Calcium-sensitive Protease Research Articles

An Activity-Based Nanosensor for Traumatic Brain Injury.

Currently, traumatic brain injury (TBI) is detected by medical imaging; however, medical imaging requires expensive capital equipment, is time- and resource-intensive, and is poor at predicting patient prognosis. To date, direct measurement of elevated protease activity has yet to be utilized to detect TBI. In this work, we engineered an activity-based nanosensor for TBI (TBI-ABN) that responds to increased protease activity initiated after brain injury. We establish that a calcium-sensitive protease, calpain-1, is active in the injured brain hours within injury. We then optimize the molecular weight of a nanoscale polymeric carrier to infiltrate into the injured brain tissue with minimal renal filtration. A calpain-1 substrate that generates a fluorescent signal upon cleavage was attached to this nanoscale polymeric carrier to generate an engineered TBI-ABN. When applied intravenously to a mouse model of TBI, our engineered sensor is observed to locally activate in the injured brain tissue. This TBI-ABN is the first demonstration of a sensor that responds to protease activity to detect TBI.

Endogenous HAX-1 Regulates SERCA Activity and Oxidation Dependent Stability

HS-associated protein X-1 (HAX-1) has been identified as a novel binding partner of both SERCA2a and phospholamban (PLN) in the heart. We observed that the expression levels of HAX-1 are decreased in human failing hearts and experimental ischemia/reperfusion injury. To elucidate the impact of such reduction in endogenous HAX-1, a mouse model with inducible cardiac-specific ablation of HAX-1 was generated. Ablation of HAX-1 resulted in a hypercontractile phenotype in whole hearts and isolated cardiomyocytes. The underlying mechanisms included enhanced Ca-transient kinetics and SR Ca uptake affinity through relief of the PLN inhibitory effects. Despite the increases in Ca-cycling, HAX-1 ablation diminished contractile recovery and increased infarct size and apoptosis in hearts subjected to ex vivo ischemia/reperfusion. This was associated with enhanced degradation of SERCA2a by the calcium sensitive protease, calpain. Modulation of SERCA2a degradation by HAX-1 was also observed in isolated cardiomyocytes and SR microsomes. The ROS scavenger thioredoxin1 in cardiomyocytes and reducing conditions in SR microsomes attenuated degradation of SERCA2a and abolished the effects of HAX-1 deficiency, suggesting the involvement of a redox mechanism. Indeed, SERCA2a oxidation was assessed by two complementary free cysteine labelling methods and was found to be uniquely increased in HAX-1 deficient hearts. The SERCA2a inhibitor thapsigargin, which stabilizes a specific conformation of the enzyme, eliminated the HAX-1 variations in free cysteine labelling of SERCA2a. This suggests that HAX-1 alters the conformation of SERCA2a thereby changing the reactivity/availability of specific cysteines to oxidation. Additionally, HAX-1 levels inversely correlated to ROS production at the ER/SR compartment, quantified by genetically encoded HyPer indicators. These findings indicate that endogenous HAX-1 prevents SERCA2a cysteine oxidation, by a change in structure and/or a change in ROS, resulting in decreased susceptibility of SERCA2a to degradation. Thus, restoring the decreased HAX-1 levels in the stressed heart may hold therapeutic potential.

Abstract 12139: Calpain Inhibition Attenuates Contractile Dysfunction Induced by Cardiac Dyssynchrony

Introduction: Cardiac dyssynchrony confers poor prognosis in heart failure, but whether dyssynchrony is merely a marker for contractile dysfunction, or actively contributes to it, is uncertain. In vivo, dyssynchrony causes local muscle lengthening during electrical activation. In skeletal muscle, forced lengthening during contraction (LEN-CON) activates the calcium sensitive protease calpain and causes contractile dysfunction and sarcomere disorganization. Hypothesis: Calpain inhibition attenuates contractile dysfunction and sarcomere disorganization in cardiac muscle subjected to LEN-CON. Methods: Rat right ventricular trabeculae (cross sectional area 0.06±.04 mm 2 ) at fixed diastolic length L 0 were paced at 1 Hz for 1 hr and allowed to shorten against normal load (50% initial isometric force F 0 , CONTROL n=4), or forced to lengthen to 110% L 0 during each contraction without (UNTREATED n=12) or with (TREATED n=14) calpain inhibitor (calpeptin 1 μM). After 1 hr, isometric force was remeasured (F 1Hr ), and sarcomere length, length dispersion and organization in trabeculae fixed in diastole at L 0 and stained for α-actinin were quantified by Fourier power spectrum analysis of digitally deconvolved photomicrographs. Results: In CONTROL, isometric force was unchanged after 1 hr (F 1Hr /F 0 94±18%, mean±SD). In UNTREATED, 1 hr LEN-CON caused dysfunction (F 1Hr /F 0 50±9%, p<.001 vs CONTROL). In TREATED, dysfunction from LEN-CON was attenuated (F 1Hr /F 0 63±16%, p=.02 vs UNTREATED). Sarcomere length after LEN-CON was similar in UNTREATED and TREATED (2.21±.20 vs 2.12±.09 μm, p=NS), but TREATED showed less length dispersion and better preserved sarcomere organization (FIG) by Fourier analysis (p<.05). Conclusions: Lengthening contraction causes dysfunction and sarcomere disorganization in cardiac muscle, at least in part through calpain activation. Calpain inhibition may have potential to ameliorate contractile dysfunction due to dyssynchrony in vivo.

Role of the calpain on the development of diabetes mellitus and its chronic complications.

Diabetes mellitus (DM) is associated with acute and chronic complications that cause major morbidity and significant mortality. Calpains, a family of Ca(2+)-dependent cytosolic cysteine proteases, can modulate their substrates' structure and function through limited proteolytic activity. Calpain is a ubiquitous calcium-sensitive protease that is essential for normal physiologic function. However, alterations in calcium homeostasis lead to pathologic activation of calpain in diabetes mellitus. Since not much is known on the relationship between calpain and diabetes mellitus, this review outlines the contribution of calpain to chronic complications of diabetes mellitus, such as diabetic cardiomyopathy, diabetic nephropathy and diabetic retinopathy.

Alterations of Ca2+-responsive proteins within cholinergic neurons in aging and Alzheimer's disease

The molecular basis of selective neuronal vulnerability in Alzheimer's disease (AD) remains poorly understood. Using basal forebrain cholinergic neurons (BFCNs) as a model and immunohistochemistry, we have demonstrated significant age-related loss of the calcium-binding protein calbindin-D28K (CB) from BFCN, which was associated with tangle formation and degeneration in AD. Here, we determined alterations in RNA and protein for CB and the Ca2+-responsive proteins Ca2+/calmodulin-dependent protein kinase I (CaMKI), growth-associated protein-43 (GAP43), and calpain in the BF. We observed progressive downregulation of CB and CaMKI RNA in laser-captured BFCN in the normal-aged-AD continuum. We also detected progressive loss of CB, CaMKIδ, and GAP43 proteins in BF homogenates in aging and AD. Activated μ-calpain, a calcium-sensitive protease that degrades CaMKI and GAP43, was significantly increased in the normal aged BF and was 10 times higher in AD BF. Overactivation of μ-calpain was confirmed using proteolytic fragments of its substrate spectrin. Substantial age- and AD-related alterations in Ca2+-sensing proteins most likely contribute to selective vulnerability of BFCN to degeneration in AD.

Ovarian Cancer G protein-Coupled Receptor 1 Is Involved in Acid-Induced Apoptosis of Endplate Chondrocytes in Intervertebral Discs

Ovarian cancer G protein-coupled receptor 1 (OGR1) has been shown to be a receptor for protons. We investigated the role of proton-sensing G protein-coupled receptors in the apoptosis of endplate chondrocytes induced by extracellular acid. The expression of proton-sensing G protein-coupled receptors was examined in rat lumbar endplate chondrocytes. Knockdown of OGR1 was achieved by transfecting chondrocytes with specific short hairpin RNA (shRNA) for OGR1. Apoptotic changes were evaluated by DNA fragmentation ELISA, electron microscopy, and flow cytometry. Intracellular calcium ([Ca(2+) ]i) was analyzed with laser scanning confocal microscopy. The mechanism of OGR1 in acid-induced apoptosis of endplate chondrocytes was also investigated. We found that OGR1 was predominantly expressed in rat endplate chondrocytes, and its expression was highly upregulated in response to acidosis. Knocking down OGR1 with shRNAs effectively attenuated acid-induced apoptosis of endplate chondrocytes and increased [Ca(2+) ]i. Blocking OGR1-mediated [Ca(2+) ]i elevation inhibited acid-induced calcium-sensitive proteases such as calpain and calcineurin, and also inhibited the activation of Bid, Bad, and Caspase 3 and cleavage of poly (ADP-ribose) polymerase (PARP). OGR1-mediated [Ca(2+) ]i elevation has a crucial role in apoptosis of endplate chondrocytes by regulating activation of calcium-sensitive proteases and their downstream signaling.

Introduction of calpain inhibitors in traumatic brain injury: A novel approach?

Traumatic brain injury (TBI) is a major cause of death and disability throughout the world. In recent years, researchers focused on the pathological significance of calcium accumulation in the brain after TBI. Neuronal calcium homeostasis disturbances may result in the activation of calpain a ubiquitous calcium-sensitive protease. The calpain family has a well-established causal role in neuronal cell death following acute brain injury: their activation has been observed to progressively increase after either contusive or diffuse brain trauma in animals, suggesting calpain to be a mediator of early neuronal damage.We hypothesize that pretreatment with the calpain inhibitors in population at objective risk (military soldiers’ pre combat) in appropriate dose would open therapeutic time window expected to prevent and reduce extensive brain damage by providing optimal TBI neuroprotection. Additional therapeutic strategy for TBI, based on calpain modulating actions such as pretreatment with calpain inhibitors has been proposed.Since calpain overexpression has been well established in acute neuronal injury and further subsequent neurodegeneration, from a clinical viewpoint, we speculate that if this hypothesis proves correct pretreatment inhibitors introduction may become a therapeutic option for different brain pathologies to be approached and treated with.

Calpain cleaves and activates the TRPC5 channel to participate in semaphorin 3A-induced neuronal growth cone collapse

The nonselective cation channel transient receptor potential canonical (TRPC)5 is found predominantly in the brain and has been proposed to regulate neuronal processes and growth cones. Here, we demonstrate that semaphorin 3A-mediated growth cone collapse is reduced in hippocampal neurons from TRPC5 null mice. This reduction is reproduced by inhibition of the calcium-sensitive protease calpain in wild-type neurons but not in TRPC5(-/-) neurons. We show that calpain-1 and calpain-2 cleave and functionally activate TRPC5. Mutation of a critical threonine at position 857 inhibits calpain-2 cleavage of the channel. Finally, we show that the truncated TRPC5 predicted to result from calpain cleavage is functionally active. These results indicate that semaphorin 3A initiates growth cone collapse via activation of calpain that in turn potentiates TRPC5 activity. Thus, TRPC5 acts downstream of semaphorin signaling to cause changes in neuronal growth cone morphology and nervous system development.

Spectrin Breakdown Products (SBDPs) as Potential Biomarkers for Neurodegenerative Diseases

The world's human population ages rapidly thanks to the great advance in modern medicine. While more and more body system diseases become treatable and curable, age-related neurodegenerative diseases remain poorly understood mechanistically, and are desperately in need of preventive and therapeutic interventions. Biomarker development consists of a key part of concerted effort in combating neurodegenerative diseases. In many chronic neurodegenerative conditions, neuronal damage/death occurs long before the onset of disease symptoms, and abnormal proteolysis may either play an active role or be a companying event of neuronal injury. Increased spectrin cleavage yielding elevated spectrin breakdown products (SBDPs) by calcium-sensitive proteases such as calpain and caspases has been established in conditions associated with acute neuronal damage such as traumatic brain injury (TBI). Here we review literature regarding spectrin expression and metabolism in the brain, and propose a potential use of SBDPs as biomarkers for neurodegenerative diseases such as Alzheimer's diseases.

Dysfunctional mitochondria uphold calpain activation: Contribution to Parkinson's disease pathology

Calpain is a ubiquitous calcium-sensitive protease that is essential for normal physiologic neuronal function. However, mitochondrial-mediated-calcium homeostasis alterations may lead to its pathologic activation that jeopardizes neuronal structure and function. Here, we provide evidence to support a role for the involvement of calpain 1 in mitochondrial-induced neurodegeneration in a Parkinson's disease (PD) cellular model. We show that dysfunctional mitochondria increases cytosolic calcium, thereby, inducing calpain activation. Interestingly, its inhibition significantly attenuated the accumulation of alpha-synuclein oligomers and contributed to an increase of insoluble alpha-synuclein aggregates, known to be cytoprotective. Moreover, our data corroborate that calpain-1 overactivation in our mitochondrial-deficient cells promote caspase-3 activation. Overall, our findings further clarify the crucial role of dysfunctional mitochondria in the control of molecular mechanisms occurring in PD brain cells, providing a potentially novel correlation between the degradation of calpain substrates suggesting a putative role of calpain and calpain inhibition as a therapeutic tool in PD.

Calpain-Mediated Signaling Mechanisms in Neuronal Injury and Neurodegeneration

Calpain is a ubiquitous calcium-sensitive protease that is essential for normal physiologic neuronal function. However, alterations in calcium homeostasis lead to persistent, pathologic activation of calpain in a number of neurodegenerative diseases. Pathologic activation of calpain results in the cleavage of a number of neuronal substrates that negatively affect neuronal structure and function, leading to inhibition of essential neuronal survival mechanisms. In this review, we examine the mechanistic underpinnings of calcium dysregulation resulting in calpain activation in the acute neurodegenerative diseases such as cerebral ischemia and in the chronic neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, prion-related encephalopathy, and amylotrophic lateral sclerosis. The premise of this paper is that analysis of the signaling and transcriptional consequences of calpain-mediated cleavage of its various substrates for any neurodegenerative disease can be extrapolated to all of the neurodegenerative diseases vulnerable to calcium dysregulation.

Endothelial Cell Calpain Activity Facilitates Lymphocyte Diapedesis

Lymphocyte infiltration of tissue is a cardinal feature of solid-organ allograft rejection. Vascular endothelial cells (EC) participate in lymphocyte recruitment through the display of adhesion molecules and chemokines to promote leukocyte extravasation. Moreover, EC reorganize the cytoskeleton and cytoskeleton-associated structures during leukocyte diapedesis. We examined the role of EC (Ca+2)i and the calcium-sensitive protease, calpain, during lymphocyte diapedesis through a human EC monolayer under physiologic shear stress in vitro. We observed that lymphocyte transendothelial migration (TEM) was inhibited by chelating EC cytosolic calcium, or depleting EC endoplasmic reticulum calcium stores by inhibition of the endoplasmic reticulum Ca ATPase. Further, inhibition of EC phospholiase C also decreased lymphocyte TEM. We determined that EC constitutively exhibit calpain activity, using fluorescence generation from a calpain substrate to report calpain activity in individual live cells. Moreover, EC adjacent to a transmigrating lymphocyte showed increased calpain activity. Further, lymphocyte TEM was inhibited by agents that block calpain activity. Inhibition of lymphocyte TEM occurs at the lumenal EC surface and correlates with impaired development of intercellular adhesion molecule 1 (ICAM-1)-rich docking structures by the EC. We conclude EC calcium and calpain activity facilitates lymphocyte TEM, and participates in the assembly of the docking structure.

Phospholipase C-δ1 rescues intracellular Ca 2+ overload in ischemic heart and hypoxic neonatal cardiomyocytes

Ischemia and simulated ischemic conditions cause intracellular Ca 2+ overload in the myocardium. The relationship between ischemia injury and Ca 2+ overload has not been fully characterized. The aim of the present study was to investigate the expression and characteristics of PLC isozymes in myocardial infarction-induced cardiac remodeling and heart failure. In normal rat heart tissue, PLC-δ1 (about 44 ng/mg of heart tissue) was most abundant isozymes compared to PLC-γ1 (6.8 ng/mg) and PLC-β1 (0.4 ng/mg). In ischemic heart and hypoxic neonatal cardiomyocytes, PLC-δ1, but not PLC-β1 and PLC-γ1, was selectively degraded, a response that could be inhibited by the calpain inhibitor, calpastatin, and by the caspase inhibitor, zVAD-fmk. Overexpression of the PLC-δ1 in hypoxic neonatal cardiomyocytes rescued intracellular Ca 2+ overload by ischemic conditions. In the border zone and scar region of infarcted myocardium, and in hypoxic neonatal cardiomyocytes, the selective degradation of PLC-δ1 by the calcium sensitive proteases may play important roles in intracellular Ca 2+ regulations under the ischemic conditions. It is suggested that PLC isozyme-changes may contribute to the alterations in calcium homeostasis in myocardial ischemia.

The pathogenic activation of calpain: a marker and mediator of cellular toxicity and disease states.

Over-activation of calpain, a ubiquitous calcium-sensitive protease, has been linked to a variety of degenerative conditions in the brain and several other tissues. Dozens of substrates for calpain have been identified and several of these have been used to measure activation of the protease in the context of experimentally induced and naturally occurring pathologies. Calpain-mediated cleavage of the cytoskeletal protein spectrin, in particular, results in a set of large breakdown products (BDPs) that are unique in that they are unusually stable. Over the last 15 years, measurements of BDPs in experimental models of stroke-type excitotoxicity, hypoxia/ischemia, vasospasm, epilepsy, toxin exposure, brain injury, kidney malfunction, and genetic defects, have established that calpain activation is an early and causal event in the degeneration that ensues from acute, definable insults. The BDPs also have been found to increase with normal ageing and in patients with Alzheimer's disease, and the calpain activity may be involved in related apoptotic processes in conjunction with the caspase family of proteases. Thus, it has become increasingly clear that regardless of the mode of disturbance in calcium homeostasis or the cell type involved, calpain is critical to the development of pathology and therefore a distinct and powerful therapeutic target. The recent development of antibodies that recognize the site at which spectrin is cleaved has greatly facilitated the temporal and spatial resolution of calpain activation in situ. Accordingly, sensitive spectrin breakdown assays now are utilized to identify potential toxic side-effects of compounds and to develop calpain inhibitors for a wide range of indications including stroke, cerebral vasospasm, and kidney failure.

Calcium-activated proteolysis in rat neocortex induced by transient focal ischemia

Ischemia-induced elevation of intracellular calcium triggers a cascade of events which is considered to play a major role in neuronal death. One candidate to participate in this process is the calcium-sensitive protease, calpain. This protease is activated by calcium, and is capable of degrading critical cytoskeletal and regulatory proteins. In order to further elucidate the role of calpain in focal ischemic damage, the present study investigated the proteolysis of spectrin, a preferred substrate for calpain, in response to transient focal ischemia. Ischemia was induced by occluding reversibly both carotid arteries and the left middle cerebral artery for three hours in Sprague-Dawley rats. Western blotting techniques were used to identify and quantify the amounts of spectrin breakdown products (BDPs) in neocortical samples from the area destined for infarction, the peri-infarct area, and the contralateral hemisphere. Substantial increases in spectrin proteolysis were observed within the first few hours of ischemia in the areas that will undergo infarction. The increase in spectrin BDPs in these areas reached a plateau around the end of the 3 h ischemic period. In the peri-infarct zone, the levels of spectrin BDPs increased in a biphasic manner. A small to moderate increase was observed by the second hour of ischemia, followed by a larger increase between the 6th and 24th hours post-ischemia. The contralateral neocortex showed a significant increase in BDPs at 2 h after the initiation of ischemia. A smaller increase in BDPs was observed thereafter. Substantial calpain-mediated proteolysis thus occurs within 1 h of focal cerebral ischemia in areas destined for infarction, while the responses in the peri-infarct area and contralateral cortex are weaker and delayed. The rapid and sustained proteolytic responses in vulnerable areas suggest that calcium-activated proteolysis is an early indicator of cell damage in focal ischemia. In addition, the findings suggest that therapeutic interventions targeting this mechanism should be initiated within a few hours after ischemia and maintained for at least 24 h.

Adrenalectomy attenuates kainic acid-induced spectrin proteolysis and heat shock protein 70 induction in hippocampus and cortex.

Glucocorticoids have been shown to exacerbate the damaging effects of a variety of neurotoxic insults in the hippocampus and other brain areas. Evidence suggests that the endangering effects of glucocorticoids may be due to augmenting the cascade of events, such as elevations in intracellular calcium levels, because of excitatory amino acid (EAA) receptor stimulation. A potential mechanism responsible for EAA-induced neuronal damage is activation of calcium-sensitive proteases, such as calpain, which then proteolytically degrade cytoskeleton structural proteins, such as spectrin. The present study was designed to determine if glucocorticoids can regulate the spectrin proteolysis produced by the EAA agonist, kainic acid. Rats were adrenalectomized (ADX) or sham operated and 7 days later injected with kainic acid (10 mg/kg). Twenty-four hours later rats were killed and tissues obtained for western blot analyses of the intact spectrin molecule and the proteolytically derived breakdown products. Kainic acid produced an approximate sevenfold increase in the 145-155-kDa spectrin breakdown products in the hippocampus relative to ADX or sham rats injected with vehicle. ADX attenuated the kainic acid-induced increase in breakdown products by 43%. In a similar way, kainic acid produced a large 10-fold increase in spectrin breakdown products in the frontal cortex, which was also significantly attenuated (-80%) by ADX. Induction of heat shock protein 70 (hsp70) by neurotoxic insults has been suggested to be a sensitive indicator of cellular stress in neurons. Kainic acid induced large amounts of hsp70 in both hippocampus and frontal cortex of sham-operated rats that was markedly attenuated (85-95%) by ADX.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of proteolysis protects hippocampal neurons from ischemia.

Intense proteolysis of cytoskeletal proteins occurs in brain within minutes of transient ischemia, possibly because of the activation of calcium-sensitive proteases (calpains). This proteolytic event precedes overt signs of neuronal degeneration, is most pronounced in regions of selective neuronal vulnerability, and could have significant consequences for the integrity of cellular function. The present studies demonstrate that (i) the early phase of enhanced proteolysis is a direct response to hypoxia rather than other actions of ischemia, (ii) it is possible to pharmacologically inhibit the in vivo proteolytic response to ischemia, (iii) inhibition of proteolysis is associated with a marked reduction in the extent of neuronal death, and (iv) protected neurons exhibit normal-appearing electrophysiological responses and retain their capacity for expressing long-term potentiation, a form of physiological plasticity thought to be involved in memory function. These observations indicate that calcium-activated proteolysis is an important component of the post-ischemic neurodegenerative response and that targeting this response may be a viable therapeutic strategy for preserving both the structure and function of vulnerable neurons.

Development of hippocampal long-term potentiation is reduced by recently introduced calpain inhibitors

The effects of two recently synthesized inhibitors of calpains, calpain inhibitor I (CiI) and calpain inhibitor II (CiII) were tested on the development of long-term potentiation (LTP) in region CA1 of rat hippocampus. Slices maintained in 100 μM of CiI or CiII showed an initial degree of potentiation after theta burst stimulation that, in contrast to controls, slowly decayed across time. The effects of CiI and CiII appeared to be independent of possible actions on the physiological mechanisms that take place during the induction stage of LTP. Since these inhibitors are more potent and specific than leupeptin in blocking calpain activity, their effects on LTP can be more convincingly ascribed to a selective blockade of the calcium-sensitive protease. Accordingly, the results favor the idea that a proteolytic event of the kind found after N-methyl- d-aspartate receptor activation is an intermediary step in the development of LTP.

Long-term potentiation: Persisting problems and recent results

In this paper we discuss recent experimental results pertinent to three unresolved issues regarding the long-term potentiation (LTP) effect, the nature of its enduring substrates, the biochemical mechanisms that produce it, and its potential role in memory. LTP appears to be triggered by a postsynaptic influx of calcium and is associated with alterations in the shape of dendritic spines and probably the formation of new synapses. We discuss the possibility that morphological reorganization also modifies membrane surface chemistry of synaptic elements. Evidence is presented that LTP is not associated with changes in presynaptic calcium currents. Activation of protein kinase C is shown to be insufficient for the induction of LTP, although it may play a modulatory role. The hypothesis that activation of a calcium-sensitive protease (calpain) is pivotal to the establishment of LTP is supported by experiments showing that a calpain inhibitor, leupeptin, blocks LTP. Furthermore, activation of NMDA receptors, an event implicated in LTP induction, is accompanied by calcium-sensitive proteolysis of spectrin, a major dendritic cytoskeletal protein. The finding that stimulation patterns designed to mimic naturally-occurring cell discharge patterns are highly effective for LTP induction greatly strengthens the hypothesis that LTP actually occurs during the encoding of information in cortical systems. Potential contributions of LTP to learning are explored using computer simulations of a simple cortical network.

Chronic administration of a thiol-proteinase inhibitor blocks long-term potentiation of synaptic responses

It has been proposed that activation of a calcium-sensitive protease (calpain) is a crucial step in the induction of long-term potentiation (LTP). To test this hypothesis, we used chronic recording techniques to measure the effects of intraventricular infusion of leupeptin, a calpain inhibitor, on LTP in the hippocampus. Rats implanted bilaterally with stimulating electrodes in the Schaffer-commissural system and one recording electrode in the apical dentrites of field CA1 were fitted with osmotic mini-pumps delivering either leupeptin (20 mg/ml) or saline at a rate of 0.5 μl/h into the lateral ventricle. Short bursts of high-frequency stimulation with the bursts delivered at 5/s were used to induce LTP in those animals which had stable responses for several days. Rats in the saline group ( n = 11) exhibited an immediate LTP effect that remained in place over successive days of testing, while only 3 of 13 leupeptin treated animals showed evidence of LTP 24 h after high-frequency stimulation, and in only one of those was a sizeable effect recorded over several days. The average change in responses at the 24-h test point was +33% for the controls and +4% for the leupeptin group ( P < 0.01). The block of LTP induction was reversible, since high-frequency stimulation applied after disconnecting the pumps led to a robust LTP effect that lasted for several days in 6 of 7 animals tested. There were no detectable differences in baseline responses in the presence and absence of leupeptin.