- Email: [email protected]
Thrombin regulation of synaptic plasticity: Implications for physiology and pathology
Thrombin regulation of synaptic plasticity: Implications for physiology and pathology? Nicola Maggio, Zeev Itsekson, Dan Dominissini, Ilan Blatt, Ninette Amariglio, Gideon Rechavi, David Tanne, Joab Chapman PII: DOI: Reference:
S0014-4886(13)00064-2 doi: 10.1016/j.expneurol.2013.02.011 YEXNR 11387
To appear in:
Experimental Neurology
Received date: Revised date: Accepted date:
16 November 2012 24 January 2013 18 February 2013
Please cite this article as: Maggio, Nicola, Itsekson, Zeev, Dominissini, Dan, Blatt, Ilan, Amariglio, Ninette, Rechavi, Gideon, Tanne, David, Chapman, Joab, Thrombin regulation of synaptic plasticity: Implications for physiology and pathology?, Experimental Neurology (2013), doi: 10.1016/j.expneurol.2013.02.011
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Experimental Neurology Regular Article
PT
24/01/2013
RI
Title: Thrombin regulation of synaptic plasticity: implications for physiology and pathology? Authors: Nicola Maggio1,2*, Zeev Itsekson2, Dan Dominissini3,4, Ilan Blatt2,5, Ninette Amariglio3,
SC
Gideon Rechavi3,4, David Tanne2,5 and Joab Chapman2,5.
NU
Affiliations:
1- Talpiot Medical Leadership Program, The Chaim Sheba Medical Center, 52621 Tel
MA
HaShomer, Israel.
2- Department of Neurology, The J. Sagol Neuroscience Center, The Chaim Sheba Medical Center, 52621 Tel HaShomer, Israel.
D
3- Cancer Research Center, The Sheba Medical Center, 52621 Tel HaShomer, Israel.
TE
4- Sackler School of Medicine, Tel Aviv University, 69978 Tel Aviv, Israel. 5- Department of Neurology, Sackler School of Medicine, Tel Aviv University, 69978 Tel
AC CE P
Aviv, Israel.
*Correspondence should be addressed to: Nicola Maggio, MD, PhD, Department of Neurology, The Chaim Sheba Medical Center, 52621 Tel HaShomer, Israel. Phone: +972547296984. Fax: +97235304752. email: [email protected]
ACCEPTED MANUSCRIPT Abstract: Thrombin, a serine protease involved in the coagulation cascade has been recently shown to affect neuronal function following blood brain barrier breakdown. Several lines of evidence have
PT
shown that thrombin may exist in the brain parenchyma under normal physiological conditions, yet its role in normal brain functions and synaptic transmission has not been established. In an
RI
attempt to shed light on the physiological functions of thrombin and Protease Activated
SC
Receptor 1 (PAR1) in the brain, we studied the effects of thrombin and a PAR1 agonist on long term potentiation (LTP) in mice hippocampal slices. Surprisingly, different concentrations of thrombin affect LTP through different molecular routes converging on PAR1. High thrombin
NU
concentrations induced a NMDA dependent, slow onset LTP, whereas low concentrations of thrombin promoted a VGCCs, mGluR-5 dependent LTP through activated Protein C (aPC).
MA
Remarkably, aPC facilitated LTP by activating PAR1 through an Endothelial Protein C Receptor (EPCR)-mediated mechanism which involves intracellular calcium stores. These findings reveal a novel mechanism by which PAR1 may regulate the threshold for synaptic plasticity in the
TE
Highlights:
AC CE P
pathological conditions.
D
hippocampus and provide additional insights into the role of this receptor in normal and
Thrombin levels regulate LTP through diverse molecular routes converging on PAR1.
[Thrombin]high induces a slow onset LTP.
[Thrombin]low enhances LTP by activating Protein C.
Protein C mediates LTP through VGCCs and mGluRs.
Keywords: Thrombin, PAR1, LTP, synaptic plasticity, hippocampus, extracellular proteases. Acknowledgements: The authors wish to acknowledge Dr. Eduard Korkotian for help with the imaging of the histological sections and Dr. Menahem Segal for comments on early drafts of the manuscript. NM is the recipient of a Talpiot Fellowship at the Sheba Medical Center. The funding agency did not have a role in study design; collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication. Conflict of interest: The authors declare no competing financial interests.
ACCEPTED MANUSCRIPT Introduction Cerebrovascular events induced by either ischemia or hemorrhage lead to blood brain barrier (BBB) breakdown and exposure of the brain to blood constituents (Yang and Rosenberg, 2011).
PT
Among others, thrombin, a serine protease involved in the coagulation cascade, has been shown to contribute to the stroke pathology following these conditions (Chen, et al., 2012,
RI
Wang, et al., 2012). High concentrations of thrombin in the brain also perturb normal physiology by saturating synaptic plasticity and inducing seizures (Isaeva, et al., 2012, Maggio, et al., 2012,
SC
Maggio, et al., 2008). These effects depend on the activation of the thrombin receptor, the Protease Activated Receptor 1 (PAR1) and consequent potentiation of NMDA receptors
NU
functions (Gingrich, et al., 2000, Maggio, et al., 2008). Previous studies, however, have shown that thrombin may exist in the brain parenchyma in normal physiological conditions (Turgeon, et
MA
al., 2000). Indeed, the mRNAs for both the thrombin precursor prothrombin and factor Xa, the enzyme converting prothrombin into thrombin, have been detected in several areas of the forebrain (Dihanich, et al., 1991, Shikamoto and Morita, 1999). Nevertheless, both the
D
mechanisms leading to the thrombin production in the brain under physiological conditions as
TE
well as its role in normal brain functions and synaptic transmission have not yet been completely clarified.
PAR1 belongs to a family of seven transmembrane domain, G protein-coupled receptors whose
AC CE P
activation requires the cleavage of a peptide bond at the N-terminal extracellular side which binds the second extracellular loop of the same receptor thus activating it (Sokolova and Reiser, 2008). In the brain PAR1 is expressed both in neurons and astrocytes (Junge, et al., 2004, Luo, et al., 2007). Its activation has been shown to modulate synaptic transmission and plasticity (Lee, et al., 2007), yet the specific contribution of the astrocytic vs. the neuronal receptor remains under investigation. In addition, while in peripheral organs PAR1 has been shown to be activated by a pool of proteases, e.g. activated Protein C (aPC), leading to different outcomes, (Mosnier, et al., 2007) the role of other PAR1 agonists in the brain has not been fully investigated. In an attempt to shed light on the physiological vs. pathological functions of thrombin and PAR1 in the brain, we studied the effects of different concentrations of thrombin and PAR1 agonist (PAR1-AP) on long term potentiation (LTP) using hippocampal slices. Surprisingly, we found that diverse thrombin concentrations differently regulate the threshold for synaptic plasticity in the hippocampus. These data provide additional insights into the role of this receptor in normal and pathological conditions.
ACCEPTED MANUSCRIPT Methods Drugs: The following drugs were prepared from frozen stocks: thrombin (Sigma Aldrich, Rehovot, Israel and Enzyme Research Laboratories, Swansea, England); PAR1 agonist (PAR-
PT
1AP, SFLLRN, Sigma Aldrich, Rehovot, Israel); PAR1 antagonist (SCH79797, Tocris Bioscience, Bristol, United Kingdom); Plasmin (Sigma Aldrich, Rehovot, Israel); activated
RI
Protein C (aPC, Sigma Aldrich, Rehovot, Israel); inactivated Protein C (iPC, Enzyme Research Laboratories, Swansea, England); APV (Sigma Aldrich, Rehovot, Israel); Nifedipine (Sigma
SC
Aldrich, Rehovot, Israel); MPEP (Tocris Bioscience, Bristol, United Kingdom); Thapsigargin (Alomone Labs, Jerusalem, Israel); Cyclopiazonic Acid (CPA, Alomone Labs, Jerusalem,
NU
Israel); -NAPAP (Sigma Aldrich, Rehovot, Israel). As the specific activity of the α-thrombin from various vendors ranged between 2700 and 3200 NIH U/mg by comparison to Lot K of the
MA
NIH standard, we estimated the concentration of active α-thrombin that corresponds to 1 U/ml activity, by calculating a conversion factor using pure α-thrombin (3200 U/mg), as previously described (Gingrich, et al., 2000). In this lot, manufacturer reported this protein to be >95% α-
D
thrombin as determined by gel electrophoresis, hence a solution with 1 U/ml α-thrombin should be 9 nM by a molecular weight for thrombin of 36.7 kDa. For the sake of simplicity, we used a
TE
conversion factor of 1 U/ml = 10 nM α-thrombin throughout the text to estimate the concentration of active α-thrombin (henceforth referred to as thrombin) from various vendors.
AC CE P
Electrophysiology: Animal handling was approved by the Institutional Animal Care and Use Committee, which adheres to the National law, and NIH rules. Briefly, 4-5 months old male C57BL/6 mice were rapidly decapitated and 350 μm coronal dorsal hippocampal slices were used. Slices were incubated for 1.5 h in a humidified, carbogenated (5% CO2 and 95% O2) gas atmosphere at 33 ± 1°C and were perfused with artificial CSF [containing (in mM) 124 NaCl, 2 KCl, 26 NaHCO3, 1.24 KH2PO4, 2.5 CaCl2, 2 MgSO4, and 10 glucose, pH 7.4] in a standard interface chamber. Recordings were made with a glass pipette containing 0.75 M NaCl (4 MΩ) placed in the stratum radiatum CA1. Stimulation was evoked using a Master 8 pulse stimulator (A.M.P.I., Jerusalem, Israel) and was delivered through two sets of bipolar nichrome electrodes placed on either side of the recording electrode such that two independent stimulation channels were used for each slice. The use of two parallel pathways allowed comparison of the effects of drug application in the same slice (Maggio and Segal, 2007). LTP was induced by highfrequency stimulation consisting of 100 pulses at twice the test intensity, delivered at a frequency of 100 Hz (HFS; 100 Hz, 1 s). Before applying the tetanic stimulation, baseline values were recorded at a frequency of 0.033 Hz. Responses were digitized at 5 kHz and stored on a computer. Off-line analysis and data acquisition were performed using the Spike 2 software
ACCEPTED MANUSCRIPT (CED, Cambridge, England). All numerical data are expressed as mean ± SEM, and EPSP slope changes after tetanic stimulation were calculated with respect to baseline. There were no systematic differences in the magnitudes of the baseline responses in the different conditions.
PT
All values reported refer to 30 min before tetanic stimulation. Unless otherwise indicated, statistical evaluations were performed by applying a Student's t test for paired and unpaired
RI
data, as the case may be (Origin 8.0). p values of <0.05 were considered a significant difference between means.
SC
Immunohistochemistry: The following antibodies were used for immunodetection: mouse antibodies raised against Neuronal nuclear antigen (NeuN) (1:100; Millipore, Billerica, MA,
NU
USA); rabbit antibodies to proteases activated receptor-1 (PAR1) (1:50; Abcam, Cambridge, UK); goat antibodies to endothelial protein C receptor (EPCR) (P20, 1:100; Santa Cruz Biotech.,
MA
CA, USA). Hippocampal sections (25m) were blocked in 10% normal serum in 0.01M PBS/ 0.25% Triton for 1 hr at room temperature (RT). After 48 hr. incubation at 4°C with the primary antibody (NeuN, PAR-1, and EPCR with 2% normal serum), sections were exposed to the
D
appropriate secondary antibody (1:500, DyLight flourophores -594; 488; 633, Thermo-Scientific,
TE
Rockford, IL, USA) for 1 hr and finally mounted and coverslipped with Flouromount (Southern Biotechnology). Slides were imaged with a Zeiss LSM 510 confocal microscope and data were
AC CE P
acquired and analyzed using a computer assisted image analysis system. Intracerebroventricular (ICV) injections: Mice were anesthetized with an intraperitoneal (IP) injection of ketamine (100 mg/kg) and xylazine (20 mg/kg). The skull was carefully exposed, and a small hole was drilled above the right lateral ventricle (2 mm lateral to the midline and 2.5 mm posterior to the bregma). A rat monoclonal antibody blocking EPCR (R252 clone, Sigma Aldrich, Rehovot, Israel) was injected at a concentration of 30 g/ml (2 mm depth) using a 27-gauge needle attached to a Hamilton syringe. Following the slow infusion of 1 l of antibody solution, the needle was withdrawn, and the skin over the scalp was sutured. Control mice were injected with a vehicle solution. Hippocampal slices were cut 36 hours following the procedure. Results Thrombin Induces Slow Onset LTP in CA1 Thrombin concentrations rise following BBB breakdown (Chen, et al., 2010, Chen, et al., 2012). Exposure of mice hippocampal slices to high concentration of thrombin (1U/ml thrombin; [Thrombin]high) for 12 minutes produced a gradual increase in population EPSP recorded in stratum radiatum of region CA1 of the hippocampus. This gradual change was specific to the
ACCEPTED MANUSCRIPT EPSP, because no parallel change was noticed in the presynaptic volley produced in response to the stimulation (Fig.1A). The increase in EPSP slope rose gradually over 40 min of recording, reaching a plateau level, which was 73% above control (n=11 slices; 1.73±0.78; P < 0.01),
PT
similar to that induced by tetanic stimulation (HFS) of the alternate pathway prior to drug application. Application of 2, 3, 5 and 10U/ml thrombin produced the same effect (data not
RI
shown). A comparable, slow-rising, persistent increase in population EPSP was seen after bath application of 1μM SFLLRN, a PAR1 receptor agonist ([PAR1-AP]high; n=11 slices; 1.62±0.071;
SC
P<0.01; Fig.1B). To verify that the effect of thrombin was mediated through activation of a genuine PAR1 receptor, the selective PAR1 antagonist SCH79797 (SCH) was used. SCH had
NU
no effect on established tetanic LTP (Fig.1C) (n=11; 1.84±0.079), yet the response to thrombin, tested in the non tetanized pathway, was completely abolished (0.99±0.066). In order to
MA
address whether thrombin-induced slow onset LTP shared a downstream mechanism with the conventional LTP, thrombin was applied in the presence of the NMDA receptor antagonist 2(R)APV (APV, 50μM). Under these conditions, thrombin was unable to produce LTP (0.97±0.062 at
D
25min following thrombin application; n=11 slices) (Fig.1D). Likely, the effect of thrombin was
TE
abolished in presence of its specific inhibitor, -NAPAP (Suppl. Fig.1A). These experiments confirm the results we previously obtained in rats (Maggio, et al., 2008), namely that the slow
AC CE P
onset LTP induced by [Thrombin]high involves NMDA receptors. The Effects of Thrombin and PAR-1AP are Concentration Dependent Thrombin produces a variety of effects in the brain being detrimental at high concentrations and protective at low concentrations (Hua, et al., 2009, Striggow, et al., 2000). In order to test whether the effects of thrombin on LTP are concentration dependent, we exposed the hippocampal slices to 100mU/ml thrombin ([Thrombin]low) for 15 minutes. In these experiments, thrombin was bath applied following the delivery of HFS at the first control pathway. At this concentration, thrombin did not affect the established LTP, yet it failed to induce a slow onset potentiation of EPSP at the second pathway (1.01±0.068 at 50min of recordings; Fig.2A). Furthermore, HFS at the second pathway, 20 minutes after drug removal, evoked an LTP of similar level to the one in the first, control pathway (n=12 slices; 1.66±0.059 vs. 1.64±0.063, respectively; P=0.43; Fig.2A). Thrombin concentrations ranging between 500mU/ml to 1mU/mL led to similar results (data not shown). Interestingly, however, exposure of slices to a lower concentration of PAR1-AP resulted in a novel, different phenomenon. Following the induction of LTP by HFS at the first pathway, the slices were exposed to 100nM PAR1-AP ([PAR1-AP]low) for 15 minutes. [PAR1-AP]low did not enhance synaptic transmission per se, yet 20 minutes after
ACCEPTED MANUSCRIPT drug withdrawal an enhanced LTP could be evoked by HFS at the second pathway (n=12 slices; 2.23±0.086; P<0.001 over LTP at the first pathway; Fig.2B). Similar data were obtained using concentrations of PAR1-AP ranging between 200nM to 10nM (data not shown). In order
PT
to verify that the genuine activation of PAR1 is required to produce an enhancement of LTP, [PAR1-AP]low was bath applied in presence of SCH. Remarkably, in this condition HFS at the
RI
second pathway generated a normal level of LTP comparable to the one obtained in the control pathway (n=12 slices; 1.79±0.77 vs. 1.75±0.084, respectively; P=0.39; Fig.2C). These
SC
experiments show that exposure to [PAR1-AP]low enhances LTP through a PAR1 mediated mechanism. This effect is specific to [PAR1-AP]low and is not shared by [Thrombin]low.
NU
activated Protein C (aPC) and not Plasmin Mimics the Effects of [PAR1-AP]low on LTP PAR1-AP consists of a short amino-acid sequence and its mechanism of PAR1 activation (i.e.
MA
direct interaction with its ligand binding site) differs from the one of endogenous proteases. In this respect, [PAR1-AP]low–mediated enhancement of LTP could be due to a peculiar property of
D
the drug which at specific concentrations may act as a general agonist, thus causing this effect. In order to exclude this possibility, we tested whether other endogenous PAR1 agonists could
TE
mimic the effects of [PAR1-AP]low on LTP.
AC CE P
Plasmin has been shown to activate PAR1 both in peripheral tissues and in the brain where it potentiates NMDA receptors currents (Mannaioni, et al., 2008). In hippocampal slices, application of 1U/ml plasmin ([Plasmin]high) for 15 minutes induced a gradual increase in population EPSP at stratum radiatum CA1 over 40 minutes of recording. This potentiation reached a plateau level (1.75±0.059; n=11 slices; Fig.3A) similar to the one HFS induced in the alternate path prior to drug application (1.81±0.053; P=0.46; Fig.3A). Like [Thrombin]low, exposure to 100mU/ml plasmin ([Plasmin]low) for 15 minutes neither generated a slow onset LTP nor enhanced LTP evoked by HFS at a second pathway following drug removal (1.77±0.073; n=11 slices; Fig.3B). Finally, as in the case of [Thrombin]high, the [Plasmin]high-mediated slow onset LTP was due to PAR1 activation as it was completely blocked in presence of SCH (1.02±0.076; n=11 slices; Fig.3C). These experiments show that plasmin and thrombin share similar outcomes on LTP and do not mimic the effects of [PAR1-AP]low. In peripheral tissues as well as in the blood coagulation cascade, it has been shown that activation of PAR1 by aPC results in different, somewhat opposite effects than those of thrombin induced PAR1 activation (Feistritzer and Riewald, 2005, Ludeman, et al., 2005, Riewald and Ruf, 2005). Hence, we tested whether aPC could mediate the enhancement of LTP
ACCEPTED MANUSCRIPT induced by [PAR1-AP]low. Following the induction of LTP in the first pathway, 1M aPC was bath applied for 15 minutes (Fig. 3D). Surprisingly, aPC did not increase basal synaptic transmission, however an enhanced LTP was evoked when HFS was delivered at the second pathway after
PT
the washout of the drug (1.81±0.074 and 2.21±0.065 at 75min of recordings for the first and second pathway respectively; P< 0.001; n=12 slices; Fig.3D). Application of 100nM aPC led to
RI
the same result (Fig.3E). PAR1 activation was in charge of this effect as aPC in presence of SCH was not able to induce an enhanced LTP (1.83±0.083 and 1.84±0.076 at 75min of
SC
recordings for the first and second pathway respectively; P=0.54; n=12 slices; Fig.3F). These experiments indicate that aPC mimics the effects of [PAR1-AP]low and causes enhanced LTP in
NU
stratum radiatum of CA1.
The Endothelial Protein C Receptor (EPCR) is Expressed in the Hippocampus,
MA
Colocalizes with PAR1 and Mediates the Effects of aPC. aPC is known to activate PAR1 upon binding its own receptor EPCR (Rezaie, Riewald and Ruf,
D
2005). Recently, studies addressing a possible neuroprotective role of aPC following glutamate toxicity have shown that EPCR is expressed in cortical and hippocampal neurons in cell cultures
TE
(Gorbacheva, et al., 2009). Besides, to our knowledge, the exact location of this receptor in the hippocampus has not been studied so far. As a first attempt to test whether EPCR may be
AC CE P
involved in the aPC induced enhancement of LTP, we qualitatively mapped its expression in the hippocampus using immunofluorescence. EPCR was found to be widely distributed in the whole hippocampus (Fig.4A). In CA1 it was localized both in neurons (Fig.4B) and astrocytes (Fig.4C). EPCR has been reported to activate PAR1 through a physical interaction between the two receptors (Riewald and Ruf, 2005), and indeed a confocal colocalization study found that EPCR and PAR1 were highly co-expressed in the same CA1 cells (Fig.4D). A second step towards the understanding of the role of EPCR in aPC- mediated phenomena consists of testing the ability of aPC to enhance LTP in presence of EPCR blockers. Taking into account that a pharmacological approach to reliably block these receptors has not been developed as yet, we tried to tackle this issue by ICV injections of EPCR blocking antibodies (R-252) and consequent use of hippocampal slices from these animals. In these experiments, slices were cut 36 hours after recovery from the ICV injection procedures. EPCR blockade did not influence the ability of the slice to undergo normal LTP. Applying HFS to the first pathway resulted in a full blown LTP (1.68±0.064; n=13 slices; Fig.5A). Remarkably, however, HFS delivered at the second pathway in presence of 100nM aPC evoked a potentiation similar to the one in the control pathway (1.70±0.066; P=0.49; n=13 slices; Fig.5A), therefore inhibiting the aPC effect. Differently, EPCR
ACCEPTED MANUSCRIPT block did not impair the effects of PAR1-AP. In this setting, [PAR1-AP]low still enhanced LTP compared to control conditions (1.70±0.065 and 2.18±0.074 at 75min of recordings for the first and second pathway respectively; P<0.001; n=13 slices; Fig.5B). Likely, [PAR1-AP]high
PT
produced a slow onset LTP in EPCR blockade condition. These experiments indicate that EPCR mediates the effects of aPC acting most likely upstream to PAR1.
RI
aPC Induces NMDA Independent, VGCCs and mGluR-5 Dependent LTP
SC
In order to understand the mechanisms leading to aPC mediated LTP, slices were treated with aPC in presence of the NMDA blocker APV. 50M APV were bath applied right after HFS on the
NU
first pathway. 15 minutes later 100nM aPC was added to the medium. Interestingly, under NMDA blocking condition aPC was still able to evoke an enhanced LTP when HFS was delivered at the second path (1.83±0.076 and 2.21±0.074; at 75min of recordings for the first
MA
and second pathway respectively; P<0.001; n=12 slices; Fig.6A). Considerably, APV was able to block LTP in the first, control pathway, but did not impair the aPC enhanced LTP (0.99±0.071
D
and 2.22±0.078; at 75min of recordings for the first and second pathway respectively; P<0.001; n=9 slices; Fig.6B). We then tested whether two additional key players in synaptic plasticity
TE
such as VGCCs or mGluR-5 would have a role in mediating aPC induced LTP. Surprisingly both VGCCs and mGluR-5 antagonists blocked the ability of aPC to induce enhanced LTP. In one
AC CE P
experiment, bath applying aPC in presence of 20M nifedipine led to a normal potentiation following HFS delivery to a second path (1.82±0.081 and 1.78±0.082; at 75min of recordings for the first and second pathway respectively; P= 0.34; n=12 slices; Fig.6C). Similarly, a normal level of potentiation was evoked following HFS at the second pathway when aPC was bath applied in presence of 10M of the mGluR-5 antagonist MPEP (1.79±0.085 and 1.84±0.082 at 75min of recordings for the first and second pathway respectively; P=0.31; n=12 slices; Fig.6D). These experiments show that aPC induces an NMDA- independent, VGCCs and mGluR-5 dependent LTP. Calcium Stores are Involved in aPC Mediated LTP aPC has been shown to modulate calcium release from intracellular stores through a EPCRPAR1 dependent mechanism (Domotor, et al., 2003). In order to test whether aPC mediated LTP is dependent on calcium stores, we bath applied aPC with Thapsigargin, an inhibitor of endoplasmatic reticulum Ca2+ ATPases (Maggio and Segal, 2007). Remarkably, aPC in presence of 1M Thapsigargin was not able to facilitate LTP; HFS at the second pathway evoked a lower LTP than that at the control path (1.87±0.083 and 1.53±0.086 at the onset
ACCEPTED MANUSCRIPT following HFS for the first and second pathway, respectively; n=14 slices, P<0.001; Fig.7A). In addition, thapsigargin even affected the LTP evoked at the first, control pathway as a slow decrease in the established synaptic potentiation was observed following the exposure of the
PT
slice to this drug (1.66±0.076 at 80min compared to 1.87±0.083 right after HFS; P<0.001; Fig.7A). We further explored these observations using cyclopiazonic acid (CPA), a blocker of
RI
endoplasmic reticulum Ca2+ ATPases. Bath application of 20M CPA blocked the potentiation facilitated by aPC (1.87±0.086 and 1.52±0.082 at the onset following HFS for the first and
SC
second pathway, respectively; n=14 slices, P<0.001; Fig.7B). In addition, similarly to thapsigargin, CPA impaired the already established LTP at the control pathway. The notable
NU
advantage of CPA is the possibility to wash it out from the medium. This allows the refill of intracellular calcium stores and the recovery of their function. Remarkably, delivering HFS in
MA
presence of CPA and aPC resulted in a potentiation of 55% over baseline (Fig.7C). However, HFS applied in presence of aPC following CPA removal facilitated LTP (1.47±0.057 and 2.19±0.082 at 150 minutes of recording for the first and second pathway respectively; P<0.001;
D
Fig.7C). These experiments indicate that calcium stores are involved in aPC mediated LTP and
TE
confirm the role of these organelles both in the establishment and maintenance of LTP in normal conditions (Maggio and Segal, 2007).
AC CE P
Thrombin Enhances LTP by Promoting the Formation of aPC. In the coagulation cascade, thrombin contributes to the generation of aPC (Griffin, et al., 2007), this initiates a feedback loop that ultimately results in the attenuation of the thrombin signaling (Griffin, et al., 2007). In this respect, we assumed that [Thrombin]low could as well promote the formation of aPC in hippocampal slices. Indeed, if this might be the case, [Thrombin] low could facilitate LTP through an aPC mediated mechanism. In order to test this hypothesis, we exposed slices to inactivated Protein C (iPC), a compound that can be converted to aPC in presence of thrombin. In the first set of experiments, [Thrombin]low did not show any notable effect on LTP: application of this drug following HFS at the control pathway did not result in a facilitation of LTP when a second HFS was delivered at the second pathway (1.76±0.075 and 1.77±0.086 at 20min following delivery of HFS at the first and second pathway respectively; P=0.62; n=14; Fig.8A). Then, in a different set of slices, we bath applied 1M iPC following HFS at the first pathway. In this conditions, HFS delivered at the second pathway also produced a potentiation of similar levels to the one in the control path (1.75±0.083 and 1.74±0.076 at 20min following delivery of HFS at the first and second pathway respectively; P= 0.66; n=14; Fig.8B). Strikingly, however exposure of slices to [Thrombin]low and iPC led to a different outcome. Here
ACCEPTED MANUSCRIPT different solutions of [Thrombin]low and iPC were simultaneously perfused in the slice such to avoid possible iPC activation in the perfusing solution. In these experiments, HFS to the second channel in presence of [Thrombin]low and iPC led to facilitation of LTP compared to control
PT
conditions (1.77±0.085 and 2.28±0.065 at 20min following delivery of HFS at the first and second pathway respectively; P<0.001; n=14; Fig.8C). Interestingly, [Thrombin]low in presence of
RI
-NAPAP was unable to produce this result (Suppl. Fig. 1B). This effect is peculiar to [Thrombin]low and not shared by [Thrombin]high. Notably, perfusing [Thrombin]high in presence of
SC
APV neither evoked an enhanced LTP (Suppl. Fig.2) nor facilitated LTP following simultaneous perfusion with iPC. These experiments demonstrate that [Thrombin]low is likely to facilitate LTP
NU
by promoting aPC formation in hippocampal slices. Discussion
MA
In this study, we addressed the role of thrombin in modulating LTP. Interestingly, we observed that different concentrations of thrombin led to diverse outcomes (Fig.9). [Thrombin]high induced a NMDA dependent, slow onset LTP while [Thrombin]low promoted a VGCCs, mGluR-5
D
dependent LTP through the activation of aPC (Fig.9). aPC facilitated LTP by activating PAR1
TE
through an EPCR-mediated mechanism and involvement of intracellular calcium stores. The highest concentrations of thrombin in the brain occur during brain hemorrhages (Gong, et
AC CE P
al., 2008, Hua, et al., 2009, Hua, et al., 2007) however high levels of thrombin have been reported following ischemic stroke (Chen, et al., 2010, Chen, et al., 2012). In these settings, BBB opening and rupture of brain endothelial cells activate the coagulation cascade leading to intracerebral production of thrombin (Chodobski, et al., 2012). Interestingly, neurological deficits in acute ischemic stroke animal models may be attenuated following thrombin blockade (Chen, et al., 2010, Chen, et al., 2012, Karabiyikoglu, et al., 2004). Our present data as well as our previous report (Maggio, et al., 2008) show that [Thrombin]high evokes a NMDA dependent LTP which saturates the ability of a neuronal network to undergo further NMDA dependent potentiation. This result might explain the inability to recover from a cerebrovascular trauma: the saturation of synaptic connectivity by thrombin-activated mechanisms does not allow the brain to use LTP-like plastic processes for either acquisition of new “memories” or adaptation to new motor plans after the insult (Maggio, et al., 2008). Definitely, additional experiments are required to explore the duration of thrombin action in the brain as well as the ways to overcome thrombin-related cognitive deficits. In this respect, it could be interesting to follow on whether patients discharged on dabigatran, a new thrombin direct inhibitor, would have better long term cognitive outcomes following stroke than other patients released on alternative anticoagulants.
ACCEPTED MANUSCRIPT There are suggestions that thrombin may be produced during normal synaptic transmission (Turgeon, et al., 2000), yet its exact amount at the synaptic cleft under physiological conditions is unknown. In the blood stream, thrombin has a short half life and its concentration is tightly
PT
regulated by multiple processes involving endogenous proteases (Siller-Matula, et al., 2011). Definitely, a similar system might exist in the brain in order to balance thrombin concentrations
RI
at the synapse during physiological events. Therefore, depending on the availability of this system, different concentrations of thrombin might arise at the synapse following a determined
SC
synaptic event and hence differently influence LTP (Komai et al., 2000; Maggio et al. 2008). In this study, we also showed the unique role of aPC in regulating LTP. Interestingly, the effects
NU
of aPC are concentration independent, therefore the presence of aPC at the synaptic cleft might shift by itself the threshold of synaptic plasticity towards novel LTP facilitation in a previously
MA
potentiated network. While the aPC receptor EPCR seems to be widely expressed in the hippocampus, it is not entirely clear whether in the brain the production of aPC exclusively depends on the thrombin-mediated conversion of iPC or it may be locally synthesized de novo.
D
In the blood stream, PC seems to circulate in its inactive, zymogen form and the switch into its
TE
active state occurs following its interaction with thrombin (Griffin, et al., 2007). Clarifying this issue in the brain might lead to important findings for the further understanding of the mechanisms regulating synaptic plasticity. However, as the case may be, this study, our
AC CE P
previous (Maggio, et al., 2008) as well as other evidences (Dihanich, et al., 1991, Gingrich, et al., 2000, Shikamoto and Morita, 1999) point towards a role of clotting factors in the regulation of synaptic plasticity. Lately, aPC has received attentions due to its cytoprotective and anticoagulant properties which may be of benefit in stroke therapy (Griffin, et al., 2012, Wang, et al., 2012, Zlokovic and Griffin, 2011). Our data further promote these positive properties of aPC. Precisely, the evidence that in the brain aPC enhances LTP through alternative pathways requiring VGCCs and mGLUR-5 may result in better cognitive outcomes in cases where NMDA receptors are saturated by thrombin. Definitely, more data on the role of aPC in stroke are needed to confirm this hypothesis. The evidence that both thrombin and aPC may exert opposite effects acting on the same receptor, i.e. PAR1, is puzzling indeed. In endothelial cells, it has been shown that PAR1 activation either by thrombin or aPC may lead to different, opposite outcomes due to the stimulation of Gq/G12/13 protein cascade in the former case and Gi in the latter (Riewald and Ruf, 2005). There might be a similar situation in the brain where PAR1 has been shown to interact with multiple G proteins (McCoy, et al., 2012). An additional hypothesis to explain these results may take into account the efficacy of different concentrations of PAR1-AP in producing diverse
ACCEPTED MANUSCRIPT effects. Specifically, it might be that two types of PAR1 bearing different affinities for the PAR1AP ligand might exist at the synapse. A low affinity PAR1 could directly be activated by thrombin and mediate the [Thrombin]high induced slow onset LTP while a high affinity receptor, not
PT
promptly cleaved by thrombin, might be in charge of the aPC-EPCR effects. These two different receptors could be linked to different G proteins and be expressed in diverse cell types
RI
(astrocytes vs. neurons). Additional pharmacological and molecular studies are needed in order to validate this hypothesis.
SC
In the present study, we have not clearly addressed whether the effects of thrombin and aPC are mediated by the neuronal and/or astrocytic PAR1. EPCR localization by itself does not add
NU
any further hint to this quest as it seems that both EPCR and PAR1 are colocalized in the same cells in the whole hippocampus. Nevertheless, the cellular localization of the PAR1 signaling
MA
seems to be a fundamental issue in order to fully understand the contribution of this receptor to synaptic plasticity. On the one hand, PAR1 knock out animals might not be an appropriate tool to utilize in solving this issue as signals from both cell types are being deleted in these animals.
D
On the other hand, the use of molecular techniques that will downregulate PAR1 expression in
TE
specific cell types is more promising and might lead to new fundamental insights into the role of this receptor in synaptic plasticity.
In summary, we have shown that thrombin may differently regulate synaptic plasticity based on
AC CE P
its concentration levels at the synaptic cleft. We believe this represents a novel mechanism by which PAR1 may regulate the threshold for synaptic plasticity in the hippocampus and provides additional insights on the role of this receptor in physiological vs. pathological conditions.
References
1. 2.
3. 4. 5.
Chen, B., Cheng, Q., Yang, K., and Lyden, P. D., 2010. Thrombin mediates severe neurovascular injury during ischemia. Stroke 41, 2348-2352. Chen, B., Friedman, B., Whitney, M. A., Winkle, J. A., Lei, I. F., Olson, E. S., Cheng, Q., Pereira, B., Zhao, L., Tsien, R. Y., and Lyden, P. D., 2012. Thrombin activity associated with neuronal damage during acute focal ischemia. J Neurosci 32, 7622-7631. Chodobski, A., Zink, B. J., and Szmydynger-Chodobska, J., 2012. Blood-brain barrier pathophysiology in traumatic brain injury. Transl Stroke Res 2, 492-516. Dihanich, M., Kaser, M., Reinhard, E., Cunningham, D., and Monard, D., 1991. Prothrombin mRNA is expressed by cells of the nervous system. Neuron 6, 575-581. Domotor, E., Benzakour, O., Griffin, J. H., Yule, D., Fukudome, K., and Zlokovic, B. V., 2003. Activated protein C alters cytosolic calcium flux in human brain endothelium via binding to endothelial protein C receptor and activation of protease activated receptor-1. Blood 101, 4797-4801.
ACCEPTED MANUSCRIPT
12. 13. 14. 15.
16.
17.
18. 19.
20.
21.
22.
23. 24.
PT
RI
SC
11.
NU
10.
MA
9.
D
8.
TE
7.
Feistritzer, C., and Riewald, M., 2005. Endothelial barrier protection by activated protein C through PAR1-dependent sphingosine 1-phosphate receptor-1 crossactivation. Blood 105, 3178-3184. Gingrich, M. B., Junge, C. E., Lyuboslavsky, P., and Traynelis, S. F., 2000. Potentiation of NMDA receptor function by the serine protease thrombin. J Neurosci 20, 4582-4595. Gong, Y., Xi, G., Hu, H., Gu, Y., Huang, F., Keep, R. F., and Hua, Y., 2008. Increase in brain thrombin activity after experimental intracerebral hemorrhage. Acta Neurochir Suppl 105, 47-50. Gorbacheva, L., Davidova, O., Sokolova, E., Ishiwata, S., Pinelis, V., Strukova, S., and Reiser, G., 2009. Endothelial protein C receptor is expressed in rat cortical and hippocampal neurons and is necessary for protective effect of activated protein C at glutamate excitotoxicity. J Neurochem 111, 967-975. Griffin, J. H., Fernandez, J. A., Gale, A. J., and Mosnier, L. O., 2007. Activated protein C. J Thromb Haemost 5 Suppl 1, 73-80. Griffin, J. H., Zlokovic, B. V., and Mosnier, L. O., 2012. Protein C anticoagulant and cytoprotective pathways. Int J Hematol 95, 333-345. Hua, Y., Keep, R. F., Gu, Y., and Xi, G., 2009. Thrombin and brain recovery after intracerebral hemorrhage. Stroke 40, S88-89. Hua, Y., Keep, R. F., Hoff, J. T., and Xi, G., 2007. Brain injury after intracerebral hemorrhage: the role of thrombin and iron. Stroke 38, 759-762. Isaeva, E., Hernan, A., Isaev, D., and Holmes, G. L., 2012. Thrombin facilitates seizures through activation of persistent sodium current. Ann Neurol 72, 192-198. Junge, C. E., Lee, C. J., Hubbard, K. B., Zhang, Z., Olson, J. J., Hepler, J. R., Brat, D. J., and Traynelis, S. F., 2004. Protease-activated receptor-1 in human brain: localization and functional expression in astrocytes. Exp Neurol 188, 94-103. Karabiyikoglu, M., Hua, Y., Keep, R. F., Ennis, S. R., and Xi, G., 2004. Intracerebral hirudin injection attenuates ischemic damage and neurologic deficits without altering local cerebral blood flow. J Cereb Blood Flow Metab 24, 159-166. Komai, S., Matsuyama T., Matsumoto K., Kato K., Kobayashi M., Imamura K., Yoshida S., Ugawa S., Shiosaka S., 2000. Neuropsin regulates an early phase of schaffercollateral long term potentiation in the murine hippocampus. Eur. J. Neurosc.; 12(4): 1479-86. Lee, C. J., Mannaioni, G., Yuan, H., Woo, D. H., Gingrich, M. B., and Traynelis, S. F., 2007. Astrocytic control of synaptic NMDA receptors. J Physiol 581, 1057-1081. Ludeman, M. J., Kataoka, H., Srinivasan, Y., Esmon, N. L., Esmon, C. T., and Coughlin, S. R., 2005. PAR1 cleavage and signaling in response to activated protein C and thrombin. J Biol Chem 280, 13122-13128. Luo, W., Wang, Y., and Reiser, G., 2007. Protease-activated receptors in the brain: receptor expression, activation, and functions in neurodegeneration and neuroprotection. Brain Res Rev 56, 331-345. Maggio, N., Cavaliere, C., Papa, M., Blatt, I., Chapman, J., and Segal, M., 2012. Thrombin regulation of synaptic transmission: Implications for seizure onset. Neurobiol Dis. Maggio, N., and Segal, M., 2007. Striking variations in corticosteroid modulation of longterm potentiation along the septotemporal axis of the hippocampus. J Neurosci 27, 5757-5765. Maggio, N., and Segal, M., 2007. Unique regulation of long term potentiation in the rat ventral hippocampus. Hippocampus 17, 10-25. Maggio, N., Shavit, E., Chapman, J., and Segal, M., 2008. Thrombin induces long-term potentiation of reactivity to afferent stimulation and facilitates epileptic seizures in rat
AC CE P
6.
ACCEPTED MANUSCRIPT
28.
29.
30.
33.
34. 35.
36.
37. 38.
PT
RI
AC CE P
32.
TE
D
31.
SC
27.
NU
26.
MA
25.
hippocampal slices: toward understanding the functional consequences of cerebrovascular insults. J Neurosci 28, 732-736. Mannaioni, G., Orr, A. G., Hamill, C. E., Yuan, H., Pedone, K. H., McCoy, K. L., Berlinguer Palmini, R., Junge, C. E., Lee, C. J., Yepes, M., Hepler, J. R., and Traynelis, S. F., 2008. Plasmin potentiates synaptic N-methyl-D-aspartate receptor function in hippocampal neurons through activation of protease-activated receptor-1. J Biol Chem 283, 20600-20611. McCoy, K. L., Gyoneva, S., Vellano, C. P., Smrcka, A. V., Traynelis, S. F., and Hepler, J. R., 2012. Protease-activated receptor 1 (PAR1) coupling to G(q/11) but not to G(i/o) or G(12/13) is mediated by discrete amino acids within the receptor second intracellular loop. Cell Signal 24, 1351-1360. Mosnier, L. O., Zlokovic, B. V., and Griffin, J. H., 2007. The cytoprotective protein C pathway. Blood 109, 3161-3172. Rezaie, A. R., The occupancy of endothelial protein C receptor by its ligand modulates the par-1 dependent signaling specificity of coagulation proteases. IUBMB Life 63, 390396. Riewald, M., and Ruf, W., 2005. Protease-activated receptor-1 signaling by activated protein C in cytokine-perturbed endothelial cells is distinct from thrombin signaling. J Biol Chem 280, 19808-19814. Shikamoto, Y., and Morita, T., 1999. Expression of factor X in both the rat brain and cells of the central nervous system. FEBS Lett 463, 387-389. Siller-Matula, J. M., Schwameis, M., Blann, A., Mannhalter, C., and Jilma, B., 2011. Thrombin as a multi-functional enzyme. Focus on in vitro and in vivo effects. Thromb Haemost 106, 1020-1033. Sokolova, E., and Reiser, G., 2008. Prothrombin/thrombin and the thrombin receptors PAR-1 and PAR-4 in the brain: localization, expression and participation in neurodegenerative diseases. Thromb Haemost 100, 576-581. Striggow, F., Riek, M., Breder, J., Henrich-Noack, P., Reymann, K. G., and Reiser, G., 2000. The protease thrombin is an endogenous mediator of hippocampal neuroprotection against ischemia at low concentrations but causes degeneration at high concentrations. Proc Natl Acad Sci U S A 97, 2264-2269. Turgeon, V. L., Salman, N., and Houenou, L. J., 2000. Thrombin: a neuronal cell modulator. Thromb Res 99, 417-427. Wang, J., Jin, H., Hua, Y., Keep, R. F., and Xi, G., 2012. Role of protease-activated receptor-1 in brain injury after experimental global cerebral ischemia. Stroke 43, 24762482. Wang, Y., Zhang, Z., Chow, N., Davis, T. P., Griffin, J. H., Chopp, M., and Zlokovic, B. V., 2012. An activated protein C analog with reduced anticoagulant activity extends the therapeutic window of tissue plasminogen activator for ischemic stroke in rodents. Stroke 43, 2444-2449. Yang, Y., and Rosenberg, G. A., 2011. Blood-brain barrier breakdown in acute and chronic cerebrovascular disease. Stroke 42, 3323-3328. Zlokovic, B. V., and Griffin, J. H., 2011. Cytoprotective protein C pathways and implications for stroke and neurological disorders. Trends Neurosci 34, 198-209.
Figures Legends:
ACCEPTED MANUSCRIPT Figure 1: [Thrombin]high induce slow onset, NMDA-dependent LTP in hippocampal slices. (A, B) Short application (12 min) of thrombin (1U/ml, [Thrombin]high; A) as well as of PAR1-AP (1M, [PAR1-AP]high; B) produces a gradual increase in population EPSP in stratum radiatum CA1
PT
without affecting population synaptic volleys (traces on top). (C) Thrombin induced slow onset LTP is blocked by the PAR1 antagonist SCH79797 (1M). (D) The NMDA antagonist APV
RI
(50M) totally blocks thrombin induced slow onset LTP. Averaged EPSP are plotted versus time. Representative traces at indicated times (a,b) are shown on top of each section. Upward
SC
arrows indicate the time of HFS.
Figure 2: Thrombin and PAR1-AP effects on LTP are concentration dependent. (A) Short
NU
application (15min) of thrombin (100mU/ml, [Thrombin]low) does not induce a slow onset LTP. As well, it does not affect the level of a tetanus induced LTP evoked at the second pathway 20
MA
minutes after drug removal. (B) Short application (15min) of PAR1-AP (100nM, [PAR1-AP]low) does not induce a slow onset LTP, however it enhances the height of a tetanus induced LTP evoked at the second pathway 20 minutes after drug removal. (C) The PAR1 antagonist SCH
D
79797 blocks the [PAR1-AP]low–mediated enhancement of LTP. Averaged EPSP are plotted
TE
versus time. Representative traces at indicated times (a,b) are shown on top of each section. Upward arrows indicate the time of HFS.
AC CE P
Figure 3: aPC shares the effects of [PAR1-AP]low on LTP. (A) Short application (15min) of plasmin (1U/ml, [Plasmin]high) induces a gradual increase in population EPSP in stratum radiatum CA1 without affecting population synaptic volleys (traces on top). (B) Short application (15min) of plasmin (100mU/ml) nor induces a slow onset LTP neither affects the level of a tetanus induced LTP evoked at the second pathway 20 minutes after drug removal. (C) The PAR1 antagonist SCH 79797 blocks the [Plasmin]high–slow onset LTP. (D, E) Short application (15min) of aPC (1M D; 100nM E) does not induce a slow onset LTP, however it enhances the level of a tetanus induced LTP evoked at the second pathway 20 minutes following drug removal. (F) The PAR1 antagonist SCH 79797 blocks the aPC mediated enhancement of LTP. Averaged EPSP are plotted versus time. Representative traces at indicated times (a,b) are shown on top of each section. Upward arrows indicate the time of HFS. Figure 4: The aPC receptor, EPCR, is expressed on neurons, on astrocytes and colocalizes with PAR1. (A) EPCR is widely expressed throughout the hippocampal fields (NeuN, red, EPCR, green; 10x) and is localized in neurons (B, NeuN, red, EPCR, green; 40x) and astrocytes (C, GFAP, red, EPCR, green; 40x). (D) PAR1 (red) and EPCR (green) colocalize in CA1 (40x). The white arrow in A indicates the area of CA1 where the pictures in B, C, D were taken.
ACCEPTED MANUSCRIPT Figure 5: EPCR blockade prevents the aPC-mediated, but not the [PAR1-AP]low-mediated enhancement of LTP. In slices from anti-EPCR ICV-injected animals application of aPC does not enhance LTP (A). Contrarily, in this setting both [PAR1-AP]high (B) and [PAR1-AP]low (C) are
PT
able to induce a slow onset LTP or to enhance the level of a tetanus induced LTP evoked at the second pathway, respectively. Averaged EPSP are plotted versus time. Representative traces
RI
at indicated times (a,b) are shown on top of each section. Upward arrows indicate the time of HFS.
SC
Figure 6: aPC induces a NMDA-independent, VGCCs and mGluR5-dependent LTP. (A) The NMDA antagonist APV (50M) does not block the aPC-induced LTP. (B) APV blocks LTP in the
NU
control pathway, but it does not affect aPC-induced LTP. (C) The Voltage Gated Calcium Channels blocker nifedipine (20M) inhibits the aPC-induced LTP. (D) The mGluR5 blocker
MA
MPEP (10M) hampers the aPC- induced LTP. Averaged EPSP are plotted versus time. Representative traces at indicated times (a,b) are shown on top of each section. Upward arrows indicate the time of HFS.
D
Figure 7: Calcium stores are involved in aPC induced LTP. (A, B) Both the irreversible
TE
(thapsigargin, 1M; A) and the reversible (cyclopiazonic acid, CPA, 20M; B) SERCA pump inhibitors prevent the aPC-induced LTP. (C) HFS in presence of aPC after CPA washout
AC CE P
restores aPC-induced LTP. Averaged EPSP are plotted versus time. Representative traces at indicated times (a,b) are shown on top of each section. Upward arrows indicate the time of HFS. Figure 8: [Thrombin]low enhance LTP through aPC. (A, B) [Thrombin]low (A) as well as inactivated Protein C (iPC; B) nor induce a slow onset LTP neither affect the level of a tetanus induced LTP evoked at the second pathway. (C) [Thrombin]low in presence of iPC enhance the level of a tetanus induced LTP evoked at the second pathway. Averaged EPSP are plotted versus time. Representative traces at indicated times (a,b) are shown on top of each section. Upward arrows indicate the time of HFS. Figure 9: Diverse thrombin concentrations differently regulate LTP. [Thrombin]high induce a NMDA dependent, slow onset LTP while [Thrombin]low promote a VGCCs, mGluRs dependent LTP through the activation of aPC. aPC facilitates LTP by activating PAR-1 through an EPCRmediated mechanism and involvement of intracellular calcium stores. Supplementary Figure 1: The thrombin inhibitor -NAPAP blocks the effects of thrombin on LTP. (A) [Thrombin]high in presence of 1M -NAPAP did not cause a slow onset LTP. Notably, -NAPAP did not have any effect on the tetanus-induced LTP. (B) [Thrombin]low in presence of 1M -NAPAP did not result in an enhanced LTP upon perfusion of iPC. Averaged EPSP are
ACCEPTED MANUSCRIPT plotted versus time. Representative traces at indicated times (a,b) are shown on top of each section. Upward arrows indicate the time of HFS. Supplementary Figure 2: Different thrombin concentrations have dissimilar effects on LTP. (A)
PT
In presence of APV, [Thrombin]high neither caused a slow onset LTP, nor evoked an enhanced LTP. (B) In presence of APV and iPC, [Thrombin]high did not enhance LTP upon delivery of
RI
tetanic stimulation. Averaged EPSP are plotted versus time. Representative traces at indicated
AC CE P
TE
D
MA
NU
SC
times (a,b) are shown on top of each section. Upward arrows indicate the time of HFS.
AC CE P
TE
D
MA
NU
SC
RI
PT
ACCEPTED MANUSCRIPT
Figure 1
NU
SC
RI
PT
ACCEPTED MANUSCRIPT
AC CE P
TE
D
MA
Figure 2
Figure 3
AC CE P
TE
D
MA
NU
SC
RI
PT
ACCEPTED MANUSCRIPT
AC CE P
Figure 4
TE
D
MA
NU
SC
RI
PT
ACCEPTED MANUSCRIPT
MA
NU
SC
RI
PT
ACCEPTED MANUSCRIPT
AC CE P
TE
D
Figure 5
MA
NU
SC
RI
PT
ACCEPTED MANUSCRIPT
AC CE P
TE
D
Figure 6
NU
SC
RI
PT
ACCEPTED MANUSCRIPT
AC CE P
TE
D
MA
Figure 7
NU
SC
RI
PT
ACCEPTED MANUSCRIPT
AC CE P
TE
D
MA
Figure 8
NU
SC
RI
PT
ACCEPTED MANUSCRIPT
AC CE P
TE
D
MA
Figure 9
ACCEPTED MANUSCRIPT Highlights: Thrombin levels regulate LTP through diverse molecular routes converging on PAR1.
[Thrombin]high induces a slow onset LTP.
[Thrombin]low enhances LTP by activating Protein C.
Protein C mediates LTP through VGCCs and mGluRs.
AC CE P
TE
D
MA
NU
SC
RI
PT
S0014-4886(13)00064-2 doi: 10.1016/j.expneurol.2013.02.011 YEXNR 11387
To appear in:
Experimental Neurology
Received date: Revised date: Accepted date:
16 November 2012 24 January 2013 18 February 2013
Please cite this article as: Maggio, Nicola, Itsekson, Zeev, Dominissini, Dan, Blatt, Ilan, Amariglio, Ninette, Rechavi, Gideon, Tanne, David, Chapman, Joab, Thrombin regulation of synaptic plasticity: Implications for physiology and pathology?, Experimental Neurology (2013), doi: 10.1016/j.expneurol.2013.02.011
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Experimental Neurology Regular Article
PT
24/01/2013
RI
Title: Thrombin regulation of synaptic plasticity: implications for physiology and pathology? Authors: Nicola Maggio1,2*, Zeev Itsekson2, Dan Dominissini3,4, Ilan Blatt2,5, Ninette Amariglio3,
SC
Gideon Rechavi3,4, David Tanne2,5 and Joab Chapman2,5.
NU
Affiliations:
1- Talpiot Medical Leadership Program, The Chaim Sheba Medical Center, 52621 Tel
MA
HaShomer, Israel.
2- Department of Neurology, The J. Sagol Neuroscience Center, The Chaim Sheba Medical Center, 52621 Tel HaShomer, Israel.
D
3- Cancer Research Center, The Sheba Medical Center, 52621 Tel HaShomer, Israel.
TE
4- Sackler School of Medicine, Tel Aviv University, 69978 Tel Aviv, Israel. 5- Department of Neurology, Sackler School of Medicine, Tel Aviv University, 69978 Tel
AC CE P
Aviv, Israel.
*Correspondence should be addressed to: Nicola Maggio, MD, PhD, Department of Neurology, The Chaim Sheba Medical Center, 52621 Tel HaShomer, Israel. Phone: +972547296984. Fax: +97235304752. email: [email protected]
ACCEPTED MANUSCRIPT Abstract: Thrombin, a serine protease involved in the coagulation cascade has been recently shown to affect neuronal function following blood brain barrier breakdown. Several lines of evidence have
PT
shown that thrombin may exist in the brain parenchyma under normal physiological conditions, yet its role in normal brain functions and synaptic transmission has not been established. In an
RI
attempt to shed light on the physiological functions of thrombin and Protease Activated
SC
Receptor 1 (PAR1) in the brain, we studied the effects of thrombin and a PAR1 agonist on long term potentiation (LTP) in mice hippocampal slices. Surprisingly, different concentrations of thrombin affect LTP through different molecular routes converging on PAR1. High thrombin
NU
concentrations induced a NMDA dependent, slow onset LTP, whereas low concentrations of thrombin promoted a VGCCs, mGluR-5 dependent LTP through activated Protein C (aPC).
MA
Remarkably, aPC facilitated LTP by activating PAR1 through an Endothelial Protein C Receptor (EPCR)-mediated mechanism which involves intracellular calcium stores. These findings reveal a novel mechanism by which PAR1 may regulate the threshold for synaptic plasticity in the
TE
Highlights:
AC CE P
pathological conditions.
D
hippocampus and provide additional insights into the role of this receptor in normal and
Thrombin levels regulate LTP through diverse molecular routes converging on PAR1.
[Thrombin]high induces a slow onset LTP.
[Thrombin]low enhances LTP by activating Protein C.
Protein C mediates LTP through VGCCs and mGluRs.
Keywords: Thrombin, PAR1, LTP, synaptic plasticity, hippocampus, extracellular proteases. Acknowledgements: The authors wish to acknowledge Dr. Eduard Korkotian for help with the imaging of the histological sections and Dr. Menahem Segal for comments on early drafts of the manuscript. NM is the recipient of a Talpiot Fellowship at the Sheba Medical Center. The funding agency did not have a role in study design; collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication. Conflict of interest: The authors declare no competing financial interests.
ACCEPTED MANUSCRIPT Introduction Cerebrovascular events induced by either ischemia or hemorrhage lead to blood brain barrier (BBB) breakdown and exposure of the brain to blood constituents (Yang and Rosenberg, 2011).
PT
Among others, thrombin, a serine protease involved in the coagulation cascade, has been shown to contribute to the stroke pathology following these conditions (Chen, et al., 2012,
RI
Wang, et al., 2012). High concentrations of thrombin in the brain also perturb normal physiology by saturating synaptic plasticity and inducing seizures (Isaeva, et al., 2012, Maggio, et al., 2012,
SC
Maggio, et al., 2008). These effects depend on the activation of the thrombin receptor, the Protease Activated Receptor 1 (PAR1) and consequent potentiation of NMDA receptors
NU
functions (Gingrich, et al., 2000, Maggio, et al., 2008). Previous studies, however, have shown that thrombin may exist in the brain parenchyma in normal physiological conditions (Turgeon, et
MA
al., 2000). Indeed, the mRNAs for both the thrombin precursor prothrombin and factor Xa, the enzyme converting prothrombin into thrombin, have been detected in several areas of the forebrain (Dihanich, et al., 1991, Shikamoto and Morita, 1999). Nevertheless, both the
D
mechanisms leading to the thrombin production in the brain under physiological conditions as
TE
well as its role in normal brain functions and synaptic transmission have not yet been completely clarified.
PAR1 belongs to a family of seven transmembrane domain, G protein-coupled receptors whose
AC CE P
activation requires the cleavage of a peptide bond at the N-terminal extracellular side which binds the second extracellular loop of the same receptor thus activating it (Sokolova and Reiser, 2008). In the brain PAR1 is expressed both in neurons and astrocytes (Junge, et al., 2004, Luo, et al., 2007). Its activation has been shown to modulate synaptic transmission and plasticity (Lee, et al., 2007), yet the specific contribution of the astrocytic vs. the neuronal receptor remains under investigation. In addition, while in peripheral organs PAR1 has been shown to be activated by a pool of proteases, e.g. activated Protein C (aPC), leading to different outcomes, (Mosnier, et al., 2007) the role of other PAR1 agonists in the brain has not been fully investigated. In an attempt to shed light on the physiological vs. pathological functions of thrombin and PAR1 in the brain, we studied the effects of different concentrations of thrombin and PAR1 agonist (PAR1-AP) on long term potentiation (LTP) using hippocampal slices. Surprisingly, we found that diverse thrombin concentrations differently regulate the threshold for synaptic plasticity in the hippocampus. These data provide additional insights into the role of this receptor in normal and pathological conditions.
ACCEPTED MANUSCRIPT Methods Drugs: The following drugs were prepared from frozen stocks: thrombin (Sigma Aldrich, Rehovot, Israel and Enzyme Research Laboratories, Swansea, England); PAR1 agonist (PAR-
PT
1AP, SFLLRN, Sigma Aldrich, Rehovot, Israel); PAR1 antagonist (SCH79797, Tocris Bioscience, Bristol, United Kingdom); Plasmin (Sigma Aldrich, Rehovot, Israel); activated
RI
Protein C (aPC, Sigma Aldrich, Rehovot, Israel); inactivated Protein C (iPC, Enzyme Research Laboratories, Swansea, England); APV (Sigma Aldrich, Rehovot, Israel); Nifedipine (Sigma
SC
Aldrich, Rehovot, Israel); MPEP (Tocris Bioscience, Bristol, United Kingdom); Thapsigargin (Alomone Labs, Jerusalem, Israel); Cyclopiazonic Acid (CPA, Alomone Labs, Jerusalem,
NU
Israel); -NAPAP (Sigma Aldrich, Rehovot, Israel). As the specific activity of the α-thrombin from various vendors ranged between 2700 and 3200 NIH U/mg by comparison to Lot K of the
MA
NIH standard, we estimated the concentration of active α-thrombin that corresponds to 1 U/ml activity, by calculating a conversion factor using pure α-thrombin (3200 U/mg), as previously described (Gingrich, et al., 2000). In this lot, manufacturer reported this protein to be >95% α-
D
thrombin as determined by gel electrophoresis, hence a solution with 1 U/ml α-thrombin should be 9 nM by a molecular weight for thrombin of 36.7 kDa. For the sake of simplicity, we used a
TE
conversion factor of 1 U/ml = 10 nM α-thrombin throughout the text to estimate the concentration of active α-thrombin (henceforth referred to as thrombin) from various vendors.
AC CE P
Electrophysiology: Animal handling was approved by the Institutional Animal Care and Use Committee, which adheres to the National law, and NIH rules. Briefly, 4-5 months old male C57BL/6 mice were rapidly decapitated and 350 μm coronal dorsal hippocampal slices were used. Slices were incubated for 1.5 h in a humidified, carbogenated (5% CO2 and 95% O2) gas atmosphere at 33 ± 1°C and were perfused with artificial CSF [containing (in mM) 124 NaCl, 2 KCl, 26 NaHCO3, 1.24 KH2PO4, 2.5 CaCl2, 2 MgSO4, and 10 glucose, pH 7.4] in a standard interface chamber. Recordings were made with a glass pipette containing 0.75 M NaCl (4 MΩ) placed in the stratum radiatum CA1. Stimulation was evoked using a Master 8 pulse stimulator (A.M.P.I., Jerusalem, Israel) and was delivered through two sets of bipolar nichrome electrodes placed on either side of the recording electrode such that two independent stimulation channels were used for each slice. The use of two parallel pathways allowed comparison of the effects of drug application in the same slice (Maggio and Segal, 2007). LTP was induced by highfrequency stimulation consisting of 100 pulses at twice the test intensity, delivered at a frequency of 100 Hz (HFS; 100 Hz, 1 s). Before applying the tetanic stimulation, baseline values were recorded at a frequency of 0.033 Hz. Responses were digitized at 5 kHz and stored on a computer. Off-line analysis and data acquisition were performed using the Spike 2 software
ACCEPTED MANUSCRIPT (CED, Cambridge, England). All numerical data are expressed as mean ± SEM, and EPSP slope changes after tetanic stimulation were calculated with respect to baseline. There were no systematic differences in the magnitudes of the baseline responses in the different conditions.
PT
All values reported refer to 30 min before tetanic stimulation. Unless otherwise indicated, statistical evaluations were performed by applying a Student's t test for paired and unpaired
RI
data, as the case may be (Origin 8.0). p values of <0.05 were considered a significant difference between means.
SC
Immunohistochemistry: The following antibodies were used for immunodetection: mouse antibodies raised against Neuronal nuclear antigen (NeuN) (1:100; Millipore, Billerica, MA,
NU
USA); rabbit antibodies to proteases activated receptor-1 (PAR1) (1:50; Abcam, Cambridge, UK); goat antibodies to endothelial protein C receptor (EPCR) (P20, 1:100; Santa Cruz Biotech.,
MA
CA, USA). Hippocampal sections (25m) were blocked in 10% normal serum in 0.01M PBS/ 0.25% Triton for 1 hr at room temperature (RT). After 48 hr. incubation at 4°C with the primary antibody (NeuN, PAR-1, and EPCR with 2% normal serum), sections were exposed to the
D
appropriate secondary antibody (1:500, DyLight flourophores -594; 488; 633, Thermo-Scientific,
TE
Rockford, IL, USA) for 1 hr and finally mounted and coverslipped with Flouromount (Southern Biotechnology). Slides were imaged with a Zeiss LSM 510 confocal microscope and data were
AC CE P
acquired and analyzed using a computer assisted image analysis system. Intracerebroventricular (ICV) injections: Mice were anesthetized with an intraperitoneal (IP) injection of ketamine (100 mg/kg) and xylazine (20 mg/kg). The skull was carefully exposed, and a small hole was drilled above the right lateral ventricle (2 mm lateral to the midline and 2.5 mm posterior to the bregma). A rat monoclonal antibody blocking EPCR (R252 clone, Sigma Aldrich, Rehovot, Israel) was injected at a concentration of 30 g/ml (2 mm depth) using a 27-gauge needle attached to a Hamilton syringe. Following the slow infusion of 1 l of antibody solution, the needle was withdrawn, and the skin over the scalp was sutured. Control mice were injected with a vehicle solution. Hippocampal slices were cut 36 hours following the procedure. Results Thrombin Induces Slow Onset LTP in CA1 Thrombin concentrations rise following BBB breakdown (Chen, et al., 2010, Chen, et al., 2012). Exposure of mice hippocampal slices to high concentration of thrombin (1U/ml thrombin; [Thrombin]high) for 12 minutes produced a gradual increase in population EPSP recorded in stratum radiatum of region CA1 of the hippocampus. This gradual change was specific to the
ACCEPTED MANUSCRIPT EPSP, because no parallel change was noticed in the presynaptic volley produced in response to the stimulation (Fig.1A). The increase in EPSP slope rose gradually over 40 min of recording, reaching a plateau level, which was 73% above control (n=11 slices; 1.73±0.78; P < 0.01),
PT
similar to that induced by tetanic stimulation (HFS) of the alternate pathway prior to drug application. Application of 2, 3, 5 and 10U/ml thrombin produced the same effect (data not
RI
shown). A comparable, slow-rising, persistent increase in population EPSP was seen after bath application of 1μM SFLLRN, a PAR1 receptor agonist ([PAR1-AP]high; n=11 slices; 1.62±0.071;
SC
P<0.01; Fig.1B). To verify that the effect of thrombin was mediated through activation of a genuine PAR1 receptor, the selective PAR1 antagonist SCH79797 (SCH) was used. SCH had
NU
no effect on established tetanic LTP (Fig.1C) (n=11; 1.84±0.079), yet the response to thrombin, tested in the non tetanized pathway, was completely abolished (0.99±0.066). In order to
MA
address whether thrombin-induced slow onset LTP shared a downstream mechanism with the conventional LTP, thrombin was applied in the presence of the NMDA receptor antagonist 2(R)APV (APV, 50μM). Under these conditions, thrombin was unable to produce LTP (0.97±0.062 at
D
25min following thrombin application; n=11 slices) (Fig.1D). Likely, the effect of thrombin was
TE
abolished in presence of its specific inhibitor, -NAPAP (Suppl. Fig.1A). These experiments confirm the results we previously obtained in rats (Maggio, et al., 2008), namely that the slow
AC CE P
onset LTP induced by [Thrombin]high involves NMDA receptors. The Effects of Thrombin and PAR-1AP are Concentration Dependent Thrombin produces a variety of effects in the brain being detrimental at high concentrations and protective at low concentrations (Hua, et al., 2009, Striggow, et al., 2000). In order to test whether the effects of thrombin on LTP are concentration dependent, we exposed the hippocampal slices to 100mU/ml thrombin ([Thrombin]low) for 15 minutes. In these experiments, thrombin was bath applied following the delivery of HFS at the first control pathway. At this concentration, thrombin did not affect the established LTP, yet it failed to induce a slow onset potentiation of EPSP at the second pathway (1.01±0.068 at 50min of recordings; Fig.2A). Furthermore, HFS at the second pathway, 20 minutes after drug removal, evoked an LTP of similar level to the one in the first, control pathway (n=12 slices; 1.66±0.059 vs. 1.64±0.063, respectively; P=0.43; Fig.2A). Thrombin concentrations ranging between 500mU/ml to 1mU/mL led to similar results (data not shown). Interestingly, however, exposure of slices to a lower concentration of PAR1-AP resulted in a novel, different phenomenon. Following the induction of LTP by HFS at the first pathway, the slices were exposed to 100nM PAR1-AP ([PAR1-AP]low) for 15 minutes. [PAR1-AP]low did not enhance synaptic transmission per se, yet 20 minutes after
ACCEPTED MANUSCRIPT drug withdrawal an enhanced LTP could be evoked by HFS at the second pathway (n=12 slices; 2.23±0.086; P<0.001 over LTP at the first pathway; Fig.2B). Similar data were obtained using concentrations of PAR1-AP ranging between 200nM to 10nM (data not shown). In order
PT
to verify that the genuine activation of PAR1 is required to produce an enhancement of LTP, [PAR1-AP]low was bath applied in presence of SCH. Remarkably, in this condition HFS at the
RI
second pathway generated a normal level of LTP comparable to the one obtained in the control pathway (n=12 slices; 1.79±0.77 vs. 1.75±0.084, respectively; P=0.39; Fig.2C). These
SC
experiments show that exposure to [PAR1-AP]low enhances LTP through a PAR1 mediated mechanism. This effect is specific to [PAR1-AP]low and is not shared by [Thrombin]low.
NU
activated Protein C (aPC) and not Plasmin Mimics the Effects of [PAR1-AP]low on LTP PAR1-AP consists of a short amino-acid sequence and its mechanism of PAR1 activation (i.e.
MA
direct interaction with its ligand binding site) differs from the one of endogenous proteases. In this respect, [PAR1-AP]low–mediated enhancement of LTP could be due to a peculiar property of
D
the drug which at specific concentrations may act as a general agonist, thus causing this effect. In order to exclude this possibility, we tested whether other endogenous PAR1 agonists could
TE
mimic the effects of [PAR1-AP]low on LTP.
AC CE P
Plasmin has been shown to activate PAR1 both in peripheral tissues and in the brain where it potentiates NMDA receptors currents (Mannaioni, et al., 2008). In hippocampal slices, application of 1U/ml plasmin ([Plasmin]high) for 15 minutes induced a gradual increase in population EPSP at stratum radiatum CA1 over 40 minutes of recording. This potentiation reached a plateau level (1.75±0.059; n=11 slices; Fig.3A) similar to the one HFS induced in the alternate path prior to drug application (1.81±0.053; P=0.46; Fig.3A). Like [Thrombin]low, exposure to 100mU/ml plasmin ([Plasmin]low) for 15 minutes neither generated a slow onset LTP nor enhanced LTP evoked by HFS at a second pathway following drug removal (1.77±0.073; n=11 slices; Fig.3B). Finally, as in the case of [Thrombin]high, the [Plasmin]high-mediated slow onset LTP was due to PAR1 activation as it was completely blocked in presence of SCH (1.02±0.076; n=11 slices; Fig.3C). These experiments show that plasmin and thrombin share similar outcomes on LTP and do not mimic the effects of [PAR1-AP]low. In peripheral tissues as well as in the blood coagulation cascade, it has been shown that activation of PAR1 by aPC results in different, somewhat opposite effects than those of thrombin induced PAR1 activation (Feistritzer and Riewald, 2005, Ludeman, et al., 2005, Riewald and Ruf, 2005). Hence, we tested whether aPC could mediate the enhancement of LTP
ACCEPTED MANUSCRIPT induced by [PAR1-AP]low. Following the induction of LTP in the first pathway, 1M aPC was bath applied for 15 minutes (Fig. 3D). Surprisingly, aPC did not increase basal synaptic transmission, however an enhanced LTP was evoked when HFS was delivered at the second pathway after
PT
the washout of the drug (1.81±0.074 and 2.21±0.065 at 75min of recordings for the first and second pathway respectively; P< 0.001; n=12 slices; Fig.3D). Application of 100nM aPC led to
RI
the same result (Fig.3E). PAR1 activation was in charge of this effect as aPC in presence of SCH was not able to induce an enhanced LTP (1.83±0.083 and 1.84±0.076 at 75min of
SC
recordings for the first and second pathway respectively; P=0.54; n=12 slices; Fig.3F). These experiments indicate that aPC mimics the effects of [PAR1-AP]low and causes enhanced LTP in
NU
stratum radiatum of CA1.
The Endothelial Protein C Receptor (EPCR) is Expressed in the Hippocampus,
MA
Colocalizes with PAR1 and Mediates the Effects of aPC. aPC is known to activate PAR1 upon binding its own receptor EPCR (Rezaie, Riewald and Ruf,
D
2005). Recently, studies addressing a possible neuroprotective role of aPC following glutamate toxicity have shown that EPCR is expressed in cortical and hippocampal neurons in cell cultures
TE
(Gorbacheva, et al., 2009). Besides, to our knowledge, the exact location of this receptor in the hippocampus has not been studied so far. As a first attempt to test whether EPCR may be
AC CE P
involved in the aPC induced enhancement of LTP, we qualitatively mapped its expression in the hippocampus using immunofluorescence. EPCR was found to be widely distributed in the whole hippocampus (Fig.4A). In CA1 it was localized both in neurons (Fig.4B) and astrocytes (Fig.4C). EPCR has been reported to activate PAR1 through a physical interaction between the two receptors (Riewald and Ruf, 2005), and indeed a confocal colocalization study found that EPCR and PAR1 were highly co-expressed in the same CA1 cells (Fig.4D). A second step towards the understanding of the role of EPCR in aPC- mediated phenomena consists of testing the ability of aPC to enhance LTP in presence of EPCR blockers. Taking into account that a pharmacological approach to reliably block these receptors has not been developed as yet, we tried to tackle this issue by ICV injections of EPCR blocking antibodies (R-252) and consequent use of hippocampal slices from these animals. In these experiments, slices were cut 36 hours after recovery from the ICV injection procedures. EPCR blockade did not influence the ability of the slice to undergo normal LTP. Applying HFS to the first pathway resulted in a full blown LTP (1.68±0.064; n=13 slices; Fig.5A). Remarkably, however, HFS delivered at the second pathway in presence of 100nM aPC evoked a potentiation similar to the one in the control pathway (1.70±0.066; P=0.49; n=13 slices; Fig.5A), therefore inhibiting the aPC effect. Differently, EPCR
ACCEPTED MANUSCRIPT block did not impair the effects of PAR1-AP. In this setting, [PAR1-AP]low still enhanced LTP compared to control conditions (1.70±0.065 and 2.18±0.074 at 75min of recordings for the first and second pathway respectively; P<0.001; n=13 slices; Fig.5B). Likely, [PAR1-AP]high
PT
produced a slow onset LTP in EPCR blockade condition. These experiments indicate that EPCR mediates the effects of aPC acting most likely upstream to PAR1.
RI
aPC Induces NMDA Independent, VGCCs and mGluR-5 Dependent LTP
SC
In order to understand the mechanisms leading to aPC mediated LTP, slices were treated with aPC in presence of the NMDA blocker APV. 50M APV were bath applied right after HFS on the
NU
first pathway. 15 minutes later 100nM aPC was added to the medium. Interestingly, under NMDA blocking condition aPC was still able to evoke an enhanced LTP when HFS was delivered at the second path (1.83±0.076 and 2.21±0.074; at 75min of recordings for the first
MA
and second pathway respectively; P<0.001; n=12 slices; Fig.6A). Considerably, APV was able to block LTP in the first, control pathway, but did not impair the aPC enhanced LTP (0.99±0.071
D
and 2.22±0.078; at 75min of recordings for the first and second pathway respectively; P<0.001; n=9 slices; Fig.6B). We then tested whether two additional key players in synaptic plasticity
TE
such as VGCCs or mGluR-5 would have a role in mediating aPC induced LTP. Surprisingly both VGCCs and mGluR-5 antagonists blocked the ability of aPC to induce enhanced LTP. In one
AC CE P
experiment, bath applying aPC in presence of 20M nifedipine led to a normal potentiation following HFS delivery to a second path (1.82±0.081 and 1.78±0.082; at 75min of recordings for the first and second pathway respectively; P= 0.34; n=12 slices; Fig.6C). Similarly, a normal level of potentiation was evoked following HFS at the second pathway when aPC was bath applied in presence of 10M of the mGluR-5 antagonist MPEP (1.79±0.085 and 1.84±0.082 at 75min of recordings for the first and second pathway respectively; P=0.31; n=12 slices; Fig.6D). These experiments show that aPC induces an NMDA- independent, VGCCs and mGluR-5 dependent LTP. Calcium Stores are Involved in aPC Mediated LTP aPC has been shown to modulate calcium release from intracellular stores through a EPCRPAR1 dependent mechanism (Domotor, et al., 2003). In order to test whether aPC mediated LTP is dependent on calcium stores, we bath applied aPC with Thapsigargin, an inhibitor of endoplasmatic reticulum Ca2+ ATPases (Maggio and Segal, 2007). Remarkably, aPC in presence of 1M Thapsigargin was not able to facilitate LTP; HFS at the second pathway evoked a lower LTP than that at the control path (1.87±0.083 and 1.53±0.086 at the onset
ACCEPTED MANUSCRIPT following HFS for the first and second pathway, respectively; n=14 slices, P<0.001; Fig.7A). In addition, thapsigargin even affected the LTP evoked at the first, control pathway as a slow decrease in the established synaptic potentiation was observed following the exposure of the
PT
slice to this drug (1.66±0.076 at 80min compared to 1.87±0.083 right after HFS; P<0.001; Fig.7A). We further explored these observations using cyclopiazonic acid (CPA), a blocker of
RI
endoplasmic reticulum Ca2+ ATPases. Bath application of 20M CPA blocked the potentiation facilitated by aPC (1.87±0.086 and 1.52±0.082 at the onset following HFS for the first and
SC
second pathway, respectively; n=14 slices, P<0.001; Fig.7B). In addition, similarly to thapsigargin, CPA impaired the already established LTP at the control pathway. The notable
NU
advantage of CPA is the possibility to wash it out from the medium. This allows the refill of intracellular calcium stores and the recovery of their function. Remarkably, delivering HFS in
MA
presence of CPA and aPC resulted in a potentiation of 55% over baseline (Fig.7C). However, HFS applied in presence of aPC following CPA removal facilitated LTP (1.47±0.057 and 2.19±0.082 at 150 minutes of recording for the first and second pathway respectively; P<0.001;
D
Fig.7C). These experiments indicate that calcium stores are involved in aPC mediated LTP and
TE
confirm the role of these organelles both in the establishment and maintenance of LTP in normal conditions (Maggio and Segal, 2007).
AC CE P
Thrombin Enhances LTP by Promoting the Formation of aPC. In the coagulation cascade, thrombin contributes to the generation of aPC (Griffin, et al., 2007), this initiates a feedback loop that ultimately results in the attenuation of the thrombin signaling (Griffin, et al., 2007). In this respect, we assumed that [Thrombin]low could as well promote the formation of aPC in hippocampal slices. Indeed, if this might be the case, [Thrombin] low could facilitate LTP through an aPC mediated mechanism. In order to test this hypothesis, we exposed slices to inactivated Protein C (iPC), a compound that can be converted to aPC in presence of thrombin. In the first set of experiments, [Thrombin]low did not show any notable effect on LTP: application of this drug following HFS at the control pathway did not result in a facilitation of LTP when a second HFS was delivered at the second pathway (1.76±0.075 and 1.77±0.086 at 20min following delivery of HFS at the first and second pathway respectively; P=0.62; n=14; Fig.8A). Then, in a different set of slices, we bath applied 1M iPC following HFS at the first pathway. In this conditions, HFS delivered at the second pathway also produced a potentiation of similar levels to the one in the control path (1.75±0.083 and 1.74±0.076 at 20min following delivery of HFS at the first and second pathway respectively; P= 0.66; n=14; Fig.8B). Strikingly, however exposure of slices to [Thrombin]low and iPC led to a different outcome. Here
ACCEPTED MANUSCRIPT different solutions of [Thrombin]low and iPC were simultaneously perfused in the slice such to avoid possible iPC activation in the perfusing solution. In these experiments, HFS to the second channel in presence of [Thrombin]low and iPC led to facilitation of LTP compared to control
PT
conditions (1.77±0.085 and 2.28±0.065 at 20min following delivery of HFS at the first and second pathway respectively; P<0.001; n=14; Fig.8C). Interestingly, [Thrombin]low in presence of
RI
-NAPAP was unable to produce this result (Suppl. Fig. 1B). This effect is peculiar to [Thrombin]low and not shared by [Thrombin]high. Notably, perfusing [Thrombin]high in presence of
SC
APV neither evoked an enhanced LTP (Suppl. Fig.2) nor facilitated LTP following simultaneous perfusion with iPC. These experiments demonstrate that [Thrombin]low is likely to facilitate LTP
NU
by promoting aPC formation in hippocampal slices. Discussion
MA
In this study, we addressed the role of thrombin in modulating LTP. Interestingly, we observed that different concentrations of thrombin led to diverse outcomes (Fig.9). [Thrombin]high induced a NMDA dependent, slow onset LTP while [Thrombin]low promoted a VGCCs, mGluR-5
D
dependent LTP through the activation of aPC (Fig.9). aPC facilitated LTP by activating PAR1
TE
through an EPCR-mediated mechanism and involvement of intracellular calcium stores. The highest concentrations of thrombin in the brain occur during brain hemorrhages (Gong, et
AC CE P
al., 2008, Hua, et al., 2009, Hua, et al., 2007) however high levels of thrombin have been reported following ischemic stroke (Chen, et al., 2010, Chen, et al., 2012). In these settings, BBB opening and rupture of brain endothelial cells activate the coagulation cascade leading to intracerebral production of thrombin (Chodobski, et al., 2012). Interestingly, neurological deficits in acute ischemic stroke animal models may be attenuated following thrombin blockade (Chen, et al., 2010, Chen, et al., 2012, Karabiyikoglu, et al., 2004). Our present data as well as our previous report (Maggio, et al., 2008) show that [Thrombin]high evokes a NMDA dependent LTP which saturates the ability of a neuronal network to undergo further NMDA dependent potentiation. This result might explain the inability to recover from a cerebrovascular trauma: the saturation of synaptic connectivity by thrombin-activated mechanisms does not allow the brain to use LTP-like plastic processes for either acquisition of new “memories” or adaptation to new motor plans after the insult (Maggio, et al., 2008). Definitely, additional experiments are required to explore the duration of thrombin action in the brain as well as the ways to overcome thrombin-related cognitive deficits. In this respect, it could be interesting to follow on whether patients discharged on dabigatran, a new thrombin direct inhibitor, would have better long term cognitive outcomes following stroke than other patients released on alternative anticoagulants.
ACCEPTED MANUSCRIPT There are suggestions that thrombin may be produced during normal synaptic transmission (Turgeon, et al., 2000), yet its exact amount at the synaptic cleft under physiological conditions is unknown. In the blood stream, thrombin has a short half life and its concentration is tightly
PT
regulated by multiple processes involving endogenous proteases (Siller-Matula, et al., 2011). Definitely, a similar system might exist in the brain in order to balance thrombin concentrations
RI
at the synapse during physiological events. Therefore, depending on the availability of this system, different concentrations of thrombin might arise at the synapse following a determined
SC
synaptic event and hence differently influence LTP (Komai et al., 2000; Maggio et al. 2008). In this study, we also showed the unique role of aPC in regulating LTP. Interestingly, the effects
NU
of aPC are concentration independent, therefore the presence of aPC at the synaptic cleft might shift by itself the threshold of synaptic plasticity towards novel LTP facilitation in a previously
MA
potentiated network. While the aPC receptor EPCR seems to be widely expressed in the hippocampus, it is not entirely clear whether in the brain the production of aPC exclusively depends on the thrombin-mediated conversion of iPC or it may be locally synthesized de novo.
D
In the blood stream, PC seems to circulate in its inactive, zymogen form and the switch into its
TE
active state occurs following its interaction with thrombin (Griffin, et al., 2007). Clarifying this issue in the brain might lead to important findings for the further understanding of the mechanisms regulating synaptic plasticity. However, as the case may be, this study, our
AC CE P
previous (Maggio, et al., 2008) as well as other evidences (Dihanich, et al., 1991, Gingrich, et al., 2000, Shikamoto and Morita, 1999) point towards a role of clotting factors in the regulation of synaptic plasticity. Lately, aPC has received attentions due to its cytoprotective and anticoagulant properties which may be of benefit in stroke therapy (Griffin, et al., 2012, Wang, et al., 2012, Zlokovic and Griffin, 2011). Our data further promote these positive properties of aPC. Precisely, the evidence that in the brain aPC enhances LTP through alternative pathways requiring VGCCs and mGLUR-5 may result in better cognitive outcomes in cases where NMDA receptors are saturated by thrombin. Definitely, more data on the role of aPC in stroke are needed to confirm this hypothesis. The evidence that both thrombin and aPC may exert opposite effects acting on the same receptor, i.e. PAR1, is puzzling indeed. In endothelial cells, it has been shown that PAR1 activation either by thrombin or aPC may lead to different, opposite outcomes due to the stimulation of Gq/G12/13 protein cascade in the former case and Gi in the latter (Riewald and Ruf, 2005). There might be a similar situation in the brain where PAR1 has been shown to interact with multiple G proteins (McCoy, et al., 2012). An additional hypothesis to explain these results may take into account the efficacy of different concentrations of PAR1-AP in producing diverse
ACCEPTED MANUSCRIPT effects. Specifically, it might be that two types of PAR1 bearing different affinities for the PAR1AP ligand might exist at the synapse. A low affinity PAR1 could directly be activated by thrombin and mediate the [Thrombin]high induced slow onset LTP while a high affinity receptor, not
PT
promptly cleaved by thrombin, might be in charge of the aPC-EPCR effects. These two different receptors could be linked to different G proteins and be expressed in diverse cell types
RI
(astrocytes vs. neurons). Additional pharmacological and molecular studies are needed in order to validate this hypothesis.
SC
In the present study, we have not clearly addressed whether the effects of thrombin and aPC are mediated by the neuronal and/or astrocytic PAR1. EPCR localization by itself does not add
NU
any further hint to this quest as it seems that both EPCR and PAR1 are colocalized in the same cells in the whole hippocampus. Nevertheless, the cellular localization of the PAR1 signaling
MA
seems to be a fundamental issue in order to fully understand the contribution of this receptor to synaptic plasticity. On the one hand, PAR1 knock out animals might not be an appropriate tool to utilize in solving this issue as signals from both cell types are being deleted in these animals.
D
On the other hand, the use of molecular techniques that will downregulate PAR1 expression in
TE
specific cell types is more promising and might lead to new fundamental insights into the role of this receptor in synaptic plasticity.
In summary, we have shown that thrombin may differently regulate synaptic plasticity based on
AC CE P
its concentration levels at the synaptic cleft. We believe this represents a novel mechanism by which PAR1 may regulate the threshold for synaptic plasticity in the hippocampus and provides additional insights on the role of this receptor in physiological vs. pathological conditions.
References
1. 2.
3. 4. 5.
Chen, B., Cheng, Q., Yang, K., and Lyden, P. D., 2010. Thrombin mediates severe neurovascular injury during ischemia. Stroke 41, 2348-2352. Chen, B., Friedman, B., Whitney, M. A., Winkle, J. A., Lei, I. F., Olson, E. S., Cheng, Q., Pereira, B., Zhao, L., Tsien, R. Y., and Lyden, P. D., 2012. Thrombin activity associated with neuronal damage during acute focal ischemia. J Neurosci 32, 7622-7631. Chodobski, A., Zink, B. J., and Szmydynger-Chodobska, J., 2012. Blood-brain barrier pathophysiology in traumatic brain injury. Transl Stroke Res 2, 492-516. Dihanich, M., Kaser, M., Reinhard, E., Cunningham, D., and Monard, D., 1991. Prothrombin mRNA is expressed by cells of the nervous system. Neuron 6, 575-581. Domotor, E., Benzakour, O., Griffin, J. H., Yule, D., Fukudome, K., and Zlokovic, B. V., 2003. Activated protein C alters cytosolic calcium flux in human brain endothelium via binding to endothelial protein C receptor and activation of protease activated receptor-1. Blood 101, 4797-4801.
ACCEPTED MANUSCRIPT
12. 13. 14. 15.
16.
17.
18. 19.
20.
21.
22.
23. 24.
PT
RI
SC
11.
NU
10.
MA
9.
D
8.
TE
7.
Feistritzer, C., and Riewald, M., 2005. Endothelial barrier protection by activated protein C through PAR1-dependent sphingosine 1-phosphate receptor-1 crossactivation. Blood 105, 3178-3184. Gingrich, M. B., Junge, C. E., Lyuboslavsky, P., and Traynelis, S. F., 2000. Potentiation of NMDA receptor function by the serine protease thrombin. J Neurosci 20, 4582-4595. Gong, Y., Xi, G., Hu, H., Gu, Y., Huang, F., Keep, R. F., and Hua, Y., 2008. Increase in brain thrombin activity after experimental intracerebral hemorrhage. Acta Neurochir Suppl 105, 47-50. Gorbacheva, L., Davidova, O., Sokolova, E., Ishiwata, S., Pinelis, V., Strukova, S., and Reiser, G., 2009. Endothelial protein C receptor is expressed in rat cortical and hippocampal neurons and is necessary for protective effect of activated protein C at glutamate excitotoxicity. J Neurochem 111, 967-975. Griffin, J. H., Fernandez, J. A., Gale, A. J., and Mosnier, L. O., 2007. Activated protein C. J Thromb Haemost 5 Suppl 1, 73-80. Griffin, J. H., Zlokovic, B. V., and Mosnier, L. O., 2012. Protein C anticoagulant and cytoprotective pathways. Int J Hematol 95, 333-345. Hua, Y., Keep, R. F., Gu, Y., and Xi, G., 2009. Thrombin and brain recovery after intracerebral hemorrhage. Stroke 40, S88-89. Hua, Y., Keep, R. F., Hoff, J. T., and Xi, G., 2007. Brain injury after intracerebral hemorrhage: the role of thrombin and iron. Stroke 38, 759-762. Isaeva, E., Hernan, A., Isaev, D., and Holmes, G. L., 2012. Thrombin facilitates seizures through activation of persistent sodium current. Ann Neurol 72, 192-198. Junge, C. E., Lee, C. J., Hubbard, K. B., Zhang, Z., Olson, J. J., Hepler, J. R., Brat, D. J., and Traynelis, S. F., 2004. Protease-activated receptor-1 in human brain: localization and functional expression in astrocytes. Exp Neurol 188, 94-103. Karabiyikoglu, M., Hua, Y., Keep, R. F., Ennis, S. R., and Xi, G., 2004. Intracerebral hirudin injection attenuates ischemic damage and neurologic deficits without altering local cerebral blood flow. J Cereb Blood Flow Metab 24, 159-166. Komai, S., Matsuyama T., Matsumoto K., Kato K., Kobayashi M., Imamura K., Yoshida S., Ugawa S., Shiosaka S., 2000. Neuropsin regulates an early phase of schaffercollateral long term potentiation in the murine hippocampus. Eur. J. Neurosc.; 12(4): 1479-86. Lee, C. J., Mannaioni, G., Yuan, H., Woo, D. H., Gingrich, M. B., and Traynelis, S. F., 2007. Astrocytic control of synaptic NMDA receptors. J Physiol 581, 1057-1081. Ludeman, M. J., Kataoka, H., Srinivasan, Y., Esmon, N. L., Esmon, C. T., and Coughlin, S. R., 2005. PAR1 cleavage and signaling in response to activated protein C and thrombin. J Biol Chem 280, 13122-13128. Luo, W., Wang, Y., and Reiser, G., 2007. Protease-activated receptors in the brain: receptor expression, activation, and functions in neurodegeneration and neuroprotection. Brain Res Rev 56, 331-345. Maggio, N., Cavaliere, C., Papa, M., Blatt, I., Chapman, J., and Segal, M., 2012. Thrombin regulation of synaptic transmission: Implications for seizure onset. Neurobiol Dis. Maggio, N., and Segal, M., 2007. Striking variations in corticosteroid modulation of longterm potentiation along the septotemporal axis of the hippocampus. J Neurosci 27, 5757-5765. Maggio, N., and Segal, M., 2007. Unique regulation of long term potentiation in the rat ventral hippocampus. Hippocampus 17, 10-25. Maggio, N., Shavit, E., Chapman, J., and Segal, M., 2008. Thrombin induces long-term potentiation of reactivity to afferent stimulation and facilitates epileptic seizures in rat
AC CE P
6.
ACCEPTED MANUSCRIPT
28.
29.
30.
33.
34. 35.
36.
37. 38.
PT
RI
AC CE P
32.
TE
D
31.
SC
27.
NU
26.
MA
25.
hippocampal slices: toward understanding the functional consequences of cerebrovascular insults. J Neurosci 28, 732-736. Mannaioni, G., Orr, A. G., Hamill, C. E., Yuan, H., Pedone, K. H., McCoy, K. L., Berlinguer Palmini, R., Junge, C. E., Lee, C. J., Yepes, M., Hepler, J. R., and Traynelis, S. F., 2008. Plasmin potentiates synaptic N-methyl-D-aspartate receptor function in hippocampal neurons through activation of protease-activated receptor-1. J Biol Chem 283, 20600-20611. McCoy, K. L., Gyoneva, S., Vellano, C. P., Smrcka, A. V., Traynelis, S. F., and Hepler, J. R., 2012. Protease-activated receptor 1 (PAR1) coupling to G(q/11) but not to G(i/o) or G(12/13) is mediated by discrete amino acids within the receptor second intracellular loop. Cell Signal 24, 1351-1360. Mosnier, L. O., Zlokovic, B. V., and Griffin, J. H., 2007. The cytoprotective protein C pathway. Blood 109, 3161-3172. Rezaie, A. R., The occupancy of endothelial protein C receptor by its ligand modulates the par-1 dependent signaling specificity of coagulation proteases. IUBMB Life 63, 390396. Riewald, M., and Ruf, W., 2005. Protease-activated receptor-1 signaling by activated protein C in cytokine-perturbed endothelial cells is distinct from thrombin signaling. J Biol Chem 280, 19808-19814. Shikamoto, Y., and Morita, T., 1999. Expression of factor X in both the rat brain and cells of the central nervous system. FEBS Lett 463, 387-389. Siller-Matula, J. M., Schwameis, M., Blann, A., Mannhalter, C., and Jilma, B., 2011. Thrombin as a multi-functional enzyme. Focus on in vitro and in vivo effects. Thromb Haemost 106, 1020-1033. Sokolova, E., and Reiser, G., 2008. Prothrombin/thrombin and the thrombin receptors PAR-1 and PAR-4 in the brain: localization, expression and participation in neurodegenerative diseases. Thromb Haemost 100, 576-581. Striggow, F., Riek, M., Breder, J., Henrich-Noack, P., Reymann, K. G., and Reiser, G., 2000. The protease thrombin is an endogenous mediator of hippocampal neuroprotection against ischemia at low concentrations but causes degeneration at high concentrations. Proc Natl Acad Sci U S A 97, 2264-2269. Turgeon, V. L., Salman, N., and Houenou, L. J., 2000. Thrombin: a neuronal cell modulator. Thromb Res 99, 417-427. Wang, J., Jin, H., Hua, Y., Keep, R. F., and Xi, G., 2012. Role of protease-activated receptor-1 in brain injury after experimental global cerebral ischemia. Stroke 43, 24762482. Wang, Y., Zhang, Z., Chow, N., Davis, T. P., Griffin, J. H., Chopp, M., and Zlokovic, B. V., 2012. An activated protein C analog with reduced anticoagulant activity extends the therapeutic window of tissue plasminogen activator for ischemic stroke in rodents. Stroke 43, 2444-2449. Yang, Y., and Rosenberg, G. A., 2011. Blood-brain barrier breakdown in acute and chronic cerebrovascular disease. Stroke 42, 3323-3328. Zlokovic, B. V., and Griffin, J. H., 2011. Cytoprotective protein C pathways and implications for stroke and neurological disorders. Trends Neurosci 34, 198-209.
Figures Legends:
ACCEPTED MANUSCRIPT Figure 1: [Thrombin]high induce slow onset, NMDA-dependent LTP in hippocampal slices. (A, B) Short application (12 min) of thrombin (1U/ml, [Thrombin]high; A) as well as of PAR1-AP (1M, [PAR1-AP]high; B) produces a gradual increase in population EPSP in stratum radiatum CA1
PT
without affecting population synaptic volleys (traces on top). (C) Thrombin induced slow onset LTP is blocked by the PAR1 antagonist SCH79797 (1M). (D) The NMDA antagonist APV
RI
(50M) totally blocks thrombin induced slow onset LTP. Averaged EPSP are plotted versus time. Representative traces at indicated times (a,b) are shown on top of each section. Upward
SC
arrows indicate the time of HFS.
Figure 2: Thrombin and PAR1-AP effects on LTP are concentration dependent. (A) Short
NU
application (15min) of thrombin (100mU/ml, [Thrombin]low) does not induce a slow onset LTP. As well, it does not affect the level of a tetanus induced LTP evoked at the second pathway 20
MA
minutes after drug removal. (B) Short application (15min) of PAR1-AP (100nM, [PAR1-AP]low) does not induce a slow onset LTP, however it enhances the height of a tetanus induced LTP evoked at the second pathway 20 minutes after drug removal. (C) The PAR1 antagonist SCH
D
79797 blocks the [PAR1-AP]low–mediated enhancement of LTP. Averaged EPSP are plotted
TE
versus time. Representative traces at indicated times (a,b) are shown on top of each section. Upward arrows indicate the time of HFS.
AC CE P
Figure 3: aPC shares the effects of [PAR1-AP]low on LTP. (A) Short application (15min) of plasmin (1U/ml, [Plasmin]high) induces a gradual increase in population EPSP in stratum radiatum CA1 without affecting population synaptic volleys (traces on top). (B) Short application (15min) of plasmin (100mU/ml) nor induces a slow onset LTP neither affects the level of a tetanus induced LTP evoked at the second pathway 20 minutes after drug removal. (C) The PAR1 antagonist SCH 79797 blocks the [Plasmin]high–slow onset LTP. (D, E) Short application (15min) of aPC (1M D; 100nM E) does not induce a slow onset LTP, however it enhances the level of a tetanus induced LTP evoked at the second pathway 20 minutes following drug removal. (F) The PAR1 antagonist SCH 79797 blocks the aPC mediated enhancement of LTP. Averaged EPSP are plotted versus time. Representative traces at indicated times (a,b) are shown on top of each section. Upward arrows indicate the time of HFS. Figure 4: The aPC receptor, EPCR, is expressed on neurons, on astrocytes and colocalizes with PAR1. (A) EPCR is widely expressed throughout the hippocampal fields (NeuN, red, EPCR, green; 10x) and is localized in neurons (B, NeuN, red, EPCR, green; 40x) and astrocytes (C, GFAP, red, EPCR, green; 40x). (D) PAR1 (red) and EPCR (green) colocalize in CA1 (40x). The white arrow in A indicates the area of CA1 where the pictures in B, C, D were taken.
ACCEPTED MANUSCRIPT Figure 5: EPCR blockade prevents the aPC-mediated, but not the [PAR1-AP]low-mediated enhancement of LTP. In slices from anti-EPCR ICV-injected animals application of aPC does not enhance LTP (A). Contrarily, in this setting both [PAR1-AP]high (B) and [PAR1-AP]low (C) are
PT
able to induce a slow onset LTP or to enhance the level of a tetanus induced LTP evoked at the second pathway, respectively. Averaged EPSP are plotted versus time. Representative traces
RI
at indicated times (a,b) are shown on top of each section. Upward arrows indicate the time of HFS.
SC
Figure 6: aPC induces a NMDA-independent, VGCCs and mGluR5-dependent LTP. (A) The NMDA antagonist APV (50M) does not block the aPC-induced LTP. (B) APV blocks LTP in the
NU
control pathway, but it does not affect aPC-induced LTP. (C) The Voltage Gated Calcium Channels blocker nifedipine (20M) inhibits the aPC-induced LTP. (D) The mGluR5 blocker
MA
MPEP (10M) hampers the aPC- induced LTP. Averaged EPSP are plotted versus time. Representative traces at indicated times (a,b) are shown on top of each section. Upward arrows indicate the time of HFS.
D
Figure 7: Calcium stores are involved in aPC induced LTP. (A, B) Both the irreversible
TE
(thapsigargin, 1M; A) and the reversible (cyclopiazonic acid, CPA, 20M; B) SERCA pump inhibitors prevent the aPC-induced LTP. (C) HFS in presence of aPC after CPA washout
AC CE P
restores aPC-induced LTP. Averaged EPSP are plotted versus time. Representative traces at indicated times (a,b) are shown on top of each section. Upward arrows indicate the time of HFS. Figure 8: [Thrombin]low enhance LTP through aPC. (A, B) [Thrombin]low (A) as well as inactivated Protein C (iPC; B) nor induce a slow onset LTP neither affect the level of a tetanus induced LTP evoked at the second pathway. (C) [Thrombin]low in presence of iPC enhance the level of a tetanus induced LTP evoked at the second pathway. Averaged EPSP are plotted versus time. Representative traces at indicated times (a,b) are shown on top of each section. Upward arrows indicate the time of HFS. Figure 9: Diverse thrombin concentrations differently regulate LTP. [Thrombin]high induce a NMDA dependent, slow onset LTP while [Thrombin]low promote a VGCCs, mGluRs dependent LTP through the activation of aPC. aPC facilitates LTP by activating PAR-1 through an EPCRmediated mechanism and involvement of intracellular calcium stores. Supplementary Figure 1: The thrombin inhibitor -NAPAP blocks the effects of thrombin on LTP. (A) [Thrombin]high in presence of 1M -NAPAP did not cause a slow onset LTP. Notably, -NAPAP did not have any effect on the tetanus-induced LTP. (B) [Thrombin]low in presence of 1M -NAPAP did not result in an enhanced LTP upon perfusion of iPC. Averaged EPSP are
ACCEPTED MANUSCRIPT plotted versus time. Representative traces at indicated times (a,b) are shown on top of each section. Upward arrows indicate the time of HFS. Supplementary Figure 2: Different thrombin concentrations have dissimilar effects on LTP. (A)
PT
In presence of APV, [Thrombin]high neither caused a slow onset LTP, nor evoked an enhanced LTP. (B) In presence of APV and iPC, [Thrombin]high did not enhance LTP upon delivery of
RI
tetanic stimulation. Averaged EPSP are plotted versus time. Representative traces at indicated
AC CE P
TE
D
MA
NU
SC
times (a,b) are shown on top of each section. Upward arrows indicate the time of HFS.
AC CE P
TE
D
MA
NU
SC
RI
PT
ACCEPTED MANUSCRIPT
Figure 1
NU
SC
RI
PT
ACCEPTED MANUSCRIPT
AC CE P
TE
D
MA
Figure 2
Figure 3
AC CE P
TE
D
MA
NU
SC
RI
PT
ACCEPTED MANUSCRIPT
AC CE P
Figure 4
TE
D
MA
NU
SC
RI
PT
ACCEPTED MANUSCRIPT
MA
NU
SC
RI
PT
ACCEPTED MANUSCRIPT
AC CE P
TE
D
Figure 5
MA
NU
SC
RI
PT
ACCEPTED MANUSCRIPT
AC CE P
TE
D
Figure 6
NU
SC
RI
PT
ACCEPTED MANUSCRIPT
AC CE P
TE
D
MA
Figure 7
NU
SC
RI
PT
ACCEPTED MANUSCRIPT
AC CE P
TE
D
MA
Figure 8
NU
SC
RI
PT
ACCEPTED MANUSCRIPT
AC CE P
TE
D
MA
Figure 9
ACCEPTED MANUSCRIPT Highlights: Thrombin levels regulate LTP through diverse molecular routes converging on PAR1.
[Thrombin]high induces a slow onset LTP.
[Thrombin]low enhances LTP by activating Protein C.
Protein C mediates LTP through VGCCs and mGluRs.
AC CE P
TE
D
MA
NU
SC
RI
PT