Repeated risperidone treatment increases the expression of NCAM and PSA-NCAM protein in the rat medial prefrontal cortex

European Neuropsychopharmacology (2009) 19, 125–137

w w w. e l s e v i e r. c o m / l o c a t e / e u r o n e u r o

Repeated risperidone treatment increases the expression of NCAM and PSA-NCAM protein in the rat medial prefrontal cortex Marzena Maćkowiak ⁎, Dorota Dudys, Agnieszka Chocyk, Krzysztof Wedzony Laboratory of Pharmacology and Brain Biostructure, Institute of Pharmacology, Polish Academy of Sciences, Smętna 12, 31-343 Kraków, Poland

Received 27 June 2008; received in revised form 2 October 2008; accepted 21 October 2008

KEYWORDS Antipsychotics; NCAM; PSA-NCAM; Prefrontal cortex; Risperidone; Schizophrenia

Abstract The present study investigates whether the anti-schizophrenic drug risperidone may evoke changes in the expression of NCAM/PSA-NCAM proteins, an indispensable element in the remodeling of synaptic arrangements, in the medial prefrontal cortex (mPFC). Rats were treated with risperidone (0.2 mg/kg, i.p.) either once or repeatedly (once a day, for 21 days). The expression of NCAM and PSA-NCAM proteins was analyzed via western blot and immunohistochemistry at intervals of 3 h and 3, 6, and 9 days after the single or the last risperidone dose. Repeated (but not acute) administration of risperidone was found to increase the expression of NCAM-180, NCAM-140 and PSA-NCAM proteins at 3 or 6 days after treatment. PSA-NCAM immunoreactivity was found in cell bodies, perisomaticlike sites, and in the neuropil of the mPFC. Neither single nor repeated risperidone administration changed the number of PSA-NCAM neurons in the mPFC. In contrast, the repeated risperidone treatment increased the number of PSA-NCAM perisomatic-like sites and the length density of PSANCAM positive neuropil at 3 days after the last injection. The data obtained indicate that risperidone, given repeatedly, may promote the remodeling of the structure of presumably GABAergic interneurons and that it may evoke the rearrangement of the synaptic contact in the mPFC. © 2008 Elsevier B.V. and ECNP. All rights reserved.

1. Introduction Dysfunction of the medial prefrontal cortex (mPFC) is a prominent feature of pathophysiology in schizophrenia. Impairments of cognitive function have been related to ⁎ Corresponding author. Institute of Pharmacology, Polish Academy of Sciences, 31-343 Kraków, 12 Smętna Street, Poland. Tel.: +48 12 6623241; fax: +48 12 6374500. E-mail address: [email protected] (M. Maćkowiak).

abnormalities in synaptic connectivity, efficacy of neurotransmission and metabolism in the mPFC (Lewis and Moghaddam, 2006; Lewis and Gonzalez-Burgos, 2008; Moghaddam and Homayoun, 2008). With respect to the anatomy of the mPFC, postmortem studies have shown an increase in cell packing density without a change in the total number of cortical neurons, but with changes in the principal components of synapses, such as a decreased amount of cortical neuropil (i.e., the condensation of axon terminals and distal dendrites (Rajkowska et al., 1998; Selemon and

0924-977X/$ - see front matter © 2008 Elsevier B.V. and ECNP. All rights reserved. doi:10.1016/j.euroneuro.2008.10.001

126 Goldman-Rakic, 1999)). In addition, the expression of components of excitatory synapses (i.e., the number of dendritic spines in specific regions of the mPFC) have been reduced (Costa et al., 2001; Halim et al., 2003). With respect to the specific neurotransmitter, abnormalities of neocortical GABA-ergic inhibitory neurons (forming characteristic perisomatic cartridges by the chandelier class of parvalbumin-containing neurons on pyramidal cell bodies and the axonal initial segment) have been noted in schizophrenia (Volk and Lewis, 2002; Lewis et al., 2005; Daskalakis et al., 2007). Downregulation of the presynaptic GABA-ergic function in schizophrenia has been reported as a decrease in the expression of glutamic acid decarboxylase-67 (GAD-67) (Akbarian et al., 1995; Volk et al., 2000) and presynaptic GABA transporter-1 (GAT-1) (Woo et al., 1998; Pierri et al., 1999; Volk et al., 2001), followed by a reduction in the number of parvalbumin-positive interneurons (Hashimoto et al., 2003). An increase in the expression of the α1 subunit of the postsynaptic GABAA receptors in the postsynaptic site of the cartridge has also been observed in the mPFC of schizophrenic brains (Impagnatiello et al., 1998). The pathological picture of the mPFC is not only limited to intrinsic cortical elements, but should also be extended to the aberration of neuroanatomy of efferent modulator systems. Although the etiology of schizophrenia leading to anatomical and functional changes is not yet clear, it is thought to be the consequence of a complex interplay between genetic susceptibility and environmental factors that alter the developmental/constitutive plasticity of neural circuits (Tsankova et al., 2007; Duman and Newton, 2007). The above hypothesis prompted an investigation to determine if genes/proteins controlling brain plasticity are engaged in the pathophysiology of schizophrenia or if pharmacological agents ameliorating positive and negative symptoms of schizophrenia are able to alter the expression of factors that govern the plastic rearrangement in central nervous systems (Deutsch et al., 2008). Among several candidate genes/proteins inducing cortical hypoplasticity in schizophrenia, are the proteins involved in synaptogenesis (for example, the cell adhesion molecule (NCAM) (Sullivan et al., 2007) and the extracellular matrix protein, reelin (Impagnatiello et al., 1998)). NCAM is an important element of cell-to-cell and cell-to-extracellular matrix contact, involved not only in constitutive alterations of cortical anatomy (Maness and Schachner, 2007; Sullivan et al., 2007; Dalva et al., 2007), but also in neurocognitive dysfunction in schizophrenia (Sullivan et al., 2007; Atz et al., 2007). The NCAM gene is located in the middle of a genomic region relatively strongly implicated by the meta-analysis in linkage studies of the etiology of schizophrenia (Lewis et al., 2003). Moreover, NCAM proteins play an important role in neuronal process outgrowth, synapse formation, and signal transduction in the immature and the adult brain (Sytnyk et al., 2006; Maness and Schachner, 2007). The level of the soluble fraction of NCAM (NCAM-EC) derived from proteolytic cleavage (shedding) of extracellular NCAM elements, with the potential antagonistic activity on the intact extracellular membraneattached NCAM molecule, is elevated in the prefrontal cortex, hippocampus and cerebro-spinal fluid (CSF) of schizophrenics (Honer et al., 1997; Vawter et al., 1998). Moreover, as in clinical studies, transgenic mice overexpressing NCAM-EC show increased locomotor activity (modeling psychomotor

M. Maćkowiak et al. agitation) and neuroleptic dependent deficits in prepulseinduced inhibition of acoustic startle response (modeling deficits of sensorimotor gating), and they are oversensitive to the behavioral effects of stimulant drugs and NMDA receptor antagonists. Interestingly, these schizophrenic-like behaviors are accompanied by a reduction in immunoreactivity for GABAergic synapses forming perisomatic innervation of principal neurons in the mPFC and in the amygdala (Pillai-Nair et al., 2005). Thus, several lines of evidence indicate that NCAM proteins might be responsible for functional and anatomical abnormalities linked with the pathology of schizophrenia. Such abnormalities mediated by NCAM cannot be limited only to NCAM itself since it is post-translationally modified by the attachment of negatively charged sialic acid residues with a large hydrated volume (specifically polysialic acid (PSA)) to one of the protein's extracellular segments. Such sialylation attenuates the adhesive properties of neurons and enables the rearrangement of cell-to-cell and extracellular matrix-to-cell contacts (Rutishauser, 2008). Interestingly, a decrease in the PSA-NCAM protein level for the postmortem hippocampus of schizophrenics has also been reported (Barbeau et al., 1995). The anatomical studies showing that the PSA-NCAM protein is localized on the GABA-ergic interneurons in the human (Varea et al., 2007c) and rat prefrontal cortex (Varea et al., 2005) support the idea that NCAM or PSA-NCAM molecules might be connected with the GABA-ergic dysfunction observed in schizophrenia. It is so far unknown whether or not neuroleptics may also affect the level of NCAM/PSA-NCAM proteins and subsequently evoked plastic changes in the mPFC. Recent evidence indicates that the atypical neuroleptic risperidone, but not haloperidol, a typical neuroleptic changed the gene expression of cell adhesion molecules such as integrin, neural adhesion molecule F3 in the rat cortex (Feher et al., 2005). Additionally, the available data indicate that PSA-NCAM expression in the mPFC is changed by chronic antidepressant administration via enhancement of serotonin transmission (Sairanen et al., 2007; Varea et al., 2007a,b). Thus, it is of interest to investigate whether treatment using risperidone, a neuroleptic acting as a D2/5HT2 receptor antagonist, which is commonly used in the treatment of schizophrenia (Stathis et al., 1996), is capable of altering the expression of NCAM and PSA-NCAM proteins in the mPFC. Therefore, the present study investigated whether or not acute and repeated risperidone administration influences the expression of NCAM and PSA-NCAM proteins in the rat mPFC using western blot and immnunohistochemical analyses to visualize the rate of PSA-NCAM/NCAM expression and the morphology of PSA-NCAM elements in the mPFC.

2. Experimental procedures 2.1. Animals and treatment All of the experiments were carried out on male Wistar rats (Charles River). At the start of the experiments, they were 60 days old (“young adult rats”) with body weights of approximately 200–250 g. Body weight changes were monitored during experiments and at the end of the treatment typical body weights were around 330–380 g with no differences between groups. The animals were housed on an artificial light/dark cycle (12/12 h lights on at 7 a.m.) with free access to a standard laboratory diet and tap water. The experimental protocols were approved by the Committee for Laboratory Animal Welfare and

Risperidone increases PSA-NCAM in the prefrontal cortex the Ethics committee of the Institute of Pharmacology, Polish Academy of Sciences in Kraków, and they met the requirements of the European Council Guide for the Care and Use of Laboratory Animals (86/609/EEC). Risperidone (Janssen) was used at a therapeutic dose of 0.2 mg/kg, i.p., which was active in a startle response test, both in clinical (Kumari et al., 2002) and animal studies (van den and Gogos, 2007). The rats were divided into 3 groups. The first group was exposed to vehicle (1% Tween 80) once daily for 21 consecutive days. The rats in the second group were injected with vehicle once daily for 20 days and, on the 21st day, were treated with a single dose of risperidone (0.2 mg/kg, i.p). The third group of animals was exposed to risperidone (0.2 mg/kg, i.p.) once daily for 21 consecutive days. Each group contained 10 rats. Five rats from each group were sacrificed, and the prefrontal cortex was dissected for western blot experiments; the other five were used in immunohistochemical experiments. The experiments were performed at 3 h and 3, 6, and 9 days after the last risperidone injection. The number of rats for each treatment and time interval was five for both the western blot and immunohistochemical experiments. The total number of rats used in the present study was 120.

3. Western blotting The brains of five rats from each group were removed after decapitation, cooled on ice, and sliced into 1 mm coronal sections using a rodent brain matrix (Ted Pella, INC). The mPFC was punched out from coronal sections with a 15-gauge punching tube. The isolated brain region was homogenized in 10 vol (w/vl) of lyses buffer: 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 0.5% Triton X-100, 0.5% SDS, and protease inhibitors (1:200, Sigma). Protein levels were determined using a BCA Protein Assay Kit (a modification of the Lowry assay, Sigma). Samples of equal protein contents were adjusted to an equal volume with 50 mM Tris (pH 6.8), containing 2% SDS, 8% glycerol, and 2% 2-mercaptoethanol with bromophenol blue as a marker and boiled for 5 min. Protein extracts (20 μg or 60 μg of protein/lane for NCAM or PSA-NCAM analysis, respectively) were separated by 7.5% SDS–PAGE and transferred to nitrocellulose membranes using an electrophoretic transfer system (BioRad). The membranes were stained with Ponceau S to confirm the equal loading and transfer of the gels. Afterwards, the membranes were cut into two parts, from which the lower part was taken to determine the β-actin protein. The blots were then incubated overnight at 4 °C with the following primary antibodies: monoclonal mouse anti-NCAM (1:1000, recognizing NCM-180 and NCAM-140, Sigma), monoclonal mouse anti-PSA-NCAM (1:500, Chemicon), and monoclonal mouse anti β-actin (1: 5000, Sigma). Immune complexes were detected using appropriate peroxidase-conjugated secondary antibodies: anti-mouse IgG (1:1000, Roche) or anti-mouse IgM (1:100,000, Jackson ImmunoResearch). The reaction was visualized by ECL (Enhanced Chemiluminescence) (Lumi-LightPlus Western Blotting Kit, Roche). Chemiluminescence was recorded and evaluated with a luminescent image analyzer (Fugifilm LAS-1000). The relative levels of immunoreactivity were quantified using Image Gauge (Fujifilm) and Image ProPlus (Media Cybernetics) software. To normalize for small variations in loading and transfer, the ratio of NCAM or PSA-NCAM level/β-actin was calculated for each sample. Molecular weights of immunoreactive bands were calculated on the basis of the migration of molecular weight markers (Bio-Rad Laboratories) using Image Gauge (Fujifilm) software. All values are expressed as a percentage of vehicle-treated controls.

4. Immunohistochemical study 4.1. Tissue preparation The rats were deeply anesthetized with sodium pentobarbital (100 mg/kg, i.p.) and were transcardially perfused with a 0.9% NaCl, followed by a 4% paraformaldehyde in 0.1 M phosphate-

127 buffered saline (PBS). After the post-fixation period of 24 h, 50 μm thick sections were cut through the entire mPFC using a Leica VT1000 S vibratome (Mackowiak et al., 2005; Mackowiak et al., 2007).

4.2. Immunoperoxidase staining Free-floating sections were processed for a single staining of PSANCAM. Brain sections were rinsed and incubated for 1 h in a blocking buffer [5% normal goat serum (Vector Labs) and 0.3% Triton X-100 in 0.01 M PBS]. Finally, the sections were incubated (48 h at 4 °C) with primary monoclonal anti-PSA-NCAM mouse antibody (1:1000, Chemicon) diluted in 3% normal goat serum and 0.3% Triton X-100 in 0.01 M PBS. The reaction was visualized with a biotinylated antimouse, rat adsorbed (Vector Labs), the avidin-biotin horseradish peroxidase complex (Vectastain Elite ABC Kit, Vector Labs, at a concentration recommended by the manufacturer), and 3,3'diaminobenzidine tetrahydrochloride (0.02% DAB solution and 0.025% H202 solution), which produced the brown color of the immunoreactive cells of PSA-NCAM. Notably, overnight pretreatment of the PSANCAM antibody with an α-2,8-linked sialic polymer (colominic acid, Sigma), as well as primary antibody omission during the immunohistochemistry procedure prevented PSA-NCAM labeling in the mPFC.

4.3. Silver–gold intensification The DAB reaction product was intensified according to the methods of Melchitzky and Lewis (2000), as described previously in (Wedzony et al., 2005). DAB-reacted slide-mounted sections were incubated in 1% silver nitrate (pH = 7.45, 55 °C), rinsed with distilled H2O, and then incubated in 0.1% gold chloride (10 min, room temperature). Sections were rinsed again with distilled H2O and fixed in 5% sodium thiosulfate (10 min, room temperature) and then placed under cover slips. For data presentation, digital images were captured using a Photometric Coolsnap digital camera attached to a Leica microscope (Leica, DMLB) and controlled by MethaMorph software. Final photomicrographs were composed using an Adobe Photoshop program.

4.4. Quantitative evaluation of staining The number of PSA-NCAM immunopositive perisomatic-like sites and cells in the mPFC were estimated using unbiased stereological methods (West et al., 1991). In brief, every sixth section from systematic random sampling along the rostrocaudal axis of the mPFC was analyzed with a 63×/1.4 − 0.7 lens using Cast stereology system software (Olympus, Denmark). Cells appearing in the upper focal plane were omitted to prevent the counting of cell caps (− 5 μm of the topmost surface of the section). For each animal, the mean numerical density of immunopositive perisomatic-like sites and cells was calculated from the sum of the counts made within the optical dissectors and the volume of dissectors (1025 μm2 × 20 μm). The total number of PSA-NCAM-immunopositive perisomatic-like sites and cells in the subregions of the mPFC (cingulate cortex, prelimbic cortex, infralimbic cortex) were then calculated by multiplying the numerical density of immunoreactive perisomatic-like sites and cells by the reference volume of each subregion of the mPFC. The volume of the subregions of the mPFC was measured in each animal on the same series of sections using the Cavalieri method with a counting grid having an area associated with the counted points of 26,283 μm2 at 10× magnification. Additionally, the same sections were used to calculate the length density of PSA-NCAM-immunopositive neuropil in the subregions of the mPFC according to global spatial sampling methods (Larsen et al., 1998) using Cast stereology system software (Olympus, Denmark). The Integrated Optical Density (IOD) of PSA-NCAM-immunopositive perisomatic-like sites was analyzed on single-scan, black-andwhite confocal images. The images were captured using a confocal

128 laser scanning microscope DMRXA2 TCS SP2 (Leica) using a 63×/1.4 − 0.7 oil objective (Leica) driven by confocal software (Leica) and working with an argon gas laser (laser line emitted at 488 nm). The background noise of each confocal image was reduced by averaging 16 scans/line and 64 frames/image. The IOD of PSA-NCAMimmunopositive perisomatic-like sites was measured in superficial (3) and deep (5/6) cortical layers of the prelimbic and infralimbic cortices using Adobe Photoshop and Image ProPlus image analysis software. One section was examined for each rat (the third of the six sections), and three perisomatic-like sites from each layer were analyzed. The average of three measurements was then used in the statistical analysis. For additional details see Fig. 2.

M. Maćkowiak et al. 4.5. Immunofluorescence staining Other free-floating sections were developed using immunofluorescence double-labeling protocols to determine the co-localization of PSA-NCAM-positive cells with NeuN, GAD-67 or GAT-1 protein. The sections were incubated in a blocking buffer (5% normal donkey serum and 0.3% Triton X-100 in 0.01 M PBS) for 1 h. Next, the sections were incubated (48 h at 4 °C) with primary antibodies: monoclonal mouse anti-PSA-NCAM antibody (1:200, Chemicon) and monoclonal mouse anti-NeuN antibody (1:1000, Chemicon) or monoclonal mouse anti-GAD-67 antibody (1:1000, Chemicon) or polyclonal rabbit antiGAT-1 antibody (1:100, Chemicon). Antibodies were diluted in 5%

Figure 1 The effect of risperidone on the expression of NCAM and PSA-NCAM proteins in the mPFC—western blot analysis. VEH— control animals, RAC—animals injected with a single dose of risperidone (0.2 mg/kg), RCH—animals treated repeatedly with risperidone (0.2 mg/kg i.p., once daily for 21 consecutive days). Each data point represents the mean ± S.E.M; n = 5 per each treatment and time point. Asterisks indicate statistical significance when compared with the appropriate control group; the level of significance is, at least, 0.05 (ANOVA followed by the Newman–Keuls test for post hoc comparison).

Risperidone increases PSA-NCAM in the prefrontal cortex normal donkey serum and 0.3% Triton X-100 in 0.01 M PBS. The sections were then washed and incubated for 2 h in a mixture of secondary antibodies: Alexa 488-conjugated anti-mouse IgM (1:200, Invitrogen) for PSA-NCAM and Cy3-conjugated anti-mouse or antirabbit IgG (1:300, (Jackson ImmunoResearch) for NeuN and GAD-67 or GAT-1, respectively. Sections were then washed, mounted, and coverslipped. For double labeling, slices were analyzed with a confocal laser scanning microscope, DMRXA2 TCS SP2 (Leica), using a 63×/1.4 − 0.7 oil objective (Leica) driven by confocal software (Leica) using sequential scan settings. Working with argon and GreNe lasers, two laser lines emitting at 488 and 543 nm were used for exciting the Alexa 488- and Cy3-conjugated antibodies, respectively. The background noise of each confocal image was then reduced by averaging four scans/line and six frames/image. The pinhole value of one airy was used in order to obtain flat images. The relative optical density of labeling on immunofluorescence images was measured using Image ProPlus software.

129

5. Statistics The results are presented as group mean ± standard error of the mean (SEM). Statistical evaluation was performed by a two-way analysis of variance (ANOVA) or by a repeated measures ANOVA followed by the Newman–Keuls post hoc test using the Statistica program (where time and treatment were regarded as independent variables).

6. Results 6.1. The effect of risperidone on expression of NCAM and PSA-NCAM proteins; western blot study The antibody used in the present study recognized two bands of protein on the western blots, with molecular weights of approximately 180 kDa and 140 kDa (Fig. 1), which correspond

Figure 2 PSA-NCAM immunostaining in the rat mPFC. A) Distribution of PSA-NCAM immunoreactivity in the different subdivision of the mPFC; Cg1—cingulate cortex, PrL—prelimbic cortex, IL—infralimbic cortex. B) PSA-NCAM immunoreactivity in the cortical layers. C) An example of a PSA-NCAM immunoreactive cell. D, E) Examples of PSA-NCAM immunostaining in the neuropil of layers 5/6 and 3, respectively. Arrows indicate a perisomatic-like site positive for PSA-NCAM. Scale bars represent 200 μm (B), 80 μm (D, E) and 40 μm (C, F, G). F, G—the perisomatic-like sites in the prelimbic cortex on day 3 after treatment. F) vehicle-treated animals, G) repeated risperidone-treated animals (0.2 mg/kg, i.p., once a day for 21 days). A circle of F demonstrates calculation of IOD of perisomatic-like site. Each interior of perisomatic site was traced manually and saved as in outline 1 and then the size of the outline was expanded by 5 pixels, i.e. an average thickness for perisomatic sites and saved as in outline 2. IOD was calculated as a difference between intensity in the outer and inner parts of the circle. For further abbreviations in A: Acb-nucleus accumbens, CPu-caudate putamen (striatum), DP-dorsal peduncular cortex, fmiforceps minor corpus callosum, M1-primary motor cortex, M2-secondary motor cortex, Pir-piriform cortex, S1J-S1 cx, jaw region.

130 to the molecular weights of NCAM-180 and NCAM-140, respectively. Risperidone administration resulted in time-dependent changes in the expression of NCAM-180 and NCAM-140 proteins (treatment × time = F(6, 48) = 6.44; p b 0.0001) for NCAM-180 protein and F(6, 48) = 2.93; p b 0.0162 for NCAM-140 protein). Post hoc analysis revealed an increase in NCAM-180 protein expression after repeated risperidone treatment 3 and 6 days after the last dose (30%, p b 0.029 and 38%, p b 0.024, respectively) but an acute administration of risperidone did not affect the NCAM-180 expression at any time interval. In contrast, a single dose of risperidone resulted in a decrease in NCAM-140 protein level (30%, p b 0.045), observed 6 days after a single dose, and repeated risperidone treatment induced an increase in NCAM-140 protein expression (30%, p b 0.046) at 6 days after the cessation of risperidone administration (Fig. 1). PSA-NCAM protein expression was also studied after risperidone treatment. Two bands of PSA-NCAM protein with molecular weights of approximately 250 kDa and 130–150 kDa were analyzed (Fig. 1). A Similar molecular weight for the PSANCAM protein has been found by other (Heidmets et al., 2007; Singh and Kaur, 2007), but not all (Abrous et al., 1997; Wang et al., 2004), authors. Immunoblot analysis showed that alternation in the PSA-NCAM expression also depends on treatment and timing of the measurement after the last dose of risperidone (treatment × time = F(6, 48) = 5.54, p b 0.0002 for PSA-NCAM-250 and F(6, 48) = 2.16, p b 0.049 for PSA-NCAM-130/ 150). Repeated risperidone treatment increased both PSANCAM-250 and PSA-NCAM-130/150 protein levels (50% and 37%, respectively) 3 days after the last dose of risperidone (p b 0.002 and p b 0.04, respectively). A single risperidone injection did not change the expression of PSA-NCAM protein at any measured time points (Fig. 1).

M. Maćkowiak et al. and prelimbic cortices, and a much lower number was observed in the infralimbic cortex (Fig. 3). An example of a PSA-NCAM immunoreactive cell is shown in Fig. 2C. Repeated measures ANOVA revealed that neither acute nor repeated risperidone administration changed the number of PSA-NCAM immunorective cells at any measured time interval in any subregion of the mPFC (treatment × time = F(18, 130.59) = 0.3805, p b 0.98, Fig. 3). The results for the entire mPFC corresponding to the western blot data are shown in Table 1.

6.2.2. The effect of risperidone on the PSA-NCAM immunoreactive perisomatic-like sites The number of perisomatic-like sites was similar in all subdivisions of the mPFC (Fig. 4). Repeated measures ANOVA

6.2. The effect of risperidone on expression of PSANCAM protein–immunohistochemical study PSA-NCAM immunostaining was observed in the cingulate, prelimbic and infralimbic cortices of the mPFC, but the intensity of PSA-NCAM immunoreactivity was different in the subdivisions of the mPFC. The intensity of PSA-NCAM immunostaining was the highest in the prelimbic and infralimbic cortices and moderate in the cingulate cortex (Fig. 2A). With respect to PSA-NCAM, immunoreactivity in the cortical layers of mPFC, a substantial PSA-NCAM immunoreactivity was observed in layers 1 and 5/6. A moderate PSA-NCAM immunoreactivity was observed in layer 3, and almost no staining appeared in layer 2 (Fig. 2B). A similar pattern of PSA-NCAM staining was found by Varea et al. (2005). PSA-NCAM immunostaining was noticed in both the cells and neuropil (Fig. 2C, D, E). In the neuropil, apart of the PSA-NCAMpositive processes, there was also visible condensation of PSANCAM proteins forming perisomatic-like sites around the soma of certain neurons in layers 3 and 5 (Fig. 2D, E). The morphology of perisomatic-like sites and their localization in layers 3 and 5 suggest that PSA-NCAM proteins are expressed in the perisomatic region of (presumably, but not exclusively) pyramidal neurons.

6.2.1. The effect of risperidone on the PSA-NCAMimmunoreactive cells PSA-NCAM immunoreactive cells were present in all of the subregions and layers of the mPFC. A comparable number of PSA-NCAM immunoreactive cells were found in the cingulate

Figure 3 The effect of risperidone on the number of PSANCAM-positive cells in a different subdivision of the mPFC. VEH— control animals, RAC—animals injected with a single dose of risperidone (0.2 mg/kg), RCH—animals treated repeatedly with risperidone (0.2 mg/kg i.p., once daily for 21 consecutive days). A—cingulate cortex, B—prelimbic cortex, C—infralimbic cortex. Each data point represents the mean ± S.E.M; n = 5 per each treatment and time point.

Risperidone increases PSA-NCAM in the prefrontal cortex revealed that risperidone treatment resulted in the timedependent increase in the number of PSA-NCAM perisomaticlike sites in all the subregions of the mPFC (treatment × time = F(18, 130.59) = 2.18, p b 0.006). Post-hoc analysis revealed that the robust increase in the number of PSA-NCAM perisomatic-like sites was observed on day 3 after the last dose of repeated risperidone treatment in the cingulate and prelimbic cortices (p b 0.0039 and p b 0.05, respectively) and on days 3 and 6 after the last dose of repeated risperidone administration in the infralimbic cortex (p b 0.0022 and p b 0.045, respectively, Figs. 2 F, G, 4). A single dose of risperidone was ineffective in this respect. The intensity of PSA-NCAM immunoreactivity in the perisomatic-like sites, given as integrated optical density (IOD), was measured in the prelimbic and infralimbic cortices (layers 3 and 5/6) where the highest intensity in PSA-NCAM of perisomatic-like sites was observed both in control and risperidone-treated animals. It was found that risperidone administration did not affect the IOD in subregions of the mPFC (treatment × time = F(24, 158.19) = 0.93, p b 0.555). However, it was found that repeated risperidone administration enhanced the intensity of PSA-NCAM immunoreactivity in the perisomatic-like site on day 3 after the last risperidone treatment (p b 0.049) in layer 5/6 of the infralimbic cortex (Figs. 2F, G, 5). Single doses of risperidone were ineffective in this respect. The results for the entire mPFC, corresponding to the western blot data, are shown in Table 1.

6.2.3. The effect of risperidone on the PSA-NCAM immunoreactive neuropil The length density of PSA-NCAM immunoreactive processes was measured in all subregions of the mPFC. Repeated measures ANOVA revealed the enhancement of the length density of PSANCAM neuropil induced by risperidone treatment (treatment × time = F(18, 130.59) = 2.2 p b 0.0058). A significant increase was observed after repeated risperidone administration at 3 days (p b 0.0046) or 3 and 6 days (p b 0.00023 and p b 0.049) after the last risperidone injection in the prelimbic and infralimbic cortices, respectively (Fig. 6). Risperidone treatment did not

Table 1 study

131 change the length density of PSA-NCAM neuropil in the cingulate cortex, however, notable tendency to increase the length density of PSA-NCAM neuropil 3 days after the last dose of repeated risperidone administration (p = 0.073). With regard to the length density of PSA-NCAM immunoreactive processes in all of the inspected brain regions, single doses of risperidone were ineffective. The results for the entire mPFC, corresponding to the Western blot analysis, are shown in Table 1.

6.2.4. Phenotype of the PSA-NCAM immunoreactive perisomatic-like sites Double immunofluorescence labeling for PSA-NCAM and NeuN, a protein expressed in mature neurons (Mullen et al., 1992), showed that NeuN immunoreactive cells are surrounded by PSANCAM immunoreactivity creating perisomatic-like sites (Fig. 7A). Since previous data (Varea et al., 2005) indicate that PSA-NCAM expressing cells in the mPFC were NeuN immunoreactive, the relative optical densities of PSA-NCAM and NeuN immunoreactivity were measured. The results demonstrate the lack of co-localization of PSA-NCAM and NeuN protein (see an example 1, Fig. 7A), which suggests a perisomatic localization for the measured PSA-NCAM reactivity. However, the cell positive for the presence of both NeuN and PSA-NCAM proteins has also been found (see an example 2, Fig. 7A). The double labeling of PSA-NCAM and GAD-67 or GAT-1 proteins, as a marker of GABA innervation, has indicated that PSA-NCAM protein appeared around the soma and dendrites of certain neurons in a pattern similar to GABA-ergic perisomatic innervation (Fig. 7B, C). The neurons look (in shape and size) like cortical pyramidal cells. It has also been found that some of these PSA-NCAM immunoreactive puncta are positive for GAD67 or GAT-1 proteins (Fig. 7B, C).

7. Discussion The major findings of the present study indicate that risperidone, given repeatedly, but not acutely, evokes a

The effect of risperidone on the PSA-NCAM protein expression in the mPFC-summarized results of immunohistochemical

Analyzed parameters

Time after treatment

3

The number of PSA-NCAM-positive cells (10 )

VEH RAC RCH VEH The number of PSA-NCAM-positive perisomatic-like sites (103) RAC RCH VEH IOD of PSA-NCAM-positive perisomatic-like sites (103) RAC RCH The length density of PSA-NCAM-positive neuropil (10− 5 μm/μm3) VEH RAC RCH

3h

3d

6d

9d

63.3 ± 3.5 61.8 ± 8.9 64.7 ± 2.3 329.1 ± 26.5 330.9 ± 31.1 323.2 ± 28.1 371.7 ± 17.6 360.6 ± 29.2 380.2 ± 28.7 35.7 ± 2.7 30.5 ± 2.4 34.7 ± 1.6

68.8 ± 9.7 63.9 ± 7.1 60.7 ± 9.7 321.7 ± 40.8 315.6 ± 11.6 487.9 ± 22.9⁎ 368.8 ± 27.1 362.6 ± 29.2 488.4 ± 43.7⁎ 34.8 ± 1.5 37.9 ±2.2 53.8 ± 4.2⁎

69.3 ± 7.5 67.4 ± 4.5 69.9 ± 5.7 70.1 ± 5.6 63.1 ± 5.7 65.1 ± 1.3 289.8 ± 14.4 307.7 ± 83.6 325.2 ± 18.5 313.1 ± 13.9 400.1 ± 59.7⁎ 333.4 ± 40.9 380.1 ± 25.1 361.6 ± 14.6 376.6 ± 17.1 358.3 ± 21.5 399.5 ± 33.2 336.4 ±42.6 36.8 ± 1.7 33.3 ± 3.5 36.9 ± 1.4 35.9 ± 3.1 46.6 ± 4.5 31.0 ± 5.1

VEH—control animals, RAC—animals injected with a single dose of risperidone (0.2 mg/kg), RCH—animals treated repeatedly with risperidone (0.2 mg/kg i.p., once daily for 21 consecutive days). IOD—Integrated Optical Density. The results are the mean ± S.E.M; n = 5 per each treatment and time point. Asterisks indicate statistical significance when compared with appropriate control group; the level of significance is at least 0.05 (ANOVA followed by the Newman-Keuls test for post hoc comparison).

132

Figure 4 The effect of risperidone on the number of PSANCAM-positive perisomatic-like sites in the different subdivision of the mPFC. VEH—control animals, RAC—animals injected with a single dose of risperidone (0.2 mg/kg), RCH—animals treated repeatedly with risperidone (0.2 mg/kg i.p., once daily for 21 consecutive days). A—cingulate cortex, B—prelimbic cortex, C—infralimbic cortex. Each data point represents the mean ± S.E.M; n = 5 per each treatment and time point. Asterisks indicate statistical significance when compared with the appropriate control group; the level of significance is at least 0.05 (ANOVA followed by the Newman-Keuls test for post hoc comparison).

robust increase in the number and density of PSA-NACAM/ NCAM-positive perisomatic-like sites of cortical neurons and increases the number of PSA-NCAM/NCAM-positive processes (neuropil). The above changes were not followed by any changes in the number of PSA-NCAM/NCAM-positive neurons. Changes observed on the anatomical level were followed by the data, indicating that risperidone, when given in this way, increases the expression of two isoforms of

M. Maćkowiak et al. PSA-NCAM proteins (250 and 130/150 kDa, respectively) as well as the expression of NCAM backbone protein at 180 and 140 kDa. The available data, based on western blot analyses, have mostly indicated molecular weights of PSA-NCAM protein in the range 200–250 kDa (Abrous et al., 1997; Wang et al., 2004; Heidmets et al., 2007; Singh and Kaur, 2007), which is probably dependent on the lengths of the PSA chain (50–100 units) attached to NCAM backbone (Kiss and Rougon, 1997) and the NCAM isoforms. Thus, as observed in the present study as well as by others (Heidmets et al., 2007), the PSA-NCAM isoform at a molecular weight in the range of 130–150 kDa might be a product of degradation, but also might be the effect of polysialylation of the NCAM-120 present mostly in glia (Kiss and Rougon, 1997; Rutishauser, 2008) (the latter of which was not visible with the anti-NCAM antibody used in the present study). In addition, the degree of polysialylation of NCAM (and the subsequently determined molecular weight of the PSA-NCAM protein) is dependent on activation of polysialyltransferases (ST8Sia II and IV), which is lower for ST8Sia II (about 40 sialic acid residues) than for ST8Sia IV (about 60 residues) (Angata et al., 2002). Thus, depending on the activation of ST8Sia II, IV or both, the molecular weight of PSA-NCAM might really take a different value. The above changes in PSA-NCAM and NCAM proteins may suggest that, apart from the alteration of NCAM expression, risperidone has also altered the process of polysialylation of the NCAM backbone protein by changing the enzymatic activity of polysialyltransferases. Here, we observed the greatest effects in the infralimbic cortex, where the constitutive expression of PSA-NCAM/NCAM was also observed on the highest level. Such an effect is not surprising since a high constitutive expression of PSA-NCAM/ NCAM reflects the highest vulnerability to plastic changes and subsequent elongation, the spread of dendritic branches and synaptogenesis. The antibody used in the present study allows us to visualize only two isoforms of NCAM (180 and 140 kDa, respectively). NCAM-180 is restricted primarily to the postsynaptic densities of mature neurons, where it is involved in synaptic functions, including those that regulate sensory gating and fear conditioning (Wood et al., 1998; Sandi et al., 2001). NCAM-140 is preferentially expressed in immature or migrating neurons (Maness and Schachner, 2007). NCAMs, especially NCAM-180, play an important role in the molecular organization of the presynaptic terminals (Polo-Parada et al., 2004). Thus, regarding the function of both isoforms, the present data may indicate that risperidone alters the synaptic organization of the mPFC. The apparent lack of changes in the number of PSA-NCAMpositive neurons speaks against the possible appearance of neurogenesis in the mPFC after prolonged administration of risperidone. It has been shown that chronic stress alters the expression of PSA-NCAM in a brain region-dependent way (Pham et al., 2003; Cordero et al., 2005). However, it is unlikely that such stress factors are apparent in the current data. First, vehicle- and drug-treated animals received similar amounts of handling (injections), which might be regarded as an exposure to stress, while the expression of PSA-NCAM was seen to differ between the two cases. Such observations speak for risperidone, but not the stress effect. Second, the level of PSA-NCAM protein in the mPFC

Risperidone increases PSA-NCAM in the prefrontal cortex

133

Figure 5 The effect of risperidone on the Integrated Optical Density (IOD) of PSA-NCAM-positive perisomatic-like sites in the different subdivision of the mPFC. VEH—control animals, RAC—animals injected with a single dose of risperidone (0.2 mg/kg), RCH— animals treated repeatedly with risperidone (0.2 mg/kg i.p., once daily for 21 consecutive days). A—prelimbic cortex layer 3, B— prelimbic cortex layer 5/6, C—infralimbic cortex layer 3, D—infralimbic cortex layer 5/6. Each data point represents the mean ± S.E.M; n = 5 per each treatment and time point. Asterisks indicate statistical significance when compared with the appropriate control group; the level of significance is at least 0.05 (ANOVA followed by the Newman-Keuls test for post hoc comparison).

is similar to the level of untreated animals (data not shown). Finally, our previous experiments have shown that an animal's exposure to repeated injections with vehicle did not alter the level of circulating corticosterone (Czyrak et al., 2003) or expression of PSA-NCAM in the hippocampus (Mackowiak et al., 2005). It is an open question as to whether or not observed changes in the plasticity of the mPFC may represent a novel mechanism of therapeutic effects of risperidone. It is possible, however, to argue against this hypothesis. First, we observed changes in PSA-NCAM/NCAM during risperidone withdrawal, while attenuation of schizophrenic symptoms was observed during drug treatment (Llorca et al., 2008). Second, the observed effects are relatively brief, lasting up to 6 days, while therapeutic effects of risperidone have long-lasting remission (Llorca et al., 2008). It may be argued, however, that any changes leading to enhancement of PSA-NCAM/NCAM proteins may have beneficial effects on the malfunction of mPFC in the course of schizophrenia (Wood et al., 1998; Vawter et al., 1998; Pillai-Nair et al., 2005), and even a transient expression of PSA-NCAM/NCAM proteins may have a longlasting consequence for the function of the cortex since, once evoked, rearrangements of interneuron connectivity may have a long lasting consequence. It is of interest that risperidone increases, in an anatomically specific manner, the perisomatic puncta of cortical neurons. We observed here an increase in the number of surroundings as well as the enhancement in the density of perisomatic sites. In the human brain, PSA-NCAM

was found to be expressed in the somata, dendrites, and axonal processes of GABA-ergic interneurons co-expressing GAD-67, GAT-1, parvalbumin, and calbindin (Arellano et al., 2002; Varea et al., 2007c). Perisomatic-like PSA-NCAMpositive puncta have also been identified as a condensation of axon terminals of chandelier cells (i.e., parvalbuminpositive cells) in the human brain (Arellano et al., 2002). In the rat brain, PSA-NCAM has been found so far in calbindinpositive cells (Varea et al., 2005), but not parvalbuminepositive neurons and relatively little is known about the nature of PSA-NCAM-positive perisomatic innervations. It has been reported that some PSA-NCAM immunoreactive puncta in the mPFC neuropil are positive for synaptophysin (Varea et al., 2005). The above data suggest that the PSANCAM protein is located in the presynaptic part of GABAergic synapses. The double immunofluorescence staining shown in the present study confirms the above suggestion since some PSA-NCAM-immunoreactive puncta are also positive for markers of GABA-ergic transmission such as GAD-67, GAT-1. Moreover, double-labeling with NeuN, a marker of neurons (especially pyramidal neurons), has shown the perisomatic pattern of PSA-NCAM innervation. The localization of PSA-NCAM on GABA-ergic terminals offers a novel mechanism of therapeutic effects of risperidone based on the remodeling of the perisomatic surroundings of pyramidal neurons (among others). Such remodeling may restore the function of the GABA-ergic innervation, which is severely impaired in schizophrenic cases and is observed as a decrease in the GAT-1, GAD-67, and density of parvalbumin-positive neurons (Volk and

134

Figure 6 The effect of risperidone treatment on the length density of PSA-NCAM-positive neuropil in the different subdivision of the mPFC. VEH—control animals, RAC—animals injected with a single dose of risperidone (0.2 mg/kg), RCH—animals treated repeatedly with risperidone (0.2 mg/kg i.p., once daily for 21 consecutive days). A—cingulate cortex, B—prelimbic cortex, C— infralimbic cortex. Each data point represents the mean ± S.E.M; n = 5 per each treatment and time point. Asterisks indicate statistical significance when compared with the appropriate control group; the level of significance is at least 0.05 (ANOVA followed by the Newman-Keuls test for post hoc comparison).

Lewis 2002). The apparent effect indicates that risperidone may not only normalize neurotransmission, but may also restore the neuroanatomical defects associated with schizophrenia-like deficits of somatodendritic innervations of pyramidal cells. It is not known if risperidone directly affects the process of PSA-NCAM or NCAM synthesis, or if it alters NCAM protein expression by changing the intensity of

M. Maćkowiak et al. neurotransmission. A recently published study (Frasca et al., 2008) indicates that another atypical neuroleptic, olanzapine, increases the number of PSA-NCAM cells in the mPFC but not in the hippocampus. Moreover, haloperidol, a typical neuroleptic did not affect PSA-NCAM expression, either in the mPFC, or in the hippocampus (Frasca et al., 2008). As previously mentioned, we did not observe any changes in the number of PSA-NCAM-positive cells after risperidone treatment in contrast to olanzapine administration. However, the effect of olanzapine on the number of PSA-NCAM perisomatic-like sites has yet to be studied (Frasca et al., 2008). The observed discrepancy between the study of Frasca et al. and our study might be dependent upon the receptor profile of the investigated atypical neuroleptics (risperidone has a higher affinity for D2 and 5-HT2 receptors and no affinity for muscarinic receptors compared to olanzapine) (Gardner et al., 2005), the time of neuroleptic administration (14 vs. 21 days) and performed experiments (24 h vs. 3 h and 3, 6, or 9 days), or on the procedure used (i.e. immunofluorescence vs. silver-gold intensification of immunohistochemical staining). However, it seems that serotonergic components of atypical neuroleptics play a crucial role in the regulation of PSA-NCAM expression since haloperidol, an antagonist of dopaminergic receptors (mainly D2) (Gardner et al., 2005) did not affect PSA-NCAM expression in the mPFC. It is also puzzling that risperidone or olanzapine evoked similar changes as imipramine and fluoxetine (Sairanen et al., 2007; Varea et al., 2007a,b), which are antidepressant drugs designed to enhance serotonergic tone, while neuroleptics evoke the opposite effect on serotonin transmission. It is, however, conceivable that risperidone may evoke upregulation of serotonin receptors after prolonged administration, and, during withdrawal from risperidone, there is enhancement of serotonergic tone. This effect has been previously known to regulate expression of NCAM family proteins (Brezun and Daszuta, 1999). Such an idea might be supported by the fact that systemic administration of risperidone dose-dependently increases the serotonin output in the frontal cortex and dorsal raphe nucleus (Hertel et al., 1998). It may also be worthwhile to note that risperidone, enhancing the PSA-NCAM/NCAM protein level, does not change the phenotype of cortical expression, i.e., the number of PSA-NCAM-positive cells, which is in contrast with the results obtained with antidepressants or olanzapine. In conclusion, the present data indicate that neuroleptic drugs, represented in the present study by risperidone, given for a prolonged period of time, may induce plastic changes in the structure of the perisomatic region of the cortical pyramidal neurons. The above effects offer a new mechanism of action of neuroleptic drugs by remodeling the structure of perisomatic innervations of cortical neurons.

Role of the funding source This work was supported by the statutory activity of the Institute of Pharmacology, Polish Academy of Sciences, Kraków and by the Polish MNSW Scientific Network Fund. The Institute of Pharmacology and Polish MNSW had no further role in study design, in the collection and

Risperidone increases PSA-NCAM in the prefrontal cortex

135

Figure 7 Confocal laser microscope analysis of PSA-NCAM immunoreactive perisomatic-like sites in the mPFC. A) PSA-NCAM (green) and NeuN (red) in an orthogonal view (objective 63×). Arrows indicate the PSA-NCAM-positive protein surroundings NeuN immunoreactive cells. 1) An example of NeuN immunoreactive cell and PSA-NCAM perisomatic-like sites. 2) An example of cell positive for the presence of both NeuN and PSA-NCAM protein. The intensity of immunolabeling measured for the examples 1 and 2 is presented in the graph as a relative optical density. Red arrows indicate the site of the increase in NeuN immunoreactivity in 1) showing the lack of the increase in intensity of immunolabeling of PSA-NCAM and NeuN protein at the same measured point and 2) showing the increase in intensity of immunolabeling both for PSA-NCAM and NeuN protein at the same measured point. B) PSA-NCAM (green) and GAD-67 (red) immunoreactivity in an orthogonal view. C) PSA-NCAM (green) and GAT-1 (red) immunoreactivity in an orthogonal view (objective 63×). Arrows indicate examples of co-localization of PSA-NCAM immunoreactivity with GAD-67 (B) or GAT-1 (C) proteins. Scale bars represent 20 μm. Images were constructed by averaging out 10–20 optical sections taken 0.5 μm apart and were optimized individually for given better visualization of labeling antigens. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article). interpretation of data, in the writing of the report and in the decision of the paper for publication.

Acknowledgements

Contributors

This work was supported by the statutory activity of the Institute of Pharmacology, Polish Academy of Sciences, Kraków and by the Polish MNSW Scientific Network Fund.

Marzena Maćkowiak designed the experiments. Marzena Maćkowiak, Dorota Dudys and Agnieszka Chocyk performed the experiments. Marzena Maćkowiak and Krzysztof Wędzony wrote the manuscript, collected laser scanning confocal images and prepared the figures. All authors contributed to and have approved the final manuscript.

Conflict of interest The authors have no potential conflicts of interest relating to this report and no other conflicts of interest to declare.

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