Characterization and ontogeny of synapse-associated proteins in the developing facial and hypoglossal motor nuclei of the Brazilian opossum

The characterization and ontogeny of synapse-associated proteins in the developing facial and hypoglossal motor nuclei were examined in the Brazilian opossum (Monodelphis domestica). Immunohistochemical markers utilized in this study were the synaptic vesicle-associated proteins synaptophysin and synaptotagmin; a synaptic membrane protein, plasma membrane-associated protein of 25 kDa (SNAP-25); a growth cone protein, growth-associated phosphoprotein-43 (GAP-43); and the microtubule-associated proteins axonal marker τ and dendritic marker microtubule-associated protein-2 (MAP-2). In this study, we have found that during the first 10 postnatal days (1–10 PN), the facial motor nucleus lacked immunoreactivity for synaptophysin, synaptotagmin, GAP-43, τ, and SNAP-25. After 10 PN, immunoreactivity increased in the facial motor nucleus for synaptophysin, synaptotagmin, GAP-43, and τ, whereas immunoreactivity for SNAP-25 was not evident until between 15 and 25 PN. Conversely, immunoreactivity for MAP-2, was present in the facial motor nucleus from the day of birth. In contrast, the hypoglossal motor nucleus displayed immunoreactivity from 1 PN for synaptophysin, synaptotagmin, SNAP-25, GAP-43, τ, and MAP-2. These results suggest that the facial motor nucleus of the opossum may not receive afferent innervation as defined by classical synaptic markers until 15 PN and, further, that characteristic mature synapses are not present until between 15 and 25 PN. These results indicate that there may be a delay in synaptogenesis in the facial motor nucleus compared to synaptogenetic events in the hypoglossal motor nucleus. Because the facial motor nucleus is active prior to completion of synaptogenesis, we suggest that the facial motoneurons are regulated in a novel or distinct manner during this time period. © 1996 Wiley-Liss, Inc.     

Phospholipid-mediated delivery of anti-GAP-43 antibodies into neuroblastoma cells prevents neuritogenesis.

The neuronal growth-associated protein GAP-43 is expressed during axonal outgrowth and regeneration (for review, see Benowitz and Routtenberg, 1987). In the present study, we demonstrate that GAP-43 is constitutively expressed by NB2a/d1 neuroblastoma cells. The initial, most rapid outgrowth period of neuritogenesis [0-4 hr after dibutyryl adenosine 3',5'-cyclic monophosphate (dbcAMP) treatment] is accompanied by intense GAP-43 immunoreactivity along the entire length of most neurites. However, this immunoreactivity declined nearly to background levels within hours during continued neurite outgrowth and persisted only at varicosities and growth cones. GAP-43 was detectable by metabolic labeling and immunoblot analysis in undifferentiated cells, and synthetic rates and steady-state levels of GAP-43 underwent only a modest (approximately twofold) increase during dbcAMP-induced differentiation. Unlike levels observed in neurites, perikarya of undifferentiated and differentiated cells contained similar, intense levels of GAP-43 immunoreactivity. Neurite elaboration and GAP-43 immunoreactivity were unaffected by treatment with cycloheximide, suggesting that translocation of perikaryal GAP-43 pools, rather than de novo synthesis, contributes to the transient burst of GAP-43 observed in developing neurites. Phosphatidylcholine-mediated delivery of anti-GAP-43 antibodies (alpha GAP) into cells immediately before dbcAMP treatment arrested neuritogenesis but did not induce the retraction of existing neurites. These results indicate that, while GAP-43 expression is insufficient to induce neuritogenesis in NB2a/d1 cells, GAP-43 is nevertheless essential for the initial, dynamic phase of neurite outgrowth.     

Acute sublethal global hypoxia induces transient increase of GAP-43 immunoreactivity in the striatum of neonatal rats

We assessed immunoreactivity (IR) in the cerebral cortex (CC), hippocampus (Hipp), and striatum (ST) of a growth-associated protein, GAP-43, and of proteins of the synaptic vesicle fusion complex: VAMP-2, Syntaxin-1, and SNAP-25 (SNARE proteins) throughout postnatal development of rats after submitting the animals to acute global postnatal hypoxia (6.5% O(2), 70 min) at postnatal day 4 (PND4). In the CC only the IR of the SNARE protein SNAP-25 increased significantly with age. The hypoxic animals showed the same pattern of IR for SNAP-25, although with lower levels at PND11, and also a significant increase of VAMP-2. SNAP-25 (control): PND11 P < 0.001 vs. PND18, 25, and 40, SNAP-25 (hypoxic): P < 0.001 vs. PND18, 25, and 40; VAMP-2 (hypoxic): P < 0.05 PND11 vs. PND18, and P < 0.01 vs. PND25 and PND40; one-way ANOVA and Bonferroni post-test. In the Hipp, SNAP-25 and syntaxin-1 increased significantly with age, reaching a plateau at PND25 through PND40 in control animals (one-way ANOVA: syntaxin-1: P = 0.043; Bonferroni: NS; SNAP-25: P = 0.013; Bonferroni: P < 0.01 PND11 vs. PND40). Hypoxic rats showed higher levels of significance in the one-way ANOVA than controls (syntaxin-1: P = 0.009; Bonferroni: P < 0.05 PND11 vs. PND25 and P < 0.001 PND11 vs. PND40). In the ST, GAP-43 differed significantly among hypoxic and control animals and the two-way ANOVA revealed significant differences with age (F = 3.23; P = 0.037) and treatment (F = 4.84; P = 0.036). VAMP-2 expression also reached statistical significance when comparing control and treated animals (F = 6.25, P = 0.018) without changes regarding to age. Elevated plus maze test performed at PND40 indicated a lower level of anxiety in the hypoxic animals. At adulthood (12 weeks) learning, memory and locomotor abilities were identical in both groups of animals. With these results, we demonstrate that proteins of the presynaptic structures of the ST are sensitive to acute disruption of homeostatic conditions, such as a temporary decrease of the O(2) concentration. Modifications in the activity of these proteins could contribute to the long term altered responses to stress due to acute hypoxic insult in the neonatal period.     

Rapid induction by kainic acid of both axonal growth and F1/GAP-43 protein in the adult rat hippocampal granule cells

Hippocampal granule cells do not normally express the axonal growth and plasticity-associated protein F1/GAP-43 in the adult rat. Using three different methods that lead to hypersynchronous activity in limbic circuits, expression of F1/GAP-43 mRNA can be induced in granule cells which is followed by sprouting in mossy fibers, the axons of granule cells. F1/GAP-43 mRNA expression in granule cells was induced in the temporal, but not septal, hippocampus beginning at 12 hours after kainic acid (KA) subcutaneous injection (10 mg/kg). Beginning 2 days after KA treatment, mossy fiber sprouts restricted to the temporal hippocampus were observed in the supragranular layer. In the same animal we also observed that levels of protein F1/GAP-43 immunoreactivity in this layer apparently increased at this same 2 day time point and same ventral hippocampal location. F1/GAP-43 protein levels and mossy fiber sprouting showed an increase up to 10 days after KA treatment. Sprouting was at a maximum at 40 days, the longest time point studied. These events parallel axonal regeneration with one critical difference: granule cell axons are not damaged by kainate. The rapid onset of axonal growth in the adult is striking and occurs earlier than reported previously (2 days vs. 12 days). Such growth closely associated with elevated levels of protein F1/GAP-43 may occur as a result of a) reactive synaptogenesis caused by the availability of post-synaptic surface on granule cell dendrites at the supragranular layer, b) Hebbian co-activation of the post-synaptic granule cells and their presynaptic afferents, and c) loss of target-derived inhibitory growth factor. © 1996 Wiley-Liss, Inc.     

Chronological alterations of neurofilament 150 immunoreactivity in the gerbil hippocampus and dentate gyrus after transient forebrain ischemia

In this study, we observed the chronological alterations of neurofilament 150 (NF-150) immunoreactivity in the gerbil hippocampus and dentate gyrus after 5 min transient forebrain ischemia. NF-150 immunoreactivity in the sham-operated group was mainly detected in mossy fibers and in the hilar region of the dentate gyrus. NF-150 immunoreactivity and protein contents of NF-150 and RT 97 (polyphosphorylation epitopes of neurofilament) were significantly decreased at 15 min after ischemic insult. Between 30 min and 12 h after ischemic insult, NF-150 immunoreactivity and protein content were significantly increased as compared with the sham-operated group. Thereafter, NF-150 immunoreactivity and protein content started to decrease. At 12 h after ischemic insult, unlike dentate gyrus, NF-150 immunoreactivity increased in pyramidal cells of the CA1 region. Thereafter, NF-150 immunoreactivity in the CA1 region started to decrease, and 4 days after ischemic insult, NF-150 immunoreactivity nearly was similar to that of the sham-operated group. These biphasic patterns of NF-150 immunoreactivity in the hippocampus and dentate gyrus are reverse correlated with that of the intracellular calcium influx. For calcium detection in the CA1 region, we also conducted alizarin red staining. Alizarin red positive neurons were detected in some neurons at 15-30 min after ischemic insult. At 12 h after ischemia, alizarin red positive neurons were decreased. Thereafter, alizarin red positive neurons started to decrease, but alizarin positive neurons were significantly increased in dying neurons 4 days after ischemia. These results suggest that ischemia-related changes of NF-150 expression may be caused by the calcium following transient forebrain ischemia.     

Transient increase of synapsin-I immunoreactivity in the mossy fiber layer of the hippocampus after transient forebrain ischemia in the mongolian gerbil

Synapsin-I is a vesicular phosphoprotein, which regulates neurotransmitter release, neurite development, and maturation of synaptic contacts during normal development and following various brain lesions in adulthood. In the present study, we have examined by immunohistochemistry possible modifications in the expression of synapsin-I in the hippocampus of Mongolian gerbils after transient forebrain ischemia. The animals were subjected to 5 min of transient forebrain ischemia through bilateral common carotid occlusion, and were examined at different time-points post-ischemia. Transient forebrain ischemia produces cell death of the majority of CA1 pyramidal neurons of the hippocampus and polymorphic hilar neurons of the dentate gyrus. This is followed by reactive changes, including synaptic reorganization and modifications in the expression of synaptic proteins, which provide the molecular bases of synaptic plasticity. Transient decrease of synapsin-I immunoreactivity was observed in the inner zone of the molecular layer of the dentate gyrus, thus suggesting denervation and posterior reinervation in this area. In addition, a strong increase in synapsin-I immunoreactivity was observed in the hilus of the dentate gyrus and in the mossy fiber layer of the hippocampus at 2, 4 and 7 days after ischemia. Parallel increases in synaptophysin immunoreactivity were not observed, thus suggesting a selective induction of synapsin-I after ischemia. The present results indicate that synapsin-I participates in the reactive response of granule cells to transient forebrain ischemia in the hippocampus of the gerbil, and suggest a role for this protein in the plastic adaptations of the hippocampus following injury.     

Up-regulation of fast-axonally transported proteins in retinal ganglion cells of adult rats with optic–peroneal nerve grafts

Metabolic labeling and quantitative 2D gel fluorography were used to assess changes in the synthesis and transport of five fast-axonally transported and developmentally regulated proteins (GAP-43, SNAP-25, and proteins of 18, 22, and 23/24 kDa) after grafting of a peroneal nerve segment onto a transected optic nerve in adult rats. After optic nerve transection alone, only GAP-43 was up-regulated significantly compared to normal adult controls. The other proteins showed little change or were down-regulated following axotomy. By 4 weeks following optic nerve transection and peroneal nerve grafting, however, GAP-43, proteins 22 and 23/24 kDa showed a sustained up-regulation in synthesis and transport compared to normal controls; SNAP-25 and protein 18 kDa showed levels of expression similar to or slightly greater than normal controls. Increased expression of GAP-43 in retinal ganglion cells was also examined with immunocytochemistry. While a transient up-regulation of GAP-43 in retinal ganglion cells was observed following optic nerve transection, a sustained increase in GAP-43 immunoreactivity was present only in animals with nerve grafts. Backfilling of retinal ganglion cells from the grafts with horseradish peroxidase combined with GAP-43 immunocytochemistry revealed that all retinal ganglion cells with axons growing into the grafts were positive for GAP-43, but not all retinal ganglion cells showing GAP-43 immunoreactivity were extending axons into the grafts. We conclude that the presence of a nerve graft sustains the up-regulation of a number of proteins including GAP-43, and that this up-regulation is correlated with an increased potential for nerve growth, but other as yet unknown factors or conditions appear to play a role in determining if this growth potential will be realized.     

Expression of GAP-43 in the Granule Cells of Rat Hippocampus After Seizure-induced Sprouting of Mossy Fibres: In Situ Hybridization and Immunocytochemical Studies

The axonal growth-associated protein GAP-43 is believed to play some role in the synaptic remodelling that takes place in the hippocampus of adult rats after certain experimental lesions. GAP-43 mRNA is highly expressed in adult CA3 pyramidal cells but almost absent in the dentate granule cells. We analysed whether the sprouting of granule cell axons, the mossy fibres of the hippocampus, caused by kainic acid-induced seizures in adult rats was associated with any induction of GAP-43 mRNA in granule cells and with any changes in the immunostaining pattern of GAP-43 in the hippocampus. Increased GAP-43 mRNA expression was found to be induced in granule cells 18, 24 and 30 h after a systemic injection of kainic acid which induced generalized seizures in adult rats, and returned to control levels by 48 h post-treatment. No effect was observed in other regions of the hippocampus. However, when kainic acid was injected into 15-day-old rats, which responded with generalized seizures but no sprouting of mossy fibres, there was no induction of GAP-43 mRNA in the granule cells, suggesting a close relation between GAP-43 expression and sprouting of these cells. Seven days after kainic acid injections, GAP-43 immunostaining was decreased in the inner molecular layer of the dentate gyrus except for a thin supragranular band, whereas 30 days after treatment all animals showed increased GAP-43 immunoreactivity in the whole inner molecular layer. Since collaterals of mossy fibres grow in the inner molecular layer after kainic acid-induced seizures, these results support the theory that GAP-43 plays a role in synaptic remodelling in the adult central nervous system.     

Tyrosine kinase A but not phosphacan/protein tyrosine phosphatase-zeta/beta immunoreactivity and protein level changes in neurons and astrocytes in the gerbil hippocampus proper after transient forebrain ischemia

In the present study, ischemia-related changes in tyrosine kinase A (trkA) and phosphacan/protein tyrosine phosphatase-zeta/beta (PTP-zeta/beta) immunoreactivities and protein contents were examined in the hippocampus proper after transient forebrain ischemia for 5 min in a gerbil model. Our investigations showed that ischemia-induced changes occurred in trkA immunoreactivity in the hippocampal CA1 region, but not in the CA2/3 region of the hippocampus proper. In the sham-operated group, trkA immunoreactivity was barely detectable. trkA immunoreactivity increased from 30 min after ischemia and peaked at 12 h. Four days after ischemic insult, trkA immunoreactivity was observed in GFAP-immunoreactive astrocytes in the strata oriens and radiatum. In addition, we found that ischemia-related changes in trkA protein content were similar to immunohistochemical changes. On the other hand, PTP-zeta/beta immunoreactivities in the hippocampus proper were unaltered by forebrain ischemia. These results suggest that chronological changes of trkA after transient forebrain ischemia may be associated with an ischemic damage compensatory mechanism in CA1 pyramidal cells.     

Chronological changes of neurofilament 200 kDa immunoreactivity in the lateral olfactory tract after transient forebrain ischemia in gerbils

This study was carried out to investigate the transient ischemia-induced changes of neurofilament 200 kDa (NF-200) immunoreactivity and protein content in the gerbil lateral olfactory tract (LOT) after 5 min of transient forebrain ischemia. Weak NF-200 immunoreactivity was detectable in the LOT in the sham-operated group. In this group, a few somata of mitral cells showed weak NF-200 immunoreactivity. One day after transient ischemia, NF-200 immunoreactivity in the LOT was increased significantly. NF-200 immunoreactivity in the LOT by 15 days after ischemia was similar to that in the 1 day post-ischemic group. In this time period, strong NF-200 immunoreactivity was expressed in the mitral cell processes, but the immunoreactivity in the mitral cell somata was significantly decreased. Thereafter, NF-200 immunoreactivity in the LOT was decreased significantly by 30 days after ischemic insult. At this time after ischemia, NF-200 immunoreactivity in the mitral cell dendrites was significantly decreased. The result of Western blot study showed that the pattern of NF-200 expression was similar to that of immunohistochemistry after ischemia-reperfusion. Our result suggests that changes of NF-200 protein in the gerbil LOT may be related to response to ischemic damage and that the axonal transport followed transient ischemia may be disturbed.     

Expression of synaptic proteins in the developing rat cerebellum following ionizing radiation

Various proteins regulating neurotransmission release and synaptic vesicle exocytosis have been implicated in axonal elongation and synaptic maturation. In the present study, immunohistochemistry to the presynaptic membrane proteins syntaxin-I and synaptosomal-associated protein of 25 kDa (SNAP-25) synaptic vesicle-associated proteins synaptophysin and synapsin-I and the neuronal maturation and axonal growth-related protein GAP-43, has been carried out in the normal developing cerebellum and following a single dose of ionizing radiation (2 Gy gamma-rays) at postnatal day 1. Our aim has been to learn about the morphological and possible functional modalities that occur during the progression of neuronal connectivity in normal and abnormal development. Expression of all these proteins is associated with the arrival of afferents in the subcortical white matter and with the maturation of the internal granule cell layer and molecular layer during normal development. In addition, SNAP-25 and GAP-43 are strongly expressed in granule cells of the external granule cell layer, thus suggesting that these proteins are involved in cell elongation of granule cells. Apoptosis appears at 3 h and peaks at 6 h following ionizing radiation. Radiation-induced apoptosis in the external granule cell layer produces a transient decrease in the expression of SNAP-25 and GAP-43 in the external granule cell layer. The external granule cell layer recovers at 48 h and external granule cells of proliferating cells also express SNAP-25 and GAP-43, thus indicating that proliferating cells in this layer are equipped with proteins involved in cell elongation. Furthermore, expression of synaptophysin, synapsin-I, syntaxin-I and SNAP-25 is the same in the cerebellum of irradiated and normal rats from this time to adulthood (3 months). These results point to the likelihood that recovery of the cerebellar cortex occurs following a single exposure of ionizing radiation during postnatal development.     

Developmental and plasticity-related differential expression of two SNAP-25 isoforms in the rat brain

In this article we study the relationship between the expression pattern of two recently identified isoforms of the 25-kD synaptosomal-associated protein (SNAP-25a and SNAP-25b) and the morphological changes inherent to neuronal plasticity during development and kainic acid treatment. SNAP-25 has been involved in vescicle fusion in the nerve terminal, and most likely participates in different membrane fusion-related processes, such as those involved in neurotransmitter release and axonal growth. In the adult brain, SNAP-25b expression exceeded SNAP-25a in distribution and intensity, being present in most brain structures. Moderate or high levels of SNAP-25a hybridization signal were found in neurons of the olfactory bulb, the layer Va of the frontal and parietal cortices, the piriform cortex, the subiculum and the hippocampal CA4 field, the substantia nigra/pars compacta, and the pineal gland, partially overlapping SNAP-25b mRNA distribution. In restricted regions of cerebral cortex, thalamus, mammillary bodies, substantia nigra, and pineal glands the two isoforms were distributed in reciprocal fashion. During development SNAP-25a mRNA was the predominant isoform, whereas SNAP-25b expression increased postnatally. The early expression of SNAP-25a in the embryo and the decrease after P21 is suggestive of a potential involvement of this isoform in axonal growth and/or synaptogenesis. This conclusion is indirectly supported by the observation that SNAP-25a mRNA, but not SNAP-25b mRNA, was upregulated in the granule cells of the adult dentate gyrus 48 hours after kainate-induced neurotoxic damage of the hippocampal CA3-CA4 regions. Increase of SNAP-25 immunoreactivity was observed as early as 4 days after kainate injection within the mossy fiber terminals of the CA3 region, and in the newly formed mossy fiber aberrant terminals of the supragranular layer. These data suggest an isoform-specific role of SNAP-25 in neural plasticity. © 1996 Wiley-Liss, Inc.     

Transient ischemia-induced expression and changes of tyrosine kinase A in the hippocampal dentate gyrus of the gerbil

The present study examined ischemia-related changes in tyrosine kinase A (trkA) immunoreactivity and its protein content in the dentate gyrus after 5 min of transient forebrain ischemia in gerbils. One day after ischemic insult, cresyl violet-positive polymorphic cells showed ischemic degeneration. The ischemia-induced changes in trkA immunoreactivity were found in the polymorphic layer (PL) and granule cell layer (GCL) of the dentate gyrus. In the sham-operated group, trkA immunoreactivity in the dentate gyrus was very weak. From 30 min after ischemia, trkA immunoreactivity was increased in the dentate gyrus and peaked in the dentate gyrus at 12 h after ischemia-reperfusion. Thereafter, trkA immunoreactivity was decreased time-dependently after ischemia-reperfusion. Four days after ischemic insult, trkA immunoreactivity was similar to that of the sham-operated group. In addition, it was found that ischemia-related changes in trkA protein content were similar to the immunohistochemical changes. These results suggest that the chronological changes of trkA in the dentate gyrus after transient forebrain ischemia may be associated with ischemic damage in polymorphic cells of the dentate gyrus.     

Growth-associated protein-43 (GAP-43) in the regenerating periodontal Ruffini endings of the rat incisor following injury to the inferior alveolar nerve

Alterations in the levels of growth-associated protein 43 (GAP-43)-like immunoreactivity (-LI) were examined in the lingual periodontal ligament of the rat incisor following two types of injury (resection and crush) to the inferior alveolar nerve (IAN). In normal animals, GAP-43-like immunoreactive (IR) structures were observed as tree-like ramifications in the alveolar half of the lingual periodontal ligament of incisors. Under immunoelectron microscopy, GAP-43-LI appeared in the Schwann sheaths associated with periodontal Ruffini endings; neither cell bodies of the terminal Schwann cells nor axonal profiles showed GAP-43-LI. During regeneration of the periodontal Ruffini endings following resection of the IAN, GAP-43-LI appeared in the cytoplasm of the terminal Schwann cell bodies and axoplasm of the terminals. The distribution of GAP-43-LI in the Ruffini endings returned to almost normal levels on days 28 and 56 following the injury. The changes in the distribution of GAP-43-LI following the crush injury were similar to those following resection; however, expression of GAP-43-LI was slightly higher for the entire experimental period compared with the resection. The transient expression of GAP-43 in the terminal Schwann cells and axonal profiles of the periodontal Ruffini endings following nerve injury suggests that GAP-43 is closely associated with axon–Schwann cells interactions during regeneration.     

Antioxidant-like protein 1 is altered in non-pyramidal cells and expressed in astrocytes in the gerbil hippocampal CA1 region after transient forebrain ischemia

In the present study, we observed chronological changes of antioxidant-like protein 1 (AOP-1) in the gerbil hippocampal CA1 region after 5 min of transient forebrain ischemia using immunohistochemistry and western blot. AOP-1 was significantly altered in the CA1 region after transient ischemia. In the sham-operated group, AOP-1 immunoreactivity was detected in pyramidal and non-pyramidal cells of the CA1 region. At 30 min after ischemic insult, AOP-1 immunoreactivity and protein level was decreased in the CA1 region. At 12 h after ischemic insult, AOP-1 immunoreactivity and protein level was highest in this region. At this time, after ischemia, AOP-1 immunoreactivity in non-pyramidal cells was high compared to the sham-operated group. Based on double immunofluorescence study, AOP-1-immunoreactive neurons were identified as GABAergic, which were stained with GAD or parvalbumin. Thereafter, AOP-1 immunoreactivity and protein levels were decreased time-dependently. From 4 days after ischemic insult, AOP 1 immunoreactivity was generally expressed in astrocytes. Five days after ischemic insult, AOP-1 immunoreactivity and protein level was increased again to 1.4 folds compared to that of the sham-operated group. In brief, AOP-1 immunoreactivity was increased in GABAergic non-pyramidal cells in the hippocampal CA1 region at early time after ischemic insult and was expressed in astrocytes at late time after ischemia. This result suggests that AOP-1 may be important role in homeostasis of GABAergic neurons because these neurons are resistant to ischemic damage.     

Changes in immunoreactivity of HSP60 and its neuroprotective effects in the gerbil hippocampal CA1 region induced by transient ischemia

Heat shock proteins act as molecular chaperones and are involved in protein folding, refolding, transport, and translocation. In the present study, we observed changes in heat shock protein 60 (HSP60) immunoreactivity and protein level in the gerbil hippocampal CA1 region after 5 min of transient forebrain ischemia and its neuroprotective effect against ischemic damage. HSP60 immunoreactivity in the CA1 region began to increase in the stratum pyramidale at 30 min after ischemia/reperfusion, and peaked 24 h after ischemia/reperfusion. Thereafter, HSP60 immunoreactivity was decreased in the CA1 region with time. Seven days after ischemia/reperfusion, HSP60 immunoreactivity was increased again in the CA1 region: at this time point after ischemia/reperfusion, HSP60 immunoreactivity was expressed in glial cells in the ischemic CA1 region. HSP60 immunoreactive glial cells were astrocytes containing glial fibrillar acidic protein. In contrast, change in HSP60 immunoreactivity in the ischemic CA2/3 region was not significant compared with that in the ischemic CA1 region. In Western blot study, HSP60 protein level in the CA1 region was increased after ischemia/reperfusion and highest 24 h after ischemia/reperfusion. Animals treated with recombinant adenoviruses expressing Hsp60 (Ad-Hsp60) showed the neuroprotection of CA1 pyramidal neurons from ischemic damage. These results suggest that HSP60 may be associated with delayed neuronal death of CA1 pyramidal neurons after transient ischemia, and the induction of HSP60 protects the neurons from ischemic damage.     

Expression of exocytosis proteins in rat supraoptic nucleus neurones

In magnocellular neurones of the supraoptic nucleus (SON), the neuropeptides vasopressin and oxytocin are synthesised and packaged into large dense-cored vesicles (LDCVs). These vesicles undergo regulated exocytosis from nerve terminals in the posterior pituitary gland and from somata/dendrites in the SON. Regulated exocytosis of LDCVs is considered to involve the soluble N-ethylmaleimide sensitive fusion protein attachment protein receptor (SNARE) complex [comprising vesicle associated membrane protein 2 (VAMP-2), syntaxin-1 and soluble N-ethylmaleimide attachment protein-25 (SNAP-25)] and regulatory proteins [such as synaptotagmin-1, munc-18 and Ca(2+) -dependent activator protein for secretion (CAPS-1)]. Using fluorescent immunocytochemistry and confocal microscopy, in both oxytocin and vasopressin neurones, we observed VAMP-2, SNAP-25 and syntaxin-1-immunoreactivity in axon terminals. The somata and dendrites contained syntaxin-1 and other regulatory exocytosis proteins, including munc-18 and CAPS-1. However, the distribution of VAMP-2 and synaptotagmin-1 in the SON was limited to putative pre-synaptic contacts because they co-localised with synaptophysin (synaptic vesicle marker) and had no co-localisation with either oxytocin or vasopressin. SNAP-25 immunoreactivity in the SON was limited to glial cell processes and was not detected in oxytocin or vasopressin somata/dendrites. The present results indicate differences in the expression and localisation of exocytosis proteins between the axon terminals and somata/dendritic compartment. The absence of VAMP-2 and SNAP-25 immunoreactivity from the somata/dendrites suggests that there might be different SNARE protein isoforms expressed in these compartments. Alternatively, exocytosis of LDCVs from somata/dendrites may use a different mechanism from that described by the SNARE complex theory.     

Changes in presynaptic proteins, SNAP-25 and synaptophysin, in the hippocampal CA1 area in ischemic gerbils

A general consensus exists that the presynaptic terminals in the hippocampal CA1 area are resistant to ischemic stress in spite of the loss of their target cells (CA1 pyramidal neurons). We have verified this by immunostaining and Western immunoblotting using the antibodies for presynaptic proteins, synaptosomal-associated protein of 25 kDa (SNAP-25) and synaptophysin in gerbils after bilateral carotid artery ligature. In the immunohistochemical analysis, decreases in SNAP-25 and synaptophysin immunoreactivities in the strata radiatum and oriens, especially around the apical dendrite of CA1 neurons, and disappearance of SNAP-25 immunoreactivity in the alveus were observed on day 2 after ischemia. On days 7 and 14, SNAP-25-positive granular materials were expressed in the CA1 area, and intense synaptophysin immunoreactivity around surviving CA1 neurons was observed. Western immunoblot analysis revealed significant decreases of SNAP-25 and synaptophysin (about 60% of control levels) on day 2, and then increase of their proteins (130–140% of control levels) on day 14. These results indicate that presynaptic degeneration occurs in the hippocampal CA1 area after ischemia, and it precedes the delayed neuronal death of CA1 neurons. The presynaptic terminal damage may be responsible for some pathological changes in ischemic brains.     

GAP-43 mRNA and protein expression in the hippocampal and parahippocampal region during the course of epileptogenesis in rats

In order to reveal axonal rewiring in the hippocampal and parahippocampal regions after status epilepticus, we investigated the temporal evolution of growth-associated protein-43 (GAP-43) mRNA and protein expression in two rat models of mesial temporal lobe epilepsy (MTLE). Status epilepticus (SE) was induced by electrical stimulation of the angular bundle or by intraperitoneal kainic acid (KA) injections. Despite increased GAP-43 mRNA expression in dentate granule cells at 24 h after SE, GAP-43 protein expression in the inner molecular layer (IML) of the dentate gyrus decreased progressively after 24 h after SE in both models. Nevertheless robust mossy fiber sprouting (MFS) was evident in the IML of chronic epileptic rats. Remaining GAP-43 protein expression in the IML in chronic epileptic rats did not correlate with the extent of MFS, but with the number of surviving hilar neurons. In the parahippocampal region, GAP-43 mRNA expression was decreased in layer III of the medial entorhinal area (MEAIII) in parallel with extensive neuronal loss in this layer. There was a tendency of GAP-43 mRNA up-regulation in the presubiculum, a region that projects to MEAIII. With regard to this parahippocampal region, however, changes in GAP-43 mRNA expression were not followed by protein changes. The presence of the presynaptic protein GAP-43 in a neurodegenerated MEAIII indicates that fibers still project to this layer. Whether reorganization of fibers has occurred in this region after SE needs to be investigated with tools other than GAP-43.     

Postnatal maternal separation elevates the expression of the postsynaptic protein kinase C substrate RC3, but not presynaptic GAP-43, in the developing rat hippocampus

We have shown that exposure of rats to neonatal handling/maternal separation results in mossy fiber axon hypoplasia in field CA3 of the hippocampus. To better understand the molecular basis of this neuroanatomical alteration, the present study examined three developmentally regulated protein kinase C substrate mRNAs that are highly expressed in hippocampal granule cells during mossy fiber outgrowth: GAP-43, a presynaptic substrate implicated in axonal outgrowth, RC3 (neurogranin), a postsynaptic substrate implicated in calmodulin signaling, and MARCKS-like protein (MLP), which binds calmodulin and filamentous actin in neurons and glial cells. mRNA expression was examined by quantitative in situ hybridization in the developing [postnatal day 7 (P7), P13, P21, and P90] hippocampus (CA1, CA3, granule cells) in Long-Evans hooded rats: (1) reared under normal animal facility (AFR) conditions, (2) subjected to brief (15 min/day, HMS15), or (3) subjected to moderate (180 min/day) handling/maternal separation (HMS180) on P2-14. RC3 mRNA expression was consistently elevated in all of the hippocampal cell fields in HMS180 rats relative to HMS15 and/or AFR rats over postnatal development, but did not differ from HMS15 rats in adulthood. In contrast, neither GAP-43 mRNA nor MLP mRNA expression differed among AFR, HMS15, or HMS180 rats at any postnatal time point. Elevations in RC3 expression would be predicted to perturb calcium-calmodulin signaling that may, in turn, impair the formation and/or maintenance of mossy fiber-CA3 synapses during postnatal development.