Efrat Shavit Stein

Thrombin regulation of synaptic transmission and plasticity: implications for health and disease.

Thrombin, a serine protease involved in the blood coagulation cascade has been shown to affect neural function following blood-brain barrier breakdown. However, several lines of evidence exist that thrombin is also expressed in the brain under physiological conditions, suggesting an involvement of thrombin in the regulation of normal brain functions. Here, we review ours’ as well as others’ recent work on the role of thrombin in synaptic transmission and plasticity through direct or indirect activation of Protease-Activated Receptor-1 (PAR1). These studies propose a novel role of thrombin in synaptic plasticity, both in physiology as well as in neurological diseases associated with increased brain thrombin/PAR1 levels.

Thrombin induces ischemic LTP (iLTP): implications for synaptic plasticity in the acute phase of ischemic stroke

Acute brain ischemia modifies synaptic plasticity by inducing ischemic long-term potentiation (iLTP) of synaptic transmission through the activation of N-Methyl-D-aspartate receptors (NMDAR). Thrombin, a blood coagulation factor, affects synaptic plasticity in an NMDAR dependent manner. Since its activity and concentration is increased in brain tissue upon acute stroke, we sought to clarify whether thrombin could mediate iLTP through the activation of its receptor Protease-Activated receptor 1 (PAR1). Extracellular recordings were obtained in CA1 region of hippocampal slices from C57BL/6 mice. In vitro ischemia was induced by acute (3 minutes) oxygen and glucose deprivation (OGD). A specific ex vivo enzymatic assay was employed to assess thrombin activity in hippocampal slices, while OGD-induced changes in prothrombin mRNA levels were assessed by (RT)qPCR. Upon OGD, thrombin activity increased in hippocampal slices. A robust potentiation of excitatory synaptic strength was detected, which occluded the ability to induce further LTP. Inhibition of either thrombin or its receptor PAR1 blocked iLTP and restored the physiological, stimulus induced LTP. Our study provides important insights on the early changes occurring at excitatory synapses after ischemia and indicates the thrombin/PAR1 pathway as a novel target for developing therapeutic strategies to restore synaptic function in the acute phase of ischemic stroke.

Ischemic LTP: NMDA‐dependency and dorso/ventral distribution within the hippocampus

A transient ischemic episode causes a reduction in evoked EPSPs in hippocampal slices, followed by an NMDA dependent LTP. We explored the relations between ischemic LTP (iLTP) and the more conventional tetanic LTP (tLTP) in CA1 region of slices along the dorsal/ventral axis of the hippocampus. Dorsal hippocampal (DH) slices produced a much larger iLTP than their ventral hippocampal (VH) counterparts. In both regions, iLTP and tLTP shared the same NMDA mediated potentiation, such that one LTP saturated the ability of the other treatment to generate LTP. The smaller LTP in VH was correlated with a lower NMDA‐mediated EPSP, and a parallel lower density of NMDA receptors. Calcium permeable AMPA receptors did not contribute to the DH/VH disparity. We conclude that a differential distribution of NMDA receptor subunits along the septotemporal axis of the hippocampus controls the diverse ability to evoke iLTP and tLTP in the two regions and may underlie their characteristic behavioral outputs as well as their differential sensitivity to ischemia.

Increased thrombin activity following reperfusion after ischemic stroke alters synaptic transmission in the hippocampus.

Thrombin, a key player in thrombogenesis, affects cells in the brain through activation of its receptors. Low levels of thrombin activity are protective while high levels are toxic. We sought to quantify thrombin activity levels and their spatial distribution in brains of mice following reperfusion after ischemic stroke focusing on infarct, peri‐infarct and contralateral areas. In order to find out the contribution of brain‐derived thrombin, mRNA levels of both prothrombin and factor X were determined. Furthermore, we assessed the effect of thrombin levels that were measured in the ischemic brain on synaptic transmission. We found that in the brains of mice following transient middle cerebral artery occlusion, thrombin activity is elevated throughout the ischemic hemisphere, including in peri‐infarct areas (90 ± 33 and 60 ± 18 mU/mL, in the infarct and peri‐infarct areas, respectively, compared to 11 ± 3 and 12 ± 5 mU/mL, in the corresponding contralateral areas; mean ± SE; p < 0.05). Brain mRNA levels of prothrombin and, in particular, factor X are up‐regulated in the ischemic core. Hippocampal slices treated with thrombin concentrations as found in the ischemic hemisphere show altered synaptic responses. We conclude that high thrombin activity following reperfusion after ischemic stroke may cause synaptic dysfunction.

A Linear Temporal Increase in Thrombin Activity and Loss of Its Receptor in Mouse Brain following Ischemic Stroke

Background Brain thrombin activity is increased following acute ischemic stroke and may play a pathogenic role through the protease-activated receptor 1 (PAR1). In order to better assess these factors, we obtained a novel detailed temporal and spatial profile of thrombin activity in a mouse model of permanent middle cerebral artery occlusion (pMCAo). Methods Thrombin activity was measured by fluorescence spectroscopy on coronal slices taken from the ipsilateral and contralateral hemispheres 2, 5, and 24 h following pMCAo (n = 5, 6, 5 mice, respectively). Its spatial distribution was determined by punch samples taken from the ischemic core and penumbra and further confirmed using an enzyme histochemistry technique (n = 4). Levels of PAR1 were determined using western blot. Results Two hours following pMCAo, thrombin activity in the stroke core was already significantly higher than the contralateral area (11 ± 5 vs. 2 ± 1 mU/ml). At 5 and 24 h, thrombin activity continued to rise linearly (r = 0.998, p = 0.001) and to expand in the ischemic hemisphere beyond the ischemic core reaching deleterious levels of 271 ± 117 and 123 ± 14 mU/ml (mean ± SEM) in the basal ganglia and ischemic cortex, respectively. The peak elevation of thrombin activity in the ischemic core that was confirmed by fluorescence histochemistry was in good correlation with the infarcts areas. PAR1 levels in the ischemic core decreased as stroke progressed and thrombin activity increased. Conclusion In conclusion, there is a time- and space-related increase in brain thrombin activity in acute ischemic stroke that is closely related to the progression of brain damage. These results may be useful in the development of therapeutic strategies for ischemic stroke that involve the thrombin-PAR1 pathway in order to prevent secondary thrombin related brain damage.

Complex modulation by stress of the effect of seizures on Long Term Potentiation in mouse hippocampal slices

Stress has a profound effect on ability to express neuronal plasticity, learning, and memory. Likewise, epileptic seizures lead to massive changes in brain connectivity, and in ability to undergo long term changes in reactivity to afferent stimulation. In this study, we analyzed possible long lasting interactions between a stressful experience and reactivity to pilocarpine, on the ability to produce long term potentiation (LTP) in a mouse hippocampus. Pilocarpine lowers paired pulse potentiation as well as LTP in CA1 region of the mouse hippocampal slice. When stress experience precedes exposure to pilocarpine, it protects the brain from the lasting effect of pilocarpine. When stress follows pilocarpine, it exacerbates the effect of the drug, to produce a long lasting reduction in LTP. These changes are accompanied by a parallel change in blood corticosterone level. A single exposure to selective mineralo‐ or gluco‐corticosterone (MR and GR, respectively) agonists and antagonists can mimic the stress effects, indicating that GRs underlie the lasting detrimental effects of stress whereas MRs are instrumental in counteracting the effects of stress. These studies open a new avenue of understanding of the interactive effects of stress and epileptic seizures on brain plasticity.

Stress and Corticosteroids Modulate Muscarinic Long Term Potentiation (mLTP) in the Hippocampus

Stress influences synaptic plasticity, learning and memory in a steroid hormone receptor dependent manner. Based on these findings it has been proposed that stress could be a major risk factor for the development of cognitive decline and dementia. Interestingly, evidence has been provided that stress also affects muscarinic, i.e., acetylcholine (ACh)-mediated neurotransmission. To learn more about the impact of stress and steroids on synaptic plasticity, in this study, we investigated the effects of stress on muscarinic long term potentiation (mLTP). We report that multiple, unpredictable exposure to stress depresses carbachol (0.5 μM)-induced mLTP, while this effect of stress is not observed in hippocampal slices prepared from mice exposed only to a single stressful procedure. Furthermore, we demonstrate that activation of distinct steroid hormone receptors is involved in stress-mediated alterations of mLTP. Activation of mineralocorticoid receptors (MR) promotes mLTP, while glucocorticoid receptor (GR) activity impairs mLTP. These effects of multiple unpredictable stress on mLTP are long-lasting since they are detected even two weeks after the last stressful experience. Thus, multiple unpredictable events rather than a single stressful experience affect mLTP in a steroid hormone receptor dependent manner, suggesting that chronic unpredictable stress can lead to lasting alterations in hippocampal cholinergic plasticity.

Thrombin Inhibition Reduces the Expression of Brain Inflammation Markers upon Systemic LPS Treatment

Systemic inflammation and brain pathologies are known to be linked. In the periphery, the inflammation and coagulation systems are simultaneously activated upon diseases and infections. Whether this well-established interrelation also counts for neuroinflammation and coagulation factor expression in the brain is still an open question. Our aim was to study whether the interrelationship between coagulation and inflammation factors may occur in the brain in the setting of systemic inflammation. The results indicate that systemic injections of lipopolysaccharide (LPS) upregulate the expression of both inflammatory and coagulation factors in the brain. The activity of the central coagulation factor thrombin was tested by a fluorescent method and found to be significantly elevated in the hippocampus following systemic LPS injection (0.5 ± 0.15 mU/mg versus 0.2 ± 0.03 mU/mg in the control). A panel of coagulation factors and effectors (such as thrombin, FX, PAR1, EPCR, and PC) was tested in the hippocampus, isolated microglia, and N9 microglia cell by Western blot and real-time PCR and found to be modulated by LPS. One central finding is a significant increase in FX expression level following LPS induction both in vivo in the hippocampus and in vitro in N9 microglia cell line (5.5 ± 0.6- and 2.3 ± 0.1-fold of increase, resp.). Surprisingly, inhibition of thrombin activity (by a specific inhibitor NAPAP) immediately after LPS injection results in a reduction of both the inflammatory (TNFα, CXL9, and CCL1; p < 0.006) and coagulation responses (FX and PAR1; p < 0.004) in the brain. We believe that these results may have a profound clinical impact as they might indicate that reducing coagulation activity in the setting of neurological diseases involving neuroinflammation may improve disease outcome and survival.

Cannabidiol Regulates Long Term Potentiation Following Status Epilepticus: Mediation by Calcium Stores and Serotonin

Epilepsy is a devastating disease, with cognitive and emotional consequences that are not curable. In recent years, it became apparent that cannabinoids help patients to cope with epilepsy. We have studied the effects of cannabidiol (CBD) on the ability to produce long term potentiation (LTP) in stratum radiatum of CA1 region of the mouse hippocampus. Exposure to seizure-producing pilocarpine reduced the ability to generate LTP in the slice. Pre-exposure to CBD prevented this effect of pilocarpine. Furthermore, CBD caused a marked increase in ability to generate LTP, an effect that was blocked by calcium store antagonists as well as by a reduction in serotonin tone. Serotonin, possibly acting at a 5HT1A receptor, or fenfluramine (FFA), which causes release of serotonin from its native terminals, mimicked the effect of CBD. It is proposed that CBD enhances non-NMDA LTP in the slice by facilitating release of serotonin from terminals, consequently ameliorating the detrimental effects of pilocarpine.

Recovery from trauma induced amnesia correlates with normalization of thrombin activity in the mouse hippocampus

Transient amnesia is a common consequence of minimal traumatic brain injury (mTBI). However, while recent findings have addressed the mechanisms involved in its onset, the processes contributing to its recovery have not yet been addressed. Recently, we have found that thrombin is detected at high concentrations in the brain of mice after exposure to mTBI and that in such settings amnesia is rescued by either inhibiting thrombin activity or by blockade of PAR1. Here, we report that mice spontaneously recover from amnesia after two weeks from mTBI exposure. At this time point, long term potentiation was equally evoked in injured vs. control animals with thrombin concentration in the brain being normalized at this stage. These findings, which refer to the specific aspect of memory retrieval upon mTBI, together with our previous work, hint to a strong correlation between cognitive defects in the context of mTBI and thrombin concentrations in the brain. This may suggest that a possible scavenging of thrombin in the brain at early phases following mTBI may improve memory function.