PSA-NCAM expression in the rat medial prefrontal cortex

Neuroscience 136 (2005) 435– 443

PSA-NCAM EXPRESSION IN THE RAT MEDIAL PREFRONTAL CORTEX E. VAREA, J. NÁCHER,* J. M. BLASCO-IBÁÑEZ, M. Á. GÓMEZ-CLIMENT, E. CASTILLO-GÓMEZ, C. CRESPO AND F. J. MARTÍNEZ-GUIJARRO

telencephalic region comprises different regions of the cerebral cortex, with heterogeneous functional and cytoarchitectonic characteristics: dorsal anterior cingulate, prelimbic and infralimbic cortices, which correspond to human Broadman’s areas 24b, 32 and 25 respectively (Vogt and Gabriel, 1993; Conde et al., 1995; Paxinos and Watson, 1998). The rat medial prefrontal cortex (mPFC) is functionally analogous to the human/primate mPFC (Kolb, 1984; Uylings et al., 2003), which plays a crucial role in the control of cognitive function and results affected in several psychiatric disorders, such as schizophrenia or depression (Mayberg, 1997; Miller and Cohen, 2001). In recent years it has become evident that the mPFC and some other regions of the adult brain retain plastic capabilities, such as growth and branching of neurites, or remodeling of synaptic contacts. Numerous studies suggest that decreases in this neuroplasticity may underlie the symptoms of depressed or schizophrenic patients, thus indicating a new approach to the pathophysiology of these mental disorders (Duman, 2002; Costa and Silva, 2004; Frost et al., 2004). Neurons in the rat mPFC undergo dendritic remodeling or synaptic rearrangement under the influence of psychoactive drugs (amphetamine or cocaine) (Robinson and Kolb, 1997, 1999), gonadal hormones (Stewart and Kolb, 1994; Forgie and Kolb, 2003) or changes in blood pressure (Vega et al., 2004). Chronic stress and corticosterone administration also induce dendritic reorganization of mPFC pyramidal neurons (Wellman, 2001; Cook and Wellman, 2004; Radley et al., 2004, 2005; Brown et al., 2005), similar to what has been described in the hippocampus (see Mc Ewen (2000) for review), or the amygdala (Vyas et al., 2002, 2003). The neural cell adhesion molecule (NCAM) has the ability to incorporate long chains of polysialic acid, which confer it anti-adhesive properties. Consequently, the polysialylated form of the neural cell adhesion molecule (PSANCAM) plays an important role in CNS structural plasticity, allowing/preventing neurons to move or change their morphology. PSA-NCAM participates in plastic events such as axonal growth (Zhang et al., 1992) and synaptic reorganization (Seki and Rutishauser, 1998). In adult animals, this molecule is expressed in cerebral regions that are undergoing some kind of structural plasticity, such as the hypothalamo-neurohypophyseal system (Theodosis et al., 1994), the olfactory bulb (Miragall et al., 1988) the piriform and entorhinal cortices (Seki and Arai, 1991a), the amygdala (Nacher et al., 2002b) or the hippocampus (Seki and Arai, 1991b). Structural changes induced in hippocampal neurons by stress and corticosterone administration have been related to changes in PSA-NCAM expression (Sandi

Neurobiology, Cell Biology Department, Universitat de València, Dr. Moliner, 50, 46100 Burjassot, València, Spain

Abstract—The rat medial prefrontal cortex, an area considered homologous to the human prefrontal cortex, is a region in which neuronal structural plasticity has been described during adulthood. Some plastic processes such as neurite outgrowth and synaptogenesis are known to be regulated by the polysialylated form of the neural cell adhesion molecule (PSA-NCAM). Since PSA-NCAM is present in regions of the adult CNS which are undergoing structural remodeling, such as the hypothalamus or the hippocampus, we have analyzed the expression of this molecule in the medial prefrontal cortex of adult rats using immunohistochemistry. PSA-NCAM immunoreactivity was found both in cell bodies and in the neuropil of the three divisions of the medial prefrontal cortex. All cell somata expressing PSA-NCAM corresponded to neurons and 5= bromodeoxyuridine labeling after long survival times demonstrated that these neurons were not recently generated. Many of these PSA-NCAM immunoreactive neurons in the medial prefrontal cortex could be classified as interneurons on the basis of their morphology and glutamate decarboxylase, isoform 67 expression. Some of the PSANCAM immunoreactive neurons also expressed somatostatin, neuropeptide Y and calbindin-D28K. By contrast, pyramidal neurons in this cortical region did not appear to express PSA-NCAM. However, some of these principal neurons appeared surrounded by PSA-NCAM immunoreactive puncta. Some of these puncta co-expressed synaptophysin, suggesting the presence of synapses. Since the etiology of some psychiatric disorders has been related to alterations in medial prefrontal cortex structural plasticity, the study of PSANCAM expression in this region may open a new approach to the pathophysiology of these mental disorders. © 2005 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: calbindin-D28K, GAD67, interneuron, NPY, somatostatin, structural plasticity.

The rat medial prefrontal cortex is situated rostral to the genu of the corpus callosum and over the orbital, insular and medial surfaces of the rostral cerebral hemispheres (Krettek and Price, 1977; Vogt and Gabriel, 1993). This *Corresponding author. Tel: ⫹34-96-354-3241; fax: ⫹34-96-354-3241. E-mail address: [email protected] (J. Nacher). Abbreviations: CCK, cholecystokinin; GAD67, glutamate decarboxylase, isoform 67; GFAP, glial fibrillar acidic protein; mPFC, medial prefrontal cortex; NCAM, neural cell adhesion molecule; NDS, normal donkey serum; NeuN, neuronal nuclear antigen; NPY, neuropeptide Y; PB, sodium phosphate buffer; PBS, phosphate-buffered saline; PSANCAM, polysialylated form of the neural cell adhesion molecule; SST, somatostatin; VIP, vasoactive intestinal peptide; 5=BrdU, 5= bromodeoxyuridine.

0306-4522/05$30.00⫹0.00 © 2005 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2005.08.009

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et al., 2001; Pham et al., 2003; Nacher et al., 2004a). Although the expression of PSA-NCAM in the adult rodent mPFC has not been studied in detail, it is known that cortical PSA-NCAM expression is strongly downregulated after birth (Seki and Arai, 1991a). However, Seki and Arai (1993) reported scarce PSA-NCAM expressing neurons and fibers in the adult rat cingulate cortex (Seki and Arai, 1993b) and a recent report has described the existence of PSA-NCAM immunoreactive cells in the rat mPFC (Hardwick et al., 2005). In order to study the expression of PSA-NCAM in the mPFC we have analyzed: i) the presence and distribution of PSA-NCAM immunoreactivity; ii) the phenotype of PSANCAM immunoreactive somata and neuropil structures; iii) the inhibitory-interneuronal nature of PSA-NCAM immunoreactive cells and their ascription to any of the interneuronal groups already described in the rat mPFC (Gabbott et al., 1997). Although recent studies have demonstrated that the rodent mPFC is apparently devoid of postnatal neurogenesis (Madsen et al., 2005), we have tested whether PSA-NCAM immunoreactive cells in the mPFC could have been generated during adulthood, because this molecule is expressed transiently in recently generated neurons (Seki and Arai, 1993b).

EXPERIMENTAL PROCEDURES Animal treatments and histology Twenty male Sprague–Dawley rats (3 months-old, Harlan Iberica) were used in this experiment. Animals were separated in the following groups: i) eight rats were used to study PSA-NCAM expression and its colocalization with several cellular markers using immunohistochemistry; ii) 12 rats were used for double PSA-NCAM/5= bromodeoxyuridine (5=BrdU) immunohistochemistry. All the rats in this later group received four injections, one each 12 h, of 5=BrdU (Sigma-Aldrich, St. Louis, MO, USA, 50 mg/kg, i.p.) and were killed 14 days (n⫽4), 21 days (n⫽4) or 90 days (n⫽4) after the last injection. 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. The number of animals and their suffering has been minimized in all experiments described in this study. Rats 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). After perfusion, the brains were extracted and cryoprotected with 30% sucrose in PB. Coronal sections (50 ␮m) were obtained with a sliding microtome and stored at ⫺20 °C in 30% glycerol; 30% ethylene glycol, 40% PB until used.

PSA-NCAM immunohistochemistry 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, 3% 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, USA) in PBS with 0.2% Triton X-100 (Sigma) and were incubated overnight at room temperature in mouse monoclonal Men-B antiPSA-NCAM antibody (1:1400; generous gift of Dr. G. Rougon), or

mouse monoclonal 5A5 anti-PSA-NCAM antibody (1:500; generous gift of Dr. Urs Rutishauser). After washing, sections were incubated for 30 min with donkey anti-mouse IgM or donkey anti-mouse IgG biotinylated antibodies (Jackson Laboratories, 1:250), 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 (DAB, Sigma) for 4 min. PBS containing 0.2% Triton X-100 and 3% NDS was used for primary and secondary antibodies dilution. Pretreatment of the PSA-NCAM antibody with ␣-2,8-linked sialic polymer (colominic acid, Sigma) overnight, or the primary antibody omission during the immunohistochemistry prevented all the labeling in the mPFC.

Double immunofluorescence 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 neuronal, astroglial and oligodendroglial markers. We have also analyzed whether PSA-NCAM immunoreactive structures in the neuropil of the mPFC corresponded to synapses using double PSA-NCAM/synaptophysin immunohistochemistry. In general, sections were processed as described above, but the endogenous peroxidase block was omitted. The sections were incubated overnight with mouse monoclonal IgM anti-PSA-NCAM antibody (Men-B, 1:1400) and one of the following primary IgG antibodies: monoclonal mouse anti-neuronal nuclear antigen (NeuN, Chemicon Int. Inc., Temecula, CA, USA; 1:100); polyclonal rabbit antiglial fibrillar acidic protein (GFAP, Sigma 1:500), monoclonal mouse anti-glutamate decarboxylase, isoform 67 (GAD67, Chemicon Int; 1:1000); monoclonal mouse rip antibody (Developmental Studies Hybridoma Bank, 1:1000); polyclonal rabbit anti-calbindinD28K (SWANT, Bellinzona, Switzerland; 1:2000); polyclonal rabbit anti-calretinin (SWANT, 1:2500); polyclonal rabbit anti-parvalbumin (SWANT, 1:2000); monoclonal mouse anti-cholecystokinin (CCK, CURE, 1:1000); polyclonal rabbit anti-vasoactive intestinal peptide (VIP, kindly provided by Dr. T. J. Görcs, 1:1000) (Lantos et al., 1995); polyclonal rabbit anti-neuropeptide Y (NPY, kindly provided by Dr. T. J. Görcs, 1:1000) (Csiffary et al., 1990); polyclonal rabbit anti-somatostatin (SST, kindly provided by Dr. T. J. Görcs, 1:200 (Leranth and Frotscher, 1987); polyclonal rabbit anti-synaptophysin (Sigma, 1:200). After washing, sections were incubated with donkey anti-mouse IgM, donkey anti-mouse IgG or donkey anti-rabbit IgG secondary antibodies conjugated with Alexa 488 or Alexa 555 (Molecular Probes, Eugene, OR, USA; 1:200) in PBS containing 0.2% Triton X-100 and 3% NDS.

PSA-NCAM/5=BrdU immunohistochemistry In order to check whether PSA-NCAM immunoreactive cells in the mPFC were recently generated, we have performed double PSANCAM/5=BrdU immunohistochemistry in sections from rats injected with 5=BrdU and let survive for 14, 21 or 90 days. Sections were treated for 60 min at 60 °C in PB. Denaturation of DNA was achieved by treating for 30 min with 2 M HCl in PB at room temperature. Then, sections were processed as above, using the following primary antibodies: monoclonal rat IgG anti-5=BrdU (Immunological Direct, Oxford Biotechnology, Oxfordshire, UK; 1:200) and monoclonal mouse IgM anti-PSA-NCAM (Men-B, 1:1400). Secondary antibodies were anti-mouse IgM and anti-rat IgG secondary antibodies generated in donkey and conjugated with Alexa 488 or Alexa 555.

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Observation and quantification of double-labeled cells

Distribution of PSA-NCAM immunoreactive cells in the rat mPFC

All sections processed for fluorescent immunohistochemistry were mounted on slides and coverslipped using Permafluor mounting medium (Immunon/Shandon, Pittsburgh, PA, USA). Then the sections were observed under a confocal microscope (Leica, Wein, Austria; TCS-SP2). Z-series of optical sections (1 ␮m apart) were obtained using sequential scanning mode. These stacks were processed with LSM 5 Image Browser software. A 1-in-10 series of telencephalic sections from each animal was double-labeled as described. PSA-NCAM immunoreactive cells were first identified using conventional fluorescence microscopy. Then, a stack of confocal images covering all its three-dimensional extension was taken to confirm that PSA-NCAM labeling corresponded to a cell somata. Fifty PSA-NCAM immunoreactive cells were analyzed in each case to determine the co-expression of PSA-NCAM and the markers of mature neurons, interneurons or glial cells described before.

PSA-NCAM immunoreactive cells were present in all the subregions and layers of the mPFC (Fig. 2). They were more abundant in deep layers (V–VI), while their density was lower in layers II–III and very sparse in layer I. PSANCAM immunoreactive cells located in layer I were rounded and had middle-sized somata (around 20 ␮m diameter). In layers II–III two types of PSA-NCAM immunoreactive cells could be observed: a) scarce big cells (30 – 40 ␮m diameter) showing immunoreactivity in their somata and proximal processes (Fig. 3A and B), which frequently were multipolar and b) frequent small cells (less than 20 ␮m diameter) with fusiform morphology. In deep layers there were also two types of PSA-NCAM immunoreactive cells: a) scarce big cells displaying multipolar morphology (similar to those described in layer III), which were located mainly in layer V and b) more abundant small cells (Fig. 3C and D) in which a single PSA-NCAM immunoreactive process could be identified. These small cells appeared in layer V as well as in layer VI. This cellular distribution pattern was observed in all the subdivisions of mPFC. The only difference observed among the different regions was the presence of a layer of big cells with high intensity of staining in the upper limit of layer V in the infralimbic cortex. Big multipolar cells resembled those described in the piriform cortex layer III (Nacher et al., 2002a) and the hippocampal “non-granule” PSA-NCAM immunoreactive neurons (Nacher et al., 2002c).

RESULTS Distribution of PSA-NCAM immunoreactivity in the neuropil of the rat mPFC The two anti-PSA-NCAM antibodies (Men-B and 5A5) used in this study rendered a similar immunostaining pattern. However, the intensity of neuropil immunostaining and the number of PSA-NCAM immunoreactive cells were higher when using the Men-B antibody. The rat mPFC neuropil showed moderate staining for PSA-NCAM when compared with that of the adjacent cortical motor areas. This fact allowed to easily delimitate the borders between the cingulate cortex and the adjacent secondary motor cortex. There were some differences in the distribution and the intensity of PSA-NCAM immunoreactivity in the different subdivisions of the rat mPFC. However, in general, a moderate intensity of staining could be observed in layer I, faint or nearly lack of staining appeared in layer II, weak staining could be seen in layer III and moderate intensity of staining was present in layers V–VI. This neuropil staining pattern was clearly seen in the cingulate cortex (Fig. 1A). In the prelimbic and infralimbic cortices (Fig. 1B and C), the intensity of neuropil staining was higher than that of the cingulate cortex.

Phenotype of PSA-NCAM immunoreactive cells in the rat mPFC Analysis of z-series of optical sections using the confocal microscope allowed us to study the three-dimensional distribution of PSA-NCAM immunoreactivity in the whole extension of a neuron. PSA-NCAM immunoreactivity was mainly located in the cellular periphery and appeared absent from the central region of the cytoplasm and the nucleus (Fig. 4A). Immunohistochemistry for PSA-NCAM and NeuN, a protein specifically expressed in mature neurons (Mullen et

Fig. 1. Distribution of PSA-NCAM immunoreactivity in the neuropil of the rat mPFC. Panoramic views of the different subdivisions of mPFC immunostained with PSA-NCAM antibody. (A) Cingulate cortex; B: prelimbic cortex; C: infralimbic cortex. Scale bar⫽250 ␮m.

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Fig. 2. Distribution of PSA-NCAM immunoreactive cells in the rat mPFC. Diagram showing the distribution and relative amount of PSANCAM immunoreactive cells in the different subdivision of mPFC. Cg1, cingulate cortex area 1; Cg2, cingulate cortex area 2; PrL, prelimbic cortex; IL, infralimbic cortex. Each dot corresponds to three labeled cells. The number of cells is the mean of three sections from three different animals.

al., 1992), has revealed that all PSA-NCAM expressing cells studied in the mPFC were NeuN immunoreactive (Fig. 4B) and thus should be considered neurons. This fact was further supported by the lack of colocalization between PSA-NCAM and GFAP, a marker for astrocytes, and between PSA-NCAM and rip, a marker of oligodendrocytes (data not shown). The different sizes and morphological characteristics of the PSA-NCAM immunoreactive cells indicated that different subtypes of neurons might express this molecule in the mPFC. To confirm this, double immunohistochemistry with anti-PSA-NCAM and anti-GAD67 (an isoform of the enzyme responsible for the synthesis of GABA from glu-

tamate) antibodies was carried out. Thirty-five percent of PSA-NCAM immunoreactive neurons expressed GAD67. These PSA-NCAM/GAD67 immunoreactive neurons were mainly located in deep layers of mPFC, but some of them could also be found in layer III. None of the big multipolar PSA-NCAM immunoreactive neurons in layer III showed GAD67 expression (Fig. 4C). Different populations of interneurons have been identified in the rat mPFC using antibodies against calcium binding proteins and neuropeptides (Gabbott et al., 1997). We have performed double immunohistochemistry using antibodies against PSA-NCAM and different calcium binding proteins (calbindin-D28K, calretinin, parvalbumin) and neuropeptides (CCK, VIP, NPY, SST). No PSA-NCAM immunoreactive neurons co-expressing parvalbumin, calretinin, CCK or VIP were observed. By contrast, 60% of PSA-NCAM expressing neurons were intensely immunoreactive for calbindin-D28K (Fig. 4D). These double immunoreactive neurons were located most frequently in layers V and VI, although some of them could also be found in superficial layers (II–III). Twenty-five percent of PSANCAM immunoreactive neurons expressed SST (Fig. 4E). These double-labeled neurons were mainly located in deep layers of both prelimbic and cingulate cortices. Finally, 15% of PSA-NCAM immunoreactive neurons expressed NPY (Fig. 4F). These neurons were located most frequently in deep layers, especially in the cingulate cortex. The presence of different immunoreactive puncta around PSA-NCAM immunoreactive neurons has also been studied. We have found parvalbumin (Fig. 4G) or VIP (Fig. 4H) immunoreactive puncta on some PSA-NCAM immunoreactive neurons in the different subdivisions of mPFC. We also analyzed the nature of the PSA-NCAM immunostaining in the neuropil of the mPFC. In order to know whether the PSA-NCAM immunoreactive puncta in the mPFC neuropil corresponded to synapses, we performed double immunohistochemistry with an antibody against synaptophysin, a synaptic specific protein. Some of the PSA-NCAM immunoreactive puncta in the mPFC neuropil also showed synaptophysin immunoreactivity (Fig. 4I). Moreover, some of these double-labeled structures appeared around the somata and proximal dendrites of certain neurons in layers III and V (Fig. 4I), suggesting that PSA-NCAM may be expressed in synapses contacting these cells. Pyramidal neurons in mPFC layers III and V appeared faintly but distinctly labeled with calbindin-D28K antibody. We never found any of these neurons expressing PSA-NCAM in their somata. However, some of these faint calbindin-D28K immunoreactive pyramidal neurons appeared surrounded by punctate PSA-NCAM immunostaining (Fig. 4J). This immunostaining was clearly located outside the cell somata, unlike that of intense calbindinD28K immunoreactive neurons. We have also analyzed the presence of PSA-NCAM immunoreactivity in GFAP immunoreactive processes of the mPFC neuropil. Only some scarce GFAP-labeled structures showed faint PSANCAM labeling (Fig. 4K).

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Fig. 3. Morphology of PSA-NCAM immunoreactive cells in the rat mPFC. PSA-NCAM immunoreactive neurons in the rat mPFC. (A) Big multipolar neuron in the cingulate cortex layer II; B: big multipolar neuron in the cingulate cortex layer III, C: small neuron in cingulate cortex layer V; D: prelimbic cortex layer VI. Scale bar⫽20 ␮m.

PSA-NCAM expression in recently generated cells of the mPFC In order to determine whether PSA-NCAM immunoreactive neurons were recently generated, we analyzed sections from rats killed 14, 21 or 90 days after 5=BrdU administration. We did not find any cell co-localizing 5=BrdU and PSA-NCAM in any of the time points analyzed. Moreover, in agreement with previous reports (Rakic, 2002; Madsen et al., 2005) we did not find any 5=BrdU immunoreactive nuclei in the mPFC expressing NeuN.

DISCUSSION This study reports the characterization of the expression pattern of PSA-NCAM in the rat mPFC. The distribution and phenotype of PSA-NCAM expressing cells are analyzed in light of previously published data on PSA-NCAM expression in other cerebral regions, on neuronal classification in the mPFC and on mPFC circuitry. We discuss the possible implication of PSA-NCAM in the structural plasticity of prefrontal cortex. Finally, our findings are revised considering the “neuroplastic” hypothesis on the origin of certain psychiatric disorders. Phenotype of cells expressing PSA-NCAM in the mPFC This study shows that most, if not all, PSA-NCAM immunoreactive cell somata of the mPFC correspond to neurons. Although faint PSA-NCAM expression appears to be located in certain astrocytic processes in the mPFC, similar to what has been described in other cerebral regions

(Theodosis et al., 2004), we have not observed PSANCAM immunoreactive cell somata in the mPFC expressing GFAP or displaying the typical morphology of astrocytes. In a similar way, we have not detected PSA-NCAM immunoreactivity in oligodendrocytes of the mPFC. Other regions of the adult CNS in which PSA-NCAM is not restricted to mature neurons are those showing adult neurogenesis, where certain progenitor cells and immature neurons also express this molecule (Seki and Arai, 1993b). Previous studies have obtained contradictory results regarding the possibility of adult neurogenesis in the mPFC: while some authors described the presence of newly generated neurons in the mPFC of primates (Gould et al., 1999), as well as in the anterior neocortical region (Gould et al., 2001) and the cingulate cortex of rats (Dayer et al., 2005); other authors failed to find new neurons in the rodent mPFC (Kodama et al., 2004; Wang et al., 2004; Madsen et al., 2005). Our results are in agreement with these latter reports. Thus, PSANCAM immunoreactive cells in the rat mPFC are most likely mature neurons. PSA-NCAM expression in interneurons of the mPFC PSA-NCAM immunoreactive neurons in the mPFC can be grouped into big multipolar neurons and small fusiform bipolar or unipolar neurons. However, most of PSA-NCAM immunoreactive neurons in the mPFC correspond to the small subtype. Our results indicate that around one third of PSA-NCAM immunoreactive neurons also express GAD67, and thus should be considered interneurons. The lack of GAD67 immunoreactivity in the somata of some PSA-

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Fig. 4. Confocal microscopic analysis of PSA-NCAM immunoreactive elements in the mPFC. (A) Consecutive focal planes (1.95 ␮m apart) of a PSA-NCAM immunoreactive neuron. Note the typical surface labeling in the inferior and superior poles of the somata and the clear peripheral labeling in the optical sections taken from the central region of the neuron. (B) PSA-NCAM/NeuN immunoreactive neurons in the cingulate cortex layer III. (C) PSA-NCAM/GAD67 immunoreactive neurons in the prelimbic cortex layer VI. (D) PSA-NCAM/calbindin-D28K immunoreactive neuron in the cingulate cortex layer V. (E) PSA-NCAM/SST immunoreactive neuron in the cingulate cortex layer V. (F) PSA-NCAM/NPY immunoreactive neuron in the prelimbic cortex layer V. (G) Parvalbumin immunoreactive puncta surrounding a PSA-NCAM immunoreactive neuron in the prelimbic cortex layer III. (H) VIP immunoreactive puncta surrounding a PSA-NCAM immunoreactive neuron in the prelimbic cortex layer III. (I) Double immunostaining for PSA-NCAM and synaptophysin in cingulate cortex layer III. Note the presence of double labeled puncta (arrowheads). (J) Double immunostaining for PSA-NCAM and calbindin-D28K in prelimbic cortex layer III. Observe the presence of some PSA-NCAM immunoreactive puncta surrounding the pyramidal neurons that are weakly immunoreactive for calbindin-D28K. A PSA-NCAM immunoreactive somata which does not display calbindin-D28K immunoreactivity can be detected at the bottom of the figure. (K) Double immunostaining for PSA-NCAM and GFAP in the cingulate cortex layer VI. Some of the PSA-NCAM immunoreactive processes also display faint GFAP immunoreactivity. Scale bar⫽10 ␮m. All photographs in this figure correspond to single optical sections taken from z-stacks.

NCAM expressing neurons may indicate that these cells are interneurons with long axonal projections, which may have very low levels of GAD67 in their somata. This kind of interneuron has been described in the hippocampus (Toth and Freund, 1992). The elevated percentage (60%) of PSA-NCAM immunoreactive cells that display intense immunoreactivity for calbindin-D28K also indicates that the subpopulation of PSA-NCAM interneurons may be more numerous than those co-expressing GAD67, because intense calbindin-D28K expression is only found in rat mPFC interneurons (Gabbott et al., 1997). The PSA-NCAM expressing neurons described in our study are immunoreactive for calbindin-D28K (around

60%), NPY (15%) and SST (around 25%). These doublelabeled neurons were located mainly in deep layers, coinciding with the distribution of PSA-NCAM/GAD67 immunoreactive neurons. Attending to the classification of interneurons in the frontal cortex made by Kubota et al. (1994), PSA-NCAM immunoreactive interneurons would belong to the subgroup that displays immunoreactivity for calbindinD28K, SST and NPY, which has a characteristic rate of electrophysiological response (Kawaguchi and Kubota, 1996). Our results are in accordance with those of Hardwick et al. (2005) indicating lack of co-localization between PSA-NCAM and parvalbumin immunoreactive elements in the rat mPFC.

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PSA-NCAM expression in mPFC pyramidal neurons The lack of GAD67 immunoreactivity in the somata of certain PSA-NCAM expressing neurons may also indicate that some of them are principal neurons. Obviously, the big multipolar neurons located in layers III and V would be the best candidates because of their morphology and the fact that we have not found any cell somata belonging to this type co-expressing PSA-NCAM and GAD67. In any case, these big multipolar neurons expressing PSA-NCAM are rare in the rat mPFC and consequently they only could account for a low number of mPFC principal neurons. Pyramidal neurons in layers III or V, clearly identified with NeuN immunohistochemistry, were always PSA-NCAM immunonegative. Moreover, calbindin-D28K immunostaining, which labels faintly cortical pyramidal neurons, also indicates that these principal neurons do not appear to express PSA-NCAM in their somata. Nature of PSA-NCAM immunoreactive elements in the mPFC neuropil We have shown that a subset of PSA-NCAM immunoreactive puncta in the mPFC neuropil co-expressed synaptophysin. This protein is exclusively expressed in synaptic vesicles and thus its expression may indicate that certain synapses in the mPFC express PSA-NCAM. Interestingly, some PSA-NCAM immunoreactive puncta surround the soma and proximal dendrites of pyramidal neurons in the mPFC layers III and V, and many of these puncta expressed synaptophysin. These results may be particularly important because there is ample evidence that pyramidal neurons in the mPFC are capable of undergoing structural changes during adulthood, and PSA-NCAM may be mediating this plasticity through its anti-adhesive properties. PSA-NCAM immunostaining in the neuropil may correspond to processes or synaptic contacts coming from intrinsic PSA-NCAM expressing neurons (most likely interneurons) or to long projections coming from cortical or extra-cortical regions with intense PSA-NCAM expression. We have found GAD67 immunoreactive processes and puncta expressing PSA-NCAM. Electron microscopic analysis has to be performed, however, in order to clearly identify the nature of these puncta. Some of the regions projecting to the mPFC, such as the perirhinal and entorhinal cortices (Delatour and Witter, 2002), the basolateral amygdala (Bacon et al., 1996) and the piriform cortex (Datiche and Cattarelli, 1996) display PSA-NCAM immunoreactive neurons (Seki and Arai, 1991a, 1993a; Nacher et al., 2002b). Moreover, preliminary results in our laboratory indicate that certain nuclei in the thalamus, a region which sends a prominent input to the mPFC (Krettek and Price, 1977; Conde et al., 1995), display high levels of PSA-NCAM immunoreactivity during adulthood. PSA-NCAM and structural plasticity in the mPFC It is widely accepted that PSA-NCAM is a major player in structural plastic processes such as neurite extension/ retraction, synaptic rearrangement or cell migration (Rutishauser and Landmesser, 1996). Consequently, changes

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in PSA-NCAM immunoreactivity are often associated with structural reorganization processes (Sandi et al., 2001; Nacher et al., 2002a; Glass et al., 2003; Pham et al., 2003). Previous reports have demonstrated the existence of structural plasticity in neurons of the mPFC, namely their ability to reorganize their synapses and neurites under stress conditions (Cook and Wellman, 2004; Radley et al., 2004; Brown et al., 2005), or after cortisone administration (Wellman, 2001). Moreover, the morphology of these neurons is also sensitive to alterations in blood pressure (Vega et al., 2004). All these treatments induce remodeling of dendrites of pyramidal neurons of layers II–III and a recent report has also described effects of chronic stress in the remodeling of dendritic spines (Radley et al., 2005). Our study points to the PSA-NCAM expressing interneurons as the best candidates to undergo structural plastic changes promoted by the anti-adhesive properties of this molecule. All previous studies on neuronal structural plasticity in the mPFC have been circumscribed to the pyramidal neurons, but the possibility that some interneurons in the mPFC may also undergo neurite remodeling should not be excluded. In any case, since most of the data regarding structural plasticity in the mPFC have been obtained in pyramidal neurons, one would expect that these principal cells expressed PSA-NCAM. However, our present data indicate that pyramidal neurons in the mPFC do not express PSANCAM. Nevertheless, at least some of these principal neurons are surrounded by PSA-NCAM immunoreactive puncta and we have shown that some of these structures may correspond to synapses. Electron microscopic analysis is, however, necessary to confirm this hypothesis. Further analysis may be also necessary to determine whether these contacts come from intrinsic PSA-NCAM expressing interneurons or from PSA-NCAM expressing projections coming from other cortical regions or from extra-cortical origin. The presence of PSA-NCAM in certain synapses contacting mPFC pyramidal neurons may promote a transitory detachment, leading to an interruption of the synaptic input onto pyramidal neurons. These detachments may account indirectly for the structural changes observed in these principal neurons, for instance after chronic stress. An increased expression of PSA-NCAM in certain synapses contacting mPFC pyramidal neurons may detach the axonal terminal from the dendritic spine and this separation may well lead to the spine retraction observed in mPFC pyramidal neurons after chronic stress (Radley et al., 2005). It is not known whether PSA-NCAM expression in the mPFC is affected by chronic stress, but other telencephalic regions, such as the piriform cortex and the dentate gyrus show increased PSA-NCAM expression after the same chronic stress paradigm (Pham et al., 2003; Nacher et al., 2004b). Prefrontal cortex dysfunction (decrease in blood flow, reduction of volume, impairment in working memory, etc.) has been observed in post-traumatic stress disorder (Bremner et al., 1999; Shin et al., 2004), depression (Botteron et al., 2002), and schizophrenia (Knable and Weinberger, 1997). Some studies have linked these behavioral

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disorders with alterations in neuronal plasticity (Duman, 2002; Costa and Silva, 2004; Frost et al., 2004). In fact, a reduction in the number of PSA-NCAM immunoreactive neurons has been described in the hippocampus of schizophrenic patients (Barbeau et al., 1995). Changes in PSANCAM expression have been described after aversive experiences, such as chronic stress or fear, in the amygdala (Cordero et al., 2005), the hippocampus (Merino et al., 2000; Pham et al., 2003; Sandi et al., 2003), and the piriform cortex (Nacher et al., 2002a). The mPFC is also involved in fear-induced responses, specifically the prelimbic subregion has been implicated in the extinction of fear memories (Milad and Quirk, 2002). Consequently, exploring the expression of PSA-NCAM in the prefrontal cortex after aversive experiences may be relevant to the study of anxiety disorders such as PTSD. If, as our results suggest, structural plasticity in the adult mPFC is mediated by changes in PSA-NCAM expression, pharmacological interventions leading to modulate the expression of PSA-NCAM may be promising approaches to intervene in the structural plasticity dysfunction that appears to underlie some psychiatric disorders. Acknowledgments—This study was supported by the following grants: GV04A-134, GV04A-076, GVGrupos03/119, MEC BFI200301254 and MEC BFU2004-00931. We are grateful to Dr. G. Rougon and Dr. U. Rutishauser for their kindly gift of anti-PSA-NCAM antibodies. We also thank Dr. T. J. Görcs for the gift of anti-VIP, anti-SST and anti-NPY antibodies. The mouse antibody raised against CCK was provided by CURE/Digestive Diseases Research Center Antibody Core, NIH Grant DK4130. Rip antibody developed by S. Hockfield was obtained from the Developmental Studies Hybridoma Bank, developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA 52242.

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(Accepted 1 August 2005) (Available online 10 October 2005)