Selective prosaposin expression in Langerhans islets of the mouse pancreas.

The islets of Langerhans are clusters of endocrine cells surrounded by exocrine acinar cells in the pancreas. Prosaposin is a housekeeping protein required for normal lysosomal function, but its expression level is significantly different among tissues. Prosaposin also exists in various body fluids including serum. Intracellularly, prosaposin activates lysosomes and may support autophagy, and extracellularly, prosaposin promotes survival of neurons via G protein-coupled receptors. In this study, prosaposin and its mRNA expression were examined in endocrine cells of the islets as well as in exocrine acinar cells in the pancreas of mice by in situ hybridization and immunostaining. High expression levels of prosaposin were found in Alpha, Beta and Delta cells in the islets, whereas prosaposin mRNA expression was faint or negative and prosaposin immunoreactivity was negative in exocrine acinar cells. The high expression levels of prosaposin in endocrine cells may indicate that prosaposin plays a crucial role in crinophagy, which is a characteristic autophagy in peptide-secreting endocrine cells, and/or that prosaposin is secreted from pancreatic islets. Since prosaposin has been reported in serum, this study suggests a new possible function of the Langerhans islets.

Expression patterns of prosaposin and neurotransmitter-related molecules in the chick paratympanic organ.

The paratympanic organ (PTO) is a small sense organ in the middle ear of birds that contains hair cells similar to those found in vestibuloauditory organs and receives afferent fibers from the geniculate ganglion. To consider the histochemical similarities between the PTO and vestibular hair cells, we examined the expression patterns of representative molecules in vestibular hair cells, including prosaposin, G protein-coupled receptor (GPR) 37 and GPR37L1 as prosaposin receptors, vesicular glutamate transporter (vGluT) 2 and vGluT3, nicotinic acetylcholine receptor subunit α9 (nAChRα9), and glutamic acid decarboxylase (GAD) 65 and GAD67, in the postnatal day 0 chick PTO and geniculate ganglion by in situ hybridization. Prosaposin mRNA was observed in PTO hair cells, supporting cells, and geniculate ganglion cells. vGluT3 mRNA was observed in PTO hair cells, whereas vGluT2 was observed in a small number of ganglion cells. nAChRα9 mRNA was observed in a small number of PTO hair cells. The results suggest that the histochemical character of PTO hair cells is more similar to that of vestibular hair cells than that of auditory hair cells in chicks.

Expression patterns of prosaposin and its receptors, G protein-coupled receptor (GPR) 37 and GPR37L1, in the mouse olfactory organ.

Prosaposin is a glycoprotein conserved widely in vertebrates, because it is a precursor for saposins that are required for normal lysosomal function and thus for autophagy, and acts as a neurotrophic factor. Most tetrapods possess two kinds of olfactory neuroepithelia, namely, the olfactory epithelium (OE) and the vomeronasal epithelium (VNE). This study examined the expression patterns of prosaposin and its candidate receptors, G protein-coupled receptor (GPR) 37 and GPR37L1, in mouse OE and VNE by immunofluorescence and in situ hybridization. Prosaposin immunoreactivity was observed in the olfactory receptor neurons, vomeronasal receptor neurons, Bowman's gland (BG), and Jacobson's gland (JG). Prosaposin expression was mainly observed in mature neurons. Prosaposin mRNA expression was observed not only in these cells but also in the apical region of the VNE. GPR37 and GPR37L1 immunoreactivities were found only in the BG and/or the JG. Prosaposin was suggested to secrete and facilitate the autophagic activities of the neurons and modulate the mucus secretion in mouse olfactory organ.

Expression patterns of prosaposin and its receptors, G protein-coupled receptor (GPR) 37 and GPR37L1 mRNAs, in the chick inner ear.

Prosaposin is a glycoprotein that is widely conserved in vertebrates. It serves as a precursor for saposins A, B, C, and D, which are necessary activators of lysosomal sphingolipid hydrolases. It can also act as a neurotrophic factor. Prosaposin plays a crucial role in the mammalian vestibuloauditory system because it prevents progressive deafness and severe vestibular dysfunction. Prosaposin can exhibit a neurotrophic effect through the G protein-coupled receptor (GPR), and GPR37 and GPR37L1 are its candidate receptors. In this study, we examined the expression patterns of prosaposin, GPR37, and GPR37L1 mRNAs in postnatal day 0 chick vestibuloauditory organs by in situ hybridization. Prosaposin mRNA expression was observed in all vestibular end organs, the vestibular and spiral ganglions, whereas no hybridization signal was observed in the auditory organ, namely basilar papilla. While GPR37L1 mRNA expression was observed in the oligodendrocytes/Schwann cells in the vestibular ganglion, GPR37 mRNA expression was observed in the crista ampullaris base region. These findings suggest that prosaposin expression in the auditory hair cells is acquired uniquely in mammals partly due to the loss of regeneration upon maturation and improved autophagic activity in mammalian auditory hair cells. In addition, as GPR37L1 expression in the chick glial cells differed from GPR37 expression in mammalian glial cells, the roles of GPR37 and GPR37L1 for prosaposin may differ between birds and mammals.

Expression of prosaposin and its G protein-coupled receptor (GPR) 37 in mouse cochlear and vestibular nuclei.

Prosaposin is a precursor of lysosomal hydrolases activator proteins, saposins, and also acts as a secretory protein that is not processed into saposins. Prosaposin elicits neurotrophic function via G protein-coupled receptor (GPR) 37, and prosaposin deficiency causes abnormal vestibuloauditory end-organ development. In this study, immunohistochemistry was used to examine prosaposin and GPR37 expression patterns in the mouse cochlear and vestibular nuclei. Prosaposin immunoreactivity was observed in neurons and glial cells in both nuclei. GPR37 immunoreactivity was observed in only some neurons, and its immunoreactivity in the vestibular nucleus was weaker than that in the cochlear nucleus. This study suggests a possibility that prosaposin deficiency affects not only the end-organs but also the first center of the vestibuloauditory system.

Morphological variations in the transverse foramen of the axis in Japanese serows (Capricornis crispus).

The presence of transverse foramina in the axes of Japanese serows, a special national natural treasure in Japan, has been reported to be unstable, but other variations are unknown. In this study, we analysed the shape, cross-sectional area, length, and volume of the transverse foramen in the axes of 19 specimens using gross anatomy and computed tomography (CT) scan. There were four types in the transverse foramen: type 1, having the transverse foramina; type 2, having two cranial openings; type 3, sifting a caudal opening to the ventral side of the transverse process; and type 4, having no transverse foramina. Although the transverse foramina showed different types on the left and right sides in several specimens, there were no statistically significant differences in the length and volume. This variation may be related to running patterns of the vertebral artery penetrating the transverse foramina. Two goats without the transverse foramina were examined to infer a running pattern of the vertebral artery instead of Japanese serows. The vertebral artery in the goats branched in two directions (spinal and muscle), between the axis and the third cervical vertebra. This passage of the goat vertebral artery might be presumed in type 4 of Japanese serows. This study reveals the instability of the transverse foramina in the axes of Japanese serows and provides new data to compare the axes of other ruminants.

Morphometric study of the vestibuloauditory organ of the African clawed frog, Xenopus laevis.

Independent auditory end-organs appear first in amphibians in vertebrate phylogeny. In amphibians, sound detection is carried out by the amphibian papilla, basilar papilla and macula saccule. Amphibians inhabit distinct habitats and exhibit specific behaviours and sound frequency responses, so the amphibian vestibuloauditory system is an excellent model for considering the relationships between behaviour and physiological/anatomical vestibuloauditory properties. The African clawed frog, Xenopus laevis, lives in shallow water throughout its life and is thought to use sound in a higher frequency range compared with terrestrial anurans. In this study, the size of each vestibuloauditory end-organ and the distribution of ganglion cells in the vestibuloauditory ganglion were examined using haematoxylin and eosin staining and lectin histochemistry in Xenopus laevis. This study revealed that the size ratios among end-organs in Xenopus are similar to those in terrestrial anurans. Large and small cells were observed in the ganglion, but their distribution patterns are different from those in general terrestrial anurans. Lycopersicon esculentum lectin stained a large number of ganglion cells. Lectin-stained cells were found throughout the whole ganglion, but were especially abundant in the caudal part. These results suggested a unique distribution pattern of the vestibuloauditory ganglion cells in Xenopus.

Morphological features of the mouse duodenocolic fold in foetus and adult.

For the mechanism of duodenojejunal flexure (DJF) morphogenesis in mice, we consider the gut tube itself and the gut mesentery as important players. In this study, we focussed on the morphological features of the gut mesentery around the mouse duodenum, especially the duodenocolic fold at embryonic day (E) 18.5 and the adult phase. The duodenocolic fold, a sheet of the mesentery, was located between the entire ascending duodenum and the descending colon. At E18.5, in the cranial area near the DJF, the duodenocolic fold joined both the mesocolon and the mesojejunal part of the root of the mesentery. In the middle and caudal areas, the duodenocolic fold joined the mesocolon. Interestingly, along with the ascending duodenum, the duodenocolic fold contained a smooth muscle bundle. The smooth muscle bundle continued from the outer muscular layer of the middle to the caudal part of the ascending duodenum. The three-dimensional imaging of the foetal duodenocolic fold revealed that the smooth muscle bundle had short and long apexes towards the proximal and distal parts of the root of the mesentery, respectively. At the adult phase, the duodenocolic fold had a much thinner connective tissue with a larger surface area in comparison with the duodenocolic fold at E18.5. The adult duodenocolic fold also contained the smooth muscle bundle which was similar to the foetal duodenocolic fold. A part of the duodenocolic fold connecting to the mesojejunal part of the root of the mesentery seemed to be homologous to the superior duodenal fold in humans, known as the duodenojejunal fold; by contrast, most of the duodenocolic fold seemed to be homologous to the inferior duodenal fold in humans, known as the duodenomesocolic fold. The smooth muscle bundle in the mouse duodenocolic fold seemed to play a role in keeping the ascending duodenum in the abdominal cavity because the duodenum in animals did not belong to a retroperitoneal organ in contrast to humans owing to the difference in the direction of gravity on the abdominal organs between mice and humans. Moreover, the smooth muscle bundle shared common and uncommon points in its location and nerve supply to the suspensory muscle of the duodenum in humans, known as the ligament of Treitz. This study had insufficient evidence that the smooth muscle bundle of the mouse duodenocolic fold was homologous to the suspensory muscle of the duodenum in humans. In conclusion, this study revealed the detailed structure of the mouse duodenocolic fold, including the relationship between the fold and other mesenteries. Particularly, the smooth muscle bundle is a specific feature of the mouse duodenocolic fold and might play several roles in DJF morphogenesis, especially the ascending duodenum and the caudal duodenal flexure during development.

Sexually dimorphic response of colorectal motility to noxious stimuli in the colorectum in rats.

KEY POINTS: This study showed a remarkable sex difference in responses of colorectal motility to noxious stimuli in the colorectum in rats: colorectal motility was enhanced in response to intracolonic administration of a noxious stimulant, capsaicin, in male rats but not in female rats. The difference in descending neurons from the brain to spinal cord operating after noxious stimulation could be responsible for the sex difference. In male rats, serotoninergic and dopaminergic neurons are dominantly activated, both of which activate the spinal defaecation centre. In female rats, GABAergic neurons in addition to serotoninergic neurons are activated. GABA may compete for facilitative action of 5-HT in the spinal defaecation centre, and thereby colorectal motility is not enhanced in response to intracolonic administration of capsaicin. The findings provide a novel insight into pathophysiological mechanisms of sex differences in functional defaecation disorders such as irritable bowel syndrome. ABSTRACT: We previously demonstrated that noxious stimuli in the colorectum enhance colorectal motility through activation of descending pain inhibitory pathways in male rats. It can be expected that the regulatory mechanisms of colorectal motility differ in males and females owing to remarkable sex differences in descending pain inhibitory pathways. Thus, we aimed to clarify sex differences in responses of colorectal motility to noxious stimuli in rats. Colorectal motility was measured in vivo in anaesthetized rats. Administration of a noxious stimulant, capsaicin, into the colorectal lumen enhanced colorectal motility in male rats but not in female rats. Quantitative PCR and immunohistochemistry showed that TRPV1 expression levels in the dorsal root ganglia and in the colorectal mucosa were comparable in male and female rats. When a GABAA receptor inhibitor was intrathecally administered to the L6-S1 level of the spinal cord, colorectal motility was facilitated in response to intracolonic capsaicin even in female rats. The capsaicin-induced response in the presence of the GABA blocker in female rats was inhibited by intrathecal administration of 5-HT2 and -3 receptor antagonists but not by a D2-like dopamine receptor antagonist. Our findings demonstrate that intracolonic noxious stimulation activates GABAergic and serotoninergic descending neurons in female rats, whereas serotoninergic and dopaminergic neurons are dominantly activated in male rats. Thus, the difference in the descending neurons operating after noxious stimulation would be responsible for the sexually dimorphic responses of colorectal motility. Our findings provide a novel insight into pathophysiological mechanisms of sex differences in functional defaecation disorders such as irritable bowel syndrome.

Expression of the G protein-coupled receptor (GPR) 37 and GPR37L1 in the mouse digestive system.

G protein-coupled receptor (GPR) 37 and GPR37L1 are known to modulate the dopaminergic neuron activity, and recently, they are identified as candidate prosaposin receptors. Intercellular prosaposin is proteolytically processed into four saposins, each of which acts as a sphingolipid hydrolase activator in the lysosome. In contrast, extracellular prosaposin exerts a trophic effect on neurons via GPR37 and GPR37L1. In this study, the expression patterns of GPR37 and GPR37L1 in the mouse digestive system were examined immunohistochemically. The islets of Langerhans of the pancreas showed intense immunoreactivity for GPR37 and GPR37L1. Weak immunoreactivity for GPR37 and GPR37L1 was found in the nerve plexuses of the esophagus and small and large intestines. Colocalization of GPR37 and tyrosine hydroxylase immunoreactivity was observed in the neuron of the nerve plexus of the large intestine. This study suggests the possibility that prosaposin affects the function of islet-secreting cells. Also, the expression of GPR37 and GPR37L1 in the nerve plexus suggests that prosaposin exerts a trophic effect not only in the central nervous system, but also in the enteric nervous system.

Prosaposin and its receptors GRP37 and GPR37L1 show increased immunoreactivity in the facial nucleus following facial nerve transection.

Neurotrophic factor prosaposin (PS) is a precursor for saposins A, B, C, and D, which are activators for specific sphingolipid hydrolases in lysosomes. Both saposins and PS are widely contained in various tissues. The brain, skeletal muscle, and heart cells predominantly contain unprocessed PS rather than saposins. PS and PS-derived peptides stimulate neuritogenesis and increase choline acetyltransferase activity in neuroblastoma cells and prevent programmed cell death in neurons. We previously detected increases in PS immunoactivity and its mRNA in the rat facial nucleus following facial nerve transection. PS mRNA expression increased not only in facial motoneurons, but also in microglia during facial nerve regeneration. In the present study, we examined the changes in immunoreactivity of the PS receptors GPR37 and GPR37L1 in the rat facial nucleus following facial nerve transection. Following facial nerve transection, many small Iba1- and glial fibrillary acidic protein (GFAP)-positive cells with strong GPR37L1 immunoreactivity, including microglia and astrocytes, were observed predominately on the operated side. These results indicate that GPR37 mainly works in neurons, whereas GPR37L1 is predominant in microglia or astrocytes, and suggest that increased PS in damaged neurons stimulates microglia or astrocytes via PS receptor GPR37L1 to produce neurotrophic factors for neuronal recovery.

Suppression of GABAergic transmission in the spinal dorsal horn induces pain-related behaviour in a chicken model of spina bifida.

Spina bifida aperta (SBA), one of the most common congenital malformations, causes various neurological disorders. Pain is a common complaint of patients with SBA. However, little is known about the neuropathology of SBA-related pain. Because loss of g-aminobutyric acid GABAergic neurons in the spinal cord dorsal horn is associated with pain, we hypothesised the existence of crosstalk between SBA-related pain and alterations in GABAergic transmission in the spinal cord. Therefore, we investigated the kinetics of GABAergic transmission in the spinal cord dorsal horn in a chicken model of SBA. Neonatal chicks with SBA exhibited various pain-like behaviours, such as an increased number of vocalisations with elevated intensity (loudness) and frequency (pitch), reduced mobility, difficulty with locomotion, and escape reactions. Furthermore, the chicks with SBA did not respond to standard toe-pinching, indicating disruption of the spinal cord sensorimotor networks. These behavioural observations were concomitant with loss of GABAergic transmission in the spinal cord dorsal horn. We also found apoptosis of GABAergic neurons in the superficial dorsal horn in the early neonatal period, although cellular abnormalisation and propagation of neuro-degenerative signals were evident at middle to advanced gestational stages. In conclusion, ablation of GABAergic neurons induced alterations in spinal cord neuronal networks, providing novel insights into the pathophysiology of SBA-related pain-like complications.

ATP-dependent potassium channels contribute to motor regulation of esophageal striated muscle in rats.

The aim of the present study was to clarify roles of ATP-dependent potassium channels (KATP channels) in motility of the striated muscle portion in the esophagus. An isolated segment of the rat esophagus was placed in an organ bath and mechanical responses were recorded using a force transducer. Electrical stimulation of the vagus nerve evoked contractile response of striated muscle in the esophageal segment. Application of glibenclamide, an antagonist of KATP channels, increased amplitude of vagally mediated twitch contractions of the rat esophagus. On the other hand, minoxidil, an agonist of KATP channels, decreased amplitude of twitch contractions. RT-PCR revealed the expression of subunits of KATP channels in esophageal tissue. In addition, immunopositivity for subunits of KATP channels was observed in the striated muscle cells of the esophageal muscle layer. These findings indicate that KATP channels contribute to motor regulation of striated muscle in the rat esophagus.

Prosaposin and its receptors are differentially expressed in the salivary glands of male and female rats.

Salivary glands produce various neurotrophins that are thought to regulate salivary function during normal and pathological conditions. Prosaposin (PSAP) is a potent neurotrophin found in several tissues and various biological fluids and may play roles in the regulation of salivary function. However, little is known about PSAP in salivary glands. As the functions of salivary glands are diverse based on age and sex, this study examines whether PSAP and its receptors, G protein-coupled receptor 37 (GPR37) and GPR37L1, are expressed in the salivary glands of rats and whether sex and aging affect their expression. Immunohistochemical analysis revealed that PSAP and its receptors were expressed in the major salivary glands of rats, although their expression varied considerably based on the type of gland, acinar cells, age and sex. In fact, PSAP, GPR37 and GPR37L1 were predominantly expressed in granular convoluted tubule cells of the submandibular gland and the intensity of their immunoreactivity was higher in young adult female rats than age-matched male rats, which was more prominent at older ages (mature adult to menopause). On the other hand, weak PSAP, GPR37 and GPR37L1 immunoreactivity was observed mainly in the basal layer of mucous cells of the sublingual gland. Triple label immunofluorescence analysis revealed that PSAP, GPR37 and GPR37L1 were co-localized in the basal layer of acinar and ductal cells in the major salivary glands. The present findings indicate that PSAP and its receptors, GPR37 and GPR37L1, are expressed in the major salivary glands of rats and their immunoreactivities differ considerably with age and sex.

Early neonatal loss of inhibitory synaptic input to the spinal motor neurons confers spina bifida-like leg dysfunction in a chicken model.

Spina bifida aperta (SBA), one of the most common congenital malformations, causes lifelong neurological complications, particularly in terms of motor dysfunction. Fetuses with SBA exhibit voluntary leg movements in utero and during early neonatal life, but these disappear within the first few weeks after birth. However, the pathophysiological sequence underlying such motor dysfunction remains unclear. Additionally, because important insights have yet to be obtained from human cases, an appropriate animal model is essential. Here, we investigated the neuropathological mechanisms of progression of SBA-like motor dysfunctions in a neural tube surgery-induced chicken model of SBA at different pathogenesis points ranging from embryonic to posthatch ages. We found that chicks with SBA-like features lose voluntary leg movements and subsequently exhibit lower-limb paralysis within the first 2 weeks after hatching, coinciding with the synaptic change-induced disruption of spinal motor networks at the site of the SBA lesion in the lumbosacral region. Such synaptic changes reduced the ratio of inhibitory-to-excitatory inputs to motor neurons and were associated with a drastic loss of γ-aminobutyric acid (GABA)ergic inputs and upregulation of the cholinergic activities of motor neurons. Furthermore, most of the neurons in ventral horns, which appeared to be suffering from excitotoxicity during the early postnatal days, underwent apoptosis. However, the triggers of cellular abnormalization and neurodegenerative signaling were evident in the middle- to late-gestational stages, probably attributable to the amniotic fluid-induced in ovo milieu. In conclusion, we found that early neonatal loss of neurons in the ventral horn of exposed spinal cord affords novel insights into the pathophysiology of SBA-like leg dysfunction.

NeuN immunoreactivity in the brain of Xenopus laevis.

Neuronal nuclear antigen (NeuN), discovered in mice brain cell nuclei by Mullen et al. (1992), is used as an excellent marker of post-mitotic neurons in vertebrates. In this study, the expression pattern of NeuN was examined in the Xenopus brain to explore phylogenetic differences in NeuN expression. Anti-NeuN antibody showed selective staining in mouse and Xenopus brain extracts, but the number and molecular weight of the bands differed in Western blotting analysis. In immunostaining, anti-NeuN antibody showed selective staining of neurons, but not glial cells, in the Xenopus brain. Most neurons, including olfactory bulb mitral cells and cerebellar Purkinjie cells, which show no immunoreactivity in birds/mammals, showed NeuN immunoreactivity in Xenopus. This study revealed that anti-NeuN antibody is a useful marker of post-mitotic neurons in amphibians, but it also stains neurons that show no reactivity in more derived animals.

Expression of prosaposin and its receptors in the rat cerebellum after kainic acid injection.

Prosaposin (PSAP), a highly conserved glycoprotein, is a precursor of saposins A-D. Accumulating evidence suggests that PSAP is a neurotrophic factor that induces differentiation and prevents death in a variety of neuronal cells through the active region within the saposin C domain both in vivo and in vitro. Recently, GPR37 and GPR37L1 were recognized as PSAP receptors. In this study, we examined the alteration in expression of PSAP and its receptors in the cerebellum using rats injected with kainic acid (KA). The results show that PSAP was strongly expressed in the cytoplasm of Purkinje cells and interneurons in the molecular layer, and that PSAP expression in both types of neurons was markedly enhanced following KA treatment. Immunoblotting revealed that the expression of GPR37 was diminished significantly three days after KA injection compared with control rats; however, no changes were observed through immunostaining. No discernable changes were found in GPR37L1. These findings may help us to understand the role of PSAP and the GPR37 and GPR37L1 receptors in alleviating the neural damage caused by KA.

Interneurons secrete prosaposin, a neurotrophic factor, to attenuate kainic acid-induced neurotoxicity.

Prosaposin (PS) is a secretory neurotrophic factor, as well as a regulator of lysosomal enzymes. We previously reported the up-regulation of PS and the possibility of its axonal transport by GABAergic interneurons after exocitotoxicity induced by kainic acid (KA), a glutamate analog. In the present study, we performed double immunostaining with PS and three calcium binding protein markers: parvalbumin (PV), calbindin, and calretinin, for the subpopulation of GABAergic interneurons, and clarified that the increased PS around the hippocampal pyramidal neurons after KA injection existed mainly in the axons of PV positive interneurons. Electron microscopy revealed PS containing vesicles in the PV positive axon. Double immunostaining with PS and secretogranin or synapsin suggested that PS is secreted with secretogranin from synapses. Based on the results from in situ hybridization with two alternative splicing forms of PS mRNA, the increase of PS in the interneurons was due to the increase of PS + 0 (mRNA without 9-base insertion) as in the choroid plexus, but not PS + 9 (mRNA with 9-base insertion). These results were similar to those from the choroid plexus, which secretes an intact form PS + 0 to the cerebrospinal fluid. Neurons, especially PV positive GABAergic interneurons, produce and secrete the intact form of PS around hippocampal pyramidal neurons to protect them against KA neurotoxicity.

A prosaposin-derived Peptide alleviates kainic Acid-induced brain injury.

Four sphingolipid activator proteins (i.e., saposins A-D) are synthesized from a single precursor protein, prosaposin (PS), which exerts exogenous neurotrophic effects in vivo and in vitro. Kainic acid (KA) injection in rodents is a good model in which to study neurotrophic factor elevation; PS and its mRNA are increased in neurons and the choroid plexus in this animal model. An 18-mer peptide (LSELIINNATEELLIKGL; PS18) derived from the PS neurotrophic region prevents neuronal damage after ischemia, and PS18 is a potent candidate molecule for use in alleviating ischemia-induced learning disabilities and neuronal loss. KA is a glutamate analog that stimulates excitatory neurotransmitter release and induces ischemia-like neuronal degeneration; it has been used to define mechanisms involved in neurodegeneration and neuroprotection. In the present study, we demonstrate that a subcutaneous injection of 0.2 and 2.0 mg/kg PS18 significantly improved behavioral deficits of Wistar rats (n = 6 per group), and enhanced the survival of hippocampal and cortical neurons against neurotoxicity induced by 12 mg/kg KA compared with control animals. PS18 significantly protected hippocampal synapses against KA-induced destruction. To evaluate the extent of PS18- and KA-induced effects in these hippocampal regions, we performed histological evaluations using semithin sections stained with toluidine blue, as well as ordinal sections stained with hematoxylin and eosin. We revealed a distinctive feature of KA-induced brain injury, which reportedly mimics ischemia, but affects a much wider area than ischemia-induced injury: KA induced neuronal degeneration not only in the CA1 region, where neurons degenerate following ischemia, but also in the CA2, CA3, and CA4 hippocampal regions.

Prosaposin overexpression following kainic acid-induced neurotoxicity.

Because excessive glutamate release is believed to play a pivotal role in numerous neuropathological disorders, such as ischemia or seizure, we aimed to investigate whether intrinsic prosaposin (PS), a neuroprotective factor when supplied exogenously in vivo or in vitro, is up-regulated after the excitotoxicity induced by kainic acid (KA), a glutamate analog. In the present study, PS immunoreactivity and its mRNA expression in the hippocampal and cortical neurons showed significant increases on day 3 after KA injection, and high PS levels were maintained even after 3 weeks. The increase in PS, but not saposins, detected by immunoblot analysis suggests that the increase in PS-like immunoreactivity after KA injection was not due to an increase in saposins as lysosomal enzymes after neuronal damage, but rather to an increase in PS as a neurotrophic factor to improve neuronal survival. Furthermore, several neurons with slender nuclei inside/outside of the pyramidal layer showed more intense PS mRNA expression than other pyramidal neurons. Based on the results from double immunostaining using anti-PS and anti-GABA antibodies, these neurons were shown to be GABAergic interneurons in the extra- and intra-pyramidal layers. In the cerebral cortex, several large neurons in the V layer showed very intense PS mRNA expression 3 days after KA injection. The choroid plexus showed intense PS mRNA expression even in the normal rat, and the intensity increased significantly after KA injection. The present study indicates that inhibitory interneurons as well as stimulated hippocampal pyramidal and cortical neurons synthesize PS for neuronal survival, and the choroid plexus is highly activated to synthesize PS, which may prevent neurons from excitotoxic neuronal damage. To the best of our knowledge, this is the first study that demonstrates axonal transport and increased production of neurotrophic factor PS after KA injection.