• fear conditioning;
  • long-term memory;
  • object recognition;
  • polyADP-ribosylation


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

PolyADP-ribosylation is a post-translational modification of nuclear proteins, catalyzed by polyADP-ribose polymerases (PARPs). In the nucleus, polyADP-ribosylation catalyzed by PARP-1 alters protein–protein and protein–DNA interactions, and is implicated in chromatin remodeling, DNA transcription, and repair. Previous results linked the activation of PARP-1 with long-term memory formation during learning in the marine mollusk Aplysia ( Science 2004, 304:1820–1822). Furthermore, PARP-1 was highly activated in mammalian cerebral neurons treated with neurotrophins and neurotrophic peptides promoting neurite outgrowth and synaptic plasticity. Here, we examine the possibility that PARP-1 activation is required for memory formation during learning in mammals. Mice were tested in two learning paradigms, object recognition and fear conditioning. PolyADP-ribosylation of PARP-1 and histone H1 were detected in their cerebral cortex and hippocampus immediately after their training session. Moreover, in both behavioral paradigms, suppression of PARP activity in the CNS during learning impaired their long-term memory formation, without damaging their short-term memory. These findings implicate PARP-1 activation in molecular processes underlying long-term memory formation during learning.

Abbreviations used:

artificial CSF


extracellular signal-regulated kinase


intertrial intervals


long-term memory


mitogen activated protein kinase kinase


object recognition test


passive avoidance


polyADP-ribose polymerases


short-term memory

PolyADP-ribosylation is a fast and energy consuming transient post-translational modification of proteins catalyzed by polyADP-ribose polymerases (PARPs) (Lautier et al. 1993; Rolli et al. 2000; Améet al. 2004; Rouleau et al. 2004; Schreiber et al. 2006). These enzymes catalyze the cleavage of NAD into nicotinamide and ADP-ribose, transfer of ADP-ribose to glutamic and aspartic residues in PARPs and their substrates, and the polymerization of ADP-riboses into long and branched negatively charged polymers (Lautier et al. 1993; Rolli et al. 2000; Améet al. 2004; Schreiber et al. 2006) that alter the interaction between proteins in the chromatin and between proteins and DNA (Tulin and Spradling 2003; Rouleau et al. 2004; Schreiber et al. 2006). Most of PARP activity in the nucleus is attributed to the activity of PARP-1, an abundant highly conserved DNA-binding protein (Rolli et al. 2000; Améet al. 2004; Schreiber et al. 2006). Its substrates include RNA polymerase II, topoisomerases, histones, and transcription factors (Ju et al. 2004; Rouleau et al. 2004; Schreiber et al. 2006; Hassa and Hottiger 2008), implicating PARP-1 activation in chromatin remodeling, DNA transcription, and repair (Masson et al. 1998; Ju et al. 2004; Rouleau et al. 2004). PARP-1 is activated in response to single strand DNA breaks, and promotes DNA repair (Szabo et al. 1996; Masson et al. 1998; Rolli et al. 2000; Kraus and Lis 2003). In addition, recent findings disclosed an alternative mechanism of PARP-1 activation, which is suppressed in the presence of damaged DNA. In this mechanism PARP-1 is activated by phosphorylated extracellular signal-regulated kinase (ERK) even in the absence of DNA (Cohen-Armon et al. 2007), suggesting additional roles of PARP-1 in processes governed by the mitogen activated protein kinase kinase (MEK)-ERK phosphorylation cascades. These include growth and differentiation, neurite outgrowth and long-term memory (LTM) formation (Kandel 2001; Inder et al. 2008). These findings and evidence implicating PARP-1 activation in LTM formation of the sea slug Aplysia (Cohen-Armon et al. 2004) urged us to examine whether PARP-1 activation mediates LTM formation in general. Here, this possibility was examined in mammals.

We found that PARP-1 is polyADP-ribosylated in the cerebral cortex and hippocampus of mice after training sessions. In addition, the ability of mice to memorize specific tasks while PARP activity in their brain is blocked was tested in two behavioral paradigms: the object recognition task (Ennaceur and Delacour 1988) and the passive avoidance (PA) task (Venault et al. 1986). The results described below provide first evidence for the dependence of LTM formation in cognitive tasks on PARP-1 activation.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Trained mice

Male imprinting control region mice (8 weeks old) weighing 25–30 g were purchased from Harlan Inc. (Jerusalem, Israel). Mice were housed in clear polycarbonate cages in groups of five mice under a controlled 12/12-h light–dark reverse cycle (light from 8:00 pm to 8:00 am) at room temperature 23°C and humidity at 55%. The mice were given free access to water and food pellets. All experimental procedures were approved by the Animal Care Committee of the Tel-Aviv University. Experiments were carried out between 12:00 pm and 16:00 pm.

Intracerebroventricular injection

Mice were anesthetized by inhaling isofluran solution and were injected in the right lateral ventricle (1 mm posterior to Bregma; 1.5 mm lateral; 2 mm depth) with the potent PARP inhibitor PJ-34 (0.2 mM, 5 μL; Alexis, Brand Enzo Life Sciences Inc., Plymouth Meeting, PA, USA) dissolved in artificial CSF (aCSF) solution. Mice in the control group were injected solely with 5 μL of aCSF solution. Injection was carried out at a rate of 1.25 μL/min with a 100 μL syringe pump (Baby Bee Bioanalytical Systems, BASi, West Lafayette, IN, USA) and a brain infusion canulla (Alzet brain infusion kit; Alzet Corp, Cupertino, CA, USA). After each injection the canulla was left in situ for an additional 20 s to avoid reflux. As PJ-34 scarcely permeated the blood–brain barrier, i.c.v. injection was the best way to increase significantly the concentration of PJ-34 in the CNS. This mode of drug administration has been used before by Schafe et al. (1999) and Ophir et al. (2003).

Two-dimensional gel electrophoresis

PolyADP-ribose polymerases-1, like other DNA binding proteins, is a basic protein (Gorg 1999). Negatively charged ADP-ribosyl moieties added to PARP-1 by polyADP-ribosylation shift its basic isoelectric point (pI) towards more acidic pH values (Lautier et al. 1993; Améet al. 2004). The shift in the pI of PARP-1 is therefore related to auto-polyADP-ribosylation of activated PARP-1. Shifts in the pI were measured by two-dimensional gel electrophoresis (Gorg 1999). For the first dimension, we used Immobiline DryStrip kit (Amersham Biosciences, Uppsala, Sweden) and polyacrylamide slab gels were used in the second dimension. Proteins were electroblotted (western blots) and PARP-1 was detected by immunolabeling (monoclonal anti-PARP-1 antibody; Serotec, Oxford, UK) on nitrocellulose membranes.

Immunoblots and autoradiography

The efficiency of i.c.v. injection with PJ-34 was assayed by measuring its effect on the [32P]polyADP-ribosylation of PARP-1. Nuclei prepared from mouse brain cortex and hippocampus were incubated (10 min, 30°C) with [32P]NAD (1000 Ci/mmole, 1 μCi/sample) and protease and phosphatase inhibitors, as described before (Homburg et al. 2000; Cohen-Armon et al. 2007). [32P]PolyADP-ribosylated nuclear proteins were extracted by high salt (0.5 M NaCl, 30 min, 4°C), separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes (western blots). [32P]polyADP-ribosylated PARP-1 in the samples was autoradiographed and immunolabeled with monoclonal anti-PARP-1 antibody (Serotec).

Behavioral methods

The PA paradigm and the object recognition test (ORT) were chosen as both were considered a one-test trial (Venault et al. 1986; Ennaceur and Delacour 1988) in which one session was sufficient to constitute the acquisition stage. Experiments were limited to one trial test parameter to avoid more than a single i.c.v. injection of PJ-34. The tested parameter of the ORT was the preference index ratio between a new and an old object (delta ratio). The tested parameter in PA was the latency to step-through a pathway leading to a ‘dangerous’ area.

The ORT was conducted according to previous reports (Ennaceur and Delacour 1988; Tang et al. 1999; Jones et al. 2001; Bozon et al. 2003; Boess et al. 2004). Briefly, in the acquisition trial (T1) mice were gently placed in an open field, and were allowed to explore two identical objects. In the retrieval trial (T2), one of the familiar objects was replaced by a novel one. Trial duration was 7 min each. Objects exploration periods were measured and plotted. Delta ratio was calculated according to the equation: Delta ratio = 100 × [new object exploration time (s) − old familiar object exploration time (s)]/[total object exploration time (s)]. The intertrial intervals (ITI) were 30 min or 24 h to assess short-term memory (STM) and LTM, respectively.

Passive avoidance was conducted according to previous reports (Venault et al. 1986; Dubrovina and Tomilenko 2007). In the acquisition trial mice were gently placed in the illuminated side of two compartments box. After 30 s the guillotine door which connected both compartments was opened and mice were allowed to step through the dark compartment after which the guillotine was closed preceding an electric foot shock (0.7 mA, 2 s), delivered through the grid floor. The latency to step-through the dark compartment and the responses to the electric shock were recorded. Immediately afterwards, the mouse was removed from the dark compartment and returned to its home cage. For each mouse, two extinction (with no shock) trials were performed in different ITI (30 min and 24 h from acquisition) to test short and long memory retention. In the extinction trials, the mouse was placed again in the illuminated compartment and allowed to step into the dark compartment. The latency of step-through was recorded.

Statistics analysis

Student’s t-tests were performed for statistical analysis.


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

PolyADP-ribosylation in the cerebral cortex and hippocampus of trained mice

Mice were trained to recognize new objects in their arena (object recognition) and killed 5 min after the acquisition session (T1; Materials and methods). Because activated PARP-1 is auto-polyADP-ribosylated, PARP activation was assayed by measuring the shift in its isoelectric point. For this purpose, nuclear proteins were extracted from the brain cortex and hippocampus of trained and naïve mice, and changes in their isoelectric point were assayed by electrofocusing using 2-D gel electrophoresis (Fig. 1a). We found that PARP-1 was activated, and auto-polyADP-ribosylated in the trained mice. In accordance, its prominent substrate, linker histone H1, was also polyADP-ribosylated in the same brain regions (Fig. 1a). Because polyADP-ribosylation is mainly attributed to PARP-1 activity (Rolli et al. 2000; Améet al. 2004; Schreiber et al. 2006), PARP-1 activation and auto-polyADP-ribosylation was accompanied by polyADP-ribosylation of other PARP-1 substrates. These proteins were immunolabeled by antibody directed against ADP-ribose polymers (Fig. 1b). PolyADP-ribosylated proteins were detected in the cerebral cortex and hippocampus but not in the brainstem of mice exposed to new objects in their arena (Fig. 1b). Thus, a possible involvement of PARP-1 activation in a learning process was suggested.


Figure 1.  An enhanced PARP-1 activity in the cerebral cortex and hippocampus of trained mice. (a) Mice (n = 6) were exposed to new objects in their arena (object recognition task) and were killed immediately after exposure. Nuclear proteins were extracted from their cerebral cortex and hippocampus as well as from the cortex and hippocampus of naive (untrained) mice (n = 6). The nuclear proteins extracted from the pooled brain regions were separated by 2-D gel electrophoresis (300 μg total protein was loaded in each sample), electroblotted, and immunolabeled for PARP-1 (Serotec) and its prominent substrate linker histone H1 (Upstate Biotech, NY, USA). PolyADP-ribosylation is indicated by the shift in the pI of PARP-1 and H1 towards lower pH values (Materials and methods). (b) Upper panel: nuclear proteins were extracted from the three brain regions: cerebral cortex, hippocampus, and brainstem of three trained (object recognition) and three untrained naïve mice. Nuclear proteins in the pooled samples were separated by polyacrylamide gel electrophoresis and electoblotted (western blot). PolyADP-ribosylated proteins were immunolabeled with antibody directed against ADP-ribose moieties (PAR; Alexis, CA). Immunolabeled PARP-1 in each sample (Serotec) was used as the loading control. PAR labeling of nuclear proteins in the cerebral cortex, hippocampus, and brainstem was calculated from the scanned blots (Lower panel).

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Intracerebroventrical administration of the PARP inhibitor PJ-34 attenuated polyADP-ribosylation in the brain cortex and hippocampus

To find out whether polyADP-ribosylation was involved in learning, mice were injected before ‘training’ by i.c.v. injection with PJ-34 in aCSF (Materials and methods). PJ-34 is a potent PARP inhibitor that permeates the cell membrane but scarcely permeates the blood–brain barrier. Control mice were similarly injected with vehicle containing aCSF only. Because NAD is not permeable in the cell membrane, we measured the activity of PARP-1 in nuclei isolated from the brain cortex and hippocampus and incubated with [32P]NAD (Fig. 2). To verify that PJ-34 is effective during the acquisition trial (Materials and methods), we killed the injected mice 45 min after i.c.v. injection (the time period preceding training; Materials and methods). Results showed that the auto-[32P]polyADP-ribosylation of PARP-1 in isolated nuclei from brain cortex and hippocampus was markedly suppressed 45 min after i.c.v. injection with PJ-34 (Fig. 2).


Figure 2.  Verifying PARP inhibition in the cortex and hippocampus of mice. PolyADP-ribosylation of PARP-1 was suppressed by PJ-34 injected in aCSF into the brain ventricles (i.c.v.). The injected solution in control mice was aCSF. PJ-34 was dissolved in aCSF (PJ-34, 5 μL, 0.2 mM). PolyADP-ribosylation was assayed 45 min after injection in nuclear protein extracts in the presence of [32P]NAD. Autoradiograms of [32P]polyADP-ribosylated PARP-1 (upper panel) and immunolabeled PARP-1 (lower panel) in the cortex and hippocampus (right and left hemispheres) of i.c.v. injected mice are presented (n = 3).

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Object recognition task

Three groups of mice were subjected to the ORT 45 min post i.c.v. injection: naïve untreated mice, mice injected with aCSF that were the control group to mice injected with PJ-34 dissolved in aCSF, and the experimental group. Mice were tested for their ability to recognize new objects in their arena after switching a familiar object with a new one (Materials and methods). In the acquisition trial (T1; Materials and methods), there was neither preference for any of the two objects, ‘familiar’ or ‘new’ object (Fig. 3) nor differences in the total exploration time (data not shown). After the T1 session, mice were divided into subgroups for STM and LTM tests. The two subgroups differed in their intertrial intervals (ITI; 30 min for STM, and 24 h for LTM, respectively). The retrieval trial (T2) was conducted after the ITI in each subgroup (Fig. 3). In each group of mice, we measured changes in the increment of time spent with the new object versus the time spent with the familiar object (Materials and methods). Results show that naïve mice (not injected) showed significant preference for the new object, regardless of the ITI period, i.e., after 30 min or after 24 h (Fig. 3). Similarly, mice injected with aCSF preferred the new object in the retrieval trial (Fig. 3). However, PJ-34 injected mice did not show any significant preference for the new object in the test assessing LTM (24 h after the first trial; Fig. 3) while PJ-34 treatment did not impair their STM for 30 min after trail. On the contrary, STM was significantly improved, relatively to STM in aCSF injected mice (Fig. 3). This finding could result from the neuroprotective effect of PARP-1 inhibition, preventing cell death in regions damaged by the i.c.v. injection (Jagtap and Szabo 2005; Komjati et al. 2005).


Figure 3.  The effect of PJ-34 on the object recognition learning task (ORT). Mice were treated by two trial sessions, acquisition, and retrieval. Exploration time of a familiar and a novel object was recorded and the delta ratio values were plotted. Delta ratio was calculated according to the equation: Delta ratio = 100 × [exploration time of new object (s) − exploration time of familiar object (s)]/[total object exploration time (s)]. Independent t-test was used (*p = 0.02). Error bars indicate the SEM. Number of mice in each trail is indicated.

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It should be noted that PARP inhibition with PJ-34 did not affect survival or overall behavior of the tested mice. Exploration time during training or other tested behavioral parameters (i.e., pilomotor, shaking, freezing, grooming, and defecating) were not affected by the treatment with PJ-34 (data not shown). We therefore concluded according to these findings that the LTM for familiar objects in the arena was impaired in the trained mice as a consequence of PJ-34 i.c.v. injection, while their STM was not impaired at all, and seemed to be even improved.

Fear conditioning tested by the passive avoidance task

Mice were tested for fear conditioning by using the PA task. Three groups of mice were subjected to three trials: acquisition trial and two extinction trials conducted 30 min and 24 h after the acquisition trial (Materials and methods). These trials tested the latencies to stepping through a dark compartment, which was accompanied by an electrical shock.

Mice that did not receive foot electric shock while stepping through the dark compartment showed similar latencies to step-through the dark compartment in the acquisition and extinction trials. Mice that were not injected with PJ-34 or vehicle but experienced foot shock on stepping through the dark compartment (Control) showed an increased latency scoring on extinction trials (trials without an electric shock) relative to acquisition trial (Fig. 4). The other two groups included vehicle or PJ-34 injected mice subjected to electric foot shock after entering the dark compartment. In one of the experimental groups mice were injected with PJ-34 (EXP-PJ). In the other group (EXP-aCSF) mice were injected with aCSF.


Figure 4.  The effect of PARP inhibition on fear conditioning. Passive avoidance training of mice was preceded by injection (i.c.v.) with (aCSF; EXP-aC), or with PJ-34 in aCSF (5 μL; 0.2 mM PJ-34; EXP-PJ). Mice were subjected to three trial sessions, acquisition session, and two extinction sessions. Control mice (Control; n = 11) were not injected, but experienced electric shock while stepping through the dark compartment. Similarly, injected mice, EXP-aC (n = 20) and EXP-PJ (n = 19) experienced electric shock while stepping through the dark compartment in the acquisition trial. The latency to step through the dark compartment after 30 min and after 24 h was measured. Average values were calculated by using independent parametric t-test. Error bars indicate the SEM.

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Results showed that PJ-34 injection had a significantly negative effect on fear memory retention tested 24 h post-acquisition, with no similar effect on their short memory tested 30 min post-acquisition (Fig. 4). Step through latency comparison between both extinction trials revealed a significantly longer latency period of the EXP-aCSF mice, similarly to the longer latency of naïve (not injected) mice in the 30 min post-acquisition and the 24 h post-acquisition tests (Control). In these groups of mice, the step-through latency was significantly enhanced at the second extinction trial. This enhancement could result from fear memory retention by a mechanism explained according to the two-process theory (Domjan 1997). According to this theory, after acquisition the conditioned context may acquire aversive traits, turning the first extinction trial into an additional fear acquisition trial.

In contrast, the step through latency was not enhanced at the second extinction trial in mice injected with PJ-34 (EXP-PJ). These mice showed no change in their step-through latencies when both extinction trials were compared (i.e., there was no significant change in the latency periods after 24 h in relation to those measured 30 min after the acquisition trial; Fig. 4).

Mice in all the tested groups did not differ in their detectable behavioral parameters in the acquisition trial, including the frequency they explored the lighted compartment, pilomotor, shaking, grooming, and freezing scores. Thus, reduced fear memory retention in mice injected with PJ-34 may implicate polyADP-ribosylation in the molecular mechanisms underlying LTM of fear conditioning in mammals.


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Our findings indicate that PARP-1 is activated in the cerebral cortex and hippocampus of mice exposed to new objects in their arena (Fig. 1). Furthermore, PARP inhibition during training by i.c.v. injected PARP inhibitor, prevented the formation of LTM in the trained mice, while their STM was not impaired (Figs 2–4). These results were consistent with previously reported effect of polyADP-ribosylation on the LTM of Aplysia trained to avoid an inedible food (Cohen-Armon et al. 2004). These findings are also in accordance with impaired learning abilities of PARP-1 knockout mice outlined by Fontin-Lozano A., Gruart A., Valenzuela-Harrington M., Taylor J. S., Delgado-Garcia J., Carrion A. in the 2005 Annual Meeting of the Society for Neuroscience (Activation of apoptosis-related proteins is required for consolidation of object recognition memory in mice. Abs No. 67.16). However, in this case it is important to emphasize that PARP-1 knockout mice may lack, apart from PARP-1, additional enzymes and transcription factors regulating memory formation; i.e., a suppressed expression of numerous genes was detected in PARP-1-deficient mouse embryonic fibroblasts, including genes expressing proteins that are involved in the MEK-ERK signaling cascades (Ogino et al. 2007).

Accumulating findings indicate a pivotal role of ERK2 and Elk1 phosphorylation in the activation of histone acetyltransferases, phosphorylation of transcription factors, and expression of immediate early genes that are implicated in the formation of LTM (Impey et al. 1999; Cammarota et al. 2000; Kandel 2001; Selcher et al. 2001; Sweatt 2001; Mazzucchelli et al. 2002; Bozon et al. 2003; Li et al. 2003; Purcell et al. 2003; Buchwalter et al. 2004; Korzus et al. 2004; Martin and Sun 2004; Sng et al. 2004; Fischer et al. 2007; Thomson et al. 1999; Guzowski et al. 2001).

The recently proposed mechanism of PARP-1 activation by phosphorylated ERK2 in the absence of DNA breaks (Cohen-Armon 2007; Cohen-Armon et al. 2007) highlighted alternative roles of PARP-1 activation in processes governed by the MEK-ERK phosphorylation cascade. Our results indicated that polyADP-ribosylated PARP-1 and phosphorylated ERK interacted with each other by a positive feedback mechanism, enhancing and prolonging PARP-1 polyADP-ribosylation and up-regulating ERK activity in the nucleus, including phosphorylation of ERK target transcription factor Elk1 (Cohen-Armon 2007; Cohen-Armon et al. 2007). Elk1 phosphorylation up-regulated the CREB binding protein-histone acetyltransferases activity (and thereby histone acetylation) and the expression of Elk1 target genes, including immediate early genes that are implicated in LTM (Herdegen and Leah 1998; Buchwalter et al. 2004). Core histone acetylation pending on PARP-1 activation and expression of immediate early genes, c-fos, Arc, and zif pending on PARP-1 activation were both observed in electrically stimulated brain cortical neurons and in neurons briefly exposed to nerve growth factors (Cohen-Armon et al. 2007; Cohen-Armon 2008). Chromatin relaxation by polyADP-ribosylation of the PARP-1 prominent substrate histone H1 (Fig. 1a) that renders the DNA more accessible to transcription factors (Kraus and Lis 2003; Rouleau et al. 2004) may further facilitate PARP-1 up-regulation of immediate early genes expression in memory processing during learning. This mechanism may also underlie the dependence of neurite outgrowth on PARP-1 activation (Visochek et al. 2005).

Calcium-induced PARP-1 activation (Homburg et al. 2000) may also implicate polyADP-ribosylation in memory formation. This could be because of the activation of c-AMP and transcription factor cAMP-response element binding protein, both implicated in LTM formation (Herdegen and Leah 1998; Ferguson and Storm 2004; Irvine et al. 2006; Won and Silva 2008; Higashida et al. 2007).

PARP-1 activation by the neurotrophic/neuroprotective peptide (Visochek et al. 2005) that provided neuroprotection and glial protection in vitro and increased cognitive functions in vivo (Gozes et al. 2000, 2005; Gozes and Divinski 2004; Divinski et al. 2006; Gozes 2007) also associated PARP-1 activation with both neuroprotection and memory processes. In conclusion, the most substantial evidence for PARP-1 activation being involved in memory formation is the activation of PARP-1 during learning, and the fact that inhibition of polyADP-ribosylation prevents the formation of LTM both in the slug Aplysia (Cohen-Armon et al. 2004) and in mice (Figs 3 and 4). Future studies are aimed at deciphering PARP-1 pending molecular mechanisms in the chromatin that are implicated in memory processing during learning.


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This work is in partial fulfillment of the requirements for a PhD thesis of Mr. Shmuel Goldberg. This work was supported by The Israel Science Foundation, The National Institute for Psychobiology in Israel, The Israeli Ministry of Health Kurt-Lion Foundation, and the US-Israel Binational Science Foundation, The Elton Laboratory, The Lily and Avraham Gildor Chair, The Adams Super Center for Brain Studies, and The Sagol fellowships program. Professor Illana Gozes serves as the Chief Scientific Officer of Allon Therapeutics Inc.;


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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