PSA-NCAM is Expressed in Immature, but not Recently Generated, Neurons in the Adult Cat Cerebral Cortex Layer II

Abstract

Neuronal production persists during adulthood in the dentate gyrus and the olfactory bulb, where substantial numbers of immature neurons can be found. These cells can also be found in the paleocortex layer II of adult rodents, but in this case most of them have been generated during embryogenesis. Recent reports have described the presence of similar cells, with a wider distribution, in the cerebral cortex of adult cats and primates and have suggested that they may develop into interneurons. The objective of this study is to verify this hypothesis and to explore the origin of these immature neurons in adult cats. We have analyzed their distribution using immunohistochemical analysis of the polysialylated form of the neural cell adhesion molecule (PSA-NCAM) and their phenotype using markers of mature neurons and different interneuronal populations. Additionally, we have explored the origin of these cells administering 5'bromodeoxyuridine (5'BrdU) during adulthood. Immature neurons were widely dispersed in the cerebral cortex layers II and upper III, being specially abundant in the piriform and entorhinal cortices, in the ventral portions of the frontal and temporoparietal lobes, but relatively scarce in dorsal regions, such as the primary visual areas. Only a small fraction of PSA-NCAM expressing cells in layer II expressed the mature neuronal marker NeuN and virtually none of them expressed calcium binding proteins or neuropeptides. By contrast, most, if not all of these cells expressed the transcription factor Tbr-1, specifically expressed by pallium-derived principal neurons, but not CAMKII, a marker of mature excitatory neurons. Absence of PSA-NCAM/5'BrdU colocalization suggests that, as in rats, these cells were not generated during adulthood. Together, these results indicate that immature neurons in the adult cat cerebral cortex layer II are not recently generated and that they may differentiate into principal neurons.

Keywords: adult neurogenesis; interneuron; neuronal differentiation; principal neuron; structural plasticity.

Figure 1

Camera lucida drawings showing three representative 50 μm coronal sections covering the rostral to caudal extent of the cat cerebral cortex, located +5, ™1, and ™12 mm from Bregma (Reinoso-Suarez, 1961). Rectangles in the bottom of the figure (a–f) are schematic representations of the distribution of PSA-NCAM expressing cells in the squared regions of the three coronal sections. The somata of PSA-NCAM expressing S cells in layer II are indicated by black dots and those of L cells by red squares. Scale bar: 5 mm.

Figure 2

Distribution of PSA-NCAM IR cells in the cat cerebral cortex. (A) Panoramic view of PSA-NCAM expression in the cat temporal cortex showing the presence of a dense band of immunostained cells in layer II. Note the characteristic vertical processes located in this cortical region. Inset in top right of the photograph shows two vertical processes to which three PSA-NCAM expressing small round somata are apposed. (B,C) Detailed view of PSA-NCAM immunoreactive cells in layer II of cat temporal cortex. Observe the different morphologies displayed by S cells: tangled (arrowheads) and semilunar–pyramidal transition cells (asterisks). (D) PSA-NCAM expressing L cells in deep layers of the cerebral cortex displaying multipolar or bipolar morphology. Scale bars: 300 μm for (A), 60 μm for (B–D). Inset in (A) is a 3× magnification of the boxed area.

Figure 3

Immature phenotype of PSA-NCAM expressing neurons in the cat cerebral cortex layer II. PSA-NCAM expressing cells in layer II (asterisks) co-express the immature neuronal markers doublecortin (DCX) (A) and the cyclic nucleotide-gated cation channel 3 (CNGA3) (B). Some PSA-NCAM expressing cells in layer II (asterisks) express weakly NeuN, a nuclear marker expressed by mature neurons (C). All the images in this figure are 2D projections of three consecutive confocal planes located 1 μm apart. Scale bar: 25 μm.

Figure 4

Polysialylated form of the neural cell adhesion molecule expressing neurons in the cat cerebral cortex layer II are immature excitatory neurons. (A) PSA-NCAM expressing cells the cat cerebral cortex layer II co-express Tbr-1, a transcription factor specifically expressed by pallium-derived principal neurons). (B) However, none of them express Ca(2+)/CaM-dependent protein kinase II (CAMKII), a marker of mature excitatory neurons. All the images in this figure are 2D projections of three consecutive confocal planes located 1 μm apart. Scale bar: 30 μm.

Figure 5

Polysialylated form of the neural cell adhesion molecule expressing neurons in layer II are not interneurons. Lack of colocalization between PSA-NCAM and different markers of interneurons in the cat cerebral cortex layer II: (A) glutamic acid decarboxylase 67 isoform (GAD67), (B) D-28 K Calbindin (Cb), (C) calretinin (Cr), (D) parvalbumin (Pv), (E) vaso-intestinal peptide (VIP), (F) neuropeptide Y (NPY), (G) cholecystokinin (CCK), (H) somatostatin (SST), or (I,J): neural isoform of the nitric oxide synthase (nNOS). All the images in this figure are 2D projections of three consecutive confocal planes located 1 μm apart. Scale bar: 25 μm for (A,E–H); 50 μm for (B–D,J); 75 μm for (I).

Figure 6

Phenotype of PSA-NCAM expressing neurons in deep layers of the cat cerebral cortex. Neurons expressing PSA-NCAM in deep layers (III–VI) of the cat cerebral cortex co-express NeuN (A) but they do not co-express Ca(2+)/CaM-dependent protein kinase II (CAMKII) (B). Many of these cells co-express glutamic acid decarboxylase 67 isoform (GAD67) (C) and the calcium binding proteins D-28k calbindin (Cb) (D) and calretinin (Cb) (E), but none of them express parvalbumin (Pv). All the images in this figure are 2D projections of three consecutive confocal planes located 1 μm apart. Scale bar: 25 μm.