Differential expression of several molecules of the extracellular matrix in functionally and developmentally distinct regions of rat spinal cord

We have examined the regional distribution of several chondroitin sulfate proteoglycans (neurocan, brevican, versican, aggrecan, phosphacan), of their glycosaminoglycan moieties, and of tenascin-R in the spinal cord of adult rat. The relationships of these molecules with glial and neuronal populations, identified with appropriate markers, were investigated by using multiple fluorescence labeling combined with confocal microscopy. The results showed that the distribution of the examined molecules was similar at all spinal cord levels but displayed area-specific differences along the dorso-ventral axis, delimiting functionally and developmentally distinct areas. In the gray matter, laminae I and II lacked perineuronal nets (PNNs) of extracellular matrix and contained low levels of chondroitin sulfate glycosaminoglycans (CS-GAGs), brevican, and tenascin-R, possibly favoring the maintenance of local neuroplastic properties. Conversely, CS-GAGs, brevican, and phosphacan were abundant, with numerous thick PNNs, in laminae III-VIII and X. Motor neurons (lamina IX) were surrounded by PNNs that contained all molecules investigated but displayed various amounts of CS-GAGs. Double-labeling experiments showed that the presence of PNNs could not be unequivocally related to specific classes of neurons, such as motor neurons or interneurons identified by their expression of calcium-binding proteins (parvalbumin, calbindin, calretinin). However, a good correlation was found between PNNs rich in CS-GAGs and the neuronal expression of the Kv3.1b subunit of the potassium channel, a marker of fast-firing neurons. This observation confirms the correlation between the electrophysiological properties of these neurons and the specific composition of their microenvironment.     

Transient expression of Bis protein in midline radial glia in developing rat brainstem and spinal cord

Bis (Bcl-2 interacting death suppressor) has been reported to contribute to the differentiation and maturation of specific neuronal populations in the developing rat forebrain, in addition to its well-established functions as a stress or survival-related protein. In the present study, we have analyzed the expression of Bis in the rat brainstem and cervical spinal cord during development by using immunohistochemistry. Bis immunoreactivity was detected in radial glial cells flanking the midline from embryonic day 14. During embryonic and early postnatal development, Bis expression persisted in differentiating radial glia at the midline but disappeared first in the spinal cord by postnatal day 7 (P7) and later also in the brainstem by P14. Bis expression was restricted to a subpopulation of the midline radial glia, i.e., the dorsal midline of the midbrain and spinal cord and the ventral midline of the hindbrain, which were double- or triple-labeled with vimentin and nestin, markers for radial glia, and S100B. However, these markers also labeled all radial glia including the ventral midline glia in the midbrain and spinal cord, with Bis being absent from these structures. In addition, the dorsal midline glia in the midbrain and spinal cord expressed Bis prior to the timing of expression for radial glial markers. Therefore, our results demonstrate the early and transient expression of Bis in the subpopulation of midline glia in the developing brainstem and spinal cord, suggesting that Bis has a unique role in association with the radial glial cells in the developing central nervous system. This research was supported by a grant (10029970) from the Ministry of Knowledge Economy, The Republic of Korea and by a grant (M103KV010010-08 K2201-01010) from Brain Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology, The Republic of Korea.     

Dynamics of the transganglionic movement of horseradish peroxidase in primary sensory neurons

Summary The dynamics of horseradish peroxidase (HRP) transport in primary sensory neurons were studied in rats by demonstration of the reaction product in spinal nerves, spinal ganglia, dorsal roots and in the spinal cord at different survival times after application of the enzyme to the transected sciatic nerve and to the spinal cord. Using tetramethylbenzidine as the chromogen according to Mesulam (1978), transganglionic transport of HRP was shown in both the disto-proximal direction after peripheral application, and proximo-distal direction after central application. Significant differences in staining intensity between the central and peripheral processes of primary sensory neurons were found after all survival times used in this study. After peripheral application the number of labeled axons and the staining intensity were higher in spinal nerves than in dorsal roots; an inverse situation occurred after central application. These differences as well as the time sequences in staining of different parts of primary sensory neurons suggest that HRP applied to a peripheral nerve and to the spinal cord, respectively, enters the perikarya of spinal ganglion cells in any case before continuing its movement in a cellulifugal direction. Lysosomal degradation of the major portion of the applied HRP is supposed. However, in the post-perikaryal portion of a considerable number of neurons HRP-transport still occurs to a varying extent, thus resulting in labeling of nerve endings. In some neurons a post-perikaryal transport could not be detected light microscopically. The transport rates differ: the calculated transport rate of disto-proximal, cellulipetal movement in the fastest transporting neurons was 7.5 mm/h, that of the disto-proximal cellulifugal movement 2.5 to 3 mm/h.This work was partly supported by the Hartmann Müller-Stiftung I want to thank Miss Regula Eichholzer for the technical assistance     

Cells capable of uptake of horseradish peroxidase in some circumventricular organs of the cat and rat

Summary The structure of mesenchymal cells distributed in some of the hypendymal organs of the circumventricular system in the cat and rat was demonstrated after intravenous injection of high doses of horseradish peroxidase. These cellular elements were observed in the vicinity of blood vessels of the organon vasculosum laminae terminalis, subfornical organ and area postrema. Electron-microscopically, these cells located between the basal laminae of the brain parenchyma and the blood capillaries show long cellular processes encircling fenestrated capillaries. Light and electron-microscopic examination revealed that this cell type is identical with the horseradish peroxidase-uptake cells, previously reported in the vicinity of the hypophysial portal system. Such phagocytic cells may be considered as a cellular component intervening between the brain parenchyma and the blood stream, playing a role in selective barrier functions in the above-mentioned circumventricular organs where a blood-brain barrier in the classical sense of the definition is lacking.This work was supported by grant No. 437002 from the Ministry of Education, Science and Culture, Japan     

P2X receptors in the rat uterine cervix,lumbosacral dorsal root ganglia,and spinal cord during pregnancy

ATP, an intracellular energy source, is released from cells during tissue stress, damage, or inflammation. The P2X subtype of the ATP receptor is expressed in rat dorsal root ganglion (DRG) cells, spinal cord dorsal horn, and axons in peripheral tissues. ATP binding to P2X receptors on nociceptors generates signals that can be interpreted as pain from damaged tissue. We have hypothesized that tissue stress or damage in the uterine cervix during late pregnancy and parturition can lead to ATP release and sensory signaling via P2X receptors. Consequently, we have examined sensory pathways from the cervix in nonpregnant and pregnant rats for the presence of purinoceptors. Antiserum against the P2X₃-receptor subtype showed P2X₃- receptor immunoreactivity in axon-like structures of the cervix, in small and medium-sized neurons in the L6/S1 DRG, and in lamina II of the L6/S1 spinal cord segments. Retrograde tracing confirmed the projections of axons of P2X₃-receptor-immunoreactive DRG neurons to the cervix. Some P2X₃-receptor-positive DRG neurons also expressed estrogen receptor- immunoreactivity and expressed the phosphorylated form of cyclic AMP response-element-binding protein at parturition. Western blots showed a trend toward increases of P2X₃-receptor protein between pregnancy (day 10) and parturition (day 22–23) in the cervix, but no significant changes in the DRG or spinal cord. Since serum estrogen rises over pregnancy, estrogen may influence purinoceptors in these DRG neurons. We suggest that receptors responsive to ATP are expressed in uterine cervical afferent nerves that transmit sensory information to the spinal cord at parturition.     

Conformation change of horseradish peroxidase in lipid membrane

The electrochemical behavior of horseradish peroxidase (HRP) in the dimyristoyl phosphatidylcholine (DMPC) bilayer on the glassy carbon (GC) electrode was studied by cyclic voltammetry. The direct electron transfer of HRP was observed in the DMPC bilayer. Only a small cathodic peak was observed for HRP on the bare GC electrode. The electron transfer of HRP in the DMPC membrane is facilitated by DMPC membrane. UV–Vis and circular dichroism (CD) spectroscopy were used to study the interaction between HRP and DMPC membrane. On binding to the DMPC membrane the secondary structure of HRP remains unchanged while there is a substantial change in the conformation of the heme active site. Tapping mode atomic force microscopy (AFM) was first applied for the investigation on the structure of HRP adsorbed on supported phospholipid bilayer on the mica and on the bare mica. HRP molecules adsorb and aggregate on the mica without DMPC bilayer. The aggregation indicates an attractive interaction among the adsorbed molecules. The molecules are randomly distributed in the DMPC bilayer. The adsorption of HRP in the DMPC bilayer changes drastically the domains and defects in the DMPC bilayer due to a strong interaction between HRP and DMPC films.     

Suramin-induced mucopolysaccharidosis in rat incisor

Two weeks after a single injection of suramin, the secretory and post-secretory ameloblasts of the rat incisor were filled with large lysosome-like vacuoles. At the light-microscope level, these vacuoles were positively stained with Alcian blue when MgCl₂ was used at a critical electrolyte concentration varying between 0.1 and 0.3 M, whereas no staining appeared when MgCl₂ varied between 0.7 and 0.9 M. Hyaluronidase digestion markedly reduced but did not totally abolish the staining, indicating that glycosaminoglycans were accumulated inside these vacuoles. Examination of these cells with the electron microscope revealed a polymorphic population of large vesicles, filled to various degrees with cetylpyridinium chloride (CPC)-positive and malachite green aldehyde (MGA)-positive material. The same pattern was observed in secretory odontoblasts but to a lesser extent. In the extracellular matrix, suramin-induced alterations appeared as large defects occurring during enamel formation. In predentin and dentin, the number and/or size of electron-dense aggregates resulting from CPC and MGA fixation, were enhanced in the suramin-injected rats. These aggregates were largely reduced or suppressed respectively by hyaluronidase digestion and chloroform/methanol treatment of the sections. The accumulation of glycosaminoglycans and phospholipids reported here inside ameloblasts and odontoblasts and in predentin and dentin supports the occurrence of suramin-induced mucopolysaccharidosis and lipidosis in this experimental animal model.     

Caspase activation throughout the first wave of spermatogenesis in the rat

Early in postnatal life, the first wave of spermatogenesis is accompanied by an initial wave of germ cell apoptosis. This may reflect an adjustment in the number of germ cells that can be adequately maintained by Sertoli cells. Two major pathways (intrinsic and extrinsic) are involved in the process of caspase activation and apoptosis in mammalian cells. The extrinsic pathway is characterized by the oligomerization of death receptors such as FAS or tumor necrosis factor, followed by the activation of caspase-8 and caspase-3. The intrinsic pathway involves the activation of procaspase-9, which in turn activates caspase-3. Extensive information is available concerning apoptotic inducers and their possible mechanisms in the adult rat. However, no data exist regarding the molecular and cellular mechanisms governing physiological cell death during puberty in the male rat. We have studied caspase activation throughout the first wave of spermatogenesis in the rat under physiological conditions, by combining the TUNEL procedure with the localization of active caspases in germ cells. We observed TUNEL-positive germ cells in rats of 5–40 days of age, the highest number being found in 25-day-old rats. TUNEL-positive and caspase-3-positive germ cells appeared as long chains of interconnected germ cells in 25-day-old rats. Caspase activation was assayed by either immunohistochemistry with antibodies against active caspase-3, -8, and -9, or by determining enzymatic activity in seminiferous tubules extracts. Both techniques showed activation of caspase-3, -8, and -9 in 25-day-old rats and low enzymatic activity at other ages. Confocal scanning laser microscopy indicated that active caspase-3, -8, and -9 co-localized with TUNEL-positive cells. Thus, caspase-3, -8, and -9 are active in apoptotic germ cells during the first wave of rat spermatogenesis. The extrinsic pathway of apoptosis may therefore play an important role in germ cell apoptosis during puberty in the rat.This work was financed by a research grant from FONDECYT (1040800) to R.D.M.     

Calmodulin in rat enterocyte: an immunogold electron-microscope study

Immunogold labeling of ultrathin sections of the epithelium of rat small intestine has been used to obtain insights into the ultrastructural localization and possible function of calmodulin in the enterocyte. Calmodulin is found mainly overlying the periphery of the microvillous core, in agreement with the location of the 110-kDa calmodulin complex. Extremely small amounts of calmodulin can be detected along the interdigitating basolateral membrane. This immunogold electron-microscope study suggests that calmodulin plays an important role in regulating the mechanochemical activity of myosin I but not in processes associated with the basolateral membrane of rat enterocyte.     

The uptake of horseradish peroxidase by cortical synapses in rat brain

Summary Horseradish peroxidase (HRP) was introduced directly into the cerebral cortex of adult rats, which were allowed to survive for 60 min before perfusion fixation. After the tissue had been incubated to demonstrate HRP at the LM and EM levels, blocks of cortical tissue were taken at varying distances from the injection site. These eight blocks of tissue constituted a time sequence for HRP diffusion.Qualitative examination of the presynaptic terminals showed that the most commonly encountered profiles are the plain synaptic vesicles, many of which accumulate tracer. In some terminals labelled vesicles are lined-up in tubular fashion. Other profiles commonly labelled are coated vesicles, tubular and vacuolar cisternae, and plain and coated pinocytotic vesicles.Quantitative analyses based on the number of terminals containing labelled profiles demonstrate an early rise in the rate of labelling of both plain synaptic vesicles and coated vesicles, after which synaptic vesicle labelling rises slowly towards a plateau. By contrast, there is a late parallel increase in the rate of labelling of coated vesicles and cisternae. A more detailed analysis, based on the actual numbers of labelled and total profiles within each presynaptic terminal, highlight early and late periods of rapid labelling for plain synaptic vesicles, coated vesicles and cisternae. A further aspect of HRP incorporation studied, concerns its uptake into four delineated regions of the presynaptic terminal.Our data indicate that membrane uptake into the presynaptic terminal is accomplished mainly via coated vesicles, although plain synaptic vesicles may also be involved. Coated vesicles, in turn, appear to give rise directly to plain synaptic vesicles, with some coalescing to produce vacuolar cisternae. The latter are involved in a two-way interchange of membrane with tubular cisternae, plain synaptic vesicles and coated vesicles. An additional source of plain synaptic vesicles are the tubular cisternae. Exocytosis of plain synaptic vesicles constitutes the mechanism by which transmitter is released from the presynaptic terminal.Supported by the Nuffield Foundation. We are grateful to Mr. M. Austin for help with the photography     

Effects of mutations in the helix G region of horseradish peroxidase

Horseradish peroxidase (HRP) has long attracted intense research interest and is used in many biotechnological fields, including diagnostics, biosensors and biocatalysis. Enhancement of HRP catalytic activity and/or stability would further increase its usefulness. Based on prior art, we substituted solvent-exposed lysine and glutamic acid residues near the proximal helix G (Lys 232, 241; Glu 238, 239) and between helices F and F' (Lys 174). Three single mutants (K232N, K232F, K241N) demonstrated increased stabilities against heat (up to 2-fold) and solvents (up to 4-fold). Stability gains are likely due to improved hydrogen bonding and space-fill characteristics introduced by the relevant substitution. Two double mutants showed stability gains but most double mutations were non-additive and non-synergistic. Substitutions of Lys 174 or Glu 238 were destabilising. Unexpectedly, notable alterations in steady-state V(m)/E values occurred with reducing substrate ABTS (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)), despite the distance of the mutated positions from the active site.     

The T-tubule system in the myocardia of the sand rat and mouse as demonstrated by horseradish peroxidase

Summary The transverse tubule (T-tubule) system in papillary muscles of the sand rat and the mouse were studied with the aid of a diffusion tracer (horseradish peroxidase). The T-tubule system in the sand rat showed a typical mammalian pattern with sarcolemmal tubules invaginating at the Z-band level of the sarcomere. These tubules follow a transverse direction in the cell with frequent longitudinal side-branches which connect tubules at different Z-band levels. In the mouse myocardium, the T-tubules also start as lateral invaginations from the sarcolemma at the Z-band levels. In the cell interior, however, the tubules ramify and brake up into a complicated system of spirally running tubules. These spirals, of relative small diameter (400Å–700Å), frequently expand and form lobulated cisternae at the Z-band levels.     

Immunolocalisation of P2X and P2Y nucleotide receptors in the rat nasal mucosa

Purinoceptor subtypes were localised to various tissue types present within the nasal cavity of the rat, using immunohistochemical methods. P2X₃ receptor immunoreactivity was localised in the primary olfactory neurones located both in the olfactory epithelium and vomeronasal organs (VNO) and also on subepithelial nerve fibres in the respiratory region. P2X₅ receptor immunoreactivity was found in the squamous, respiratory and olfactory epithelial cells of the rat nasal mucosa. P2X₇ receptor immunoreactivity was also expressed in epithelial cells and colocalised with caspase 9 (an apoptotic marker), suggesting an association with apoptosis and epithelial turnover. P2Y₁ receptor immunoreactivity was found within the respiratory epithelium and submucosal glandular tissue. P2Y₂ receptor immunoreactivity was localised to the mucus-secreting cells within the VNO. The possible functional roles of these receptors are discussed.     

Implantation in vitro: co-culture of rat blastocyst and epithelial cell vesicles

Implantation of blastocysts involves conversion of maternal and embryonic cell surfaces from a nonadhesive to an adhesive state in response to the internally driven developmental program or to externally generated factors. However, the intricacies of the cellular and subcellular changes that promote the attachment are not known, because these changes are difficult to determine in situ because of the nonaccessibility of the site. To overcome this, an in vitro model of implantation was developed by co-culturing rat blastocysts and uterine epithelial cells of the same gestational age (day 5 postcoitum; plug day as day 1) in drops hanging from the lid of a Petri dish. The system was used to study the changes on the surface membranes of the cells of the trophectoderm and uterine epithelium and to evaluate the antiadhesive activity of the newly designed test substances. The isolated epithelial cell vesicles were co-cultured with zona-free blastocysts in the microdrops (40–50 µl) hanging from the lid of a 60-mm Petri dish. The lid was placed over the lower dish, which was presaturated with the medium. The culture was examined 48 h later to determine the site of adhesion of epithelial cell vesicles with the trophoblasts lining the blastocyst. The cell-cell adhesion was monitored on a computerized image analyzer. To validate the adhesion of blastocysts and epithelial cell vesicles in co-culture, the expression of a cell adhesion molecule, uvomorulin, was studied using immunocytochemical technique after incubating with antiuvomorulin antibody. Intense staining was noted on the membrane surfaces at the site of attachment of the blastocyst and cell vesicles.The authors express their sincere thanks to the Ministry of Health and Family Welfare, Government of India, for their financial support     

The subsets of keratinocytes responsible for covering open wounds in neonatal rat skin

Full-thickness excisional wounds were made on the dorsal skin of 1-day-old rats to elucidate from where the cells move into the defect and what kinds of cells they are. Immunohistochemical analyses of the wound sites revealed that the following two subsets of keratinocytes were the major contributors to reepithelialization: first, the cells at the forefront of the migrating epithelium, termed leading edge cells, which expressed K14 keratin, known as basal cell-specific keratin, but not K6 or K10 keratins, so that they had probably moved from the basal cell layer; and, second, the cells tentatively termed immature spinous cells, which expressed K14 and K6 but not K10, and formed an ingrowth region following the leading edge cells. These two kinds of cells moved to the open wound area, as a multilayered cell sheet. Fluorescent phalloidin staining experiments indicated that actin filaments became concentrated in the leading edge cells within 6 h postwounding (PW), whereas weak signals of actin filaments were detected in the immature spinous cells. Taken together, the present findings support the view that wound covering in neonatal rat skin is accomplished by a movement en masse of keratinocytes from the bottom half of the surrounding epidermis.     

The expression of the gene coding for parathyroid hormone-related protein (PTHrP) during tooth development in the rat

By means of in situ hybridisation studies, it is shown that parathyroid hormone-related protein (PTHrP) mRNA is strongly expressed in the developing enamel organs of rat teeth. In particular, the cervical loop hybridises strongly with the PTHrP probe and expression is maintained at this site throughout life in the permanently erupting incisor teeth. In mature molar teeth, expression is downregulated to low levels and confined to the epithelial cell rests of Malassez and/or cementoblasts which may derive from these. The gene is also expressed at low levels in the tissue overlying the erupting molars and, thereafter, in the junctional epithelia and connective tissue cells of the epithelial attachment on all tooth surfaces. The premise that PTHrP may undergo post-translational processing and that the resultant products could act in different ways raises the possibility of its exerting multiple paracrine actions during tooth development. These could include the control of cell division and local vascular dilation during development.     

Production of an endothelial cell migratory signal in rat endometrium during early pregnancy

Rat endometrial explants were cultured in a three-dimensional collagen/endothelial cell matrix to measure angiogenic activity, as represented by migration of vascular endothelial cells towards the explants. Minimal endothelial cell migratory activity was observed with endometrial explants taken during the four-day oestrous cycle and days 3 and 4 of pregnancy. In contrast, a surge of endothelial cell migration occurred in response to endometrial explants taken from day-5-pregnant rats. Activity was found in explants taken approximately 5 h prior to implantation, but returned to minimal levels by day 6 of pregnancy. Endothelial cell migration remained minimal in response to both implantation and intersite tissue explants taken from days 6 and 7 of pregnancy. Endometrium from ovariectomised rats produced no endothelial cell migratory activity as measured by this technique. However, near preimplantation levels of endothelial cell migratory activity could be induced in ovariectomised rat endometrium by administering progesterone for 72 hours. Oestrogen given in conjunction with progesterone had no additional effect. These results demonstrate the presence of an endometrial signal that controls endothelial cell migration, and demonstrate this activity can be induced by progesterone without the addition of oestrogen.