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Published as doi: 10.1096/fj.06-7856com.
 (The FASEB Journal. 2007;21:2033-2041.)
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D-cycloserine improves functional recovery and reinstates long-term potentiation (LTP) in a mouse model of closed head injury

Rami Yaka*,1, Anat Biegon{dagger}, Nikolaos Grigoriadis{ddagger}, Constantina Simeonidou§, Savvas Grigoriadis||, Alexander G. Alexandrovich*, Henri Matzner*, Johanna Schumann*, Victoria Trembovler*, Jeanna Tsenter*,¶ and Esther Shohami*

* Department of Pharmacology, School of Pharmacy, Hebrew University, Jerusalem, Israel;

{dagger} Brookhaven National Laboratory, Upton, New York, USA;

{ddagger} Department of Neurology, AHEPA University Hospital, Greece;

§ Department of Physiology, Faculty of Medicine, Aristotle University of Thessaloniki, Greece;

|| Department of Neurosurgery, Hadassah University Hospital, Jerusalem, Israel; and

Department of Rehabilitation, Hadassah Medical Center, Jerusalem, Israel

1Correspondence: Department of Pharmacology, School of Pharmacy, The Hebrew University of Jerusalem, POB 12065, Jerusalem 91120, Israel. E-mail: yaka{at}md.huji.ac.il


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Traumatic brain injury triggers a massive glutamate efflux, activation of NMDA receptor channels, and cell death. Recently, we reported that NMDA receptors in mice are down-regulated from hours to days following closed head injury (CHI), and treatment with NMDA improved recovery of motor and cognitive functions up to 14 d post-injury. Here we show that a single injection of a low dose of D-cycloserine (DCS), a partial NMDA receptor agonist, in CHI mice 24 h post-injury, resulted in a faster and greater recovery of motor and memory functions as assessed by neurological severity score and object recognition tests, respectively. Moreover, DCS treatment of CHI mice led to a significant improvement of hippocampal long-term potentiation (LTP) in the CA1 region that was completely blunted in CHI control mice. However, DCS did not improve CHI-induced impairment in synaptic glutamate release measured by paired pulse facilitation (PPF) ratio in hippocampal CA1 region. Finally, CHI-induced reduction of brain-derived neurotrophic factor (BDNF) was fully restored following DCS treatment. Since DCS is in clinical use for other indications, the present study offers a novel approach to treat human brain injury.—Yaka, R., Biegon, A., Grigoriadis, N., Simeonidou, C., Grigoriadis, S., Alexandrovich, A. G., Matzner, H., Schumann, J., Trembovler, V., Tsenter, J., Shohami, E. D-cycloserine improves functional recovery and reinstates long-term potentiation (LTP) in a mouse model of closed head injury.


Key Words: traumatic brain injury • NMDA receptors • synaptic plasticity • BDNF


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
TRAUMATIC BRAIN INJURY (TBI) is a leading cause of mortality and morbidity among young people in the western world (1) . Yet, to date, no pharmacological agent has been approved to treat TBI patients. Traumatic and ischemic brain injury triggers a large, transient increase in glutamate efflux in the brain of experimental animals as well as humans (2 3 4 5 6 7) . A key role for glutamate activity has been ascribed to the glutamate-gated N-methyl-D-aspartate receptors (NMDAR) ion (calcium) channel. When glutamate binds at the extracellular side of the receptor along with glycine, binding as a cofactor, the channel is opened, leading to calcium influx into the cell. Excessive glutamate release in the brain (8 9 10) is implicated in excitotoxic neuronal death following brain ischemia and trauma, and numerous studies show that competitive or noncompetitive inhibitors of NMDAR improved outcome in animal models of TBI and stroke (10 11 12) . Thus, it has long been thought that hyperactivation of NMDAR underlies cognitive and neurological decline after head injury. However, the NMDAR antagonists appeared to be beneficial only within a fairly narrow therapeutic window, such that treatment initiation more than 30 min after injury was no longer protective (10 11 12) .

It is generally believed that activity-dependent synaptic processes that modify the strength of hippocampal glutamatergic synapses, known as long-term potentiation (LTP) and long-term depression (LTD), are critical for spatial learning and memory (13) . Such processes are the means by which the hippocampus can regulate the storage of information and require activation of NMDARs (14) . TBI produces chronic cognitive learning/memory deficits that are thought to be mediated, in part, by impaired hippocampal function. Experimental TBI results in a chronic inability of the hippocampal CA1 neurons to maintain synaptic plasticity and LTP (15 16 17 18) , and a significant amelioration of LTP impairment was demonstrated after treatment with cyclosporine A (19) . It is, therefore, suggested that alterations in hippocampal synaptic plasticity may be responsible for learning and memory deficits after TBI.

Recent data from diverse brain injury models, including closed head injury (CHI), middle cerebral artery occlusion, retinal exposure to glutamate, and endotoxin (LPS)-induced neuroinflammation, demonstrate a loss of NMDAR, which is evident a few hours after injury and lasts 24 h or more (20 21 22 23 24 25) . Thus, the time of onset and the duration of NMDAR hypofunction, rather than hyperfunction, are important factors determining the outcome of NMDAR blockade. These observations have prompted us to explore the effect of stimulation of NMDAR with the full agonist NMDA at 1 and 2 d after CHI. Indeed, we found that this treatment paradigm resulted in a progressive and significant amelioration of neurological and cognitive deficits, measured 14 d post-injury (20) . Thus, our results fully support the notion that NMDAR activation may prove beneficial to TBI patients.

The present study had two major goals: first, to determine whether CHI leads to impaired LTP and whether it is associated with decline cognitive functions; second, to investigate the effect of D-cycloserine (DCS), a partial agonist of the NMDAR-associated glycine site, on LTP, motor and cognitive function, and neuronal cell death. DCS was selected because it has been shown to improve cognitive function in various animal models (26 27 28 29) . DCS has a good safety profile, and it is already in use in humans in several different indications (30 31 32 33) . These properties could significantly facilitate the clinical application of DCS to human TBI patients.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and DCS treatment
Male Sabra mice, 8–10 wk old, (strain of the Hebrew University) were used in the study. Animals were maintained and treated according to the regulations of the Animal Care Committee of the Hebrew University. Food and water were provided ad libitum. D-cycloserine (Sigma, St. Louis, MO, USA) was dissolved in saline and intraperitoneally (i.p.) injected (10 mg/kg, 100 µl/10gr) 24 h post-injury. Vehicle (saline) was i.p. injected in control CHI mice.

Trauma model
CHI was induced under ether anesthesia, confirmed by loss of pupillary and corneal reflexes, using a weight-drop device as described previously (12 , 34 , 35) . Briefly, after induction of isoflurane anesthesia, a midline longitudinal incision was performed, the skin was retracted, and the skull was exposed. The left anterior frontal area was identified and a tipped TeflonTM cone was placed 1 mm lateral to the midline, in the midcoronal plane. The head was fixed and a 75 g weight was dropped on the cone from a height of 18 cm, resulting in a focal injury to the left hemisphere. After trauma, the mice received supporting oxygenation with 95% O2 for no longer than 2 min and were then brought back to their cages. Sham mice were anesthetized, their scalps were incised, but trauma was not induced. Normal body temperature was maintained throughout the whole period of anesthesia.

Neurobehavioral evaluation
Neurological recovery
Mice were evaluated by a blinded examiner, using a set of 10 tasks, collectively called Neurological Severity Score (NSS, see Fig. 1 A), which tests reflexes, alertness, coordination and motor abilities. One point is awarded for failure to perform a particular task; thus a normal mouse scores 0 (34) . NSS was evaluated at 1h (NSS1h) to define severity of injury, and then every other day during 22 d. The extent of recovery was calculated as the difference between NSS1h and that at any other time, as shown: {Delta}NSS = NSS (1h) – NSS (24h).


Figure 1
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  		Figure 1. DCS facilitates motor and cognitive functions after CHI. A) Neurological Severity Score tests reflexes, alertness, coordination, and motor abilities. One point is awarded for failure to perform a particular task; thus a normal mouse scores 0. The extent of recovery is calculated as the difference between NSS at 1 h post-injury and that at any other time: {Delta}NSS = NSS (1 h) – NSS (t). NSS at 1 h in the range of 8–10 reflects severe injury. Mice (n=18) were subjected to moderate-severe CHI (NSS1h=6.8±0.4). After 24 h, they were treated with either DCS (10 mg/kg, intraperitoneally) or vehicle (n=9/group). NSS was assessed during 22 d thereafter. {Delta}NSS = NSS (1 h) – NSS (t) represents functional recovery. *P < 0.05; #P < 0.02 vs. vehicle-treated mice, Mann-Whitney test. B) Object recognition (OR) was evaluated on the same mice (n=9/group), on days 3, 9, and 16. Four hours after baseline evaluation (baseline) the mice were reintroduced in the same cages to a novel object that replaced the "familiar" one, and the time spent near each of the objects was monitored (test). Memory impairment of the CHI+vehicle-treated mice is shown by the inability to distinguish between the novel and "familiar" object. The results of naive animals on day 3 were also included as control. DCS-treated mice regained the ability to explore the novel object from day 9 and on. *P < 0.02; vs. vehicle-treated mice, t test.

Recognition memory
Object recognition test (ORT) which tests memory function (36 , 37) was performed by a blinded evaluator on days 3, 9 and 16 after CHI as described (20) . Mice were placed in a test-cage with two identical objects and the cumulative time spent by the mouse at each of the objects was recorded during a 5 min interval. Four hours later, the mice were reintroduced into the cage, where one of the two objects was replaced by a new one, and the time spent at each of the objects was again recorded. The basic measure is the percent of the total time spent in exploring an object, whereby healthy rodents spend more time exploring a new object than a familiar, i.e., "memorized" object.

Electrophysiology
Coronal hippocampal slices (300–400 µm) were prepared from sham, CHI-vehicle or CHI-DCS male Sabra mice 16 d following CHI. Slices were maintained for at least 2 h in artificial cerebrospinal fluid (aCSF) that contained 126 mM NaCl, 1.2 mM KCl, 1.2 mM NaH2PO4, 1.2 mM MgCl2, 2.4 mM CaCl2, 18 mM NaHCO3, and 11 mM glucose saturated with 95% O2, 5% CO2 at 25°C. After recovery, slices were submerged and continuously superfused with aCSF at 25°C. Field excitatory postsynaptic potentials (fEPSPs) were recorded from stratum-radiatum of the CA1 region with glass microelectrodes filled with aCSF. Picrotoxin (100 µM) was added to the bath solution to block GABAA receptor-mediated inhibitory postsynaptic potentials (fIPSPs). To evoke fEPSPs, Schaffer collateral/commissural afferents were stimulated with 0.1-Hz pulses using steel bipolar microelectrodes at intensities adjusted to produce an evoked response that was 40–50% that of the maximum-recorded fEPSP for each recording. Following stable baseline (from which the last 10 min are shown), LTP was induced by high frequency stimulation (100 Hz, 1 s duration, 2 trains at 10 s inter train intervals) at the same intensity as the test stimulus, and synaptic responses were monitored for 50 min after LTP. For measuring paired pulse ratio (PPR), paired stimuli were given with an interstimulus interval of 50 msec, and the ratio between the second and the first amplitude of the fEPSPs was calculated. Data were collected using Multiclamp 700B amplifier (Molecular Devices, CA), filtered at 2 kHz and digitized at 5–10 kHz. Compiled data were analyzed and expressed as the mean percent of fEPSP slope ± S.E.M over the base-line levels. Results in the text and figure are presented as the mean ± SEM. Group comparisons were compared using a t test, either paired or unpaired as appropriate; P < 0.05 was taken as indicating statistical significance.

Immunohistochemistry and Western blot analysis
At 16 d after CHI, a subgroup of injured and sham-operated mice were anesthetized with a lethal dose of anesthetic and perfused via the ascending aorta with PBS followed by cold 4% paraformaldehyde in PBS. Brain tissue was postfixed in the same fixative for 24 h at 4°C and embedded in paraffin, thereafter. Coronal brain sections (6 µm thick) were serially taken at 200 µm intervals throughout the neuraxis, between –1.06 mm and –2.30 mm from bregma (38) . Correspondent slides with adhered sections were selected for various histological techniques, thereafter, in the following order: immunohistochemistry for astrocytes, microglia, BDNF, and synaptophysin.

Adjacent sections were double stained for BDNF and synapthopysin. In brief, antigen retrieval was performed in citrate buffer (pH.6) and the endogenous peroxidase was blocked with H2O2 (0.3% in PBS). Sections were then incubated in blocking buffer for 1h. Following incubation overnight at 4°C with anti-BDNF antibody (N-20, Santa Cruz), sections were incubated with goat anti-rabbit IgG, Rhodamine-conjugated (Jackson, ImmunoResearch Laboratories, West Grove, PA USA), as a secondary antibody. Following intensive washes, sections were incubated overnight at 4°C with antisynaptophysin mouse monoclonal antibody (Clone SY38, DakoCytomation, Denmark) and goat anti-mouse IgG, FITC – conjugated (Jackson, ImmunoResearch Laboratories), as a secondary antibody, thereafter. In a number of sections, either the first or the second primary antibody was omitted for the detection of non-specific binding of either secondary antibody used.

A series of adjacent sections were then treated with primary antibody against glial fibrillary acidic protein (GFAP) (DakoCytomation, Denmark) and then with goat anti-rabbit (Vector, Burlingame, CA, USA) as secondary antibody. Immunoreactions were visualized with the avidin–biotin complex (Vectastain) and the peroxidase reaction was visualized with diaminobenzidine (DAB) (Vector) as chromogen. In adjacent sections, endogenous biotin blocking was performed by using the blocking Kit Avidin/Biotin (Vector) and sections were incubated with Lycopersicon esulentum tomato lectin (Sigma) which stains microglia and macrophages. The immunoreactions were visualized with LSAB-2 System-HRP (DakoCytomation, Denmark) and DAB (Vector) was used as chromogen. Sections were finally counterstained with hematoxylin. For all captured images, each square subdivision was counted if it contained at least one labeled element, defined as a labeled cell body and/or its processes. The tissue section, which exhibited the greatest number of either BDNF or synaptophysin positive structures, was selected for statistical analysis (39) . The scores of the areas studied represented subjective assessments of fluorescence intensity and numbers of labeled cells, as follows: 0: no labeling observed in any square subdivision; 1 (+): 1–10 square subdivisions positive; 2 (++): 11–20 square subdivisions positive; 3 (+++): >20 square subdivisions positive. The scores either for astrogliosis or microglia represented subjective assessments of staining intensity and numbers of labeled cells, as follows: 0: no labeling observed; 1 (+): small number of cells that are only weakly labeled; 2 (++): moderate number of cells that are clearly labeled; 3 (+++): large number of intensely labeled cells present (40) . Two independent observers performed all quantitative assessments. In cases where significant discrepancies were seen between the two observers, the evaluation was repeated by a third one. Sections were examined under either light or fluorescent microscope (Zeiss Axioplan 2, Thornwood, NY, USA).

For Western blot analysis, 16 d post CHI, mice were decapitated, the brains removed, and the hippocampus rapidly dissected and frozen in liquid nitrogen. The tissues were homogenized in homogenization buffer containing 320 mM sucrose, 1% SDS, 10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM EGTA and protease inhibitor cocktail (Sigma). Homogenates were centrifuged at 1000g for 5 min to remove nuclei and large debris and the supernatant and the amounts of protein determined by BCA (Pierce). Equal amounts of protein (50 µg/lane) were separated on 15% SDS-PAGE and transferred to a nitrocellulose membrane. The membranes were probed with anti-BDNF (Chemicon, Temecula, CA, USA), and anti-tubulin (Sigma). Immunoreactivity was detected with enhanced chemiluminescence (Pierce, Rockford, IL, USA) and processed using the NIH Image software.

Statistical analysis
Data are expressed as mean ± SEM. A commercial statistics package (Graph Pad Prism version 3.03) was used for determining statistical significance. Significance was determined using the nonparametric Mann-Whitney test for {Delta}NSS values and one way analysis of variance (ANOVA) followed by student’s t tests for ORT results. Paired pulse ratio was compared using Student’s t test. Histological and immunohistochemical data were semi quantitative and statistics were used for original data sets. Thus, where appropriate, the Pearson’s {chi}2 or Fisher’s exact test were applied as mentioned in the text. Analysis of the differences in BDNF levels (densitometric scans normalized to tubulin) from Western blot was performed by Student’s t test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
DCS facilitates recovery of motor function
One hour after CHI the NSS was evaluated (see Fig. 1A ) and mice were randomized into two groups such that severity of injury was comparable between the vehicle- and DCS-treated mice. Mice with similar initial NSS (mean ± SEM 6.78±0.40, and 6.99±0.39; n=9/group) were then assigned to receive vehicle (saline) or DCS, respectively. Prior to treatment initiation, 24 h after CHI, NSS of both groups was also similar, as expected (6.56±0.29 and 6.78±0.36, respectively). Neurological recovery was followed for 3 more weeks, and the trend toward better recovery of the DCS-treated group, seen at day 8, became highly significant at day 15 and 22 (15 d P < 0.05; 22 d P < 0.02 vs. vehicle treated mice; Fig. 1A ).

DCS facilitates recovery of cognitive function
DCS treated mice also performed significantly better than vehicle treated controls in the object recognition test from day 9 and at 16 and 23 d after CHI, at a dose which had no effect on performance in naive animals. Both groups of mice spent a similar proportion (~50%) of time exploring two identical objects in an observation cage at baseline. Four hours after baseline exposure to the identical objects, the mice were reintroduced into the cage in which one of the two "old" objects was replaced by a novel object, similar in size, color and material to the original one. The vehicle-treated CHI mice spent a similar proportion of their time with the old and new object (Fig. 1B ), with no significant preference for the novel object, while the DCS-treated CHI mice increased their exploration of the novel, compared to the familiar object, such that at day 3 a trend (P=0.1) toward longer exploration time was already noted. From day 9 and on, the DCS-treated mice spent the same proportion of the exploration time at the novel object (~75%) as intact untreated mice, significantly longer than the vehicle-treated animals (Fig. 1B ; P<0.02 vs. vehicle-treated mice).

Impaired LTP following CHI was recovered in DCS-injected mice
Previous studies have shown that TBI results in alteration of synaptic plasticity in the hippocampus that may be responsible for TBI-induced deficits in learning and memory (15 , 16 , 18 , 41 42 43) . If the impairment in the object recognition test following CHI and the improved ability of CHI mice to perform this task after DCS injection (Fig. 1B ) are due to changes in synaptic plasticity, it is likely that hippocampal long term potentiation (LTP) after CHI will be impaired and that injection of DCS will recover the impaired LTP. Field excitatory post synaptic potentials (fEPSPs) were recorded in the CA1 region of the hippocampus from slices that were prepared from animals 16 d post DCS injection, when maximal recovery in the object recognition test was observed (Fig. 1B ). Recordings were made in sham mice, CHI mice that were injected with vehicle (CHI+Vehicle) and CHI mice that were injected with DCS (CHI+DCS). When a stable base line was established (Fig. 2 A, traces 1, 3 and 5), LTP was induced by tetanic stimulation of afferent fibers (Fig. 2B ). The magnitude of LTP in sham slices was 35% ± 0.05 SEM while in CHI slices we observed no potentiation (Fig. 2A,B traces 1,2 vs. 5,6). However, in CHI+DCS slices the magnitude of potentiation recovered to 19% ± 0.08. Thus, LTP that was blunted in CHI mice was partially recovered in DCS injected mice. Although the recovery in the magnitude of LTP in DCS injected mice is most likely due to an enhancement of NMDAR activity by DCS, it is also possible that a change in the presynaptic transmitter release probability may be involved in the modulation of LTP. We thus examined the paired-pulse facilitation (PPF) to assess whether CHI may affect the presynaptic release mechanisms. PPF induced at an interstimulus interval of 50 msec was significantly different between sham and CHI+Vehicle mice or sham and CHI+DCS mice (Fig. 2C ; sham 1.45±0.03; CHI+Vehicle 0.84±0.01; P < 0.001, CHI+DCS mice (sham 1.45±0.03; CHI+DCS 0.93±0.02; P<0.001, t test), suggesting that presynaptic release probability is affected by CHI but DCS has no effect on the alterations in presynaptic release probability.


Figure 2
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  		Figure 2. Impaired hippocampal LTP in CHI mice was improved by DCS. A) Sample traces of fEPSPs recorded in the CA1 region of hippocampal slices prepared 16 d post-injury from sham, CHI+Vehicle or CHI+DCS mice at the time indicated in (B). The stimulus artifacts are truncated. B) The averaged time course of LTP in sham (n=10 slices, 5 mice), CHI+Vehicle (n=12 slices, 7 mice) and CHI+DCS (n=13 slices, 7 mice) mice. Initial fEPSPs slopes were measured, and the values were normalized in each experiment using the averaged slope value measured during the control period (time, –10 to 0 min). Tetanic stimulation (100 Hz, 1 s, 2 trains 10 s apart) was applied at time 0. C) paired pulse facilitation (PPF) of synaptic responses in the CA1 region was induced by delivering afferent fiber stimulation twice at an interstimulus interval of 50 msec at stimulus intensity of 50% of maximal response. The facilitation ratios were calculated by dividing the amplitude of the second fEPSPs by that of the first fEPSPs. Ten consecutive traces were averaged from each slice, and the averaged amplitude from all slices is presented as mean ± SEM (sham, 4 mice, 12 slices; CHI+Vehicle, 5 mice, 13 slices; CHI+DCS, 4 mice, 16 slices). There was a significant difference in PPF between the sham mice and CHI+Vehicle (sham 1.45±0.03; CHI+PBS 0.84±0.01; P<0.001, t test) or CHI+DCS mice (sham 1.45±0.03; CHI+DCS 0.93±0.02; P<0.001, t test).

Immunohistochemistry of CA1 hippocampal area
To determine whether DCS affected neuronal or glial cell number or morphology, neuronal degeneration was assessed with FluoroJade B, glial activation with glial fibrillary acidic protein (GFAP) and microglia with lectin in animals sacrificed 16 d post CHI. CHI induced over the frontal cortex, did not lead to neurodegenration at the CA1 area of the injured hippocampus when compared to the contralateral control side (data not shown). DCS treatment had also no effect on neuronal cell morphology either at the injured or contralateral control CA1 area, both in injured and sham operated animals. However, activated astrocytes were detected at the injured CA1 area in both groups of injured animals (Fig. 3 C,D), and DCS did not induce any significant change either in their number or morphology. Activated microglia cells were not detected at the CA1 area in all groups of animals (Fig. 3A,B ). These results suggest that DCS-induced alterations did not involve cell death.


Figure 3
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  		Figure 3. DCS does not affect CHI-induced activation of astrocytes and microglia at the CA1 area. Sixteen days after CHI, mice were sacrificed, and their brains processed for immunohistochemistry as described in the methods. Microglia, stained with lectin (A, B) and activated astrocytes, stained with GFAP (C, D) are shown at the CA1 area of the hippocampus of the injured hemisphere in DCS (A, C) and vehicle (B, D) treated animals. No difference could be detected in the number or distribution of either cellular type between the two groups of animals. Scale bar = 100 µm.

DCS ameliorates CHI-induced reduction of BDNF but not synaptophysin
Since stimulation of NMDAR was shown to increase release of brain-derived neurotrophic factor (BDNF) from hippocampal neurons in culture, and this trophic factor plays a critical role in long-term potentiation (LTP) and affects neuronal survival (44) , we stained CA1 area taken from mice 16 d post CHI for BDNF (Fig. 4 Ad, g). In addition, we stained the same area for synaptophysin, a major integral transmembrane protein of synaptic vesicles, which serves as a functional marker of the brain (Fig. 4Ab, e, h ) and the nuclear marker DAPI (Fig. 4Ac, f, i ). Both BDNF expression and synaptophysin immunoreactive presynaptic buttons were dramatically reduced in the injured CA1 areas in either group of animals. However, the number of BDNF positive cells was significantly higher following DCS treatment (Fig. 4Ag ; P<0.005 vs. vehicle-treated mice), whereas no significant preservation of the synaptophysin immunoreactivity was detected (Fig. 4Ah ). To further support our immunohistochemical findings quantitatively, we performed Western blot analysis on hippocampal homogenates prepared from 16 d post CHI mice and determined the levels of BDNF using specific antibodies. As shown in Fig. 4B , DCS restored the decrease in protein levels of BDNF found in vehicle-treated mice, almost to the levels found in sham untreated mice (P<0.05 vs. vehicle treated mice). Taken together, these results suggest that DCS restored CHI-induced reduction of BDNF but did not affect the reduction of synaptophysin, and are in line with our electrophysiological data.


Figure 4
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  		Figure 4. DCS restored CHI-induced reduction in BDNF. A) Sixteen days after CHI, mice were sacrificed, and their brains processed for immunohistochemistry as described in the methods. BDNF (a, d, g) and synaptophysin (b, e, h see white arrows) immunoreactivity at the sham-operated (a–c) and injured (d–i) CA1 area (c, f, i the corresponding nuclei of the cells stained with DAPI) in sham (c), CHI+Vehicle (f) and CHI+DCS (i) treated animals. DCS preserved a significantly higher number of BDNF-positive cells following CHI, whereas the reduced synaptophysin immunoreactivity was not similarly affected. Histogram depicts the level of fluorescence intensities of BDNF immunoreactivity in the CA1 hippocampal region of CHI+Vehicle or CHI+DCS treated animals quantified as described in the methods. P < 0.0001, {chi}2 test. B) Total homogenates (50 µgr/lane) were prepared from 16 d old sham (n=3), CHI+Vehicle (n=4) and CHI+DCS (n=4) mice, and resolved on 15% SDS-PAGE. The membranes were probed with anti-BDNF and antitubulin antibodies to control equal protein loading. Histogram depicts the levels of BDNF normalized to tubulin and presented as the mean ± SEM percentage of sham control (*P<0.01, 53.9±9.1%, significantly lower from sham controls, #P<0.01, 95.17±9.1%, significantly higher from vehicle treated mice, t-test).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results of the present study show, for the first time to our knowledge, that long-term potentiation (LTP) of synaptic transmission in the hippocampus, which is markedly impaired after CHI can be rescued by DCS, a coagonist of the NMDAR, when given as a single dose even as late as 24 h post-injury. The effect of DCS involves post-synaptic modifications since DCS did not affect the impairment of synaptic release reflected by paired pulse depression seen in CHI mice. The improved LTP was associated with recovery of cognitive function, pointing to the role of declined LTP in impaired cognition after CHI. In addition, DCS treatment significantly facilitated the recovery of motor functions and restored the decreased levels of the neurotrophic factor BDNF.

The present study is based on two observations, made in our previous report (20) , namely: 1) A progressive decrease in the functional NMDAR density occurs between 1 and 24 h post-injury and lasts for at least a week. 2) Administration of the NMDAR agonist NMDA to mice, 1 and 2 d after injury, significantly improves general neurological and cognitive function up to 2 wk. These observations led us to challenge the dogma that hyperactivation of the NMDAR underlies cognitive and neurological impairments after head injury and to propose that stimulation, rather than inhibition of NMDARs in the subacute post-injury phase, will be beneficial to head-injured patients. The present results, thus, not only corroborate our findings on the benefit of NMDAR activation as a treatment, they also point to a possible clinical application of a drug, currently used as "cognitive enhancer" in patients with schizophrenia or Alzheimer’s diseases (30 , 32) . The current findings fully agree with those of Temple and Hamm (1996), who showed that daily injections of DCS during 15 d post-injury ameliorated TBI-associated cognitive deficits (26) .

Earlier reports demonstrated that TBI results in a chronic inability of the CA1 hippocampal synapses to maintain synaptic plasticity (15 16 17 18 19) . Similarly, the present results show that mice, failing to perform in the ORT, also display impaired LTP when recorded in hippocampal slices isolated 14–16 d after injury. Interestingly, a single dose of DCS, at 24 h after injury, a time of maximal down-regulation of the NMDAR, was sufficient to activate the receptors such as to ameliorate the cognitive function (in vivo) as well as the LTP (in vitro), assessed 2 wk later. It is well established that the induction of hippocampal long-term potentiation (LTP), the leading cellular model of learning and memory, largely depends on NMDAR activation (14) . Therefore, it is reasonable to assume that the recovery in LTP following DCS injection reflects the improvement in the functionality of NMDARs and can be thought as the underlying molecular basis for the improved hippocampal-related cognitive function. As was previously reported using a fluid percussion injury model (45) , TBI resulted in impairment in synaptic release machinery measured by PPR 14 d following injury. Further, TBI induced a remarkable decrease in the presynaptic protein synaptophysin, a synaptic vesicle membrane protein that participate in the fusion of synaptic vesicles to the presynaptic membrane (46) . However, in DCS injected mice both PPR and the levels of synaptophysin remained unchanged, suggesting that DCS effects are mainly postsynaptic. However, since the magnitude of LTP measured in DCS treated mice did not recover to control sham levels, we cannot exclude the possibility that damage to the presynaptic inputs contributes to the impaired LTP in CA1, which results in a partial recovery of LTP in DCS injected mice. Further, LTP, as indicated by a change in the fEPSP slope, can occur from several mechanisms, which are NMDAR-independent, and may be augmented by other factors. Therefore, we are currently conducting an in depth investigation to reveal the underlying molecular mechanism by which DCS leads to the recovery of LTP.

Brain derived-neurotrophic factor (BDNF) belongs to the neurotrophin family and regulates the survival, differentiation, and maintenance of function in different neuronal populations (44 , 47) . BDNF is a critical regulator of transcription-dependent adaptive neuronal responses, such as LTP (48 , 49) . Earlier studies have shown that activation of NMDARs lead to increased expression of BDNF (50 , 51) . This effect appears to involve neuronal rather than astrocytic release of BDNF (52 , 53) . Therefore, the results of the present study raise the following hypothesis as for the consequences of DCS injection; first, DCS activates NMDARs and as a result improves channel function, either by increasing the number of channels on the membrane or increasing the activity of existing channels, or both. Second, increased NMDARs channel activity leads to an increase in BDNF levels, which initiates a positive feedback loop to further enhance the function of hippocampal NMDARs.

In conclusion, we propose that LTP is one of the mechanisms through which cognitive performance is impaired after CHI, and that activation of NMDAR at the subacute phase may rescue their function. The extended loss of NMDAR function provides a therapeutic window of at least 24 h for the treatment of cognitive and neurological deficits after head injury and may offer benefit to brain-injured patients.


   ACKNOWLEDGMENTS
 
The authors would like to thank Mrs. Olga Touloumi for her technical assistance. This work was supported by the National Institute of Health grant R01 NS 050285-01 A2 (A.B.)

Received for publication December 14, 2006. Accepted for publication January 25, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Waxweiler, R. J., Thurman, D., Sniezek, J., Sosin, D., O’Neil, J. (1995) Monitoring the impact of traumatic brain injury: a review and update. J. Neurotrauma 12,509-516[Medline]
  2. Benveniste, H., Drejer, J., Schousboe, A., Diemer, N. H. (1984) Elevation of the extracellular concentrations of glutamate and aspartate in rat hippocampus during transient cerebral ischemia monitored by intracerebral microdialysis. J. Neurochem. 43,1369-1374[Medline]
  3. Bullock, R., Zauner, A., Woodward, J., Young, H. F. (1995) Massive persistent release of excitatory amino acids following human occlusive stroke. Stroke 26,2187-2189[Abstract/Free Full Text]
  4. Bullock, R., Zauner, A., Myseros, J. S., Marmarou, A., Woodward, J. J., Young, H. F. (1995) Evidence for prolonged release of excitatory amino acids in severe human head trauma. Relationship to clinical events. Ann. N. Y. Acad. Sci. 765,290-297; discussion 298[Medline]
  5. Davalos, A., Castillo, J., Serena, J., Noya, M. (1997) Duration of glutamate release after acute ischemic stroke. Stroke 28,708-710[Abstract/Free Full Text]
  6. Globus, M. Y., Busto, R., Dietrich, W. D., Martinez, E., Valdes, I., Ginsberg, M. D. (1988) Effect of ischemia on the in vivo release of striatal dopamine, glutamate, and gamma-aminobutyric acid studied by intracerebral microdialysis. J. Neurochem. 51,1455-1464[Medline]
  7. Nilsson, P., Hillered, L., Ponten, U., Ungerstedt, U. (1990) Changes in cortical extracellular levels of energy-related metabolites and amino acids following concussive brain injury in rats. J. Cereb. Blood Flow Metab. 10,631-637[Medline]
  8. Wallace, M. C., Teasdale, G. M., McCulloch, J. (1992) Autoradiographic analysis of 3H-MK-801 (dizocilpine) in vivo uptake and in vitro binding after focal cerebral ischemia in the rat. J. Neurosurg. 76,127-133[Medline]
  9. Owens, J., Tebbutt, A. A., McGregor, A. L., Kodama, K., Magar, S. S., Perlman, M. E., Robins, D. J., Durant, G. J., McCulloch, J. (2000) Synthesis and binding characteristics of N-(1-naphthyl)-N'-(3-[(125)I]-iodophenyl)-N'-methylguanidine ([(125)I]-CNS 1261): a potential SPECT agent for imaging NMDA receptor activation. Nucl. Med. Biol. 27,557-564[CrossRef][Medline]
  10. Di, X., Bullock, R. (1996) Effect of the novel high-affinity glycine-site N-methyl-D-aspartate antagonist ACEA-1021 on 125I-MK-801 binding after subdural hematoma in the rat: an in vivo autoradiographic study. J. Neurosurg. 85,655-661[Medline]
  11. Kroppenstedt, S. N., Schneider, G. H., Thomale, U. W., Unterberg, A. W. (1998) Protective effects of aptiganel HCl (Cerestat) following controlled cortical impact injury in the rat. J. Neurotrauma 15,191-197[Medline]
  12. Chen, M., Bullock, R., Graham, D. I., Frey, P., Lowe, D., McCulloch, J. (1991) Evaluation of a competitive NMDA antagonist (D-CPPene) in feline focal cerebral ischemia. Ann. Neurol. 30,62-70[CrossRef][Medline]
  13. Bliss, T. V., Collingridge, G. L. (1993) A synaptic model of memory: long-term potentiation in the hippocampus. Nature 361,31-39[CrossRef][Medline]
  14. Malenka, R. C., Nicoll, R. A. (1999) Long-term potentiation—a decade of progress?. Science 285,1870-1874[Abstract/Free Full Text]
  15. Sanders, M. J., Sick, T. J., Perez-Pinzon, M. A., Dietrich, W. D., Green, E. J. (2000) Chronic failure in the maintenance of long-term potentiation following fluid percussion injury in the rat. Brain Res. 861,69-76[CrossRef][Medline]
  16. Sick, T. J., Perez-Pinzon, M. A., Feng, Z. Z. (1998) Impaired expression of long-term potentiation in hippocampal slices 4 and 48 h following mild fluid-percussion brain injury in vivo. Brain Res. 785,287-292[CrossRef][Medline]
  17. D’Ambrosio, R., Maris, D. O., Grady, M. S., Winn, H. R., Janigro, D. (1998) Selective loss of hippocampal long-term potentiation, but not depression, following fluid percussion injury. Brain Res. 786,64-79[CrossRef][Medline]
  18. Schwarzbach, E., Bonislawski, D. P., Xiong, G., Cohen, A. S. (2006) Mechanisms underlying the inability to induce area CA1 LTP in the mouse after traumatic brain injury. Hippocampus 16,541-550[CrossRef][Medline]
  19. Albensi, B. C., Alasti, N., Mueller, A. L. (2000) Long-term potentiation in the presence of NMDA receptor antagonist arylalkylamine spider toxins. J. Neurosci. Res. 62,177-185[CrossRef][Medline]
  20. Biegon, A., Fry, P. A., Paden, C. M., Alexandrovich, A., Tsenter, J., Shohami, E. (2004) Dynamic changes in N-methyl-D-aspartate receptors after closed head injury in mice: Implications for treatment of neurological and cognitive deficits. Proc. Natl. Acad. Sci. U. S. A. 101,5117-5122[Abstract/Free Full Text]
  21. Biegon, A., Alvarado, M., Budinger, T. F., Grossman, R., Hensley, K., West, M. S., Kotake, Y., Ono, M., Floyd, R. A. (2002) Region-selective effects of neuroinflammation and antioxidant treatment on peripheral benzodiazepine receptors and NMDA receptors in the rat brain. J. Neurochem. 82,924-934[CrossRef][Medline]
  22. Grossman, R., Shohami, E., Alexandrovich, A., Yatsiv, I., Kloog, Y., Biegon, A. (2003) Increase in peripheral benzodiazepine receptors and loss of glutamate NMDA receptors in a mouse model of closed head injury: a quantitative autoradiographic study. Neuroimage 20,1971-1981[CrossRef][Medline]
  23. Miller, L. P., Lyeth, B. G., Jenkins, L. W., Oleniak, L., Panchision, D., Hamm, R. J., Phillips, L. L., Dixon, C. E., Clifton, G. L., Hayes, R. L. (1990) Excitatory amino acid receptor subtype binding following traumatic brain injury. Brain Res. 526,103-107[CrossRef][Medline]
  24. Sihver, S., Marklund, N., Hillered, L., Langstrom, B., Watanabe, Y., Bergstrom, M. (2001) Changes in mACh, NMDA and GABA(A) receptor binding after lateral fluid-percussion injury: in vitro autoradiography of rat brain frozen sections. J. Neurochem. 78,417-423[CrossRef][Medline]
  25. Friedman, W. J. (2001) Cytokines regulate expression of the type 1 interleukin-1 receptor in rat hippocampal neurons and glia. Exp. Neurol. 168,23-31[CrossRef][Medline]
  26. Temple, M. D., Hamm, R. J. (1996) Chronic, post-injury administration of D-cycloserine, an NMDA partial agonist, enhances cognitive performance following experimental brain injury. Brain Res. 741,246-251[CrossRef][Medline]
  27. Andersen, J. M., Lindberg, V., Myhrer, T. (2002) Effects of scopolamine and D-cycloserine on non-spatial reference memory in rats. Behav. Brain Res. 129,211-216[CrossRef][Medline]
  28. Lelong, V., Dauphin, F., Boulouard, M. (2001) RS 67333 and D-cycloserine accelerate learning acquisition in the rat. Neuropharmacology 41,517-522[CrossRef][Medline]
  29. Schneider, J. S., Tinker, J. P., Van Velson, M., Giardiniere, M. (2000) Effects of the partial glycine agonist D-cycloserine on cognitive functioning in chronic low dose MPTP-treated monkeys. Brain Res. 860,190-194[CrossRef][Medline]
  30. Duncan, E. J., Szilagyi, S., Schwartz, M. P., Bugarski-Kirola, D., Kunzova, A., Negi, S., Stephanides, M., Efferen, T. R., Angrist, B., Peselow, E., Corwin, J., Gonzenbach, S., Rotrosen, J. P. (2004) Effects of D-cycloserine on negative symptoms in schizophrenia. Schizophr. Res. 71,239-248[CrossRef][Medline]
  31. Heresco-Levy, U., Kremer, I., Javitt, D. C., Goichman, R., Reshef, A., Blanaru, M., Cohen, T. (2002) Pilot-controlled trial of D-cycloserine for the treatment of post-traumatic stress disorder. Int. J. Neuropsychopharmacol. 5,301-307[CrossRef][Medline]
  32. Laake, K., Oeksengaard, A. R. (2002) D-cycloserine for Alzheimer’s disease. Cochrane Database Syst. Rev. ,CD003153
  33. Nitsche, M. A., Jaussi, W., Liebetanz, D., Lang, N., Tergau, F., Paulus, W. (2004) Consolidation of human motor cortical neuroplasticity by D-cycloserine. Neuropsychopharmacology 29,1573-1578[CrossRef][Medline]
  34. Beni-Adani, L., Gozes, I., Cohen, Y., Assaf, Y., Steingart, R. A., Brenneman, D. E., Eizenberg, O., Trembolver, V., Shohami, E. (2001) A peptide derived from activity-dependent neuroprotective protein (ADNP) ameliorates injury response in closed head injury in mice. J. Pharmacol. Exp. Ther. 296,57-63[Abstract/Free Full Text]
  35. Yatsiv, I., Morganti-Kossmann, M. C., Perez, D., Dinarello, C. A., Novick, D., Rubinstein, M., Otto, V. I., Rancan, M., Kossmann, T., Redaelli, C. A., Trentz, O., Shohami, E., Stahel, P. F. (2002) Elevated intracranial IL-18 in humans and mice after traumatic brain injury and evidence of neuroprotective effects of IL-18-binding protein after experimental closed head injury. J. Cereb. Blood Flow Metab. 22,971-978[CrossRef][Medline]
  36. Ennaceur, A., Delacour, J. (1988) A new one-trial test for neurobiological studies of memory in rats. 1: Behavioral data. Behav. Brain Res. 31,47-59[CrossRef][Medline]
  37. Ennaceur, A., Michalikova, S., Bradford, A., Ahmed, S. (2005) Detailed analysis of the behavior of Lister and Wistar rats in anxiety, object recognition and object location tasks. Behav. Brain Res. 159,247-266[CrossRef][Medline]
  38. Paxinos, G., Franklin, K. B. J. (2000) The mouse brain in stereotaxic coordinates Academic San Diego, CA, USA.
  39. Sato, M., Chang, E., Igarashi, T., Noble, L. J. (2001) Neuronal injury and loss after traumatic brain injury: time course and regional variability. Brain Res. 917,45-54[CrossRef][Medline]
  40. Simic, G., Lucassen, P. J., Krsnik, Z., Kruslin, B., Kostovic, I., Winblad, B., Bogdanovi, (2000) nNOS expression in reactive astrocytes correlates with increased cell death related DNA damage in the hippocampus and entorhinal cortex in Alzheimer’s disease. Exp. Neurol. 165,12-26[CrossRef][Medline]
  41. Albensi, B. C., Sullivan, P. G., Thompson, M. B., Scheff, S. W., Mattson, M. P. (2000) Cyclosporin ameliorates traumatic brain-injury-induced alterations of hippocampal synaptic plasticity. Exp. Neurol. 162,385-389[CrossRef][Medline]
  42. Miyazaki, S., Katayama, Y., Lyeth, B. G., Jenkins, L. W., DeWitt, D. S., Goldberg, S. J., Newlon, P. G., Hayes, R. L. (1992) Enduring suppression of hippocampal long-term potentiation following traumatic brain injury in rat. Brain Res. 585,335-339[CrossRef][Medline]
  43. Reeves, T. M., Lyeth, B. G., Povlishock, J. T. (1995) Long-term potentiation deficits and excitability changes following traumatic brain injury. Exp. Brain Res. 106,248-256[Medline]
  44. Sharma, H. S. (2006) Post-traumatic application of brain-derived neurotrophic factor and glia-derived neurotrophic factor on the rat spinal cord enhances neuroprotection and improves motor function. Acta Neurochir. Suppl. 96,329-334[Medline]
  45. Cao, R., Hasuo, H., Ooba, S., Akasu, T., Zhang, X. (2006) Facilitation of glutamatergic synaptic transmission in hippocampal CA1 area of rats with traumatic brain injury. Neurosci Lett. 401,136-141[CrossRef][Medline]
  46. Thiel, G. (1993) Synapsin I, synapsin II, and synaptophysin: marker proteins of synaptic vesicles. Brain Pathol. 3,87-95[Medline]
  47. Bibel, M., Barde, Y. A. (2000) Neurotrophins: key regulators of cell fate and cell shape in the vertebrate nervous system. Genes Dev. 14,2919-2937[Free Full Text]
  48. Korte, M., Carroll, P., Wolf, E., Brem, G., Thoenen, H., Bonhoeffer, T. (1995) Hippocampal long-term potentiation is impaired in mice lacking brain-derived neurotrophic factor. Proc. Natl. Acad. Sci U. S. A. 92,8856-8860[Abstract/Free Full Text]
  49. Korte, M., Staiger, V., Griesbeck, O., Thoenen, H., Bonhoeffer, T. (1996) The involvement of brain-derived neurotrophic factor in hippocampal long-term potentiation revealed by gene targeting experiments. J. Physiol. Paris 90,157-164[CrossRef][Medline]
  50. Takasu, M. A., Dalva, M. B., Zigmond, R. E., Greenberg, M. E. (2002) Modulation of NMDA receptor-dependent calcium influx and gene expression through EphB receptors. Science 295,491-495[Abstract/Free Full Text]
  51. Yaka, R., He, D. Y., Phamluong, K., Ron, D. (2003) Pituitary adenylate cyclase-activating polypeptide (PACAP(1–38)) enhances N-methyl-D-aspartate receptor function and brain-derived neurotrophic factor expression via RACK1. J. Biol. Chem. 278,9630-9638[Abstract/Free Full Text]
  52. Zafra, F., Castren, E., Thoenen, H., Lindholm, D. (1991) Interplay between glutamate and gamma-aminobutyric acid transmitter systems in the physiological regulation of brain-derived neurotrophic factor and nerve growth factor synthesis in hippocampal neurons. Proc. Natl. Acad. Sci. U. S. A. 88,10037-10041[Abstract/Free Full Text]
  53. Zafra, F., Lindholm, D., Castren, E., Hartikka, J., Thoenen, H. (1992) Regulation of brain-derived neurotrophic factor and nerve growth factor mRNA in primary cultures of hippocampal neurons and astrocytes. J. Neurosci. 12,4793-4799[Abstract]




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