Compartments of the crural fascia: clinically relevant ultrasound, anatomical and histological findings.

PURPOSE: Compartment syndrome is a surgical emergency that can occur in any part of the body and can cause cell necrosis when maintained over time. The resulting defects can affect the nerves, muscle cells, bone tissue, and other connective tissues inside the compartment, and fasciotomy has to be performed. The anatomical and histological characteristics of the leg make acute, chronic, and exertional compartment syndrome more likely in this limb. For these reasons, knowledge of the ultrasound, anatomical, and histological features of the crural fascia can help in the treatment of leg compartment syndrome. METHODS: Twenty-one cryopreserved lower limbs from adult cadavers and from one 29-week-old fetus were obtained from the dissection room. They were examined by ultrasound and a subsequent anatomical dissection and microscopy to study the crural fascia and its relationship with the different muscles. Anthropometric measurements were taken of the distances from the head of the fibula and lateral malleolus to the origin of the tibialis anterior muscle in the crural fascia, the exit of the superficial fibular nerve, and the fascia covering the deep posterior muscles of the leg. RESULTS: The crural fascia has very important clinical relationships, which can be identified by ultrasound, as the origin of the tibialis anterior muscle at 16.25 cm from the head of the fibula and the exit of the superficial fibular nerve that crosses this fascia at 21.25 cm from the head of the fibula. Furthermore, the presence of a septum that fixes the deep posterior muscles of the leg and the vessels and nerve can be seen by ultrasound and can explain the possible development of a posterior compartmental syndrome of the leg. Awareness of these features will help to keep these structures safe during the surgical treatment of compartment syndrome. CONCLUSION: The ultrasound study allows identification of anatomical structures in the leg and, thus, avoids damage to them during surgery for compartmental syndromes.

Compartments of the antebrachial fascia of the forearm: clinically relevant ultrasound, anatomical and histological findings.

PURPOSE: Acute compartment syndrome is defined as a limb-threatening condition caused by bleeding or oedema in a closed muscle compartment surrounded by fascia or bone. It is most commonly encountered in the forearm, which has three compartments: posterior, anterior and lateral. These are surrounded and closed in by the antebrachial fascia, formed by dense connective tissue that facilities their study on ultrasound and is key to fasciotomy treatment. The purpose of this study was to broaden existing ultrasound, anatomical and histological knowledge of the fascia of the forearm to facilitate their identification on ultrasound, with possible clinical and therapeutic applications. METHODS: The study was performed in 50 cryopreserved upper limbs from adult cadavers from the dissection room of the Faculty of Medicine and Health Sciences. They were examined on ultrasound and subsequent anatomical dissection and microscopy to study the fascia and its relationship with different muscles of the forearm compartments. RESULTS: Distinct anatomical relationships were observed on ultrasound and dissection between the fascia and the extensor carpi ulnaris, extensor digiti minimi, and anconeus muscle in the posterior compartment, and the flexor carpi radialis and flexor carpi ulnaris in the anterior compartment. They were isolated by the antebrachial fascia and had distinct relationships with the neurovascular structures. CONCLUSION: These results demonstrate that high-definition ultrasound enables us to locate the antebrachial fascia and particular muscles with a distinct relationship with neurovascular structures. This helps better identify these structures, facilitating diagnosis of any pathology in the area, with potential therapeutic and clinical applications.

CD34+ circulating progenitor cells after different training programs.

Circulating progenitor cells (CPC) are bone marrow-derived cells that are mobilized into the circulation. While exercise is a powerful mediator of hematopoiesis, CPC levels increase, and reports of their activation after different types of exercise are contradictory. Moreover, few studies have compared the possible effects of different training programs on CPC concentrations. 43 physically active healthy male subjects (age 22±2.4 years) were assigned to 4 different training groups: aerobic, resistance, mixed and control. Except for the control group, all participants trained for 6 weeks. Peripheral blood samples were collected through an antecubital vein, and CPC CD34(+) was analyzed on different days: pre-training, post-training, and 3 weeks after finishing the training period. While no significant differences in CPC were observed either within or between the different training groups, there was a tendency towards higher values post-training and large intra- and intergroup dispersion. We detected an inverse linear relationship between pre-training values and % of CPC changes post-training (p<0.001). In the CPC values 3 weeks after training this inverse relationship was maintained, though to a lower extent (p<0.001). No changes in CPC CD34(+) were detected after 6 weeks of different training groups, or after 3 weeks of follow-up.

The homeobox gene mirror links EGF signalling to embryonic dorso-ventral axis formation through notch activation.

Recent studies in vertebrates and Drosophila melanogaster have revealed that Fringe-mediated activation of the Notch pathway has a role in patterning cell layers during organogenesis. In these processes, a homeobox-containing transcription factor is responsible for spatially regulating fringe (fng) expression and thus directing activation of the Notch pathway along the fng expression border. Here we show that this may be a general mechanism for patterning epithelial cell layers. At three stages in Drosophila oogenesis, mirror (mirr) and fng have complementary expression patterns in the follicle-cell epithelial layer, and at all three stages loss of mirr enlarges, and ectopic expression of mirr restricts, fng expression, with consequences for follicle-cell patterning. These morphological changes are similar to those caused by Notch mutations. Ectopic expression of mirr in the posterior follicle cells induces a stripe of rhomboid (rho) expression and represses pipe (pip), a gene with a role in the establishment of the dorsal-ventral axis, at a distance. Ectopic Notch activation has a similar long-range effect on pip. Our results suggest that Mirror and Notch induce secretion of diffusible morphogens and we have identified TGF-beta (encoded by dpp) as such a molecule in germarium. We also found that mirr expression in dorsal follicle cells is induced by the EGF-receptor (EGFR) pathway and that mirr then represses pip expression in all but the ventral follicle cells, connecting EGFR activation in the dorsal follicle cells to repression of pip in the dorsal and lateral follicle cells. Our results suggest that the differentiation of ventral follicle cells is not a direct consequence of germline signalling, but depends on long-range signals from dorsal follicle cells, and provide a link between early and late events in Drosophila embryonic dorsal-ventral axis formation.

Clostridial neurotoxins: new tools for dissecting exocytosis.

Tetanus toxin and botulinal toxins are potent inhibitors of neuronal exocytosis. Within the past five years the protein sequences of all eight neurotoxins have been determined, their mode of action as metalloproteases has been established, and their intraneuronal targets have been identified. The toxins act by selectively proteolysing the synaptic vesicle protein synaptobrevin (VAMP) or the presynaptic membrane proteins syntaxin (HPC-1) and SNAP-25. These three proteins form the core of a complex that mediates fusion of carrier vesicles to target membranes. Tetanus and botulinal neurotoxins could serve in the future as tools to study membrane trafficking events, or even higher brain functions such as behaviour and learning.

The t-SNAREs syntaxin 1 and SNAP-25 are present on organelles that participate in synaptic vesicle recycling.

Syntaxin 1 and synaptosome-associated protein of 25 kD (SNAP-25) are neuronal plasmalemma proteins that appear to be essential for exocytosis of synaptic vesicles (SVs). Both proteins form a complex with synaptobrevin, an intrinsic membrane protein of SVs. This binding is thought to be responsible for vesicle docking and apparently precedes membrane fusion. According to the current concept, syntaxin 1 and SNAP-25 are members of larger protein families, collectively designated as target-SNAP receptors (t-SNAREs), whose specific localization to subcellular membranes define where transport vesicles bind and fuse. Here we demonstrate that major pools of syntaxin 1 and SNAP-25 recycle with SVs. Both proteins cofractionate with SVs and clathrin-coated vesicles upon subcellular fractionation. Using recombinant proteins as standards for quantitation, we found that syntaxin 1 and SNAP-25 each comprise approximately 3% of the total protein in highly purified SVs. Thus, both proteins are significant components of SVs although less abundant than synaptobrevin (8.7% of the total protein). Immunoisolation of vesicles using synaptophysin and syntaxin specific antibodies revealed that most SVs contain syntaxin 1. The widespread distribution of both syntaxin 1 and SNAP-25 on SVs was further confirmed by immunogold electron microscopy. Botulinum neurotoxin C1, a toxin that blocks exocytosis by proteolyzing syntaxin 1, preferentially cleaves vesicular syntaxin 1. We conclude that t-SNAREs participate in SV recycling in what may be functionally distinct forms.

Calcium enhances binding of Clostridium perfringens epsilon toxin to sulfatide.

Epsilon toxin (Etx) from Clostridium perfringens is synthesized as a very low-active prototoxin form (proEtx) that becomes active upon proteolytic activation and has the capacity to cross the blood-brain barrier (BBB), thereby producing severe neurological effects. The identity and requirements of host receptors of Etx remain a matter of controversy. In the present study, we analysed the binding of proEtx or Etx to liposomes containing distearoylphosphatidylcholine (DSPC), cholesterol and sulfatide, or alternatively to detergent-solubilized lipids, using surface plasmon resonance (SPR). We also tested the influence of calcium on Etx or proEtx binding. Our findings show that the presence of sulfatide in liposomes increases both Etx and proEtx binding, and Etx binding is enhanced by calcium. These results were corroborated when SPR was conducted with immobilized toxin, since detergent-solubilized sulfatide increases its binding to Etx in the presence of calcium, but not to proEtx. Moreover, binding affinity is also affected, since the treatment of liposomes with sulfatase causes the dissociation rate constants (KD) in both proEtx and Etx to increase, especially in the case of proEtx in the presence of calcium. In addition, protein-lipid overlay assays corroborated the calcium-induced enhancement of Etx binding to sulfatide, and to lipids extracted from sulfatide-enriched rat brain lipid rafts. In conclusion, the present work highlights the role of sulfatide as an important element in the pathophysiology of Etx and reveals the influence of calcium in the interaction of Etx, but not of proEtx, with the target membrane.

Analysis and detection of functional outliers in water quality parameters from different automated monitoring stations in the Nalón river basin (Northern Spain).

The purposes and intent of the authorities in establishing water quality standards are to provide enhancement of water quality and prevention of pollution to protect the public health or welfare in accordance with the public interest for drinking water supplies, conservation of fish, wildlife and other beneficial aquatic life, and agricultural, industrial, recreational, and other reasonable and necessary uses as well as to maintain and improve the biological integrity of the waters. In this way, water quality controls involve a large number of variables and observations, often subject to some outliers. An outlier is an observation that is numerically distant from the rest of the data or that appears to deviate markedly from other members of the sample in which it occurs. An interesting analysis is to find those observations that produce measurements that are different from the pattern established in the sample. Therefore, identification of atypical observations is an important concern in water quality monitoring and a difficult task because of the multivariate nature of water quality data. Our study provides a new method for detecting outliers in water quality monitoring parameters, using turbidity, conductivity and ammonium ion as indicator variables. Until now, methods were based on considering the different parameters as a vector whose components were their concentration values. This innovative approach lies in considering water quality monitoring over time as continuous curves instead of discrete points, that is to say, the dataset of the problem are considered as a time-dependent function and not as a set of discrete values in different time instants. This new methodology, which is based on the concept of functional depth, was applied to the detection of outliers in water quality monitoring samples in the Nalón river basin with success. Results of this study were discussed here in terms of origin, causes, etc. Finally, the conclusions as well as advantages of the functional method are exposed.

Expression of synaptosomal-associated protein SNAP-25 in endocrine anterior pituitary cells.

A growing body of evidence indicates that the fundamental molecular mechanism of exocytosis in the secretory pathway may be structurally similar in all eukaryotic cells. The synaptosomal-associated protein of 25 kDa (SNAP-25) is a plasma membrane protein involved in regulated exocytosis in neurons. In order to compare exocytotic components in neurons and endocrine cells, we have analyzed the expression of SNAP-25 in the rat anterior pituitary. Western blotting analysis documented the presence of SNAP-25 in anterior pituitary homogenates and cultured anterior pituitary cells. In addition to SNAP-25, other neuronal proteins involved in exocytosis (syntaxin, VAMP/synaptobrevin and Rab3A) were also detected in the anterior pituitary. The specific expression of SNAP-25 mRNA in anterior pituitary cells was also corroborated by Northern analysis. SNAP-25 immunoreactivity was located at the plasma membrane of endocrine anterior pituitary cells. Characteristically, patches of fine punctate deposits exhibited intense SNAP-25 immunoreactivity. Double-labeling immunocytochemistry revealed that SNAP-25 was mainly associated with gonadotroph cell populations. Furthermore, we demonstrate that in the anterior pituitary, SNAP-25 is selectively cleaved by clostridial neurotoxins. In conclusion, our results establish the presence of SNAP-25 in secretory anterior pituitary cells and suggest a potential role of this protein in the secretion of adenohypophysial hormone.

Dystrophic neurites of senile plaques are defective in proteins involved in exocytosis and neurotransmission.

Dystrophic neurites are major components of neuritic (both immature and mature) senile plaques in Alzheimer disease. Previous studies have shown strong immunoreactivity for different neuropeptides, and chromogranin A, a protein associated with dense-core vesicles, in dystrophic neurites. In the present study, antibodies to synaptophysin, synapsin, Rab3a and synaptotagmin (synaptic vesicle proteins), and SNAP-25 (synaptosomal-associated protein of 25 kD) and syntaxin (presynaptic plasma membrane proteins) have been used to learn about the dystrophic neurite equipment of proteins that are necessary for the docking and fusion of synaptic vesicles, and then for exocytosis and neurotransmission. The present results have shown that, although most neuritic senile plaques have chromogranin A- and SNAP-25-immunoreactive dystrophic neurites, only a percentage of them contain synaptophysin, and a minority contain synaptotagmin and Rab3a. Dystrophic neurites do not contain synapsin and syntaxin. These results show that dystrophic neurites of senile plaques are defective in proteins that control exocytosis and neurotransmission.

BDNF and TrkB co-localize in CA1 neurons resistant to transient forebrain ischemia in the adult gerbil.

Delayed cell death of projection cells in the CA1 area of the hippocampus is produced in the adult gerbil following 5 minutes (min) of transient forebrain ischemia. Parvalbumin-immunoreactive local-circuit neurons are resistant to the ischemic insult. Brain-Derived Neurotrophic Factor (BDNF) immunoreactivity is localized in all neurons of the CA1 area in control gerbils. However, TrkB immunoreactivity is observed in a minority of BDNF-immunoreactive neurons in the CA1 area. The number of BDNF-immunoreactive cells in CA1 is dramatically reduced in ischemic gerbils as early as 24 h after ischemia, but the number of TrkB-immunoreactive cells in the CA1 area is maintained following ischemia. Moreover, about 90% of BDNF-immunoreactive cells and about 85% of TrkB-immunoreactive cells in ischemic gerbils co-localize the calcium-binding protein parvalbumin. Finally, BDNF and TrkB are coexpressed in about 95% of CA1 neurons surviving the ischemic insult. These results indicate that a subpopulation of CA1 hippocampal neurons coexpressing TrkB, parvalbumin and BDNF is resistant to transient forebrain ischemia in the gerbil. These results also suggest that a subpopulation of CA1 hippocampal neurons in the gerbil hippocampus is endowed with a putative BDNF/TrkB autocrine regulatory loop that may be involved in both cell survival and synaptic remodeling of the damaged gerbil hippocampus following transient forebrain ischemia.

Characterization of two distinct intracellular GLUT4 membrane populations in muscle fiber. Differential protein composition and sensitivity to insulin.

A major objective for the understanding of muscle glucose disposal is the elucidation of the intracellular trafficking pathway of GLUT4 glucose carriers in the muscle fiber. In this report, we provide functional and biochemical characterization of two distinct intracellular GLUT4 vesicle pools obtained from rat skeletal muscle. The two pools showed a differential response to insulin; thus, one showed a marked decrease in GLUT4 levels but the other did not. They also showed a markedly different protein composition as detected by quantitative vesicle immunoisolation analysis. The GLUT4 pool showing no response to insulin contained SCAMP proteins and the vSNARE proteins VAMP2 and cellubrevin, whereas only VAMP2 was found in the insulin-recruitable GLUT4 pool. SDS-PAGE and further silver staining of the immunoprecipitates revealed discrete polypeptide bands associated to the insulin-sensitive pool, and all these polypeptide bands were found in the insulin-insensitive population. Furthermore, some polypeptide bands were exclusive to the insulin-insensitive population. The presence of cellubrevin and SCAMP proteins, endosomal markers, suggest that the insulin-insensitive GLUT4 membrane population belongs to an endosomal compartment. In addition, we favor the view that the insulin-sensitive GLUT4 membrane pool is segregated from the endosomal GLUT4 population and is undergoes exocytosis to the cell surface in response to insulin. Intracellular GLUT4 membranes obtained from skeletal muscle contain cellubrevin, and VAMP2 and GLUT4-vesicles from cardiomyocytes also contain cellubrevin. This suggests that vSNARE proteins are key constituents of GLUT4 vesicles. The presence of the tSNARE protein SNAP25 in skeletal muscle membranes and SNAP25 and syntaxin 1A and syntaxin 1B in cardiomyocyte plasma membranes further suggest a role of the SNAREs in GLUT4 trafficking in muscle.

Differential interaction patterns in binding assays between recombinant syntaxin 1 and synaptobrevin isoforms.

Syntaxin 1 and synaptobrevin play an essential role in synaptic vesicle exocytosis. Two isoforms for each of these proteins, syntaxin 1A and 1B and synaptobrevin 1 and 2, have been found in nerve endings. Previous morphological studies have revealed a characteristic co-localization of syntaxin 1 and synaptobrevin isoforms in nervous and endocrine systems; however, the physiological significance of differential distribution is not known. In the present study an in vitro assay has been used to study a possible isoform specific interaction between syntaxin and synaptobrevin isoforms. The results show that although both syntaxin 1A and 1B may interact with synaptobrevin 1 and 2, this interaction is not uniform, showing different affinity patterns depending on the syntaxin 1/synaptobrevin isoform combination. The addition of SNAP-25 increased the binding capacity of syntaxin and synaptobrevin isoforms without affecting specific interactions.

Colchicine administration in the rat central nervous system induces SNAP-25 expression.

he arrest of axonal transport by colchicine administration has been extensively used in immunocytochemical studies to increase the levels of neuroactive compounds in neuronal somata. In order to study the accumulation rates of a variety of proteins with location and physiological action at the synaptic terminal, we analysed, by immunocytochemical methods, the neuronal cell body content of these synaptic proteins in colchicine-injected rats. In sham-injected animals, all synaptic proteins tested were essentially observed in nerve fibres and terminal boutons. After colchicine administration, intense SNAP-25 immunoreactivity was found in many neuronal cell bodies throughout the CNS. In contrast, immunostaining for the rest of the synaptic proteins analysed (syntaxin 1A and 1B, synaptobrevin I and II, Rab3A, synaptophysin, synapsin I, synaptotagmin I and GAP-43) was virtually absent in neuronal cell bodies in treated animals. Furthermore, northern blot and in situ hybridization analysis revealed an increase in SNAP-25a and SNAP-25b messenger RNA isoforms in the brains of adult colchicine-administered animals. In addition, colchicine administration in five-day-old rat pups induced a notable increase in both SNAP-25 transcript isoforms. The present results indicate that in vivo colchicine administration, under conditions known to inhibit axoplasmic transport, upregulates SNAP-25 expression in the rat brain.

Tetanus toxin mechanism of action in Torpedo electromotor system: a study on different steps in the intoxication process.

The mechanism of action of tetanus toxin was characterized in the electromotor system of Torpedo marmorata either at peripheral and central nervous system. The consecutive steps of the intoxication pathway were observed: (i) [125I]tetanus toxin specifically bound to neuronal plasma membranes isolated both from electric organ and electric lobe of Torpedo, exhibiting one and two binding sites respectively; (ii) [125I]tetanus toxin was internalized into nerve terminals and retrogradely transported to the electric lobe after its injection in the electric organ; (iii) finally, intracellular effect of tetanus toxin was studied either at electric organ and electric lobe membrane fractions. In both preparations tetanus toxin cleaved synaptobrevin, as detected by immunoblotting methods. In conclusion, our findings exhibit the presence of two different populations of acceptors for tetanus toxin in central and peripheral nervous system and show that synaptobrevin cleavage may account for intracellular toxicity in Torpedo.

Calcium channel antagonist omega-conotoxin binds to intramembrane particles of isolated nerve terminals.

Voltage-sensitive calcium channels play a key role in evoked neurotransmitter release and their distribution in presynaptic membranes can be critical for fast signalling at chemical synapses. Using a biotinylated derivative of the neuronal calcium channel antagonist, omega-conotoxin, and a combination of colloidal gold labeling and freeze-fracture techniques, we have labeled calcium channels present at the membrane of nerve terminals isolated from the electric organ of Torpedo marmorata. The biotinylated blocker exerts an inhibitory action on the high potassium-evoked release of adenosine triphosphate as the native toxin does and its interaction with biological membranes is specific as shown in displacement experiments. This study shows that an antagonist specific for voltage-activated calcium channels binds to intramembrane particles in presynaptic membranes, reinforcing the idea that these particles, concentrated at neurotransmitter release sites, effectively represent calcium channels.

Characterization of presynaptic proteins involved in synaptic vesicle exocytosis in the nervous system of Torpedo marmorata.

Synaptobrevin, SNAP-25 and syntaxin (SNAP receptor proteins) are molecular components that play a key role in the exocytotic machinery of synaptic vesicles. Their presence, distribution and interactions are reported in central and peripheral nervous systems of the electric fish Torpedo marmorata. These three proteins form a protein complex in all the nervous system regions tested, including the electric lobe and the electric organ which is innervated by pure cholinergic nerve terminals. Immunoblot analysis revealed a double protein pattern of SNAP-25 in the anterior brain and cerebellum, although a single protein band corresponding to SNAP-25 was observed in the electromotor system. Moreover, SNAP-25 showed a differential distribution in the electromotor system. It was present along nerve fibres and terminals that innervated the electric organ but it was not detected in nerve terminals at the electric lobe. Immunoisolation experiments using anti-synaptobrevin antibodies showed a tissue-specific co-existence of SNAP-25 and syntaxin with synaptobrevin in the immunoisolated organelles. In conclusion, the molecular components of the exocytotic machinery are shown to be conserved in Torpedo, although some differences mainly on SNAP-25, suggest a potential diversity in the regulation of neurosecretion.

Increase in reactive cholesterol in the presynaptic membrane of depolarized Torpedo synaptosomes: blockade by botulinum toxin type A.

We have investigated the redistribution of filipin-cholesterol complexes at freeze-fractured presynaptic membrane of pure cholinergic synaptosomes isolated from Torpedo electric organ during acetylcholine release. After chemical depolarization, filipin-induced lesions increase at the presynaptic membrane. These changes do not take place when synaptosomes are stimulated in a calcium-free medium. Botulinum neurotoxin type A blocks both acetylcholine release and the rearrangement of filipin-induced lesions induced by depolarization. Since botulinum neurotoxin type A does not block either membrane depolarization or calcium entry into the nerve terminal, our results suggest that the redistribution of filipin-cholesterol complexes is linked to the acetylcholine release process.

Axonal transport of ribonucleoprotein particles (vaults).

RNA was previously shown to be transported into both dendritic and axonal compartments of nerve cells, presumably involving a ribonucleoprotein particle. In order to reveal potential mechanisms of transport we investigated the axonal transport of the major vault protein of the electric ray Torpedo marmorata. This protein is the major protein component of a ribonucleoprotein particle (vault) carrying a non-translatable RNA and has a wide distribution in the animal kingdom. It is highly enriched in the cholinergic electromotor neurons and similar in size to synaptic vesicles. The axonal transport of vaults was investigated by immunofluorescence, using the anti-vault protein antibody as marker, and cytofluorimetric scanning, and was compared to that of the synaptic vesicle membrane protein SV2 and of the beta-subunit of the F1-ATPase as a marker for mitochondria. Following a crush significant axonal accumulation of SV2 proximal to the crush could first be observed after 1 h, that of mitochondria after 3 h and that of vaults after 6 h, although weekly fluorescent traces of accumulations of vault protein were observed in the confocal microscope as early as 3 h. Within the time-period investigated (up to 72 h) the accumulation of all markers increased continuously. Retrograde accumulations also occurred, and the immunofluorescence for the retrograde component, indicating recycling, was weaker than that for the anterograde component, suggesting that more than half of the vaults are degraded within the nerve terminal. High resolution immunofluorescence revealed a granular structure-in accordance with the biochemical characteristics of vaults. Of interest was the observation that the increase of vault immunoreactivity proximal to the crush accelerated with time after crushing, while that of SV2-containing particles appeared to decelerate, indicating that the crush procedure with time may have induced perikaryal alterations in the production and subsequent export to the axon of synaptic vesicles and vault protein. Our data show that ribonucleoprotein-immunoreactive particles can be actively transported within axons in situ from the soma to the nerve terminal and back. The results suggest that the transport of vaults is driven by fast axonal transport motors like the SV2-containing vesicles and mitochondria. Vaults exhibit an anterograde and a retrograde transport component, similar to that observed for the vesicular organelles carrying SV2 and for mitochondria. Although the function of vaults is still unknown studies of the axonal transport of this organelle may reveal insights into the mechanisms of cellular transport of ribonucleoprotein particles in general.

Selective early induction of synaptosomal-associated protein (molecular weight 25,000) following systemic administration of kainate at convulsant doses in the rat.

SNAP-25 (synaptosomal-associated protein of mol. wt 25,000) is an essential component for neurotransmitter release, and its expression has been related to the plastic responses that follow CNS injury. In the present study, transient induction of SNAP-25 in selected brain areas is shown by immunohistochemistry at short times after a single intraperitoneal injection of kainate at convulsant doses. Six hours after kainate injection, SNAP-25 immunoreactivity was noticed in the perikarya of certain neurons of the perirhinal and lateral cortices, polymorphic layer of the dentate gyrus, CA3 pyramidal area of the hippocampus, and thalamus. In the same areas, a strong increase in SNAP-25 immunorectivity was detected at 12 and 24 h after kainate injection in cell bodies and fibers. Four days after kainate administration, the immunostaining pattern was similar to that observed in control animals. Intraperitoneal injection of cycloheximide blocked the expression of SNAP-25, thus suggesting de novo SNAP-25 protein synthesis following kainate administration. Kainate-dependent induction of SNAP-25a messenger RNA synthesis was observed by in situ hybridization in the mentioned brain areas. Heat shock protein of mol. wt 72,000 (HSP70/72) is a chaperone whose expression is induced early under stress conditions. Its expression and distribution were compared to that of SNAP-25 after the excitotoxic insult. Brain areas overexpressing SNAP-25 and HSP70/72 overlapped. In addition, partial co-localization of both antigens was observed by double-labeling immunohistochemistry. These results provide evidence of an involvement of SNAP-25 in the reactive response that follows kainate administration, and support the role of this protein in the plastic events that take place after kainate excitotoxicity.