Abstract

New neurons in the adult brain transiently express molecules related to neuronal development, such as the polysialylated form of neural cell adhesion molecule, or doublecortin (DCX). These molecules are also expressed by a cell population in the rat paleocortex layer II, whose origin, phenotype, and function are not clearly understood. We have classified most of these cells as a new cell type termed tangled cell. Some cells with the morphology of semilunar–pyramidal transitional neurons were also found among this population, as well as some scarce cells resembling semilunar, pyramidal. and fusiform neurons. We have found that none of these cells in layer II express markers of glial cells, mature, inhibitory, or principal neurons. They appear to be in a prolonged immature state, confirmed by the coexpression of DCX, TOAD/Ulip/CRMP-4, A3 subunit of the cyclic nucleotide-gated channel, or phosphorylated cyclic adenosine monophosphate response element–binding protein. Moreover, most of them lack synaptic contacts, are covered by astroglial lamellae, and fail to express cellular activity markers, such as c-Fos or Arc, and N-methyl-d-aspartate or glucocorticoid receptors. We have found that none of these cells appear to be generated during adulthood or early youth and that most of them have been generated during embryonic development, mainly in E15.5.

Introduction

Neuronal production persists in discrete regions of the adult central nervous system (CNS). New neurons are incorporated to the granule layer of the dentate gyrus and to the olfactory bulb after being generated in the subventricular zone (SVZ) (Kempermann 2005). Recent studies have indicated the presence of neurogenesis in the adult neocortex and other brain regions, although this is still a matter of debate (please see Nowakowski and Hayes 2000; Rakic 2002b; Gould 2007 for review). Newly generated neurons can be identified by the coexpression of different molecules related to neuronal development. Immature neurons generated during adulthood express molecules related to cytoskeletal dynamics, such as doublecortin (DCX) or TOAD/Ulip/CRMP-4 (TUC-4) (rCRMP4, TOAD64) (Minturn, Geschwind, et al. 1995; Gleeson et al. 1999). These cells also express the polysialylated form of the neural cell adhesion molecule (PSA-NCAM), which is related to different neurodevelopmental processes (Seki and Arai 1993). Different reports have described a population of cells in the layer II of the paleocortex of adult rodents, specially in the piriform and lateral entorhinal cortices, which expresses PSA-NCAM, DCX, and TUC-4 (Seki and Arai 1991; Nacher et al. 2000; Fox et al. 2000; Nacher, Crespo, McEwen, et al. 2001; Varea, Castillo-Gomez, Gomez-Climent, Guirado, et al. 2007). A previous study demonstrated that many of these cells in layer II do not express the mature neuronal marker NeuN, suggesting an immature state, although no quantification was performed (Nacher et al. 2002a). Moreover, there is discrepancy on the time of origin of these cells. Although some reports have failed to find adult neurogenesis in this region (Nacher et al. 2002a; Bonfanti 2006), 3 recent studies have described the presence of newly generated neurons in the adult paleocortex (Pekcec et al. 2006; Shapiro, Ng, Kinyamu, et al. 2007; Shapiro, Ng, Zhou, Ribak, et al. 2007). In order to investigate in detail the phenotype of these putative immature cells in the paleocortex layer II, we have analyzed the expression of molecules commonly found in recently generated neurons and neuronal progenitor cells in the adult CNS, such as those found in the dentate gyrus and SVZ/olfactory bulb. We have also checked whether these cells expressed typical markers of mature neurons and specific markers of principal neurons and interneurons. The glial nature of these cells has also been studied using markers of astrocytes, oligodendrocytes, and microglia. Using electron microscopy we have observed the ultrastructural characteristics of these cells and the presence of synaptic contacts on them. Additionally, using 5′bromodeoxyuridine (BrdU) pulse-chase experiments we have analyzed whether these cells were proliferating and whether they were generated during adulthood, during early youth or during embryogenesis. We have also explored whether these cells were functional, studying the expression of molecules related to cellular activity. Because the number of cells expressing PSA-NCAM in the paleocortex layer II is regulated by N-methyl-d-aspartate (NMDA) receptors and glucocorticoids (Nacher et al. 2002a, 2004), we have also studied the expression of glucocorticoid and NMDA receptors, in order to understand whether excitatory amino acids and adrenal steroids could act directly on these cells.

Material and Methods

Animals and Treatments

Forty-three young-adult male Sprague–Dawley (SD) rats (3 months old, 300 ± 15 g, Harlan Iberica, Barcelona, Spain) were separated in different groups. 1) Eight rats were used to study PSA-NCAM expression and its colocalization with several cellular markers using immunohistochemistry. 2) Thirty rats were used to study whether PSA-NCAM immunoreactive cells in the paleocortex layer II were generated during adulthood using double PSA-NCAM/BrdU immunohistochemistry. All the rats in this group received 4 intraperitoneal (ip) injections, 1 each 12 h, of BrdU (Sigma, St Louis, MO; 50 mg/kg, in sterile saline) and were sacrificed 2, 4, 7, 14, 21, or 30 days after the last injection (n = 5 per group). 3) Five rats were used to study whether any of the PSA-NCAM immunoreactive cells in the paleocortex layer II was proliferating. These animals received an ip injection of BrdU (200 mg/kg, in sterile saline) and were sacrificed 2 h later.

A second set of 5 SD rats (45 days old, 280 ± 10 g, Harlan Iberica, Barcelona, Spain) was used to explore the possibility that PSA-NCAM expressing cells in paleocortex layer II were generated during early youth. These animals were also injected with BrdU (4 ip injections, 1 each 12 h, 50 mg/kg, in sterile saline) and were sacrificed 60 days after the last injection.

Six pregnant SD rats (Harlan Iberica) received 2 ip injections of 5′BrdU (50 mg/kg), 8 h apart, on the following days after coupling: E11.5, E13.5, E15.5 (n = 2 per group). The first 24 h after coupling were designated as embryonic day 0 (E0). Four males (3 months old, 306 ± 32 g) were selected from each offspring and processed for double PSA-NCAM/BrdU immunohistochemistry, in order to identify whether these cells in the paleocortex layer II were generated during embryogenesis.

Four CD1 mice (3 months old, 30 ± 5 g, Harlan Iberica) were used for double PSA-NCAM/calretinin (CR) fluorescence immunohistochemistry as described below.

Six SD rats (3 months old, 300 ± 50 g, Harlan Iberica) were used for PSA-NCAM, TUC-4, or CNGA-3 preembedding immunohistochemistry for electron microscopy.

Histological Procedures

Rats destined for light microscopy studies were perfused transcardially under deep chloral hydrate anesthesia, with saline and then 4% paraformaldehyde in sodium phosphate buffer 0.1 M, pH 7.4 (PB). Brains for fluorescence immunohistochemistry were cut with a vibratome (Leica VT 1000E, Leica, Nussloch, Germany) and 50-μm-thick transverse sections were collected and kept in cold PB (4 °C) before processing. Brains for conventional immunohistochemistry were cryoprotected with 30% sucrose in PB, coronal sections (50 μm) were obtained with a sliding freezing microtome (Leica SM2000R) and stored at −20 °C in 30% glycerol, 30% ethylene glycol in PB until used.

The rats processed for electron microscopy were perfused transcardially under deep chloral hydrate anesthesia, first with saline for 1 min, followed by 50 mL of 3.8% acrolein (Fluka AG, Busch, Switzerland) and then by 450 mL of 3% paraformaldehyde in PB. Brains were extracted and sliced with a vibratome as described above.

All animal experimentation was conducted in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) and was approved by the Committee on Bioethics of the Universitat de València. Every effort was made to minimize the number of animals used and their suffering.

Immunohistochemistry for Conventional Light Microscopy

Tissue was processed “free-floating” for immunohistochemistry as follows. Briefly, sections were incubated for 1 min in an antigen unmasking solution (0.01 M citrate buffer, pH 6) at 100 °C. After cooling down the sections to room temperature they were incubated with 10% methanol, 0.003% H2O2 in phosphate buffered saline (PBS) for 10 min to block endogenous peroxidase activity. After this, sections were treated for 1 h with 5% normal donkey serum (NDS) (Jackson ImmunoResearch Laboratories, West Grove, PA) in PBS with 0.2% Triton-X-100 (Sigma-Aldrich, St Louis, MO) and were incubated overnight at room temperature in anti-PSA-NCAM or anti-γ-aminobutyric acid (GABA) antibodies (please see Table1). After washing, sections were incubated for 1 h with donkey anti-mouse IgM or donkey anti-rabbit IgG biotinylated antibodies (1:250; Jackson ImmunoResearch Laboratories, West Grove, PA), followed by an avidin–biotin–peroxidase complex (ABC; Vector Laboratories, Peterborough, UK) for 30 min in PBS. Color development was achieved by incubating with 3,3′-diaminobenzidine tetrahydrochloride (Sigma-Aldrich) and 0.033% hydrogen peroxide in PB for 4 min. PBS containing 0.2% Triton-X-100 and 3% NDS was used for primary and secondary antibodies dilution.

Double Fluorescence Immunohistochemistry

In order to characterize the phenotype of PSA-NCAM immunoreactive cells, we have performed double immunohistochemistry using an anti-PSA-NCAM antibody and antibodies against different markers of immature neurons, mature neurons, interneurons, astrocytes, oligodendrocytes, microglia, and proteins related to neuronal activity (please see Table 1). Sections were processed as described above, but the endogenous peroxidase block was omitted. The first day, sections were incubated overnight at room temperature with mouse monoclonal IgM anti-PSA-NCAM antibody and 1 of the above-mentioned antibodies. The second day sections were washed and incubated for 1 h with donkey anti-mouse IgM, donkey anti-goat IgG, or donkey anti-rabbit IgG secondary antibodies conjugated with Alexa 488 or Alexa 555 (1:200; Molecular Probes, Eugene, OR) in PBS containing 0.2% Triton-X-100 and 3% NDS.

Table 1

Primary antibodiesa

Name Abbreviation Dilution Company 
Monoclonal mouse anti-α-actinin (Sarcomeric) Anti-α-actinin 1:500 Sigma-Aldrich 
Polyclonal goat for activity-regulated cytoskeleton-associated protein Anti-Arc 1:500 Santa Cruz Biotechnology, Inc. 
Polyclonal rabbit anti-γ-aminobutyric acid Anti-GABA 1:5000 Sigma-Aldrich 
Monoclonal rat anti-5′bromodeoxyuridine Anti-5′BrdU 1:200 Immunological Direct, Oxford Biotechnology 
Monoclonal mouse anti-Ca2+/calmodulin dependent protein kinase II Anti-CaMKII 1:200 Chemicon Int., Inc. 
Polyclonal rabbit anti-calbindin-D28K Anti-CB 1:2.000 SWANT 
Polyclonal rabbit anti-calretinin Anti-CR 1:2.000 SWANT 
Polyclonal rabbit c-Fos antibody Anti-c-Fos 1:2.000 Santa Cruz Biotechnology, Inc. 
Monoclonal mouse anti-cholecystokinin Anti-CCK 1:1.000 CURE 
Polyclonal rabbit anti-cyclic nucleotide-gated cation channel bAnti-CNGA-3 1:500 Alomone Labs; IL 
Polyclonal goat anti-doublecortin (C-18) Anti-DCX 1:250 Santa Cruz Biotechnology, Inc. 
Polyclonal rabbit anti-glial fibrillar acidic protein Anti-GFAP 1:500 Sigma-Aldrich 
Polyclonal rabbit anti-glucocorticoid receptor Anti-GR 1:50 ABCAM 
Monoclonal mouse anti-glutamate decarboxylase, isoform 67 Anti-GAD-67 1:500 Developmental Studies Hybridoma Bank 
Monoclonal mouse anti-microtubule associated protein2 Anti-MAP2 1:1000 Sigma-Aldrich 
Polyclonal rabbit anti-NG2 chondroitin sulfate proteoglycan Anti-NG2 1:250 Chemicon Int., Inc. 
Polyclonal rat anti-Nestin 401 Anti-Nestin 1:500 DSHB 
Monoclonal mouse anti-neuronal nuclear antigen Anti-NeuN 1:100 Chemicon Int., Inc. 
Polyclonal rabbit anti-neuropeptide Y Anti-NPY 1:1000 Provided by Dr T. J. Görcs 
Polyclonal rabbit anti-N-methyl-d-aspartate1 receptor Anti-NMDAR1 1:100 Chemicon Int., Inc. 
Polyclonal rabbit anti-parvalbumin Anti-PV 1:2.000 SWANT 
Polyclonal rabbit anti-Pax6 (C-17) Anti-Pax6 1:3.000 Provided by Dr Grant S. Mastick 
Polyclonal rabbit anti-phospho-CREB (Ser133) Anti-p-CREB 1:500 Upstate, New York, USA 
Monoclonal mouse rip antibody Anti-RIP 1:1.000 Developmental Studies Hybridoma Bank 
Monoclonal mouse anti-polysialylated form of the neural cell adhesion molecule Anti-PSA-NCAM 1:1400 Abcys, Paris, FR 
Polyclonal rabbit anti-somatostatin Anti-SST 1:200 Provided by Dr T. J. Görcs 
Polyclonal rabbit anti-TUC-4/rCRMP-4 Anti-TUC-4/rCRMP4 1:1.000 Chemicon Int., Inc. 
Polyclonal rabbit anti-vasoactive intestinal peptide Anti-VIP 1:1.000 Provided by Dr T. J. Görcs 
Name Abbreviation Dilution Company 
Monoclonal mouse anti-α-actinin (Sarcomeric) Anti-α-actinin 1:500 Sigma-Aldrich 
Polyclonal goat for activity-regulated cytoskeleton-associated protein Anti-Arc 1:500 Santa Cruz Biotechnology, Inc. 
Polyclonal rabbit anti-γ-aminobutyric acid Anti-GABA 1:5000 Sigma-Aldrich 
Monoclonal rat anti-5′bromodeoxyuridine Anti-5′BrdU 1:200 Immunological Direct, Oxford Biotechnology 
Monoclonal mouse anti-Ca2+/calmodulin dependent protein kinase II Anti-CaMKII 1:200 Chemicon Int., Inc. 
Polyclonal rabbit anti-calbindin-D28K Anti-CB 1:2.000 SWANT 
Polyclonal rabbit anti-calretinin Anti-CR 1:2.000 SWANT 
Polyclonal rabbit c-Fos antibody Anti-c-Fos 1:2.000 Santa Cruz Biotechnology, Inc. 
Monoclonal mouse anti-cholecystokinin Anti-CCK 1:1.000 CURE 
Polyclonal rabbit anti-cyclic nucleotide-gated cation channel bAnti-CNGA-3 1:500 Alomone Labs; IL 
Polyclonal goat anti-doublecortin (C-18) Anti-DCX 1:250 Santa Cruz Biotechnology, Inc. 
Polyclonal rabbit anti-glial fibrillar acidic protein Anti-GFAP 1:500 Sigma-Aldrich 
Polyclonal rabbit anti-glucocorticoid receptor Anti-GR 1:50 ABCAM 
Monoclonal mouse anti-glutamate decarboxylase, isoform 67 Anti-GAD-67 1:500 Developmental Studies Hybridoma Bank 
Monoclonal mouse anti-microtubule associated protein2 Anti-MAP2 1:1000 Sigma-Aldrich 
Polyclonal rabbit anti-NG2 chondroitin sulfate proteoglycan Anti-NG2 1:250 Chemicon Int., Inc. 
Polyclonal rat anti-Nestin 401 Anti-Nestin 1:500 DSHB 
Monoclonal mouse anti-neuronal nuclear antigen Anti-NeuN 1:100 Chemicon Int., Inc. 
Polyclonal rabbit anti-neuropeptide Y Anti-NPY 1:1000 Provided by Dr T. J. Görcs 
Polyclonal rabbit anti-N-methyl-d-aspartate1 receptor Anti-NMDAR1 1:100 Chemicon Int., Inc. 
Polyclonal rabbit anti-parvalbumin Anti-PV 1:2.000 SWANT 
Polyclonal rabbit anti-Pax6 (C-17) Anti-Pax6 1:3.000 Provided by Dr Grant S. Mastick 
Polyclonal rabbit anti-phospho-CREB (Ser133) Anti-p-CREB 1:500 Upstate, New York, USA 
Monoclonal mouse rip antibody Anti-RIP 1:1.000 Developmental Studies Hybridoma Bank 
Monoclonal mouse anti-polysialylated form of the neural cell adhesion molecule Anti-PSA-NCAM 1:1400 Abcys, Paris, FR 
Polyclonal rabbit anti-somatostatin Anti-SST 1:200 Provided by Dr T. J. Görcs 
Polyclonal rabbit anti-TUC-4/rCRMP-4 Anti-TUC-4/rCRMP4 1:1.000 Chemicon Int., Inc. 
Polyclonal rabbit anti-vasoactive intestinal peptide Anti-VIP 1:1.000 Provided by Dr T. J. Görcs 
a

Alphabetic order by name.

b

Other abbreviations: CNG3, CNGα2, CCNC1.

In order to convert monoclonal mouse primary antibodies into a species different from that of the anti-PSA-NCAM primary antibody (Lewis Carl et al. 1993) sections were incubated for 2 h with an excess of unconjugated goat anti-mouse IgG Fab fragments (1:100; Jackson ImmunoResearch Laboratories, West Grove, PA). Then, sections were washed and incubated in fluorescent secondary antibodies as described above.

N-methyl-D-aspartate 1 receptor (NR1) immunohistochemistry requires a pretreatment, which usually affects PSA-NCAM antigeneicity. For these reason, we have decided to investigate NR1 expression in DCX immunoreactive cells, as described earlier (Nacher et al. 2007). Briefly, the sections were pretreated as those used for BrdU immunohistochemistry and incubated for 48 h at 4 °C with a mixture containing anti-DCX and anti-NR1 primary antibodies (please see Table 1). Then, secondary fluorochrome-conjugated antibodies were applied as described above.

Mirror Technique

In order to analyze the coexistence of PSA-NCAM with GABA in paleocortex layer II cells, we employed the mirror technique (Kosaka et al. 1985) using alternate series of vibratome sections. The tissue was immunostained as described above, using primary antibodies against PSA-NCAM and GABA (please see Table 1) for each 2 alternate subseries. Then the sections were treated with 1% OsO4, 7% glucose in PB for 1 h, dehydrated with ascending alcohols, flat mounted with Durcupan resin (Fluka, Sigma-Aldrich), and coverslipped. PSA-NCAM immunoreactive perikarya cut in half at the surface of the sections, as well as nearby capillaries, were drawn with a camera lucida under 40× immersion oil objective. The other halves of the immunoreactive somata were located on the corresponding surface of the adjacent section (processed for GABA immunohistochemistry) using the capillaries as landmarks. Only those cells in which the 2 halves (immunoreactive or not) could be identified with the aid of interferential contrast, were evaluated (Kosaka et al. 1985).

PSA-NCAM/ BrdU Immunohistochemistry

Sections from alternate series were treated for 60 min at 60 °C in PB and denaturation of DNA was achieved by treating for 30 min with 2 M HCl in PB at room temperature. Then, sections were processed for immunohistochemistry as described above, using monoclonal rat IgG anti-BrdU in combination with anti-PSA-NCAM monoclonal mouse antibody (see Table 1). Secondary antibodies were anti-mouse IgM and anti-rat IgG secondary antibodies generated in donkey and conjugated with Alexa 488 and Alexa 555, respectively.

Observation and Quantification of Double-Labeled Cells

All sections processed for fluorescent immunohistochemistry were mounted on slides and coverslipped using DakoCytomation fluorescent mounting medium (Dako North America, Inc., Carpinteria, CA). Then the sections were observed under a confocal microscope (Leica TCS-SP). Z-series of optical sections (1 μm apart) were obtained using sequential scanning mode. These stacks were processed with LSM 5 Image Browser software. A one in 10 series of telencephalic sections from each animal was double-labeled as described. Fifty immunoreactive cells were analyzed in each case to determine the coexpression of PSA-NCAM and the markers described before.

Immunohistochemistry for Electron Microscopy

In this procedure, sections were incubated with sodium borohydride 1% in ddH2O for 10 min. Then, sections were washed in PB and the endogenous peroxidase activity was blocked as described above, without methanol. Free aldehydes were quenched using Tris buffer 0.5 N, pH 7.2 for 30 min. Subsequently, nonspecific binding sites were blocked with NDS 10% in PB with glycine 0.2% and lysine 0.2%. After washing, sections were processed using primary antibodies against PSA-NCAM, TUC-4, or CNGA-3, (please see Table 1), as described for conventional immunohistochemistry. Immunolabeling was intensified treating the sections with 1% osmium tetroxide (EMS, Hatfield, PA) in PB for 60 min, protected from light. After each step, sections were carefully rinsed in PB. Sections were dehydrated through increasing graded series of ethanol baths (30–100%, 10 min each step) and dyed with 1% uranyl acetate in 70% ethanol bath during the process of dehydration. Finally, sections were cleared in propylene oxide and flat-embedded in Durcupan. After the analysis of the sections under the light microscope, some cells were re-embedded in Durcupan, and 60-nm-thick ultrathin sections were cut with an ultramicrotome. Ultrathin sections were serially collected on formvar-coated single-slot copper grids and stained using lead citrate. Selected cells were observed and partially reconstructed from serial sections under a JEOL JEM-1010 electron microscope.

Results

Distribution and Morphology of PSA-NCAM Expressing Cells in the Paleocortex Layer II

The distribution of PSA-NCAM expressing cells in the piriform and entorhinal cortices layer II was identical to that described earlier (Nacher et al. 2002a; Varea, Castillo-Gomez, Gomez-Climent, Guirado, et al. 2007). Scattered PSA-NCAM expressing cells were also found in the layer II of the perirhinal cortex and the most ventral region of the agranular insular and ectorhinal cortices.

In the paleocortex layer II, the majority of PSA-NCAM immunoreactive cells were small (soma diameter: 8.9 ± 0.96 μm) and showed processes with highly irregular trajectories, usually restricted to layer II, although some of these processes with vertical trajectories were also found in layer I. Most of these cells appeared frequently in clusters of 2–5 and closely resembled those described previously as neurogliaform neurons (Haberly 1983) (Fig. 1A,B). However, although we have previously classified these small cells as neurogliaform neurons (Nacher et al. 2002a), we have found that none of them expressed GABA, glutamate decarboxylase, isoform 67 (GAD67), or α-actinin (please see Mature Neuronal Markers section below), which are commonly expressed by neurogliaform neurons (Price et al. 2005). In consequence, we have chosen to denominate them “tangled cells” due to their intricate neuritic trajectories.

Figure 1.

Confocal microscopic analysis of PSA-NCAM immunoreactive cells in the paleocortex layer II. (A) PSA-NCAM immunohistochemistry in the piriform cortex layer II. Note the high density of tangled cells. (B) PSA-NCAM immunohistochemistry in the entorhinal cortex layer II. A semilunar–pyramidal transition neuron can be observed (arrow) among the abundant smaller tangled cells. (C) Example of a PSA-NCAM immunolabeled semilunar cell displaying a basal process resembling an axon (arrowhead). (D) Detail of an inverted pyramidal PSA-NCAM immunoreactive cell. Note its single process directed to layer III (arrows), to which another PSA-NCAM expressing cell is attached (arrowheads). (E) Most PSA-NCAM immunoreactive tangled cells lack NeuN expression in their nuclei. However, some of these cells display faint NeuN expression in their nuclei (arrow). (F) PSA-NCAM expressing cells lacking MAP2 expression in their somata and dendrites. (G) PSA-NCAM expressing cells lacking CAMKII expression. (H) PSA-NCAM expressing cells lacking GAD67 expression in their somata. Note a GAD67 positive cell (arrow) which lacks PSA-NCAM expression. All the images in this figure are taken from unique confocal planes, except (C) and (D), which are 2D projections of reconstructions of 3 consecutive confocal planes located 1 μm apart. Scale bar: 20 μm. NeuN: neuronal nuclear antigen; MAP2: microtubule associated protein2; CAMKII: Ca2+/calmodulin dependent protein kinase II.

Figure 1.

Confocal microscopic analysis of PSA-NCAM immunoreactive cells in the paleocortex layer II. (A) PSA-NCAM immunohistochemistry in the piriform cortex layer II. Note the high density of tangled cells. (B) PSA-NCAM immunohistochemistry in the entorhinal cortex layer II. A semilunar–pyramidal transition neuron can be observed (arrow) among the abundant smaller tangled cells. (C) Example of a PSA-NCAM immunolabeled semilunar cell displaying a basal process resembling an axon (arrowhead). (D) Detail of an inverted pyramidal PSA-NCAM immunoreactive cell. Note its single process directed to layer III (arrows), to which another PSA-NCAM expressing cell is attached (arrowheads). (E) Most PSA-NCAM immunoreactive tangled cells lack NeuN expression in their nuclei. However, some of these cells display faint NeuN expression in their nuclei (arrow). (F) PSA-NCAM expressing cells lacking MAP2 expression in their somata and dendrites. (G) PSA-NCAM expressing cells lacking CAMKII expression. (H) PSA-NCAM expressing cells lacking GAD67 expression in their somata. Note a GAD67 positive cell (arrow) which lacks PSA-NCAM expression. All the images in this figure are taken from unique confocal planes, except (C) and (D), which are 2D projections of reconstructions of 3 consecutive confocal planes located 1 μm apart. Scale bar: 20 μm. NeuN: neuronal nuclear antigen; MAP2: microtubule associated protein2; CAMKII: Ca2+/calmodulin dependent protein kinase II.

Some cells resembling the semilunar–pyramidal transitional neurons described by (Haberly 1983) in the paleocortex layer II were also PSA-NCAM immunoreactive, although they appeared absent from the most rostral region of the piriform cortex. They were larger than the tangled cells (soma diameter: 14.8 ± 1.59 μm), they showed 1 or 2 long dendrites expanding into layer I and some thin basal processes (Fig. 1B). Occasionally, we have also found some PSA-NCAM expressing cells resembling semilunar neurons, in which only a thin process resembling an axon could be observed (Fig. 1C). Some scarce cells with the characteristic morphology of pyramidal neurons and some displaying fusiform morphology can be found also in the paleocortex layer II (Fig. 1D). Additionally, some small bipolar cells with short processes oriented vertically were observed in piriform cortex layer III, endopiriform nucleus and the deep layers of the lateral entorhinal cortex. These small immunolabeled cells were usually located adjacent to long immunoreactive processes that traverse vertically both layers. These vertical prolongations appeared to arise from PSA-NCAM expressing somata in layer II (Fig. 1D).

Expression of Glial Markers

Double immunohistochemistry using an anti-PSA-NCAM antibody in combination with anti-glial fibrilar acidic protein (GFAP), anti-Rip, or anti-OX42 antibodies revealed that none of PSA-NCAM expressing cells in layer II of the paleocortex of adult rats could be classified as an astrocyte, an oligodendrocyte or a microglial cell (Supplemental Fig. 1A–C).

Expression of Markers of Mature Neurons

PSA-NCAM expressing cells in paleocortex layer II rarely expressed NeuN (14 ± 1.93%, Fig. 1E). Moreover, in most of these scarce immunoreactive nuclei this expression was faint, especially in those corresponding to tangled cells. NeuN immunoreactivity was usually more intense in the nuclei of the larger PSA-NCAM expressing cell types (semilunar–pyramidal transitional, semilunar, pyramidal, and fusiform neurons). Tangled cells lack expression of MAP2 (Fig. 1F). However, faint MAP2 labeling could be found in some semilunar–pyramidal transitional neurons.

PSA-NCAM expressing cells in the paleocortex layer II did not express markers of principal neurons, such as Ca(2+)/CaM-dependent protein kinase II (CAMKII, Fig. 1G) or interneurons, such as GABA, GAD67, calbindin, parvalbumin, CR, somatostatin, neuropeptide Y, cholecystokinin, or vasointestinal peptide (Fig. 1H). Abundant cells expressing these markers can be found in the paleocortex, especially in deep layers (Supplemental Fig. 1D–J).

In order to unequivocally identify the neurogliaform phenotype of PSA-NCAM expressing cells in layer II of the adult paleocortex of rats (Nacher et al. 2002a), we performed double PSA-NCAM/α-actinin immunohistochemistry. Although some cells scattered in layers II and III expressed α-actinin, the expression of this protein was never found in any of the PSA-NCAM expressing cells in layer II (Supplemental Fig. 1K).

Expression of Markers of Immature Neurons and Neuronal Progenitor Cells

Many PSA-NCAM expressing cells, including all the subpopulations described in the precedent section, were also immunoreactive for DCX (87.4 ± 3.28%, Fig. 2A) and TUC-4 (68.7 ± 3.7%, Fig. 2G), as previously described (Nacher et al. 2000; Nacher, Crespo, McEwen, et al. 2001). Both proteins are intracytoplasmic and, consequently, allow a detailed observation of the cell morphology (Fig. 2BF). The processes of tangled cells were usually devoid of spines or excrescences, but in some of the dendrites of semilunar and semilunar–pyramidal transition neurons, located in layer I, occasional bead-like swellings, protrusions resembling thin dendritic spines, or typical spines could be observed (Fig. 2E,F). DCX immunostaining also allowed the identification of basal thin processes in these large cells, which resembled an axon (Fig. 2BD). Most, if not all, PSA-NCAM expressing cells also expressed the A3 subunit of the cyclic nucleotide-gated ion channel (CNGA-3; 97.6 ± 1.2%. Fig. 2H), which is strongly expressed by migrating neuroblasts of the rostral migratory stream (RMS) (Gutierrez-Mecinas et al. 2007). Moreover, most of PSA-NCAM expressing cells in the paleocortex layer II expressed phosphorylated cyclic adenosine monophosphate response element–binding protein (p-CREB) in their nuclei (94.6 ± 1.83%, Fig. 2I), a molecule expressed transiently in differentiating granule neurons of the adult hippocampus (Nakagawa et al. 2002). Because immature neurons in the dentate gyrus of adult mice and olfactory bulb of adult mice expressed CR (Dominguez et al. 2003; Brandt et al. 2003), we have also analyzed the expression of this calcium binding protein in PSA-NCAM expressing cells of the paleocortex layer II of adult mice. None of these cells coexpressed CR.

Figure 2.

Confocal microscopic analysis of PSA-NCAM immunoreactive cells in the paleocortex layer II. (A) Cells coexpressing PSA-NCAM and DCX. (BF) DCX expressing cells in the paleocortex layer II. (B) Typical semilunar–pyramidal transitional neuron. Observe the 2 apical dendrites (arrows) and the presence of a basal dendritic tree (arrowheads). (C) A couple of semilunar–pyramidal transition neurons (white arrows) in entorhinal cortex layer II. The blue arrow points to a tangled cell showing some characteristics of a semilunar neuron, such as the presence of 2 apical dendrites. (D) Detailed view of the cells shown on the right side of Fig. 3C. Note the presence of thick basal processes resembling dendrites and a thin process resembling an axon (arrowheads). Two typical tangled cells can be observed, one of them closely apposed to the semilunar–pyramidal transition neuron (red arrow) and the other located in the left margin of the figure (white arrow). (E, F) Entorhinal cortex layer I. High magnification view of dendritic processes from semilunar–pyramidal transition neurons. Observe the presence of several swellings, and protrusions resembling dendritic spines (arrowheads). (G) PSA-NCAM/TUC-4 immunoreactive cells. (H) Cells coexpressing PSA-NCAM and CNGA-3. (I) PSA-NCAM expressing cells displaying p-CREB expression in their nuclei. (J) Double PSA-NCAM/NG2 immunohistochemistry. Observe the absence of NG2 immunoreactivity in PSA-NCAM expressing cells. Some NG2 expressing processes can be observed in close apposition to PSA-NCAM expressing cell somata. (K) PSA-NCAM expressing cells containing BrdU immunoreactive nuclei in the piriform cortex of an adult rat injected with BrdU at E15.5. (B), (C), (D), (E), and (F) are 2D projections of reconstructions of 3 (BD) or 2 (A, B) focal planes located 1 μm apart. Scale bar: 20 μm for (AD) and (GJ). 10 μm for (E) and (F). DCX: doublecortin (C-18); TUC-4 (TOAD64 [Turned On After Division]/Ulip/CRMP); p-CREB: phospho-CREB (Ser133).

Figure 2.

Confocal microscopic analysis of PSA-NCAM immunoreactive cells in the paleocortex layer II. (A) Cells coexpressing PSA-NCAM and DCX. (BF) DCX expressing cells in the paleocortex layer II. (B) Typical semilunar–pyramidal transitional neuron. Observe the 2 apical dendrites (arrows) and the presence of a basal dendritic tree (arrowheads). (C) A couple of semilunar–pyramidal transition neurons (white arrows) in entorhinal cortex layer II. The blue arrow points to a tangled cell showing some characteristics of a semilunar neuron, such as the presence of 2 apical dendrites. (D) Detailed view of the cells shown on the right side of Fig. 3C. Note the presence of thick basal processes resembling dendrites and a thin process resembling an axon (arrowheads). Two typical tangled cells can be observed, one of them closely apposed to the semilunar–pyramidal transition neuron (red arrow) and the other located in the left margin of the figure (white arrow). (E, F) Entorhinal cortex layer I. High magnification view of dendritic processes from semilunar–pyramidal transition neurons. Observe the presence of several swellings, and protrusions resembling dendritic spines (arrowheads). (G) PSA-NCAM/TUC-4 immunoreactive cells. (H) Cells coexpressing PSA-NCAM and CNGA-3. (I) PSA-NCAM expressing cells displaying p-CREB expression in their nuclei. (J) Double PSA-NCAM/NG2 immunohistochemistry. Observe the absence of NG2 immunoreactivity in PSA-NCAM expressing cells. Some NG2 expressing processes can be observed in close apposition to PSA-NCAM expressing cell somata. (K) PSA-NCAM expressing cells containing BrdU immunoreactive nuclei in the piriform cortex of an adult rat injected with BrdU at E15.5. (B), (C), (D), (E), and (F) are 2D projections of reconstructions of 3 (BD) or 2 (A, B) focal planes located 1 μm apart. Scale bar: 20 μm for (AD) and (GJ). 10 μm for (E) and (F). DCX: doublecortin (C-18); TUC-4 (TOAD64 [Turned On After Division]/Ulip/CRMP); p-CREB: phospho-CREB (Ser133).

PSA-NCAM expressing cells in paleocortex layer II did not coexpress proteins commonly found in neuronal precursor cells of the adult CNS, such as nestin (Doetsch et al. 1997; Nacher, Rosell, et al. 2001) (Supplemental Fig. 1M) or Pax6 (Nacher et al. 2005; Maekawa et al. 2005; Hack et al. 2005) (Supplemental Fig. 1N). Most PSA-NCAM expressing cells in layer II lack NG2 expression (99 ± 0.57%, Fig. 2J). However, occasionally we have found cells in which partial colocalization of PSA-NCAM and NG2 could be observed (Supplemental Fig. 2). Interestingly, some NG2 processes were found in close apposition to the somata of PSA-NCAM expressing cells (Fig. 2J).

BrdU Labeling

Proliferating Cells

In adult animals injected with BrdU and sacrificed 2 h later, some BrdU-labeled nuclei could be found scattered in layer II. However, none of these nuclei were found inside a PSA-NCAM expressing perikaryon.

Adult Neurogenesis

Although in all the groups (adult rats injected with BrdU and sacrificed 2, 4, 7, 14, 21, or 30 days later) some scarce BrdU-labeled nuclei were found in layers II and III (many of them appeared in pairs), we never found any of them located inside a PSA-NCAM expressing soma. PSA-NCAM immunoreactive cells displaying a BrdU-labeled nucleus could be found in areas with known adult neurogenetic activity (Supplemental Fig. 1O).

Neurogenesis during Early Youth

Some BrdU-labeled nuclei were found in the paleocortex layer II of animals injected with BrdU when they were 45 days old and sacrificed when they were 105 days old, but none of them appeared inside a PSA-NCAM expressing somata.

Embryonic Neurogenesis

In the adult rats which received BrdU at E11.5, only 2 ± 1.41% of PSA-NCAM expressing somata in the paleocortex layer II displayed BrdU labeled nuclei. In animals injected at E13.5, 23.5 ± 4.5% of PSA-NCAM expressing somata contained BrdU labeled nuclei. When BrdU was administered at E15.5, 74.75 ± 4.2% of PSA-NCAM expressing cells displayed BrdU labeled nuclei (Figs 2K and 3, Supplemental Fig. 3). Big BrdU-labeled nuclei lacking peripheral PSA-NCAM expression and resembling those of pyramidal neurons, usually showed a dispersed chromatin pattern, whereas the small labeled nuclei located inside PSA-NCAM expressing somata normally showed a more condensed pattern.

Figure 3.

Graph representing the percentage of PSA-NCAM/5′BrdU double-labeled cells in the adult rats which received BrdU at E11.5, E13.5, and E15.5, respectively.

Figure 3.

Graph representing the percentage of PSA-NCAM/5′BrdU double-labeled cells in the adult rats which received BrdU at E11.5, E13.5, and E15.5, respectively.

Electron Microscopy

A detailed analysis of PSA-NCAM, TUC-4, or CNG3A expressing somata in the paleocortex layer II showed that they had only a small rim of cytoplasm surrounding the nucleus (Fig. 4AC, F and G). Many astroglial lamellae were found in close apposition to the plasma membrane both in the somata (Fig. 4AC, F, and G), as well as in their processes (Fig. 4D,J). It was common to find swellings of the extracellular space adjacent to the portions of the plasmatic membrane not covered by these glial processes (Fig. 4B,G). In the nuclei of labeled cells, chromatin appeared slightly more compacted than in that of the neighboring nonlabeled pyramidal neurons and the presence of heterochromatin clumps was also more abundant in the labeled cells (Fig. 4AC, F, and G). None of these immunoreactive cells had synapses on their somata or proximal processes in layer II. However, in layer I we found some very scarce asymmetric synapses with round clear vesicles contacting some of the labeled dendrites belonging to semilunar–pyramidal transitional cells (Fig. 4H,I).

Figure 4.

Electron micrographs of PSA-NCAM– (A, B), TUC-4– (C, D), and CNGA-3–immunolabeled cells (EI) of the paleocortex layer II. (A, B) Somata of PSA-NCAM expressing cells. The couple of cells in panel B are intimately associated. PSA-NCAM immunoreactivity is especially abundant in certain regions associated with the plasma membrane (arrowheads). Note the presence of clear structures surrounding the labeled somata (arrows), corresponding to astroglial lamellae and swellings of the extracellular space (asterisk). Several heterochromatin clumps can be observed inside the nuclei of the labeled cells. (C) Soma of a TUC-4 expressing cell showing an intensely labeled cytoplasm and a nucleus displaying abundant heterochromatin. (D) Transversal section of a proximal process from a TUC-4 expressing cell. Some mitochondria and cisternae can be observed inside the immunoreactive cytoplasm. Note that this process is also surrounded by clear structures resembling astroglial lamellae (arrows). (E) View of the resin-embedded section from which the ultrathin sections shown in (FI; squared areas) were obtained. (F) CNGA-3 immunoreactive cell surrounded by 2 unlabeled somata, probably corresponding to pyramidal neurons. (G) Detail of a couple of closely apposed tangled cells, in which the 2 nuclei, as well as the labeled cytoplasms, can be discerned. Note the presence of a swelling of the extracellular space (asterisk) and an astroglial lamella located closely to the plasma membrane (arrows). (H) Asymmetric synaptic contact (arrow) onto a CNGA-3 immunoreactive dendrite in layer I, corresponding to a semilunar–pyramidal transition neuron. (I) Detailed view of the synaptic contact (arrowheads) shown in (H). (J) Transversal section of a proximal process from a CNGA-3 immunoreactive cell surrounded by astroglial lamellae (arrows). Scale bar: 5 μm for (A), (B), and (F); 2.5 μm for (C) and (G); 1 μm for (D); 50 μm for (E); 1.5 μm for (H), 0.37 μm for (I) and (J). TUC-4 (TOAD64 [Turned On After Division]/Ulip/CRMP).

Figure 4.

Electron micrographs of PSA-NCAM– (A, B), TUC-4– (C, D), and CNGA-3–immunolabeled cells (EI) of the paleocortex layer II. (A, B) Somata of PSA-NCAM expressing cells. The couple of cells in panel B are intimately associated. PSA-NCAM immunoreactivity is especially abundant in certain regions associated with the plasma membrane (arrowheads). Note the presence of clear structures surrounding the labeled somata (arrows), corresponding to astroglial lamellae and swellings of the extracellular space (asterisk). Several heterochromatin clumps can be observed inside the nuclei of the labeled cells. (C) Soma of a TUC-4 expressing cell showing an intensely labeled cytoplasm and a nucleus displaying abundant heterochromatin. (D) Transversal section of a proximal process from a TUC-4 expressing cell. Some mitochondria and cisternae can be observed inside the immunoreactive cytoplasm. Note that this process is also surrounded by clear structures resembling astroglial lamellae (arrows). (E) View of the resin-embedded section from which the ultrathin sections shown in (FI; squared areas) were obtained. (F) CNGA-3 immunoreactive cell surrounded by 2 unlabeled somata, probably corresponding to pyramidal neurons. (G) Detail of a couple of closely apposed tangled cells, in which the 2 nuclei, as well as the labeled cytoplasms, can be discerned. Note the presence of a swelling of the extracellular space (asterisk) and an astroglial lamella located closely to the plasma membrane (arrows). (H) Asymmetric synaptic contact (arrow) onto a CNGA-3 immunoreactive dendrite in layer I, corresponding to a semilunar–pyramidal transition neuron. (I) Detailed view of the synaptic contact (arrowheads) shown in (H). (J) Transversal section of a proximal process from a CNGA-3 immunoreactive cell surrounded by astroglial lamellae (arrows). Scale bar: 5 μm for (A), (B), and (F); 2.5 μm for (C) and (G); 1 μm for (D); 50 μm for (E); 1.5 μm for (H), 0.37 μm for (I) and (J). TUC-4 (TOAD64 [Turned On After Division]/Ulip/CRMP).

Markers of Cellular Activity

None of the PSA-NCAM expressing cells studied in the paleocortex layer II expressed c-Fos (Fig. 5A). Arc expression was also virtually absent from these cells (1 ± 0.36%) (Fig. 5B).

Figure 5.

Confocal microscopic analysis of different markers on PSA-NCAM and DCX expressing cells in the paleocortex layer II. (A) PSA-NCAM expressing cells lacking c-Fos expression in their nuclei. (B) PSA-NCAM expressing cells lacking Arc expression in their nuclei. (C) PSA-NCAM immunoreactive cells lacking GR expression in their nuclei. (D) DCX expressing tangled cell lacking NR1 expression. (E) DCX expressing semilunar neuron expressing NR1 in its somata and proximal apical dendrite. All photographs in this figure correspond to single optical sections taken from z-stacks. Scale bar: 20 μm. Arc: activity-regulated cytoskeleton-associated protein.

Figure 5.

Confocal microscopic analysis of different markers on PSA-NCAM and DCX expressing cells in the paleocortex layer II. (A) PSA-NCAM expressing cells lacking c-Fos expression in their nuclei. (B) PSA-NCAM expressing cells lacking Arc expression in their nuclei. (C) PSA-NCAM immunoreactive cells lacking GR expression in their nuclei. (D) DCX expressing tangled cell lacking NR1 expression. (E) DCX expressing semilunar neuron expressing NR1 in its somata and proximal apical dendrite. All photographs in this figure correspond to single optical sections taken from z-stacks. Scale bar: 20 μm. Arc: activity-regulated cytoskeleton-associated protein.

Expression of Glucocorticoid and NMDA Receptors

PSA-NCAM expressing cells in layer II of the paleocortex of adult rats did not coexpress glucocorticoid receptor (Fig. 5C). There were differences among the expression of NR1 subunit of the NMDA receptor among different cell types in the paleocortex layer II. Although none of the DCX expressing tangled cells studied expressed this receptor (Fig. 5D), the vast majority of larger cells (semilunar–pyramidal transition, semilunar, pyramidal, and fusiform neurons) expressed NR1 (92 ± 4%) (Fig. 5E).

Discussion

On the Nomenclature of PSA-NCAM Expressing Cells in the Paleocortex Layer II

To date there is not a consensus on the identity of the PSA-NCAM expressing cells in the paleocortex layer II. Seki and Arai (1991) described some of them as semilunar neurons (Seki and Arai 1991). O'Connell et al. (1997) described most of them as small neurons and also acknowledged the presence of some scarce PSA-NCAM expressing pyramidal neurons. In a previous study we have classified these cells mainly as neurogliaform neurons and pyramidal/semilunar transitional neurons, although we also found some PSA-NCAM expressing cells with the morphology of pyramidal neurons and fusiform cells in the paleocortex layer II (Nacher et al. 2002a). The present report has confirmed these previous results. However, we demonstrate that the abundant population of small PSA-NCAM expressing cells does not correspond to neurogliaform neurons. Neurogliaform neurons are mature interneurons, which usually express GABA, GAD, or α-actinin (Price et al. 2005), and none of these markers are expressed by the small cells in layer II. Given the intricate trajectories of their processes we have chosen to denominate them “tangled cells.” It has to be noted, however, that some cells with characteristics of both tangled cells and semilunar neurons can be found, which may indicate a transition between these cell types. Although in rodents the distribution of PSA-NCAM–expressing cells in layer II appears to be restricted to the piriform, entorhinal, and perirhinal cortex (with some scarce cells located in the adjacent agranular insular and ectorhinal cortices), in mammals with larger cerebral cortices, such as rabbits (Bonfanti 2006), cats (E Varea and J Nacher, unpublished results), or primates (Kornack et al. 2005), there is a more widespread distribution of these cells, which can be found in layer II of most neocortical regions.

PSA-NCAM Expressing Cells in the Paleocortex Layer II are Immature Neurons

The present study provides evidence that the cells expressing PSA-NCAM in the paleocortex layer II are immature neurons. This evidence is based on 3 groups of experimental findings: 1) these cells do not express markers of mature neurons, 2) these cells express markers of immature neurons, and 3) their ultrastructural characteristics are typical of immature neurons.

Most of these cells lack expression of NeuN, a nuclear protein found in most mature neurons (Mullen et al. 1992), which is absent from immature neurons in the CNS neurogenic regions. It has to be noted, however, that certain mature neuronal populations, such as Purkinje cells, do not express NeuN (Mullen et al. 1992). However, none of the PSA-NCAM expressing cells in the paleocortex layer II express typical markers of interneurons or principal cells, thus excluding the possibility that they were mature but NeuN negative. The possibility that these cells were glia had to be investigated, because certain astrocytes (Theodosis et al. 1991; Shen et al. 1999) and oligodendrocyte precursors (Ben-Hur et al. 1998) express PSA-NCAM. However, the lack of expression of astroglial, oligodendroglial, and microglial markers completely excludes this hypothesis.

Despite its clear involvement in several neurodevelopmental events (see Bruses and Rutishauser 2001; Bonfanti 2006 for review), PSA-NCAM expression is not a sufficient criterion to classify a cell as an immature neuron, because some mature neurons do express this molecule in regions such as the neocortex and the hippocampus (Nacher et al. 2002a; Nacher, Blasco-Ibáñez, et al. 2002; Varea et al. 2005; Varea, Castillo-Gomez, Gomez-Climent, Blasco-Ibanez, et al. 2007). For this reason, we have investigated the expression of different proteins that are found in recently generated neurons during adulthood, both in the dentate gyrus and in the SVZ/RMS/olfactory bulb. DCX is a microtubule-associated protein expressed by migrating neuroblasts during embryonic development, which is also found in certain transiently amplifying progenitors and immature neurons in the granule cell layer and the SVZ/RMS/olfactory bulb (Brown et al. 2003). This molecule is also found in some cells in the striatum, located close to the lateral ventricle wall (Nacher, Crespo, McEwen, et al. 2001). Recent reports have clearly indicated that these cells are recently generated immature neurons, probably migrating from the SVZ (Yang et al. 2004; Dayer et al. 2005). The only other telencephalic region in which DCX-expressing cells can be found is the paleocortex layer II, indicating that these cells, also, should be considered immature neurons. In fact a recent report indicates that DCX is a protein exclusively expressed by progenitor cells or by cells restricted to the neuronal lineage (Walker et al. 2007). TUC-4 is also expressed by immature neurons during embryonic development (Minturn, Geschwind, et al. 1995) and is found in immature neurons of the adult dentate gyrus (Seki 2002) and SVZ (Bedard et al. 2002). However, this molecule is also found in other telencephalic regions where no evidence of adult neurogenesis has been found, such as the hypothalamus, and it is also present in a subset of oligodendrocytes in the adult CNS (Nacher et al. 2000). A recent report has described the presence of the CNGA-3 in migrating neuroblasts of the RMS (Gutierrez-Mecinas et al. 2007). p-CREB is also intensely expressed, although not exclusively, by immature neurons in the adult CNS (Nakagawa et al. 2002; Giachino et al. 2005).

Despite all the cautions that have to be taken into account regarding the expression of PSA-NCAM, TUC-4, CNGA-3, or p-CREB as exclusive markers of immature neurons, it is true that the coexpression of all these molecules has only been found in immature neurons of the neurogenic regions of the adult brain. In these neurogenic regions, up to 6 different cell types representing successive neurodevelopmental milestones can be identified. Following Kempermann's classification for the adult hippocampus (Kempermann et al. 2004): there is a multipotential precursor cell (type-1), which expresses nestin and GFAP. This precursor gives rise to 3 putative transiently amplifying progenitor cells, all of them lacking GFAP expression: type-2a cells express nestin alone, type-2b express nestin and DCX, and type-3 cells express DCX alone. These progenitors generate immature neurons, which retain DCX expression and start to express NeuN. These neuroblasts progressively differentiate into mature neurons, which express NeuN and lack DCX expression. If we consider these neurodevelopmental milestones, the PSA-NCAM/DCX expressing cells in the adult paleocortex layer II can only correspond to type-3 cells or to immature neurons, because they do not express GFAP or nestin, they express DCX and PSA-NCAM and only a minor portion express NeuN. However, immature neurons in mice express CR (Jankovski and Sotelo 1996; Dominguez et al. 2003; Brandt et al. 2003) and we have not found the expression of this calcium binding protein in the PSA-NCAM/DCX expressing cells of adult mice paleocortex layer II. These cells cannot, however, be considered truly type-3 progenitors, because we have found that they do not proliferate. We believe that the PSA-NCAM/DCX expressing cells in the adult paleocortex layer II are in an intermediate stage between type-3 cells and differentiating neurons. They have been generated during embryonic development, abandoned their mitotic activity and migrated to their final destination. Once there, they seem to enter a “dormant” stage and show features of early immature neurons, although they (or most of them) do not appear to have entered the final phase of their neuronal differentiation program.

Adult Neurogenesis in the Paleocortex Layer II?

Two recent studies have reported the presence of adult neurogenesis in the piriform cortex of mice (Shapiro, Ng, Zhou, Ribak, et al. 2007) and rats (Pekcec et al. 2006) and a previous report indicated the existence of newly generated neurons in the paleocortex of adult primates (Bernier et al. 2002). Additionally, during the review process of the present study, a new report on adult neurogenesis in the rodent piriform cortex has been published by Ribak's group (Shapiro, Ng, Kinyamu, et al. 2007). A previous study has described the presence of individual SVZ-derived neurons in the anterior olfactory nucleus and anterior olfactory cortex of perinatal mice (De Marchis et al. 2004). However, these cells appear to be absent from the posterior piriform cortex, where more abundant PSA-NCAM expressing cells exist. De Marchis et al. did not find evidence of such cells in the olfactory cortex of adult animals. Although we have not studied perinatal animals, our present results indicate that, if some neurogenesis exists in olfactory cortical structures during early postnatal development, this phenomenon does not continue in young animals.

Although the existence of adult neurogenesis in the cerebral cortex is still a matter of debate (see Rakic 2002a; Gould 2007 for review), different reports have failed to find evidence that DCX, TUC-4, or PSA-NCAM expressing neurons in the paleocortex layer II of adult rats and mice (Nacher et al. 2002a; Fontana et al. 2005), rabbits (Luzzati et al. 2003), or primates (Kornack et al. 2005) are being generated during adulthood. In fact, the number of recently generated neurons found in the adult paleocortex (Pekcec et al. 2006; Shapiro, Ng, Kinyamu, et al. 2007; Shapiro, Ng, Zhou, Ribak, et al. 2007) is very low when compared with the number of DCX or PSA-NCAM cells present in this region and this may be the cause by which some studies did not find evidence of adult neurogenesis. Additionally, there is reason to believe that claims of adult cortical neurogenesis outside the dentate gyrus may be based on data misinterpretation (Nowakowski and Hayes 2000; Rakic 2002b). Moreover, the presence of newly generated neurons in the entorhinal cortex, where an extremely abundant population of PSA-NCAM expressing cells exists, has not been described in the adult rodent brain. It is also important to note that the previous reports on adult neurogenesis in the rodent piriform cortex do not explicitly indicate or quantify the presence of 5′BrdU labeled cells expressing DCX or NeuN in layer II (Pekcec et al. 2006; Shapiro, Ng, Kinyamu, et al. 2007; Shapiro, Ng, Zhou, Ribak, et al. 2007).

A recent report has described the presence of DCX/NG2 expressing cells in layer II of the piriform and entorhinal cortices of adult rats (Tamura et al. 2007). Although these authors did not find evidence PSA-NCAM expression in these cells, we have observed, that a very small subpopulation of tangled cells coexpresses NG2 and PSA-NCAM. It is interesting to note that we found that all DCX expressing cells in the paleocortex layer II also express PSA-NCAM (Nacher, Crespo, McEwen, et al. 2001) and that identical results have been found in our present material, using a different anti-DCX antibody. Tamura et al. have also shown that, in the cerebral cortex, some of these DCX expressing cells may be proliferating or have been recently generated. We have not found evidence of such dividing or recently generated cells in the paleocortex layer II of our samples. Tamura et al. state, however, that these cells are very faintly labeled with DCX immunohistochemistry and, consequently, we may have missed them. In any case, our results demonstrate that most of PSA-NCAM expressing cells in the paleocortex layer II are generated during embryonic development, in a stage that appears to coincide with, or to occur slightly before, that of most layer II neurons in this region (Bayer 1986).

Although the doses of BrdU used in our study are considered nontoxic in adult animals (Cameron and McKay 2001), some possible toxic effects of BrdU cannot be totally excluded (Taupin 2007), especially in the embryos.

Are PSA-NCAM Expressing Cells in the Paleocortex Layer II Functional?

A critical question arising from our study is what the function is of PSA-NCAM expressing cells in the adult paleocortex layer II. Our double labeling experiments indicate that they are early immature neurons, which apparently have detained their differentiation program and are in a “dormant” stage. The fact that these cells never expressed activity-related molecules, such as the immediate early genes c-Fos and Arc, may be also indicative of this “dormant” stage. Their ultrastructure also gives support to this hypothesis: These cells commonly show heterochromatin clumps and they appear to be surrounded by multiple astroglial lamellae and swellings of the extracellular space adjacent to the plasmatic membrane, which isolate them from the surrounding nervous parenchyma. Moreover, they normally do not show synaptic contacts on their somata or proximal processes. These are 2 characteristics found in early immature neurons in the adult dentate gyrus (Seki and Arai 1999; Shapiro et al. 2005). Moreover, the presence of astroglial processes and the absence of synapses on DCX expressing cell somata in the piriform cortex layer II has been reported during the review process of the present study (Shapiro, Ng, Kinyamu, et al. 2007). However, it appears that a subpopulation of PSA-NCAM expressing cells in the paleocortex layer II, mostly constituted by semilunar and semilunar–pyramidal transitional neurons does receive, although sparsely, excitatory synapses in its apical dendrites located in layer I, suggesting that these cells are in a more differentiated state. Moreover, these cells show dendritic protrusions resembling spines and thin basal processes resembling axons. The fact that NeuN expression is more intense in these larger cells than in tangled cells also gives support to the hypothesis that large PSA-NCAM expressing cells in layer II are more mature than tangled cells, because similar increases in NeuN expression have been found during neuronal differentiation in the adult dentate gyrus (Kempermann et al. 2004; Marques-Mari et al. 2007). The fact that these larger cells also expressed the NR1 subunit of the NMDA receptor also supports this view, because this receptor is not expressed in early differentiating granule neurons in the adult dentate gyrus (Nacher et al. 2007). Although we have previously suggested that the expression of PSA-NCAM (Nacher et al. 2002a) in paleocortex layer II cells was linked to neuronal structural plasticity, our present results suggest that this expression, together with the astroglial covering, probably has an insulating role, which will prevent these cells from receiving synaptic inputs. We still do not have a plausible explanation for the expression of DCX or TUC-4 proteins in these cells, because during development these proteins are associated with a dynamic cytoskeleton, which is participating in neurite development or neuronal migration (Minturn, Fryer, et al. 1995; Francis et al. 1999).

The second intriguing point arisen by our study is the fate of PSA-NCAM expressing cells in the adult paleocortex layer II. Several studies have demonstrated that the number of these cells dramatically declines during aging (Abrous et al. 1997; Murphy et al. 2001; Varea, Castillo-Gomez, Gomez-Climent, Guirado, et al. 2007). This reduction is also observed when analyzing DCX or TUC-4 expression (J Nacher, unpublished observations). There are 2 possible explanations for this reduction in the expression of immature neuronal markers: 1) the cells expressing these proteins are progressively dying or 2) they are differentiating into mature neurons, which lack these markers. Both explanations are plausible. However, when observing the paleocortex layer II of 6-, 12-, or 24-month-old rats we have failed to find substantial numbers of pyknotic nuclei (J Nacher, unpublished observations). The second possibility appears more likely, and it is tempting to think that tangled cells could mature eventually into some of the larger PSA-NCAM expressing cells and then into mature neurons. This is supported by the fact that intermediate cell types between tangled and larger cells are habitually found in layer II and that, as discussed above, larger cells appear more mature than tangled cells. Then PSA-NCAM expressing cells in the paleocortex layer II may constitute a “reservoir,” which in different circumstances may complete its differentiation program. These modulations of PSA-NCAM expression in the paleocortex layer II are probably mediated by glucocorticoids (Nacher et al. 2004) and excitatory amino acids acting on NMDA receptors (Nacher et al. 2002a). However, a direct effect has to be excluded, because, as we have shown, PSA-NCAM expressing cells in layer II rarely express glucocorticoid or NMDA receptors. We still do not know the significance of these changes in PSA-NCAM expression. Although the number of PSA-NCAM expressing cells in the piriform cortex layer II increases 21 days after a single injection of NMDA receptor antagonist (Nacher et al. 2002b), we have not observed incorporation of newly generated neurons after this treatment (J Nacher, unpublished results). This suggests that PSA-NCAM expression upregulation occurs in preexisting cells, either in cells in which PSA-NCAM was already expressed, although at undetectable levels, or in cells that did not express PSA-NCAM before the treatment. Experiments that follow the fate of PSA-NCAM expressing cells after different experimental treatments and during aging will shed light on the mysterious nature of these cells.

Supplementary Material

Supplementary material can be found at: http://www.cercor.oxfordjournals.org/.

Funding

GV04A-134, GV04A-076, GVACOMP07/169, MEC2006-BFU07313/BFI, Foundation Jerome Lejeune; and MEC BFU2004-00931. Postdoctoral fellowship for stays in Research Excellence Centers located in the Comunitat Valenciana from the Generalitat Valenciana to E.V.; predoctoral fellowships (FPU) from the Spanish Ministry of Education and Science to M.A.G.-C. and E.C.-G.; and predoctoral fellowship (FPI) from the Spanish Ministry of Education and Science to R.G.

The authors are grateful to Dr Bruce McEwen and Dr Tatsunori Seki for their comments on the manuscript. Conflict of Interest: None declared.

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