Histochemical study of perineuronal nets in the retrosplenial cortex of adult rats

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Histochemical study of perineuronal nets in the retrosplenial cortex of adult rats Ramadan Sayed 1'2, Wafaa Mubarak 1'3, Aiji Ohtsuka 1, Takehito Taguchi 4 and Takuro Murakami I 1Section of Human Morphology, Graduate School of Medicine and Dentistry, Okayama University, Japan, 2Department of Anatomy and Histology, Faculty of Veterinary Medicine, Assiut University; Assiut 71526, Egypt, 3Department of Anatomy, Faculty of Medicine, Assiut University, Egypt and 4Department of Radiological Technology, Faculty of Health Sciences, School of Medicine, Okayama University, Japan

Dedication. This paper is dedicated to Dr. Mohamed Rashad Fath El-bab, Professor of Histology and the former Vice Dean for Research and Staff Affairs, Faculty of Veterinary Medicine, Assiut University, Egypt.

Summary. The retrosplenic cortex of rats, similar to many cortical or subcortical regions, is provided with special subsets of neurons that exhibited a fenestrated or reticular coat of condensed extracellular matrix on their soma, initial dendrites and proximal axon segment. This pericellular coating, currently termed "Perineuronal Nets", was detected on the surfaces of some neurons distributing throughout the cortical layers II-V. They presented direct interconnections with each other, and appeared in close association to the astroglial processes. In addition to their collagenous ligands, the perineuronal nets (PNs) were enriched with proteoglycans (PGs, sulfated glycoconjugates) and/or glycoproteins (GPs, unsulfated glycoconjugates with terminal N-acetylgalactosamine). Accordingly, the PNs were differentially identified as belonging to three categories, depending upon their organic nature or chemical composition. First, coats exclusively formed of PGs (stained with iron colloid); second, coats formed of GPs (labeled with plant lectins binding to terminal N-acetylgalactosamine); and third, complex coats formed of PG networks intermingled with glycoprotein molecules (double stained with iron colloid and lectin). Since differential distribution of protein containing substances (GPs and/or PGs) in the extracellular matrix contributes to functional terms, we suggest that these biochemical or morphological

Correspondence to: R. Sayed E-mail: [email protected]

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Ann Anat (2002) 184:333-339 © Urban & Fischer Verlag http://www.urbanfischer.de/journals/annanat

differences in the microenvironment of some retrosplenial neurons might reflect certain functional aspects concerned with processing of navigation or episodic memory.

Key words: Perineuronal nets - Glycoproteins - Proteoglycans - Retrosplenial cortex

Introduction Carbohydrate complexes such as proteoglycans and glycoproteins are main constituents of the extracellular matrix in the central nervous system (Margolis and Margolis 1993; Celio and Bliimcke 1994; Ruoslahti 1996). They are unevenly distributed in the neuropil or as lattice-like ensheathments around certain types of neurons. Recent studies of the central nervous system in mammals, avians and other lower vertebrates have shown that certain subsets of neurons exhibited perineuronal coats of PGs reactive to cationic iron colloid and aldehyde fuchsin (Murakami et al. 1994, 1996). This PGs coat is frequently intermingled with an additional extracellular chain of glycoprotein molecules (Murakami et al. 1999 b) and collagenous ligands (Murakami et al. 1999 a). The extracellular GP chains (possessing terminal N-acetylgalactosamine) are labeled with lectin Vicia villosa agglutinin 'VVA', Wisteria floribunda agglutinin 'WFA' or lectin Glycine max. agglutinin 'SBA' (Murakami et al. 1996) and that of the collagenous ligands are stainable with Gomori ammonical silver (Murakami et al. 1999 a). These investigations were systematically limited and insufficient to describe the histochemical characteristics of rat retrosplenial cortex, especially its coated neurons. 0940-9602102118414-333$15.00•0

The p r e s e n t investigation systematically supplements our previous studies and provides m o r e information on the chemical compounds, and the different types of PNs. F u r t h e r m o r e , it d e m o n s t r a t e s a spatial relationship between PNs and astrocytes. The granular and n o n - g r a n u l a r subfields of the rat retrosplenial cortex were used for this study. This cortex is involved in navigation and processing of episodic m e m o r y (Maguire 2001), as well as acquisition or extension of the trace c o n d i t i o n e d reflex (Weibel et al. 2000). The o b t a i n e d d a t a would contribute as a baseline for future quantitative or e x p e r i m e n t a l studies searching for the biological significance of p e r i c o a t e d neurons in this cortical region.

Materials and methods Animals and tissue processing. Adult Wistar rats at four months of postnatal life (n = 8) were perfused transcardially under ether anesthesia with saline, followed by 4% paraformaldehyde in 0.1 M sodium cacodylate buffer (pH 7.3). The brains were thereafter isolated and 2-3 mm-thick tissue slices were cut with a razor blade traversing the retrosplenial cortex in the sagittal and coronal planes and immersed in the aforementioned fixative for 24 hours, After fixation, specimens were dehydrated, cleared and embedded in paraffin. Serial sagittal or coronal sections (5-7 gm in thickness), traversing the retrosplenial cortex were prepared and, after deparaffinization by xylene, were treated as follows:

Lectin cytochemistry. Tissue sections were incubated with lectin Vicia villosa agglutinin "VVA" (Kosaka and Heizmann 1989), lectin Wisteria floribunda agglutinin "WFA" (H/~rtig et al. 1992) and lectin Glycine max. agglutinin "SBA" (Lath et al. 1992). The aim was to examine the spatial distribution pattern of N-acetyl-galactosamine containing glycoconjugates in the rat retrosplenial cortex. Peroxidase activity was demonstrated with diaminobenzidine (Nakagawa et al. 1986). Counter-staining with Mayer's hematoxylin was applied prior to balsam embedding. Controls for lectin-stained sections consisted of adjacent sections treated with phosphate buffer containing no agglutinin. Immunocytochemistry. For astroglial cell demonstration, using the intermediate filament protein glial fibrillary acidic protein (GFAP), we applied the protocol of Bignami and Dahl (1974). Briefly, the unspecific binding sites for immunoreagents were blocked with 5% normal goat serum in PBS. Subsequently, sections were processed with a rabbit antiserum to GFAP (1:500; RBI, Natick, MA, USA), followed by incubation with biotinylated goat anti-rabbit IgG (1 : 400; Vector Lab), then treated with HRP-conjugated streptavidin (Vector Lab), and finally the reaction was visualized by diaminobenzidine. Control experiments were performed omitting the primary antibodies. Dual staining with Iron Colloid and WFA, VVA, SBA, GFAP or Bodian. Some sections were stained with WFA, VVA or SBA and then additionally with cationic iron colloid. The aim was to allow differentiation of sulfated and unsulfated glycoconjugates. Double staining of GFAP and iron colloid staining would indicate a spatial relationship between PNs of proteoglycans and astrocytes. Repetition of the iron colloid and Bodian procedure would promote a clear resolution of PNs (Murakami et al. 1997).

Colloidal iron histochemistry. To estimate the spatial distribution of polyanionic components (sulfated glycoconjugates) incorporated into the extracellular matrix, tissue sections were incubated with a cationic iron colloid (CIC) at pH 1.5, and the reaction was demonstrated by Prussian blue (Murakami et al. 1986). Counter staining with nuclear fast red was usually applied prior to balsam embedding. Sections for control were not treated with iron colloid, but were subjected to the Prussian blue reaction.

Enzyme histochemistry. Some sections were digested with hyaluronidase "Streptomyces hyalurolyticus" (Murakami et al. 1994). Other sections were incubated with chondroitinase ABC "Proteus vulgaris" (Yoshikawa et al. 1997), endo-alpha-N-acetyl-galactosaminidase or with hyaluronidase/endo-alpha-N-acetylgalactosaminidase (Mukarami et al. 1999b). These enzyme-treated sections were then stained with CIC, lectin VVA or WFA or CIC/WFA and embedded in balsam. Control sections for hyaluronidase, chondroitinase ABC, endo-

Fig. 1. Staining patterns and distribution of perineuronal coats at the retrosplenial cortex. A: The glycoconjugates-coated neurons are distributed throughout the cortical layers II-IV. They are more frequent in layers IV and II than layer III and V. VVA-stained section counter-stained using Mayer's hematoxylin, x 33. B: The proteoglycans-coated neurons are distributed throughout the cortical layers II-V. They are more frequent in layers IV and II than layer III and V. CIC/Bodian-stained section, x 50. C: The perineuronal coats are differentially stained with CIC (blue color), lectin SBA (yellow color) or doubly labeled with CIC and SBA (green to dark-brown color), x200. Lower inset shows a sulfated proteoglycans coat (blue color) interconnected with a coat complex (light green color), x 330. Right inset shows a neuron-exhibiting coat complex. SBA/CIC-stained section counter-stained using Mayer's hematoxylin, x 330. Fig. 2. Staining patterns and distribution of the expressed epitopes on the perineuronal coats. A: Proteoglycans-coated neuron as revealed by CIC procedure and counter-stained by nuclear fast red. This neuron shows the morphological features of a pyramidal multipolar neuron. The CIC-binding sites surround the perikaryon, the initial segment of the axon (arrowhead), and the proximal parts of the dendrites. Note that the expressed epitopes exhibit a bluish, intensely stained net-like pattern. x 330. B: Proteoglycans-coated neuron as expressed by CIC/Bodian procedure. Note that section stained with this procedure reveals numerous dendritic arborizations with clearer or sharper intensity than that in section stained with CIC alone. (Arrowhead = axon), x 330. C: Glycoprotein-coated neuron stained with VVA and counter-stained with Mayer's hematoxylin. The VVA-bound epitopes ensheath the perikaryon, the initial segment of the axon (arrowhead), and the proximal parts of dendrites, x 330. Fig. 3. A, B: GFAP-immunoreactive glial cells and glial fibers are present in close association to or interlacing with the perineuronal proteoglycans nets (arrowheads). Immunostained section with GFAR doubled with CIC, and counter-stained by Mayer's hematoxylin. x 200 (A), and x 330 (B). 334

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alpha-N-acetyl-galactosaminidase or hyaluronidase/endo-alphaN-acetylgalactosaminidase digestions were incubated in the respective buffers containing no enzyme. Finally, these control sections were stained with CIC, VVA, WFA and/or the CIC/WFA combination procedure.

Photography. Photographic documentation was performed with an Olympus BX 50 transmission light microscope using Kodak technical pan TM film or Kodak TM (color) film and presented at their original magnifications.

some neurons in layers II-V. Most labeled neurons were found in layer II and IV. Notably, labeling intensity of perineuronal coats differed considerably among neurons with respect to the lectin used. They exhibited higher intensities in specimens treated with lectin VVA and WFA than with SBA. Furthermore, the staining intensity of neuropil was higher in sections stained with WFA than with VVA or SBA.

Combination staining with CIC and VVA, WFA or SBA.

Results General findings. The retrosplenial cortex in rats presented certain neuronal subsets that exhibited fenestrated pericellular coats recognized as network formations of condensed extracellular matrix (Figs. 1 and 2). This reticular structure of the extracellular matrix usually ensheathes somata, dendritic arborizations and the axon proximal segment of nerve cells (Figs. 2 A, B, C). The subpopulation of coated neurons were distributed throughout the second to fifth (II-V) cortical layers; however, they could not be revealed at the molecular (I) layer. In general, the pericoated cells exhibited the morphological characteristics of multipolar neurons. The perineuronal nets varied in thickness and staining intensity from cell to cell, and presented direct interconnection between each other as revealed from reconstruction studies of serial sections (Fig. 1 C). Moreover, glial cell somata and/or their processes were in direct contact with or interlacing perineuronal nets (Figs. 3 A, B). Control experiments. In all experiments, control incubations resulted in a significant lack of the labeling. Iron colloid histochernistry. Certain subsets of neurons in the retrosplenial cortex were ensheathed with bluish negatively charged pericellular nets of sulfated proteoglycans as indicated by iron colloid staining (Fig. 2 A and Figs. 3 A, B). The coated cells were distributed throughout the second to fifth cortical layers being relatively numerous in the fourth and second layers. Apart from iron colloid staining, similar argyrophilic nets were also demonstrated around somata, cellular processes and the axon initial segments of certain neuronal subsets, as revealed by CIC/Bodian staining (Figs. 1 B and 2B). Notably, this combination procedure relatively visualized the pericellular nets with clearer or sharper intensity than those stained with iron colloid alone. The intracellular structures of retrosplenial neurons and their associated glial cells were reacted negatively for the iron colloid and its combination with Bodian staining. Lectin cytochernistry. Special subpopulation of retrosplenial neurons were ensheathed in reticular extracellular coats as revealed by three plant lectins from Vicia villosa, Wisteria floribunda and Glycine max. agglutinin (Fig. 1 A and 2 C). This sheath surrounded the surface of

The PNs were differentially labeled by these combination procedures. Either they solely stained with CIC (blue) or lectins (yellow) or were doubly labeled with CIC and lectin (light greenish to dark brown) (Fig. 1 C). Accordingly, the perineuronal coats were classified into three categories: PG coats (solely stained with iron colloid), GP coats (solely stained with lectins) and coat complexes formed of PG networks that intermingled with glycoprotein molecules (doubly stained with lectins and iron colloid). Notably, the intensity of coat complexes varied considerably among labeling lectins. It was higher in sections stained with CIC/VVA or WFA/CIC than in those stained by SBA/CIC. Interestingly, the various colors of the complex coats properly reflected the relative quantities of unsulfated glycoconjugates (labeled with lectins) that intermingled with the sulfated proteoglycans nets (stained with CIC).

Immunocytochernistry. Immunoreacted sections from the retrosplenial cortex, stained with antibodies to the GFAP and successively stained with CIC demonstrated large numbers of immunolabeled astroglial cells and associated glial fibers. They were distributed throughout the cortical layers I-V, which appeared in close approximation to blood capillaries and pericoated or non-coated neuronal cells. Notably, direct contact or interlacing between labeled astrocytes or associated glial fibers with perineurohal coats was clearly revealed (Figs. 3 A, B). Enzyme histochernistry. In control sections, the perineuronal coats were positively stained with CIC, lectins and CIC/lectin staining. Sections treated with hyaluronidase showed that CIC staining of perineuronal PG coats was abolished (Fig. 4A) and VVA or WFA labeling of the GP coats were not erased. In the chondroitinase ABC-treated sections, the perineuronal PG coats were reactive with CIC and the VVA or WFA labeling of perineuronal GP coats was partially erased (Fig. 4 C). Sections treated with endo-alpha-N-acetyl-galactosaminidase showed that lectin labeling of the perineuronal GP coats was abolished (Fig. 4B) and CIC staining of sulfated PG coats was not abolished. Sections treated with hyaluronidase and endo-alphaN-acetyl-galactosaminidase showed that the perineurohal sulfated proteoglycan coats and glycoprotein coats were not reactive to CIC or lectins, respectively (not shown).

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A

B

C

Fig. 4. A: A retrosplenial section incubated with hyaluronidase and stained with CIC. Note that the perineuronal proteoglycan coats are not reactive for CIC staining, x 50. B: A retrosplenial section incubated with endo-alpha-N-acetylgalactosaminidase and stained with WFA. Note that the perineuronal glycoconjugates coats are not reactive for WFA staining, x 50. C: A retrosplenial section incubated with chondroitinase ABC and stained with VVA. Note that the VVA-staining of the perineuronal glycoconjugates coatings are partially erased, x 50 (Main Figure) and x 200 (Inset). Discussion The present investigation provided additional information on the chemical components of PNs and demonstrated three neuronal circuits at the retrosplenial cortex of rats: PGs-, GPs-, and PGs/GPs-coated neurons. The retrosplenial cortex in rats contains special neurons that possess reticular or net-like extracellular coatings on their surfaces that are enriched with proteoglycans and/or glycoproteins. The lattice-like or fenestrated structure of these coats has been described to ensheath somata, dendritic arborizations and axon initial segments of nerve cells (Celio and Bltimcke 1994). The PNs result from the visualization of extracellular matrix molecules that are confined to the space interposed between glial processes and the nerve cells that they outline (Celio and Bliimcke 1994). They have been visualized with various plant lectins, certain molecular markers, or other histochemical staining including iron colloid and aldehyde fuchsin (Brauer et al. 1982; Nakagawa et al. 1986; Kosaka and Heizmann 1989; Atoji et al. 1992; Lt~th et al. 1992; BrOck-

ner et al. 1993; Atoji et al. 1995; Brtickner et al. 1996 a, b; K6ppe et al. 1997). They were first described by Golgi as "Pericellular Nets" (Golgi 1893), and after their re-investigation by Brauer et al. (1982) the currently accepted terminus "Perineuronal Nets" has come into use. The ultrastructure or fine organization of these perineuronal matrices is described elsewhere (Murakami et al. 1995). It seems that the expressed epitopes in the present investigation are deposited at the perineuronal spaces of certain neuronal populations including their related synaptic buttons and intercalated glial structures (Celio and Bliimcke 1994), but not in the synaptic clefts (Hagihara et al. 1999; Ohtsuka et al. 2000). Ultrastructural investigations of PNs have demonstrated glycan components in the close vicinity of astrocyte processes, suggesting that the extracellular matrix contributes differentially to the glia-neuron interface (Derouiche et al. 1996). It is postulated that a single neuron may receive net-like contacts from several astrocytes and that a single astrocyte may contribute to a PN on more than one neuron, confirming that the extracellular matrix molecules and astrocytic pro-

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cesses are to a high degree topically associated. In addition, different proportions of both components may specify the individual neuronal microenvironment (Derouiche et al. 1996). Our previous ultrastructural and immunohistochemical study of PNs has revealed that each synaptic bouton fits into a mesh of the perineuronal network (Ohtsuka et al. 2000). The present investigation together with our previous study suggests that the windows (fenestrae) in perineuronal coats correspond or closely fit the synaptic buttons and intercalated glial structures. It may be possible that perineuronal sheaths play a significant role in permanent stabilization of neuronal-synaptic interfaces and therefore assuring signal transmission at the axo-somatic synapses (Celio and Bltimcke 1994). It is well known that lectins VVA and SBA bind selectively to terminal alpha and/or beta N-acetyl-galactosamine (Debray et al. 1981). The lectin WFA binds selectively to terminal alpha N-acetyl-galactosamine (H~irtig et al. 1992; Murakami et al. 1999b). In accordance with these studies, it is confirmed that the observed lectin binding epitope in the present investigation is a membrane bound glycoprotein with terminal N-acetylgalactosamine. In the present study, the sulfated proteoglycans coatings were solely stained with CIC and digested with hyaluronidase, but not erased by endo-alpha-N-acetylgalactosaminidase. The glycoprotein coats were solely labeled with lectins, and digested with endo-alpha-N-acetylgalactosaminidase (Murakami et al. 1999b). They were partially abolished by chondroitinase A B C but not erased by hyaluronidase. The complex coats were double stained with CIC and lectin staining and digested with hyaluronidase/endo-alpha-N-acetylgalactosaminidase. The basic components of the PNs in the retrosplenial cortex have been demonstrated to be somewhat variable. Notably, clear differences exist between individual perineuronal nets with respect to the quantity of proteoglycans and/or glycoproteins present in the microenvironment of the corresponding neurons. The differential concentration of these epitopes appears to be a significant factor in the regulation of individual neuronal micromilieu (Brt~ckner et al. 1996 a; K6ppe et al. 1997) and suggests a definite or specific function for each type. Although the coated neurons comprise a distinct population in the retrosplenial cortex of rats, their anatomical or functional significance is quite uncertain. However, the histochemical and differential characterization of perineuronal coats, as presented in the present investigation, may provide a qualitative information that has a special significance in establishing correlations with experimental data. Nevertheless, there are some putative roles for proteoglycans which include: neuronal maturation, stabilization af synapses, generation of polyanionic ion-buffering microenvironment, maintenance of extracellular spaces and concentration of growth or inhibitory factors (Matsui et al. 1999). Furthermore, the PNs play a supportive or protective role, and serve as recognition molecules between certain neurons and their surrounding cells (Celio

and Bliimcke 1994). Interestingly, the neurofibrillary changes or amyloid B deposits - characterizing Alzheimer's disease do not affect net-bearing neurons (Brackner et al. 1999). In addition, the coated neurons are not susceptible to age related changes including abnormal or hyperphosphorylation (Hfirtig et al. 2001). In conclusion, the central nervous system of vertebrates possesses certain subsets of PGs, GPs and PGs/GPs-ensheathed neurons, suggesting a significant role for each neuronal circuit. Future experimental studies should center on the molecular structure of perineuronal nets and knocking out of related genes, including their biochemical or physiological significance.

Acknowledgements. We are grateful to Prof. Dr. Gert Brt~ckner, Paul Flechsig Institute for Brain Research, University of Leipzig, Germany, for sending his helpful manuscripts related to this study. Drs. Ramadan Sayed and Wafaa Mubarak are Visiting Researchers from the Assiut University, Egypt at the Okayama University Medical School. Expenses for chemicals and experimental animals were sponsored by a departmental fund, Department of Human Anatomy, Okayama University Medical School. The technical cooperation of Mr. H. Kusano is greatly appreciated.

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Accepted February 6, 2002

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