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Synaptic microenvironments — structural plasticity, adhesion molecules, proteases and their inhibitors
Neuroscience Research 37 (2000) 85 – 89 www.elsevier.com/locate/neures
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Synaptic microenvironments — structural plasticity, adhesion molecules, proteases and their inhibitors Sadao Shiosaka *, Shigetaka Yoshida Di6ision of Structural Cell Biology, Nara Institute of Science and Technology (NAIST), 8916 -5 Takayama Ikoma Nara, 630 -0101, Japan Received 6 December 1999; accepted 8 March 2000
Abstract Proteolytic regulation might be essential in neural plasticity in mature brain as well as the developing brain. An increasing number of studies support the idea that structural changes in the synapses are closely associated with synaptic plasticity. Proteases and their inhibitors in a synaptic microenvironment are important in the regulation of dynamic changes in the extracellular matrix components associated with synaptic plasticity. In the present article, the possible roles of neuronal proteases, protease inhibitors and extracellular macromolecules are reviewed. © 2000 Elsevier Science Ireland Ltd and the Japanese Neuroscience Society. All rights reserved. Keywords: Serine protease; Synapse; Spine; CA1; Hippocampus; Protease inhibitor; ECM
An increasing number of studies support the idea that synaptic plasticity is closely associated with activity-dependent structural changes in synapses (Geinisman et al., 1991; Buchs and Muller, 1996; Agnihotri et al., 1998; Toni et al. 1999). Pericellular proteolysis of the macromolecules is involved in the remodeling or formation of synapses during early synaptogenesis and their elimination at neuromuscular junctions (Hantai et al., 1989; Festoff et al., 1991; Champaneria et al., 1992; Sanes and Lichtman, 1999 as a review). In the central nervous system, an analogous mechanism might operate to organize and rearrange the synaptic interactions during development (Oliver et al., 1989; Dent et al., 1993). The pre- and post-synaptic components of the central nervous system are composed of a number of extracellular matrix (ECM) macromolecules (Pfenninger, 1971a,b). Concerning induction and maintenance of long-term potentiation (LTP), investigation of pericellular proteolysis of the macromolecules could clarify neural plasticity. * Corresponding author. Tel.: +81-74372-5410; fax: + 81-743725419. E-mail address: [email protected] (S. Shiosaka)
In the present review article, we attempt to describe the structural remodeling and formation of synapses and to describe the roles of adhesion molecules, proteases and their inhibitors at various synaptic plasticity events.
1. Structural changes in hippocampal synapses/spines in relation to neural plasticity One of the most interesting functions of the brain is that related to learning and memory. LTP in the hippocampus is an experimental model that is thought to reflect the fundamental mechanism underlying learning and memory and has been studied using a signal transduction system downstream of glutamate receptors. Investigators have not shown as much interested, however, in extracellular molecular interactions associated with synaptic structures as in the intracellular events. Geinisman and coworkers proposed that induction of LTP was accompanied by synaptic structural changes, e.g. increase in the number of perforated synapses (Geinisman et al., 1991). They hypothesized that the turnover or conversion of small nonperforatedtype synapses into large perforated type synapses may
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explain the long-lasting enhancement of synaptic-efficacy, namely LTP. Confocal laser microscopy has been successfully applied to the time-course analysis of slice sections. Hosokawa et al. (1995) observed that small spines grew and became angularly displaced up induction of LTP, using DiI-labeled living spines. Although this observation is a strong evidence of activity-dependent structural change in synapses or spines, confocal microscopy cannot distinguish short or curved spines because of insufficient resolution (Sorra and Harris, 1998). On the other hand, single-section analysis using an electron microscope before and after a tetanic stimulation is an inaccurate method to identify such variable synapses. Nevertheless, using more sophisticated electron microscopic techniques, the spines activated by LTP were revealed to promote the formation of multiple spine synapses arising from the same dendrite (Toni et al., 1999). Thus, although there are still technical difficulties to determine the phenomena directly at present, results of recent studies indicate that structural remodeling of synapses and/or formation of new synapses could be crucial for neural plasticity.
1999). Moreover, another cadherin subfamily, CNRs, was identified by Kohmura et al. (1998). CNRs bind with Fyn which mediates the induction of LTP in the hippocampal CA1 subfield and are thus involved in synaptic plasticity similar to the classic cadherins and Arcadlin (Kohmura et al., 1998). Therefore, not only classic, but also nonclassic cadherins have important roles in activity-dependent synaptic plasticity. The synaptic localization of NCAM and L1, homophilic or heterophilic cell adhesion molecules, is still unclear. Nevertheless, electrophysiological studies have revealed that various antibodies against NCAM and L1 reduced CA1 LTP elicited by theta-burst stimulation (Luthl et al., 1994; Fields and Itoh, 1996). Several investigators have also demonstrated the importance of integrin-mediated cell-to-cell contact in neural plasticity. Integrins (a5, a5b1, and avb3), which are receptors for fibronectin and vitronectin, were shown to be present in the hippocampus, and inhibitors of integrins suppressed the induction of LTP (Staubli et al., 1998). Thus, it appears that these extracellular macromolecules, localized in the vicinity of the synaptic cleft, play a role in neural plasticity via reinforcement of cell-to-cell binding.
2. ECM macromolecules involved in neural plasticity There are two major cell-to-cell attachment mechanisms; one is homophilic binding, in which identical molecules on the surface of either cell membrane bind to each other, and the other is heterophilic binding, in which receptors on the cell membrane, such as integrin, bind to extracellular matrix molecules. Numerous adhesion macromolecules have been identified in the synaptic cleft and its vicinity, such as classic cadherins (Eand N-cadherin) (Fannon and Colman, 1996), protocadherins (Obata et al., 1998), cadherin-related neuronal receptors (CNRs) (Kohmura et al., 1998), fibronectin (Hoffman et al., 1998), laminin (Chen and Strickland, 1997), integrins (Bahr et al., 1991; Staubli et al., 1998), and NCAM (Luthl et al., 1994; Muller et al., 1996; Schuster et al., 1998). Fannon and Colman (1996) using an immunohistochemical technique reported that E- and N-cadherins are localized between the pre- and post-synaptic membranes in the CA3 subfield of the hippocampus as a primary adhesion moiety. Hippocampal slices were pretreated with antibodies against the extracellular domain of N- and E-cadherins or antagonistic peptides that inhibit cadherin dimerization, LTP was significantly reduced in both cases (Tang et al., 1998). Thus, the classic cadherins participate in modulating activity-dependent changes in the synaptic strength. A homologue of protocadherin-8, Arcadlin, which shows Ca2 + -dependent homophilic binding activity, was recently cloned and it was found that an antibody directed against it blocked LTP (Yamagata et al.,
3. Extracellular proteases in the synaptic microenvironment Antibodies against adhesion molecules and inhibitors of the binding domain can block LTP. Similarly, modification of the extracellular macromolecules might disrupt the function of adhesion molecules at the site of synaptic activity. Therefore, proteolytic modification of the macromolecules might be a significant regulation system for neural plasticity. Qian et al. (1993) performed differential screening by high-frequency stimulation of the perforant path or convulsive seizures and identified a tissue plasminogen activator (tPA) gene as the immediate-early gene induced by neuronal activity. tPA was originally known as a protease involved in blood coagulation-fibrinolysis. Targeting of the tPA gene in mice resulted in selective interference of the late-phase LTP in the hippocampus (Frey et al., 1996; Huang et al., 1996) and was characterized by stronger GABAergic transmission in the hippocampal CA1 region (Frey et al., 1996). In addition to the hippocampal function, tPA is involved in the induction of cerebellar motor learning (Seeds et al., 1995). Interestingly, tPAdeficient mice did not show impaired hippocampal-dependent learning and memory, but did show impairment in striatum-dependent learning (Huang et al., 1996). Therefore, tPA has relatively broad functions in neural plasticity in various brain areas. tPA-deficient mice are less susceptible to kainate induced seizures than wild-type mice (Tsirka et al., 1995). Chen and
S. Shiosaka, S. Yoshida / Neuroscience Research 37 (2000) 85–89
Strickland (1997) reported that such sensitization of hippocampal neurons is caused by the disruption of neuron–ECM interaction via tPA, which is a lamininmediated interaction. Since LTP and seizures have similar biochemical mechanisms (Baudry, 1986), the susceptibility to late-phase LTP of tPA-deficient hippocampus might also somewhat resemble the mechanism underlying seizures. Local proteolysis in the synaptic vicinity precedes morphological changes involved in plasticity, which modifies pre- and postsynaptic interactions. We hypothesized that a protease acts on the local synaptic microenvironment. Neuropsin was cloned as a novel serine protease from a hippocampal cDNA library (Chen et al., 1995). Neuropsin mRNA was localized predominantly in the hippocampal CA1 – 3 subfields and exhibited an activity-dependent change during epileptogenesis and LTP (Chen et al., 1995; Momota et al., 1998; Yoshida et al., 1998; Komai et al., 2000). The protease is potentially involved in the modification of the extracellular environment (Shimizu et al., 1998) and we studied effect by applying neuropsin antibodies to the lateral ventricle in mice. When a specific antibody was applied in the middle of kindling formation, these mice showed significant attenuation of epileptogenesis compared to control mice injected with non-immune IgG (Momota et al., 1998). More intriguingly, neuropsin exhibited a regulatory effect, particularly in the early phase of the Schaffer-collateral LTP (Komai et al., 2000). Application of various concentrations of recombinant neuropsin resulted in a bell-shaped doseresponse curve for the amplitude of the tetanic-stimulation-induced early-phase LTP. Thus, neuropsin appears to act as a regulatory molecule in the early phase of LTP via its proteolytic action. Besides these two serine proteases, it is still unclear whether other proteases are also involved in neural plasticity. Several new serine proteases other than tPA and neuropsin have been recently identified in the brain (Gschwend et al., 1997; Scarisbrick et al., 1997; Yamashiro et al., 1997; Little et al., 1997; Proba et al., 1998). The endogenous substrates of these proteases are still to be determined. Therefore, it is not known how these extracellular proteins are involved in neural plasticity. More recently, some metalloproteases and their inhibitors were suggested to participate in tissue remodeling in the developing cerebellum (Vaillant et al., 1999). Further studies on the distribution, substrate specificity and neural functions of each of the proteases are necessary to reveal their possible roles in brain development and plasticity. 4. Protease inhibitors in the synaptic microenvironment Neuroserpin is a secretory protease inhibitor, which
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was cloned by the group of Sonderegger (Osterwalder et al., 1996). In the adult mouse brain, neuroserpin was most strongly expressed in the neocortex, hippocampus, olfactory bulb and amygdala (Kruger et al., 1997). Its inhibitor may act as a regulator of tPA activity, because recombinant neuroserpin inhibited the amidolytic activity of tPA more efficiently than that of urokinase plasminogen activator (uPA), plasmin and thrombin (Kruger et al., 1997). Therefore, the balance in the protein expression between tPA and neuroserpin in the pericellular environment might regulate various functions of tPA as discussed above. It is noteworthy that a new disease, familial encephalopathy associated with neuroserpin inclusion bodies, was recently clinically characterized as an autosomal-dominantly inherited dementia (Davis et al., 1999). Mutant neuroserpin causes the disease, associated with conformational transition and polymerization of the serpin (Davis et al., 1999). Since tPA, a potential cognizant protease, has an effect on long-lasting LTP as mentioned above, the neuroserpin-tPA interaction may be involved in memory and learning. However, it is still open to question whether tPA-deficient mice have a memory deficit. Protease nexin-1 is another member of the serpin family and possibly control the activity of extracellular serine proteases (Guenther et al., 1985; Gloor et al., 1986). Nexin-1 is the most potent inhibitor of thrombin and inhibits plasmin, tPA and uPA to a lesser extent. The nexin-1-overexpressing and -deficient mice exhibited an enhancement and diminution in thetaburst-induced Schaffer-collateral LTP, respectively (Lu¨thi et al., 1997). It was found that decreased levels of nexin-1 seem to increase the susceptibility to seizures evoked by glutamatergic excitotoxins, concomitant with a decrease in sensitivity induced by the toxins in tPAdeficient mice (Tsirka et al., 1995; Lu¨thi et al., 1997). In nexin-1-overexpressing mice, GABAergic synaptic transmission is increased, indicating an imbalance between excitatory and inhibitory synaptic transmission caused by the presence of nexin-1 (Lu¨thi et al., 1997). Finally, it should be emphasized that the interactions between protease, and ECM macromolecules might be the basis for the presumed structural changes in the synapse/spine complex in relation to neural plasticity. Further studies are required to clarify the functional roles of neural proteases, protease inhibitors, and ECM macromolecules under various physiological conditions of the brain. In addition, it might be necessary to develop new techniques that enable visualization of the rapid movements of synapses or spine protrusions during neural plasticity at the ultrastructural level.
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Update article
Synaptic microenvironments — structural plasticity, adhesion molecules, proteases and their inhibitors Sadao Shiosaka *, Shigetaka Yoshida Di6ision of Structural Cell Biology, Nara Institute of Science and Technology (NAIST), 8916 -5 Takayama Ikoma Nara, 630 -0101, Japan Received 6 December 1999; accepted 8 March 2000
Abstract Proteolytic regulation might be essential in neural plasticity in mature brain as well as the developing brain. An increasing number of studies support the idea that structural changes in the synapses are closely associated with synaptic plasticity. Proteases and their inhibitors in a synaptic microenvironment are important in the regulation of dynamic changes in the extracellular matrix components associated with synaptic plasticity. In the present article, the possible roles of neuronal proteases, protease inhibitors and extracellular macromolecules are reviewed. © 2000 Elsevier Science Ireland Ltd and the Japanese Neuroscience Society. All rights reserved. Keywords: Serine protease; Synapse; Spine; CA1; Hippocampus; Protease inhibitor; ECM
An increasing number of studies support the idea that synaptic plasticity is closely associated with activity-dependent structural changes in synapses (Geinisman et al., 1991; Buchs and Muller, 1996; Agnihotri et al., 1998; Toni et al. 1999). Pericellular proteolysis of the macromolecules is involved in the remodeling or formation of synapses during early synaptogenesis and their elimination at neuromuscular junctions (Hantai et al., 1989; Festoff et al., 1991; Champaneria et al., 1992; Sanes and Lichtman, 1999 as a review). In the central nervous system, an analogous mechanism might operate to organize and rearrange the synaptic interactions during development (Oliver et al., 1989; Dent et al., 1993). The pre- and post-synaptic components of the central nervous system are composed of a number of extracellular matrix (ECM) macromolecules (Pfenninger, 1971a,b). Concerning induction and maintenance of long-term potentiation (LTP), investigation of pericellular proteolysis of the macromolecules could clarify neural plasticity. * Corresponding author. Tel.: +81-74372-5410; fax: + 81-743725419. E-mail address: [email protected] (S. Shiosaka)
In the present review article, we attempt to describe the structural remodeling and formation of synapses and to describe the roles of adhesion molecules, proteases and their inhibitors at various synaptic plasticity events.
1. Structural changes in hippocampal synapses/spines in relation to neural plasticity One of the most interesting functions of the brain is that related to learning and memory. LTP in the hippocampus is an experimental model that is thought to reflect the fundamental mechanism underlying learning and memory and has been studied using a signal transduction system downstream of glutamate receptors. Investigators have not shown as much interested, however, in extracellular molecular interactions associated with synaptic structures as in the intracellular events. Geinisman and coworkers proposed that induction of LTP was accompanied by synaptic structural changes, e.g. increase in the number of perforated synapses (Geinisman et al., 1991). They hypothesized that the turnover or conversion of small nonperforatedtype synapses into large perforated type synapses may
0168-0102/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd and the Japanese Neuroscience Society. All rights reserved. PII: S 0 1 6 8 - 0 1 0 2 ( 0 0 ) 0 0 1 1 5 - 2
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explain the long-lasting enhancement of synaptic-efficacy, namely LTP. Confocal laser microscopy has been successfully applied to the time-course analysis of slice sections. Hosokawa et al. (1995) observed that small spines grew and became angularly displaced up induction of LTP, using DiI-labeled living spines. Although this observation is a strong evidence of activity-dependent structural change in synapses or spines, confocal microscopy cannot distinguish short or curved spines because of insufficient resolution (Sorra and Harris, 1998). On the other hand, single-section analysis using an electron microscope before and after a tetanic stimulation is an inaccurate method to identify such variable synapses. Nevertheless, using more sophisticated electron microscopic techniques, the spines activated by LTP were revealed to promote the formation of multiple spine synapses arising from the same dendrite (Toni et al., 1999). Thus, although there are still technical difficulties to determine the phenomena directly at present, results of recent studies indicate that structural remodeling of synapses and/or formation of new synapses could be crucial for neural plasticity.
1999). Moreover, another cadherin subfamily, CNRs, was identified by Kohmura et al. (1998). CNRs bind with Fyn which mediates the induction of LTP in the hippocampal CA1 subfield and are thus involved in synaptic plasticity similar to the classic cadherins and Arcadlin (Kohmura et al., 1998). Therefore, not only classic, but also nonclassic cadherins have important roles in activity-dependent synaptic plasticity. The synaptic localization of NCAM and L1, homophilic or heterophilic cell adhesion molecules, is still unclear. Nevertheless, electrophysiological studies have revealed that various antibodies against NCAM and L1 reduced CA1 LTP elicited by theta-burst stimulation (Luthl et al., 1994; Fields and Itoh, 1996). Several investigators have also demonstrated the importance of integrin-mediated cell-to-cell contact in neural plasticity. Integrins (a5, a5b1, and avb3), which are receptors for fibronectin and vitronectin, were shown to be present in the hippocampus, and inhibitors of integrins suppressed the induction of LTP (Staubli et al., 1998). Thus, it appears that these extracellular macromolecules, localized in the vicinity of the synaptic cleft, play a role in neural plasticity via reinforcement of cell-to-cell binding.
2. ECM macromolecules involved in neural plasticity There are two major cell-to-cell attachment mechanisms; one is homophilic binding, in which identical molecules on the surface of either cell membrane bind to each other, and the other is heterophilic binding, in which receptors on the cell membrane, such as integrin, bind to extracellular matrix molecules. Numerous adhesion macromolecules have been identified in the synaptic cleft and its vicinity, such as classic cadherins (Eand N-cadherin) (Fannon and Colman, 1996), protocadherins (Obata et al., 1998), cadherin-related neuronal receptors (CNRs) (Kohmura et al., 1998), fibronectin (Hoffman et al., 1998), laminin (Chen and Strickland, 1997), integrins (Bahr et al., 1991; Staubli et al., 1998), and NCAM (Luthl et al., 1994; Muller et al., 1996; Schuster et al., 1998). Fannon and Colman (1996) using an immunohistochemical technique reported that E- and N-cadherins are localized between the pre- and post-synaptic membranes in the CA3 subfield of the hippocampus as a primary adhesion moiety. Hippocampal slices were pretreated with antibodies against the extracellular domain of N- and E-cadherins or antagonistic peptides that inhibit cadherin dimerization, LTP was significantly reduced in both cases (Tang et al., 1998). Thus, the classic cadherins participate in modulating activity-dependent changes in the synaptic strength. A homologue of protocadherin-8, Arcadlin, which shows Ca2 + -dependent homophilic binding activity, was recently cloned and it was found that an antibody directed against it blocked LTP (Yamagata et al.,
3. Extracellular proteases in the synaptic microenvironment Antibodies against adhesion molecules and inhibitors of the binding domain can block LTP. Similarly, modification of the extracellular macromolecules might disrupt the function of adhesion molecules at the site of synaptic activity. Therefore, proteolytic modification of the macromolecules might be a significant regulation system for neural plasticity. Qian et al. (1993) performed differential screening by high-frequency stimulation of the perforant path or convulsive seizures and identified a tissue plasminogen activator (tPA) gene as the immediate-early gene induced by neuronal activity. tPA was originally known as a protease involved in blood coagulation-fibrinolysis. Targeting of the tPA gene in mice resulted in selective interference of the late-phase LTP in the hippocampus (Frey et al., 1996; Huang et al., 1996) and was characterized by stronger GABAergic transmission in the hippocampal CA1 region (Frey et al., 1996). In addition to the hippocampal function, tPA is involved in the induction of cerebellar motor learning (Seeds et al., 1995). Interestingly, tPAdeficient mice did not show impaired hippocampal-dependent learning and memory, but did show impairment in striatum-dependent learning (Huang et al., 1996). Therefore, tPA has relatively broad functions in neural plasticity in various brain areas. tPA-deficient mice are less susceptible to kainate induced seizures than wild-type mice (Tsirka et al., 1995). Chen and
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Strickland (1997) reported that such sensitization of hippocampal neurons is caused by the disruption of neuron–ECM interaction via tPA, which is a lamininmediated interaction. Since LTP and seizures have similar biochemical mechanisms (Baudry, 1986), the susceptibility to late-phase LTP of tPA-deficient hippocampus might also somewhat resemble the mechanism underlying seizures. Local proteolysis in the synaptic vicinity precedes morphological changes involved in plasticity, which modifies pre- and postsynaptic interactions. We hypothesized that a protease acts on the local synaptic microenvironment. Neuropsin was cloned as a novel serine protease from a hippocampal cDNA library (Chen et al., 1995). Neuropsin mRNA was localized predominantly in the hippocampal CA1 – 3 subfields and exhibited an activity-dependent change during epileptogenesis and LTP (Chen et al., 1995; Momota et al., 1998; Yoshida et al., 1998; Komai et al., 2000). The protease is potentially involved in the modification of the extracellular environment (Shimizu et al., 1998) and we studied effect by applying neuropsin antibodies to the lateral ventricle in mice. When a specific antibody was applied in the middle of kindling formation, these mice showed significant attenuation of epileptogenesis compared to control mice injected with non-immune IgG (Momota et al., 1998). More intriguingly, neuropsin exhibited a regulatory effect, particularly in the early phase of the Schaffer-collateral LTP (Komai et al., 2000). Application of various concentrations of recombinant neuropsin resulted in a bell-shaped doseresponse curve for the amplitude of the tetanic-stimulation-induced early-phase LTP. Thus, neuropsin appears to act as a regulatory molecule in the early phase of LTP via its proteolytic action. Besides these two serine proteases, it is still unclear whether other proteases are also involved in neural plasticity. Several new serine proteases other than tPA and neuropsin have been recently identified in the brain (Gschwend et al., 1997; Scarisbrick et al., 1997; Yamashiro et al., 1997; Little et al., 1997; Proba et al., 1998). The endogenous substrates of these proteases are still to be determined. Therefore, it is not known how these extracellular proteins are involved in neural plasticity. More recently, some metalloproteases and their inhibitors were suggested to participate in tissue remodeling in the developing cerebellum (Vaillant et al., 1999). Further studies on the distribution, substrate specificity and neural functions of each of the proteases are necessary to reveal their possible roles in brain development and plasticity. 4. Protease inhibitors in the synaptic microenvironment Neuroserpin is a secretory protease inhibitor, which
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was cloned by the group of Sonderegger (Osterwalder et al., 1996). In the adult mouse brain, neuroserpin was most strongly expressed in the neocortex, hippocampus, olfactory bulb and amygdala (Kruger et al., 1997). Its inhibitor may act as a regulator of tPA activity, because recombinant neuroserpin inhibited the amidolytic activity of tPA more efficiently than that of urokinase plasminogen activator (uPA), plasmin and thrombin (Kruger et al., 1997). Therefore, the balance in the protein expression between tPA and neuroserpin in the pericellular environment might regulate various functions of tPA as discussed above. It is noteworthy that a new disease, familial encephalopathy associated with neuroserpin inclusion bodies, was recently clinically characterized as an autosomal-dominantly inherited dementia (Davis et al., 1999). Mutant neuroserpin causes the disease, associated with conformational transition and polymerization of the serpin (Davis et al., 1999). Since tPA, a potential cognizant protease, has an effect on long-lasting LTP as mentioned above, the neuroserpin-tPA interaction may be involved in memory and learning. However, it is still open to question whether tPA-deficient mice have a memory deficit. Protease nexin-1 is another member of the serpin family and possibly control the activity of extracellular serine proteases (Guenther et al., 1985; Gloor et al., 1986). Nexin-1 is the most potent inhibitor of thrombin and inhibits plasmin, tPA and uPA to a lesser extent. The nexin-1-overexpressing and -deficient mice exhibited an enhancement and diminution in thetaburst-induced Schaffer-collateral LTP, respectively (Lu¨thi et al., 1997). It was found that decreased levels of nexin-1 seem to increase the susceptibility to seizures evoked by glutamatergic excitotoxins, concomitant with a decrease in sensitivity induced by the toxins in tPAdeficient mice (Tsirka et al., 1995; Lu¨thi et al., 1997). In nexin-1-overexpressing mice, GABAergic synaptic transmission is increased, indicating an imbalance between excitatory and inhibitory synaptic transmission caused by the presence of nexin-1 (Lu¨thi et al., 1997). Finally, it should be emphasized that the interactions between protease, and ECM macromolecules might be the basis for the presumed structural changes in the synapse/spine complex in relation to neural plasticity. Further studies are required to clarify the functional roles of neural proteases, protease inhibitors, and ECM macromolecules under various physiological conditions of the brain. In addition, it might be necessary to develop new techniques that enable visualization of the rapid movements of synapses or spine protrusions during neural plasticity at the ultrastructural level.
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