trast (Nomarski) optics and a long working distance, high numerical aperture 40 x water immersion objective. Video images of individual neurons, printed using a Sony video graphic printer, were used to document the location and gross morphology of layer I neurons prior to recording.

Solutions The bath saline contained (in mM): 125 NaC1, 3.5 KCl,

2.5 CaClZ, 1.3 Mg Clz, 26 NaHC03, and 10 D-Glucose. The intracellular solution contained (in mM): 10 KC1, 125 K-isethionate or K-gluconate, 0.5 EGTA, 10 HEPES, 2 Mg-ATP, and 0.2 Na-GTP. Osmolarity and pH of the intracellular solution were adjusted to 280-290 mOsm and 7.3, respectively.

Whole-cell patch recording Patch pipettes were pulled from borosilicate capillary

tubing (KG-33 glass, 1.5 mm OD, 1.12 mm ID; Garner Glass) with the use of a Narishige PP-83 electrode puller. These pipettes, when filled with the intracellular solution, had resistances of 2-4 MR. Under visual guidance, the patch electrode was smoothly advanced to the neuron. After contact, gentle suction was applied, resulting in formation of tight seals (2 3 GR). Stronger suction was then used to

RESULTS Layer I neurons in living brain slices

Using Nomarski optics in living brain slices, neuronal somata and proximal segments of dendrites and axons were visualized in layer I, as shown in Figure 1. In PN 0-6 animals, numerous large bipolar, horizontally or obliquely oriented, cells were observed throughout layer I with no preferred location. These cells were termed Cajal-Retzius (CR) cells since they had an appearance similar to the Cajal-Retzius cells from small mammals shown in Cajal’s original drawings (DeFelipe and Jones, 1988). CR cells were not seen in the cortical plate or layers 11-VI. Vertically oriented bipolar neurons were much less frequently seen in layer 1. In addition to the CR cells, other small to medium, multipolar or irregular cells were observed in living slices.

200 F.-M. ZHOU AND J.J. HABLITZ

Fig. 1. Morphology of layer I cells in unstained living brain slices. Photomicrograph of layer I as seen during recordings. This slice was cut coronally from the frontal cortex of a postnatal day (PN) 5 rat. The pial surface was clearly visible. A typical horizontally oriented cell or

Cajal-Retzius (CR) cells was largely in focus. The soma is indicated by the arrow and the main dendrite is indicated by the arrowheads. Several obliquely oriented CR cells can also be seen. Scale bar = 30 pm.

Somata of CR cells usually had a dimension of 5-10 x 20-40 pm with the longer axis horizontally or obliquely oriented. The two poles of the somata usually became thinner with a robust dendrite originating from one pole. Even though more difficult to visualize, the axon or axon- like process was seen to emerge from the somatic pole opposite to where the dendrite arose. Axonal processes also ran horizontally or obliquely in layer I. Similar results were obtained in both coronal and sagittal slices, indicating that CR cells have a random planar orientation confined to layer I, but not limited to an anterior-posterior orientation. Our observation also suggests that rat CR cells are basically bipolar cells, not multipolar cells with processes radiating in all directions.

Starting at PN 6-7, CR cells were observed less frequently. In PN 13-21 animals, horizontal bipolar neurons or CR cells were only occasionally seen. During this relatively mature stage, the somata of most layer I neurons were multipolar, triangular or irregular, with a size (longer dimension) around 15 pm (range 10 to 20 pm).

Neuronal morphology revealed by intracellular biocytin staining

The data presented below were derived from whole-cell recordings made from more than 400 layer I neurons.

However, the mechanical seal of the recording pipette to the cell often resulted in cell bodies being detached upon pipette withdrawal. Smaller cells were more frequently destroyed or damaged. For analysis, three age groups, PN 0-7, PN 8-12, and PN 13-21, were defined based on the development of membrane properties of layer I neurons (Zhou and Hablitz, 1996a). The number of cells included in the three groups of rats was 56, 15, and 179, respectively.

Cajal-Retzius (CR) cells at P N 0-7 At this age, horizontal and oblique bipolar neurons or CR

cells were abundant. Camera lucida reconstructions of intracellularly stained CR cells are shown in Figure 2A-E. The somata, dendrites, and axons were found throughout layer I, from just under the pia to the layer 1-11 border. The somata were often fusiform, ranging from 2 0 4 0 pm in width and 5-10 pm in height. The primary dendrites were thick and appeared as a continuation of the somata, extending for up to about 200 pm. The primary dendrites often originated from one pole of the somata and gave rise to a few secondary branches along its course (Fig. 2A,B,D). Higher order dendrites were not elaborate. The dendrites were usually smooth (Fig. 2A,C,D) even though beaded dendrites were also seen (Fig. 2B). Axons usually originated from the pole of the somata opposite to where the dendrites


20 pm -

B

Fig. 2. Examples of reconstructed frontal cortex layer I Cajal- Retzius (CR) cells intracellularly labeled with biocytin. A, B, C, D, and E were from PN2,3,4,5, and 14 rats, respectively. All of these cells had a more or less horizontal bipolar profile. Note that none of these CR

originated (Fig. 2A,C,D). Sometimes, axons arose from proximal dendrites (Fig. 2B). Even in well-labeled neurons, axons did not ramify profusely and axonal collaterals did not form elaborate axonal arbors (Fig. 2B,D).

During this developmental period, in addition to the CR cells described above, neurons with other morphological features were also encountered in layer I, as shown in Figure 3. Most of these layer I neurons had a more typical nonpyramidal morphology with multipolar or irregular somata (Fig. 3A,B).

Layer I neurons in PN 8-12 rats By PN 10, CR cells were substantially less common. The

remaining CR cells had morphologies similar to those seen in PN 0-6 animals and were able to fire action potentials comparable to those recorded from other cells types at the same age (Zhou and Hablitz, 1996a). During the PN 8-12

cells formed a complex dendritic tree or axonal arbor. Arrowheads point to the axon and the arrow in C indicates a patch finger induced by the withdrawing of the patch pipette. Note the different scale for D.

period, non-CR cells gradually became the main neuronal component. Such neurons had complex morphologies not seen in PN 0-7 neurons. The most prominent change was the formation of elaborate axonal arbors. Starting at PN 10, morphologies of many layer I neurons were indistinguishable from those of older rats. Figure 4 shows layer I neurons from PN 11 and PN 12 rats displaying relatively small dendritic trees and extensive axonal arbors. As will be described below, such a dendro-axonal profile was pre- served in older rats.

Layer I neurons in PN 13-21 rats During this period, layer I neurons had extensive axonal

arbors and relatively small, simple dendritic trees. The locations, shapes, and orientations of the somata and the profiles of axonal arbors varied considerably from cell to cell. Following Bradford et al. (1978) and Fairen et al.

202 F.-M. ZHOU AND JJ. HABLITZ

B

Fig. 3. Camera lucida drawings of early postnatal non-CR neurons in layer I. A and B are neurons from frontal cortex in PN5 and PN6 rats, respectively. In the first postnatal week, these types of neurons were more frequently encountered with age. Arrowheads point to the axon.

PNll

Fig. 4. Examples of neurons with complex morphologies observed during the second postnatal week. Layer I neurons displayed a rapid postnatal morphological development, and complex layer I neurons were frequently seen after PN 10. The PN 11 and PN 12 layer I neurons

shown were from the frontal and parietal areas, respectively. Elaborate axonal arbors were observed even within the confines of the slice preparation. Arrowheads point to the axon initial segments. The arrow in the PN 11 neuron points to the patch finger.

(1984), we used the axonal and soma-dendritic profiles to classify layer I neurons. However, although useful for descriptive purpose, classification of layer I neurons according to morphological criteria alone is of limited utility as for

cortical nonpyramidal cells in general (Fairen et al., 1984). Further studies are needed to define the cytochemical nature and synaptic relationship of the individually recorded neurons such that better classifications can be


Fig. 5. Drawing of a layer I neuron with axonal arbors confined to layer I. This cell was from the frontal area of a PN 16 rat. The soma was located in the lower half of layer I. Five main dendrites radiated from the soma. The dendrites were beaded but aspiny. The dendritic tree was about 180 Fm in diameter and was restricted to layer I. The axon

achieved. The numbers given below only give a rough indi- cation of frequency of occurrence of cell types since neurons with smooth well-defined somata were chosen for recording.

Compared with PN 0-7 animals, CR cells were rare in PN 13-21 rats with only four cells labeled. However, when encountered, electrophysiological recordings indicated that such neurons had normal resting membrane potentials and were capable of repetitive firing (Zhou and Hablitz, 1996a). A successfully recovered CR cell from a PN 14 rat is shown in Figure 2E. Like the CR cells in the first postnatal week, this CR cell also had a simple horizontal bipolar morphology. The axon and dendrite arose from opposite sides of the soma and traveled parallel to the pial surface.

Layer Z neurons with axonal arbors confined to layer I. These neurons were the most commonly observed cell type in layer I in PN 13-21 animals (N = 82). Somata were found throughout layer I without any preferred location and were triangular, multipolar or irregular with dimensions of 10-20 pm (width) x 7-14 pm (height). As shown in Figure 5, three to five main dendrites usually originated from the soma and bifurcated into secondary and tertiary branches. The diameter or width of the dendritic trees was about 200 pm (ranged from 100 to 350 pm). Most of the dendritic trees were within layer I event though in a few cases dendritic branches went to layer 11. Dendrites were spineless in half of the cells in this class. In about a third of the cells, dendrites were sparsely spiny whereas the remaining cells had spiny dendrites.

Axons usually originated from the proximal portions of ventral or horizontal dendrites and, in a few cases, directly from the lower part of the somata. Initially, the axon traveled horizontally, obliquely or ventrally for up to 10 pm. It then branched into horizontal or oblique trunks. Each of these trunks gave off numerous branches which ramified profusely in layer I. Axonal branches often extended up to the pial surface but only occasionally descended into layer 11. The caliber of the axonal branches was mostly uniform along their entire length. Numerous small enlargements or varicosities, suggestive of boutons en passant, were seen along the axonal collaterals of every cell.

The extensive axonal arbors of these layer I neurons suggest that branches from nearby layer I neurons may converge. When 2 to 4 layer I neurons were labeled in single

CR cells.

originated from the bottom of the soma as indicated by the arrowhead, and then turned giving off numerous collaterals. The collaterals spread in layer I and formed an elaborate axonal arbor with a few collaterals descending to layer 11.

slices (Fig. 61, axonal branches from neighboring layer I neurons formed a dense interwoven axonal plexus. Axonal branches from neurons whose somata were several hun- dred microns apart interdigitated, presumably synapsing onto other layer I neurons and the apical dendrites of pyramidal neurons.

Layer I neurons sending axon collaterals to deeper layers. This type of cell (N = 21) was defined by having a major portion of its axon arbor in layer IIiIII and even upper layer IV. An example is shown in Figure 7. Somata of this cell type were usually located in mid and lower portions of layer I and were multipolar or irregular in shape with a dimension of about 12 x 15 pm. Three to five primary dendrites were found to originate from somata. Secondary and tertiary dendritic branches were formed but higher order branches were rare in most cases. The dendritic tree was about 200 pm in diameter and largely confined to layer I. The dendrites of 17 cells of this type were smooth (Fig. 7) or sparsely spiny, whereas the dendrites of the remaining neurons were spiny. Beaded dendrites were also seen (Fig. 7). The axon originated from a main dendrite on the lower half of the soma or directly from the bottom of the soma. Several (usually 2-4) trunks emerged from the axon initial segment. These trunks then traveled horizontally, obliquely and ventrally, repeatedly giving off collaterals and forming elaborate axonal arbors with diameters around 700 pm. The axon collaterals were usually constant in caliber for their entire length. Varicosities were always seen on the collaterals.

Cingulate cortex is considered to be a part of the limbic system (Vogt, 1985). To examine whether there was any difference in layer I neurons between cingulate and other cortices, layer I neurons in the cingulate cortex were labeled. Layer I cells with profiles similar to those found in frontal and parietal cortices were also found in the cingulate cortex. Figure 8 shows one of the two layer I neurons with major descending axonal branches recovered from cingulate cortex. In this cell, the axonal arbor was not extensive but reached layer 111. The dendrites were moder- ately beaded but spine free. The dendritic tree was small with a diameter of 100 pm, mainly restricted to layer I.

Nineteen cells were included in this group. Cell bodies were usually located in upper or

Vertical layer Z cells.

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MORPHOLOGYOFLAYERINEURONS 205

Fig. 7. An example of a layer I neuron with axonal collaterals in deep cortical layers. This cell was from the medial frontal cortex of a PN 17 rat. The soma was relatively large (12 x 18 pm) and was located in middle layer I. Four main dendrites arose from the soma. The dendrites were headed and aspiny. The dendritic tree had a diameter of about 200 pm, largely restricted to layer I and upper layer 11. The axon initial

r i segment, indicated by the arrows, originated from a primary dendrite. Axon collaterals spread widely in the horizontal and vertical direction such that a major portion of the elaborate axonal arhor was located in layer IIIIII. The diameter of the axonal arhor was about 1,500 pm, much larger than that of the dendritic tree.

Fig. 8. Camera lucida reconstruction of a PN 18 cingulate layer I neuron with axon collaterals reaching deeper layers. Layer I cells with profiles similar to those found in frontal and parietal cortices were also found in the cingulate cortex. No difference in layer I neurons between cingulate and other cortices were observed.

middle layer I. The somata had dimensions of around 10 x 15 Fm and had a distinct inverted pyramidal- or cone-like shape. The photomicrographs (Fig. 9) and the camera

lucida reconstruction (Fig. 10) show a well-filled vertical layer I cell. This type of neuron differed from the so-called inverted pyramidal neurons in that the axons originated


Fig. 9. Photomicrographs ofa biocytin-labeled vertical layer I neuron recovered from frontal cortex in a PN 15 rat. A: Low power view of the vertical layer I neuron. The pial surface is indicated by the arrowheads. Layer I is clearly identifiable by its cell-free appearance. The soma was located in upper layer I and had a dimension of 14 X 18 km. The long axis of the soma was perpendicular to the pial surface. Note the densely packed cells in layer IIIIII. B: Higher power view of the same cell. The

soma had an inverted pyramidal shape with the basal side toward the pial surface and the vertex facing downward. Four parent dendrites came off the soma; two originated from the vertex. The axon initial segment is indicated by the arrow. Inset: The area indicated by the two large arrows viewed at higher power. The sparsely spaced spines are indicated by the arrowheads. Scale bar = 100 pm in A, 50 pm in B.

from the vertex of the inverted pyramid-shaped somata. In inverted pyramidal neurons, axons come off from the basal sides of inverted pyramid-shaped somata (Feldman, 1984;

Kasper et al., 1994). Axonal collaterals formed extensive axonal arbor spreading mainly in layer I with a diameter up to more than 1,000 pm. Several primary dendrites emerged

MORPHOLOGYOFLAYERINEURONS 207

Layer I1 Fig. 10. Camera lucida reconstruction of the vertical type layer I

neuron shown in Figure 9. The dendritic tree had a diameter of about 180 pm and remained in layer I. The axonal initial segment originated from a main dendrite near the vertex of the pyramidal soma (as

from the somata and the one originating from the vertex of the somata was particularly prominent and thick. Den- drites of this cell type were smooth or sparsely spiny (Fig. 9, lower panel). The dendritic trees were about 200 km (100 to 250 pm) in diameter.

Vertical type layer I cells were also found in the cingulate cortex. Figure 11 shows a cingulate vertical type layer I cell whose soma was located just under the pial surface. The main dendrite was derived from the vertex. The dendrites were highly beaded (upper panel of Fig. 11) and sparsely spiny (lower panel of Fig. 11). The dendritic tree was small and simple even though one dendritic branch descended into layer 11. Axonal collaterals also formed an extensive axonal arbor (middle panel of Fig. 11).

Twenty-two neurons of this type were recovered in upper and middle layer I in PN 13-21 rats. The major distinguishing feature of this neuron type was that the longer axis of the somata and axonal arbors were more or less parallel to the pial surface, as shown in Figure 12. In addition, the axonal arbors often spread evenly from the two sides of the somata. Therefore, the profile of these cells often gave a horizontal bipolar impression. The axonal arbors were dense and larger than the dendritic trees. The dendrites were often smooth or sparsely spiny and/or beaded. Cells in this group differed from the neonatal horizontal bipolar cells (CR cells) by virtue of their smaller and more compact somata and larger and denser axonal arbors.

Other layer Z neurons. Thirty layer I neurons recovered from PN 13-21 animals could not be assigned to any of the above groups. Figure 13 shows a cell whose axonal arbor, particularly the axon trunk, resembles the vertical cell type whereas the shape of the soma and the location of the axonal arbor do not. Figure 14 shows another layer I neuron included in this category. The somata of neurons in this group were of medium size (about 15 x 10 pm), multipolar or irregular in shapes, and located throughout layer I. The dendrites were smooth or sparsely spiny. Like other cell types, axonal branches in these neurons formed elaborate arbors.

Bipolar layer Z neurons.

DISCUSSION The main finding of the present study is that, in the rat

neocortex, layer I neurons have extensive axonal arbors

100 pm indicated by the arrowhead), descended, and gave off several main collaterals which traveled more or less horizontally in layer I. The collaterals formed an extensive axonal arbor with a diameter of 700 Fm in layer I.

suggesting significant synaptic interactions among neurons in this and other cortical layers. Our descriptions of the dendritic and axonal arbors may underestimate the true extent of these processes since 150 to 250 pm-thick brain slices were used in the present study. Developmental changes were rapid in the first 10 postnatal days. Subse- quent to PN 13, changes were slow and limited. Therefore, the profiles of layer I neurons from PN 13-21 rats are likely to be qualitatively similar to those in mature animals.

Cajal-Retzius (CR) cells CR cells were prominent in layer I from PN 0-7 rats.

These neurons had morphologies similar to those described by Cajal (DeFelipe and Jones, 1988) and Bradford et al. (1978) using the Golgi method in the developing neocortex and by Ogawa et al. (1995) by using a CR cell specific monoclonal antibody. After PN 7, CR cells became progres- sively less numerous. Previous investigations have attrib- uted this decline to degeneration and/or transformation of CR cells (Noback and Purpura, 1961; Bradford et al., 1978; Edmunds and Parnavelas, 1982; Parnavelas and Edmunds, 1983; Derer and Derer, 1990), as originally suggested by Cajal (DeFelipe and Jones, 1988). Marin-Padilla (1984, 1988, 1990) has proposed that the number of CR cells is fixed prior to the birth of the animal such that the density of CR cells decreases as the cortex expands and matures but the total number of the CR cells remains unchanged. However, from PN 6 to PN 14, the volume of the prefrontal cortex increases by about 50% (Van Eden and Uylings, 19851, whereas the decrease of CR cells is more dramatic and disproportionate to the volume increase. Therefore, the apparent disappearance of CR cells may be a combined result of dilution, degeneration and/or transformation.

It has been suggested that CR cells initially have axons which descend into the cortical plate during corticohistogen- esis and, as the cortex grows, all of the processes undergo a progressive horizontal lengthening (Marin-Padilla, 1984). In the present study, layer I neurons, including CR cells, were not observed to send axon collaterals into deep layers in the first postnatal week. We actually saw an expansion of axonal arbors with development in layer I neurons of non-CR type cells. Our observations also show that, in addition to CR cells, other types of non-pyramidal neurons are present in layer I from the earliest postnatal time.

Layer I1 100 urn

Fig. 11. Example of a vertical-type layer I neuron stained in the cingulate cortex. A: Photomicrograph of a vertical type layer I cell intracellularly labeled in the cingulate cortex from a PN 16 rat. Arrowheads indicate the pial surface. Arrow points to the axon initial segment which is partially out of focus. Note that the axonal branches appear thin and smooth whereas dendrites are thick and varicose. Scale bar = 30 pm.

B,C: Camera lucida reconstruction of the neuron shown above. Middle panel shows the dense axonal arbor whereas the dendritic tree is shown in lower panel. The soma was located just under the pia. Because of the curvature of the brain surface, a portion of the processes appear to extend out of pial surface.


Layer I1 100 ym

Fig. 12. Camera lucida reconstruction of a layer I horizontal bipolar cell. The cell was labeled in a frontal cortical slice from a PN 16 rat. Both the soma and the axonal arbor are horizontally oriented. The features of this cell type are clearly different from that of Cajal-Retzius cells. Arrowhead points to the axonal initial segment.

Morphological diversity of PN 13-21 neurons Despite the relative homogeneity in histochemistry (Gab-

bott and Somogyi, 1986; Prieto et al., 1994a,b) and electrophysiology (Zhou and Hablitz, 1996a,b,c), the morphological properties of layer I neurons were quite heterogeneous. Based on the somadendritic and axonal profiles, we classi- fied labeled layer I neurons into six types. Many of our layer I neurons had morphologies comparable to those described in previous reports (Bradford et al., 1978, rat occipital cortex; Meyer and Ferres-Torres, 1984, cat visual cortex; Sousa-Pinto et al. 1975, cat auditory cortex). The diverse morphologies are also apparent in Cajal‘s original drawings (DeFelipe and Jones, 1988). Attempts to group these neurons have never been successful. Cajal changed his classification repeatedly and at one time suggested there were more than ten cell types in layer I (DeFelipe and Jones, 1988). However, we found that layer I neurons from frontal, parietal, and cingulate cortices had similar somadendritic and axonal profiles suggesting that these are general properties of cells in this layer.

Dendrites of layer I neurons were usually smooth or sparsely spiny even though a small number of layer I neurons were quite spiny. Layer I neurons with spines have been reported in Golgi materials (Bradford et al., 1978; Haberly, 1983; Sousa-Pinto et al., 1975; Valverde and Facal-Valverde, 1986). Local circuit inhibitory neurons with spines have also been found in the hippocampal dentate gyrus (Han et al., 1993) and neocortex (Deuchars and Thomson, 1995; Kawaguchi and Kubota, 1996). Because the layer I neurons described in the present study were relatively immature, some of these neurons may lose their spines when they are fully mature as suggested for visual cortical nonpyramidal neurons (Meyer and Ferres- Torres, 1984). Dendrites of layer I neurons did not branch extensively and the diameter of the dendritic tree was about 200 p,m. A small, relatively simple dendritic tree was also observed by Bradford et al. (1978) and Anderson et al. (1992) in cortical layer I neurons.

The relatively small dendritic trees of layer I neurons may indicate a more effective input-output transformation. We estimated that the space constant h ( was 1 to 1.1 mm, according to the relation A = (a~R,,,/2&)l’~ (Rall, 19771, where a is the radius of the dendrites (estimated to be 0.8-1 wm), R, is the specific membrane resistivity (50 kOcm2, Zhou and Hablitz, 1996b; Sprutson et a1.,1993; Major et al., 1994), and Ri is the internal resistivity (200 Ocm, Sprutson

Dendritic processes.

et al., 1993; Major et al., 1994). This predicts that the dendritic trees of layer I neurons are within 0.1 A of the somata. If dendrites have higher R,,,s (Pongracz et al., 19911, then the dendritic trees may be even more electrotonically compact. Such morphologically small and electrotonically compact dendritic trees may result in small but highly effective receptive fields. Layer I neurons may therefore effectively transform inputs from relatively restricted receptive fields into outputs to much larger areas.

Cajal had difficulties in distinguishing axon from dendrites and described multi-axon layer I neurons on several occasions in his early publica- tions (DeFelipe and Jones, 1988). Marin-Padilla (1984) once reported that there are small layer I neurons with indistinguishable axonal and dendritic processes. In the present study, we found that, in well-filled layer I neurons in PN13-21 rats, axon initial segments and axon collaterals were reliably distinguished from dendrites on the basis of their smaller uniform caliber and smooth appearance.

The elaborate axonal arbors, extending up to 1,600 pm in layer I, were the most prominent feature of layer I neurons. The extent of the axonal arbors revealed in this present study was larger than previous estimates from Golgi (Sousa- Pinto et al., 1975; Bradford et al., 1978; Marin-Padilla, 1984) and intracellular Lucifer Yellow labeling studies (Anderson et al., 1992). The dimensions of the axonal arbors of our layer I neurons were comparable to those of basket and clutch cells revealed by intracellular staining in vivo in cat visual cortex (Somogyi et al., 1983; Kisvarday et al., 1985, 1987). Consistent with our findings, an electron microscopic study has shown that axonal elements com- prise almost half of layer I volume (Vaughan and Peters, 19731, even though it remains to be established how much of these axonal elements originate from intrinsic layer I neurons. Our results are also consistent with the report by Beaulieu et al. (1994) that a single layer I GABAergic neuron makes the largest number of synapses among the cortical layers in rat visual cortex.

Axonal ramifications.

Possible functions of layer I neurons Layer I neurons have been suggested to be important in

cortical development (Marin-Padilla, 1984, 1992; Ogawa et al., 1995) and may also function in local cortical circuits. In olfactory cortex, the lateral olfactory tract, carrying primary olfactory information, terminates exclusively in layer I on the apical dendrites of pyramidal neurons and on


B Pia

Fig. 13. A layer I cell not fitting the previously described cell classifications. This neuron was from a frontal slice from a PN 14 rat. A A photomicrograph of the cell. The soma was large (18 x 15 &m), irregular, and located in middleilower layer I. The pial surface is indicated by the arrowheads. Layer I1 is distinguished by the high

density of neuronal somata. Scale bar = 30 km. B: Camera lucida reconstruction of the cell. Both dendritic and axonal branches descended to layer 11. Its axonal, but not dendritic and somatic, pattern is similar to that of vertical-type cells. Arrow indicates the axon initial segment. Arrowhead indicates the patch finger.

MORPHOLOGYOFLAYERINEURONS

B

211

Fig. 14. A subpial layer I cell from a frontal slice from a PN 17 rat. A: A photomicrograph of the cell taken under a 40x lens and Nomarski optics. The cell body had a dimension of 15 x 10 pm, was irregular, and located just below the pial surface. The pial surface is indicated by the arrowheads. The arrow points to the axon. Scale bar = 15 pm. B: A

camera lucida reconstruction of the cell shown above. Axonal branches descended into layer IIIIII. The soma and the dendrites were spiny and even the axon initial segment was sparsely spiny. The arrow points to the axon.


GABAergic layer I neurons. Layer I neurons have been suggested as possible elements mediating feedforward inhi- bition onto pyramidal neurons in piriform cortex (Haberly, 1990). Pyramidal and nonpyramidal neurons in neocortex send axon collaterals into layer I (Martin, 1984; Valverde, 1985; Rockland and Virga, 1989; Cowan and Wilson, 1994; Kisvarday et al., 1987). Because most layer I neurons are GABAergic, excitatory inputs from underlying pyramidal neurons or other areas will excite layer I inhibitory neurons. Layer I neurons may in turn inhibit pyramidal neurons by synapsing onto the dendrites and/or somata of these pyramidal neurons. Layer I neurons can also serve as a feedback or feedforward inhibitory element. Because of the extensive axonal arborization of layer I neurons, excita- tion of a single layer I neuron may result in inhibitory influence on neurons in a relatively large area. Layer I neurons may also interact with each other and/or deep layer inhibitory neurons, resulting in disinhibition. If the neurotransmitters used by layer I neurons are not homog- enous (Del Rio et al., 1995; Meyer and Gonzalez-Hernadez, 19931, then more complex interactions may occur. Further- more, Yuste et al. (1994) have suggested that the distal apical dendrites constitute a separate functional domain in pyramidal neurons. Inhibitory outputs from layer I neurons may influence activities in this compartment.

ACKNOWLEDGMENTS We thank Alison Margolies for excellent technical assis-

tance, Felicia Hester for advice, and Dr. M.J. Friedlander for use of equipment. We also thank Dr. C.E. Ribak for reading and commenting on an earlier version of this paper. This work was supported by NIH grants NS22373 and HD32901.

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Morphological properties of intracellularly labeled layer I neurons in rat neocortex